Egypt. J. Lab. Med. Vol.(27) No.1&2, February/June , 2015

THE EGYPTIAN JOURNAL OF LABORATORY MEDICINE “DAR EL HEKMA”, 42, Kasr El‐Eini Street, Cairo, Egypt

PUBLISHED BY THE EGYPTIAN SOCIETY OF LABORATORY MEDICINE

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Clinical Chemistry Mohga Zewar

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STUDY OF SERUM HEPCIDIN AS A DIAGNOSTIC TOOL OF IRON OVERLOAD IN

EGYPTIAN CHILDREN WITH BETA-THALASSEMIA MAJOR AND BETA-THALASSEMIA

INTERMEDIA

Ahmed Maher Kaddah, Amina Abdel-Salam, Marwa Salah Farhan and Reham Ragab................................

ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISM OF SPARC GENE WITH

INCREASED RISK OF HEPATOCELLULAR CARCINOMA

Dina Hisham Ahmed Soliman.........................................................................................................................

ASSOCIATION OF MONOCYTE CHEMOATTRACTANT PROTEIN 1 (MCP-1) GENE

POLYMORPHISM WITH LUPUS NEPHRITIS IN EGYPTIAN PATIENTS

Lamiaa A. Mohammad, Dina M. Atef and Amany Mustafa Abul-Saoud.......................................................

STEADY-STATE SERUM LEVELS OF VASCULAR CELL ADHESION MOLECULE-1 IN

CHILDREN WITH SICKLE CELL DISEASE:RELEVANCE TO STROKE RISK AND DISEASE

SEVERITY.

Mona Hassan El-Tagui, Mona El- Ghamrawy, Foad Abd Allah, Fadwa Said

and Ahmed Ibrahim.........................................................................................................................................

ASSOCIATION BETWEEN MDM2-SNP309 POLYMORPHISM AND TUMOUR SIZE, SITE

AND NUMBER IN HEPATOCELLULAR CARCINOMA

Noha Kamal Abdel Moamen, Hany Hussien El Sied, Marianne Fathy Ishak, Moataz Syam, Maha

Hasaballa, Afaf Ahmed Abdelhadi and Hebat Allah Mohamed Sharaf El Deen............................................

MDM2 PROMOTER POLYMORPHISM AND ITS ASSOCIATION WITH

HEPATOCELLULAR CARCINOMA IN EGYPTIAN POPULATION WITH CHRONIC VIRAL

HEPATITIS

Noha Kamal Abdel Moamen, Hany Hussien El Sied, Marianne Fathy Ishak, Moataz Syam, Maha

Hasaballa, Nihal moustafa Safwat El Assaly, Afaf Ahmed Abdelhadi and Hebat Allah Mohamed Sharaf

El Deen ............................................................................................................................................................

EVALUATION OF OSTEOPONTIN AND ALPHA L-FUCOSIDASE AS DIAGNOSTIC

MARKERS OF HEPATOCELLULAR CARCINOMA

Hanaa H. Elsaid, Lamiaa Mamoud Kamel , Abd el Motaleb Mohamed and Reham Ahmed.........................

SERUM MATRIX METALLOPROTEINASE-9 (MMP-9), TISSUE INHIBITOR OF

METALLOPROTEINASE-1(TIMP-1) AND TRANSFORMING GROWTH FACTOR-β1 (TGF-

β1) IN BRONCHIAL ASTHMA

Neveen Abd El-Moneum Hassan, Aliaё Abd-Rabou Mohamed-Hussein, Ebtsam F. Mohammed, Omnia

Abd El-Moneum Mohamed and Manal Mohamed Tammam Mahmoud .......................................................

THE EFFECT OF UGT1A6 POLYMORPHIC GENETIC VARIANTS ON THE CLINICAL

RESPONSE TO VALPROIC ACID IN EGYPTIAN CHILDREN WITH IDIOPATHIC EPILEPSY

Mohamad el-Sawy, Osama Ahmed Badary, Manal Zaghloul Mahran, Sahar Mohamed Ahmed Hassanein,

Manal El Hamamsy, Nesrine Aly Mohamed and Engi Abdel-Hady...............................................................

NON-INVASIVE METHOD FOR THE ASSESSMENT OF LIVER FIBROSIS AND PREDICTION

OF HEPATOCELLULAR CARCINOMA IN PATIENTS WITH CHRONIC ACTIVE

HEPATITIS

Nevine E. El-Abd, Sherihan H. El-Kady and Mohamed A. El-Attiq..............................................................

HAS PERIODONTAL THERAPY A ROLE ON THE EXPRESSION OF THE RECEPTOR OF

ADVANCED GLYCATION END PRODUCTS (RAGE) IN GINGIVAL TISSUES AMONG

PATIENTS WITH TYPE 2 DIABETES MELLITUS?

Nagwa Abdel Hamid Osman, Mona Salah El Din Darhous, Ashraf Ismail, Dina M. Rasheed Bahgat,

Rania Khalifa ..................................................................................................................................................

EFFECT OF MESENCHYMAL STEM CELLS ON CD4, CD8 EXPRESSION AND IL17 FROM

RHEUMATOID ARTHRITIS PATIENTS IN ALLERGENIC CO-CULTURE

Maha Abd El Rafeh ELBassuoni, Gehan Abd El Fatah, Heba Ahmed El- Esselly ........................................

HUMAN TELOMERASE REVERSE TRANSCRIPTASE MRNA AS A NOVEL TUMOR MARKER

FOR HEPATOCELLULAR CARCINOMA

Madeeha Y. Bakheet, Sherief I. Kamel, Hanan O. Mohamed, Tahra M. Sherif, Ebtsam F. Mohamed and

Dina A. Mohareb .............................................................................................................................................

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Contents Page

STUDY OF SERUM HEPCIDIN AS A DIAGNOSTIC TOOL OF IRON OVERLOAD IN EGYPTIAN CHILDREN WITH BETA-THALASSEMIA

MAJOR AND BETA-THALASSEMIA INTERMEDIAAhmed Maher Kaddah*, Amina Abdel-Salam*, Marwa Salah Farhan** and Reham Ragab*

ABSTRACTBackground and Aim: Iron homeostasis is disturbed in β-thalassemia and results in iron overload with or without transfu-

sion dependence. Iron homeostasis proved to be regulated by hepatic peptide hormone “Hepcidin”. We aimed to investigate serum hepcidin level and its potential usefulness in the diagnosis of iron overload in children with β-thalassemia major and intermedia as well as to assess the relationship between hepcidin level and various clinical and laboratory characteristics of recruited patients. Methodology: A prospective study conducted on 30 thalassemia intermedia (TI) and 30 thalassemia major (TM) patients and 60 healthy -age and sex- matched subjects as controls. All patients were subjected to history taking and complete physical examination. Complete blood count, liver function tests, serum bilirubin (total, direct), serum ferritin and serum hepcidin level was measured by Human Hepcidin, ELISA Kit. Results: Mean serum hepcidin in healthy children was 0.4 ng/ml (0.19 to 0.89 ng/ml) and it was 3.1 ng/ml in TM group and 1.4 ng/ml in TI group (p<0.001). TI group had lower hepcidin compared to TM group (p=0.034). Median hepcidin was significantly higher among those with higher number of total transfu-sions (p=0.01) and among chelated patients (p=0.002). Among the control group, serum hepcidin didn’t correlate with age (r=0.225, p=0.084), but it correlated positively with age, disease duration, transfusion frequency, total number of transfusions, total bilirubin, and serum ferritin in β-thalassemia patients. TM group had higher level of serum ferritin compared to TI group (p<0.001). Hepcidin/ ferritin ratio was <1 in all cases and was comparable between TM and TI patients (p=0.4). Well-chelated patients had lower serum hepcidin compared to poor-chelated patients (p<0.05), but Hepcidin/ ferritin ratio was comparable between both groups (p>0.05). The overall predictability of hepcidin in severe iron overload was statistically significant whencompared to hepcidin to ferritin ratio. Conclusion: Serum hepcidin is generally elevated in B-thalassemia patients in the pediatric age group, but this elevation is more evident in TM patients mostly due to the regular transfusion therapy. Serum hepcidin can be adopted as an indicator of severe iron overload. Key words: Hepcidin, Iron Overload, B-Thalassemia major, B-Thalassemia intermedia.

Departments of Pediatrics*, Clinical Pathology**, Faculty of Medicine, Cairo University, Cairo, Egypt

Egypt. J. Lab. Med. Vol.(27) No.1&2, February/June, 2015, 1 - 8

INTRODUCTION

Β-thalassemia is the most common hemoglo-bin disorder in Egypt with a carrier rate varying from 5.3 to 9% (5). The most severe form of the disease is termed β- thalassemia major (TM), TM patients require regular blood transfusions to maintain life and this increase the rate of iron accumulation. Moreover, increased gastrointes-tinal iron absorption secondary to ineffective erythropoiesis and anemia may also play a role in iron overload in transfused β-thalassemia pa-tients (28). β-thalassemia intermedia (TI) is a mild-er form where blood transfusions are not always necessary, however, they are still prone to iron overload due to progressive iron absorption from the gastrointestinal tract (29). It is well known that iron overload is the principal cause of morbidity and mortality in β- thalassemia with or without transfusion dependence (18).

Hepcidin, a 25-amino-acid (aa) peptide, hormone is a key regulator of iron homeostasis(18). Hepcidin is synthesized predominantly in hepatocytes. Other cells and tissues, including macrophages, adipocytes and brain, express low levels of hepcidin (20). Hepcidin synthesis is physiologically increased by elevated serum iron level(15), decreased by erythropoietic activity (24), and pathologically increased by inflammation (26). In addition, anemia and hypoxia can dramatically suppress hepcidin production (22). In beta thalassemia, hepcidin level is variable. If anemia and high erythropoietic activity predominates, it will result in hepcidin deficiency (10), but in the presence of iron overload, hepcidin expression will be increased (23).

In β-thalassemia, estimation of iron over-load has relied on serum ferritin measurements despite evidence that these do not always corre-late well with liver iron concentration. Informa-

Kaddah, A. M. et al.2tion on hepcidin concentrations in patients with thalassemia, may be valuable for identifying the most severely affected patients and predicting and monitoring parenchymal iron overload in patients with β-thalassemia. In the present study we aimed to investigate serum hepcidin level and its potential usefulness in the diagnosis of iron overload in children with β-thalassemia ma-jor and intermedia, and to assess the relationship between hepcidin level and various clinical and laboratory characteristics of recruited patients.

SUBJECTS AND METHODS

This was a prospective case-control study that included sixty patients with beta-thalassemia (30 diagnosed as thalassemia intermedia and 30 with thalassemia major), all presented to the Hema-tology Clinic of El-Monira Children’s Hospital, Faculty of Medicine, Cairo University, either for blood transfusion or for medical follow-up dur-ing the period of the study (from January to April 2014). Patients consisted of 27 (45%) males and 33 (55%) females. Their age ranged from 1.5- 18 years (mean age was 8.3±5.0 years) and were diagnosed as β-TM and β-TI based on conven-tional clinical and hematologic criteria. Patients older than 18 years old, those with concurrent infection, chronic inflammatory disorders, ab-normal liver functions (> 3 fold of normal) and transfusion-dependent thalassemia intermedia patients were excluded from the study. Further 60 apparently healthy subjects with matching age & sex were included and served as a control group. Consent was obtained from the patients or their legal guardians before enrollment in the study. The study protocol was approved by the Ethical Committee of Cairo University.

Data was collected by direct patient in-terviewing where detailed history-taking and thorough clinical examinations were carried out. The collected data included: age and sex, consanguinity, similar family condition, history of disease related complications, splenectomy, transfusional history including age of onset, du-ration, frequency of transfusion and total number of transfusions in life and history of drug therapy e.g. hydroxyurea, chelation.

Blood samples were withdrawn at least 2 weeks after the last transfusion to avoid possible

effects of transfusion on hepcidin level. The fol-lowing were done: Complete blood count, reticu-locyte count, alanine transaminase (ALT), aspar-tate transaminase (AST),serum bilirubin (total, direct), serum ferritin and serum hepcidin.

Serum hepcidin was determined by using an automated commercial immunoassay using Hu-man Hepcidin, ELISA Kit (EIA: E1979h; EIAab Science Co.Ltd. ). The Hepcidin ELISA Kit is a solid phase enzyme-linked immunosorbent as-say (ELISA), based on the principle of competi-tive binding and the detection Range of the used kit batch was 0.187-12 ng/mL. Reference values were obtained from the recruited control group.

Statistical method: The data was summa-rized using: mean, standard deviation, median, interquartile range (IQR), minimum and maxi-mum for quantitative variables and number and percentage for qualitative values. Statistical dif-ferences between groups were tested using Chi Square test for qualitative variables, student- t test for quantitative parametric variables while Mann Whitney test and kruskal-Wallis test were used for nonparametric quantitative variables. Correlations were done to test for linear rela-tions between variables. ROC curve analysis was done to test the validity of hepcidin versus hepcidin to ferritin ratio in detecting iron over-load, P- values less than or equal to 0.05 were considered statistically significant. The data wascoded and entered using the statistical package SPSS version 15.

RESULTS

The study included 60 β-thalassemia patients, and 60 apparently healthy subjects with matching age & sex were as a control group. Serum hep-cidin level in healthy children ranged from 0.19 to 0.89 ng/ml with a median value 0.33 ng/ml and this level was lower compared to cases with beta-thalassemia where median serum hepcidin was 1.6 ng/ml (range 0.19 to 11.23 ng/ml).

TM group had higher level of hepcidin com-pared to other two groups with statistically sig-nificant difference (p<0.001) and this differ-ence was also highly significant on comparingTM group to each group separately. All Pair-wise Multiple Comparison Procedures (Dunn’s

Study of Serum Hepcidin as a Diagnostic Tool of Iron Overload 3Method) revealed that TI group had significant-ly higher hepcidin compared to control group (p<0.001) but lower compared to that of TM group (p=0.034). Hepcidin/ ferritin ratio was be-low 1 in all cases and was comparable between TM and TI patients (p=0.4) (Table 1, 2).

Well-chelated thalassemia patients (serum ferritin (SF) > 1500 ng/ml) had significantlylower serum hepcidin compared to poor-che-lated patients (SF ≥ 1500 ng/ml) with statis-tically significant difference (p<0.05), howeverH/F ratio was comparable between both groups (p>0.05) (Table 3).

Among the control group, serum hepcidin didn’t correlate with age (r=0.225, p=0.084). Among β- thalassemia patients (n=60), se-rum hepcidin was found to correlate positively with age (r=0.4; p=0.001), disease duration (r=0.5; p<0.001), transfusion frequency (r=0.35; p=0.007), total number of transfusions (r=0.4; p=0.003), total bilirubin (r=0.3; p=0.04), and se-rum ferritin (r=0.3; p=0.027), but it didn’t show any correlation with age at diagnosis, or indices

of hemolysis. While among TI group (n=30), it correlated positively with age (r= 0.6; p<0.001), disease duration (r=0.7; p<0.001), total number of transfusions (r=0.5; p=0.007), platelet count (r=0.4; p=0.03), and direct bilirubin (r=0.4; p=0.04). In TM group (n=30) it showed posi-tive correlation with serum ferritin only (r=0.4; p=0.035).

Roc curve: As the gold standard of assess-ment of iron overload is the liver iron concentra-tion (LIC) and it wasn’t available for our cases to compare with, we tested hepcidin and Hepcidin / ferritin ratio by ROC curve, it was found that ar-eas under the curve of hepcidin (0.667) (p=0.032) while that of H/F ratio (0.568) (p=0.368) indicat-ing that overall predictability of hepcidin is sig-nificantly high when compared to H/F ratio. Thebest cut off of hepcidin was ≥1.3 ng/ml (85% sensitivity, 65% specificity, 77% PPV and 89%NPV, overall accuracy 73%) and further increas-es in hepcidin level were associated with drop in both sensitivity and specificity (Figures 1,2).

Table (1): Comparison of serum hepcidin among the studied groups:

P-valueControls (n=60)Cases (n=60)Variables

<0.001*0.4±0.2 (0.19-0.89)0.33 (0.13-0.41)

2.3±2.4 (0.19-11.23)1.6 (0.75-2.53)

Hepcidin (ng/ml):Mean±SD (Range)

Median (IQR)

p-valueControls (n=60)TM (n=30)TI (n=30)Variables

<0.001*0.4±0.2 (0.19-0.89)0.33 (0.13-0.41)

3.15±3.1 (0.25-11.23)1.9 (0.87-4.27)

1.4 ±0.87 (0.19-2.81)1.3 (0.61-2.33)

Hepcidin (ng/ml):Mean±SD (Range)

Median (IQR)

* P- value is significant if P<0.05

Table (2): Serum ferritin and Hepcidin/ferritin ratio between the studied groups:

p-value

TMN=30

TIN=30Variables

Median(IQR)

Mean±SD(range)Median (IQR)Mean±SD

(range)

<0.001*1636.51367.0-1889.0

1761±674917-4312

787.7467.0-1111.0

817±448165-1600Ferritin (ng/ml):

0.3610.0010.0007-0.003

0.002±0.0020.0001-0.006

0.00230.0009-0.004

0.002±0.0020.0001-0.008Hepcidin/ferritin ratio


Kaddah, A. M. et al.4Table (3): Comparison of hepcidin level and H/F ratio among well and poorly chelated cases:

Variables SF <1500 (n=38) SF≥1500 (n=22) P value

Serum hepcidin Mean ±SDMedian (range)

1.56±1.141.29 (0.19-4.98)

3.51±3.452.13 (0.19-11.23) 0.032*

Hepcidin/ ferritin ratio Mean ±SD

Median (range)

0.0023±0.00180.002 (0.0001-0.0077)

0.0018±0.00160.001 (0.0001-0.0056) 0.383


Figure (1): ROC curve of Hepcidine for prediction of iron overload.

Figure (2): ROC curve of Hepcidine/ferritin ratio for prediction of iron overload.

DISCUSSION

Hepcidin is a key regulator of iron homeosta-sis and mediator of anemia of inflammation (6). This peptide hormone controls dietary iron ab-sorption, plasma iron concentrations, and tissue iron distribution (3). With regard to β- thalasse-mia syndromes, hepcidin synthesis is stimulated by iron loading which in turn blocks dietary iron absorption and prevents further iron loading (20), and suppressed by anemia; both of these “op-posing” factors would be expected to regulate hepcidin production (12). However, the drive for erythropoiesis is thought to be the dominant in-fluence for hepcidin production when iron over-load and anemia coexist (22).

The previous studies carried out on β- thalas-semia patients proved that hepcidin level is vari-able and the regulation of hepcidin synthesis and production in such type of inheritable iron load-ing anemia is not that simple. Possible undeter-mined clinical, laboratory or even genetic vari-

ables might modify the rate of hepcidin synthe-sis in β- thalassemia patients (17, 27). The present study aimed to investigate serum hepcidin level and its potential usefulness in the diagnosis of iron overload in children with β- thalassemia major and intermedia as well as to elucidate the relationship between hepcidin level and various clinical and laboratory characteristics of recruit-ed patients. In order to fulfill our aim, sixty pa-tients with β-thalassemia (30 TM and 30 TI) and sixty apparently healthy - age and sex- matched subjects were included in the study and served as a control group. Our study is one of few studies that were entirely carried out on children with β-thalassemia (9, 4, 2).

Based on our results from age- and sex-matched healthy controls, the reference range for hepcidin was from 0.19 to 0.89 ng/ml with a mean value 0.4 ng/ml. However, the previous studies of healthy controls, revealed consider-able intra-individual variations in hepcidin lev-els and this then results in wide reference ranges

Study of Serum Hepcidin as a Diagnostic Tool of Iron Overload 5which then may have limitations when used for interpretation of individual hepcidin concentra-tions. Serum hepcidin among our B-thalassemia patients ranged between 0.19 and 11.23 ng/ml, median value 2.3ng/ml, and this level was high-er compared to controls. This was in agreement with previous studies (23; 9). Contrary to this, sev-eral studies reported undetectable or low serum or urinary hepcidin in β-TM subjects as com-pared to controls (25, 11, 13, 8, 4). Only 2 studies re-ported comparable hepatic hepcidin mRNA and serum hepcidin between β-thalassemia patients and healthy subjects (1, 2).

The difference in hepcidin levels between β-TI and β-TM was reported by several reports concluding that regular transfusions in β-TM induce high tissue iron accumulation and also inhibit erythropoietic drive, both of which re-sult in an increase of hepcidin in β-TM patients when compared to β-TI individuals (21, 19, 10, 23, 9). In our study, median serum hepcidin of TM pa-tients was 1.9 ng/ml and this was significantlyhigher compared to that of TI patients which was 1.3 ng/ml (p=0.03). Both groups had sig-nificantly higher hepcidin compared to controlgroup (p<0.001); this was in concordance with the previously mentioned reports.

As regards the relationship between serum hepcidin and age, we found that serum hepci-din didn’t correlate with age among the control group, but this association was marginally insig-nificant (r=0.225, p=0.084). However, among β-thalassemia patients, serum hepcidin was found to correlate positively with age (r=0.4, p=0.001) and with the disease duration (r=0.5, p<0.001), and this was the case in TI patients. This was in agreement with El Beshlawy et al., (4) who found a significant positive correlation betweenage and hepcidin among their patients; they ex-plained this occurrence by the increased number of blood transfusions, and consequently, the iron overload with age. However, this significantpositive correlation with age and disease dura-tion became marginally insignificant among TMpatients.

Moreover, serum hepcidin didn’t correlate with Hb or indicators of hemolysis such as re-ticulocytic count, total and indirect bilirubin

among the studied cases. This was in agreement with previous reports (11, 9, 4). It is explained by Vokurka et al. (30) who reported that the hemo-lysis and anemia decrease hepcidin expression only when erythropoiesis is functional (i.e. ef-fective erythropoiesis) which is not the case in thalassemia; on the other hand, if erythropoiesis is blocked, even severe anemia does not lead to a decrease of hepcidin expression, which is indeed increased. Contrary to this Camberlein et al. and Kattamis et al. (1,10) reported that hepcidin mRNA expression of TM patients was found to correlate positively with hemoglobin values.

No significant correlation was detected inthis study between liver enzymes and hepcidin level; this was in agreement with Kaddah et al.(9). This may be explained by the synthesis of hepcidin in many sites other than the liver. Serum hepcidin is independent of the degree of hepatic inflammation as measured by aminotransferaseslevel(16).

The relationship between hepcidin and iron overload among thalassemia patients is still controversial. Previous studies carried out on β- thalassemia (major and intermedia), reported that the enhanced erythropoiesis dysregulates iron homeostasis by inhibiting hepcidin synthe-sis, even in the presence of iron overload and low hepcidin will increase intestinal iron absorption and further worsens iron load (10, 7, 11) . Among the human studies, Chauhan et al. (2) showed no correlation of serum ferritin and serum hepcidin. Similarly, Kattamis et al. (10) found a lack of cor-relation between the two variables and negative correlation of serum hepcidin with serum ferritin level was reported by Hendy et al.(8). However, a few authors found a significant positive cor-relation of indices of iron overload with hepci-din. Origa et al. (23) reported a significant positivecorrelation of serum ferritin and hepatic mRNA levels.

In the present study, we found that TM group had significantly higher level of serum ferritin(p<0.001) compared to TI group and this was in agreement with two previous reports carried out on Egyptian β-thalassemia patients (9, 4). More-over, serum hepcidin levels correlated positively with serum ferritin, in all patients (r=0.3, p=0.03)

Kaddah, A. M. et al.6and TM group (r=0.4, p=0.04) but this signifi-cant correlation was lost in TI patients (r=-0.01, p=0.7). This may be explained by the fact that relatively low serum ferritin in TI patients com-pared to TM patients didn’t adequately reflect thepatients’ liver iron load, because hepcidin was reported to affect the distribution of tissue iron. Origa et al.(23) reported that non transfused TI pa-tients have liver iron concentrations (LIC) simi-lar to those of regularly transfused TM patients. In NTDT, iron was deposited in hepatocytes, whereas higher hepcidin levels in transfused pa-tients resulted in macrophage iron loading. As a consequence of this difference in cellular iron distribution, serum ferritin levels were much lower in NTDT. This means that serum ferritin is suspected to correlate to serum hepcidin in TD patients rather than NTDT patient. In our study, well-chelated thalassemia patients (SF less than 1500 ng/ml) had significantly lower serum hep-cidin compared to poor-chelated patients (SF ≥1500 ng/ml) with statistically significant dif-ference (p<0.05).

Hepcidin-to-ferritin ratio (normal ratio is close to 1), is an index of appropriateness of hepcidin expression relative to the degree of iron loading, it was found to be very low in thalas-semia major(11, 23, 10, 7, 23, 14, 4) this means that the consequences of ineffective erythropoiesis on hepcidin expression appear to overcome the con-sequences of excessive iron stores. In this work, the ratio of serum hepcidin to ferritin ratio was found to be below 1 in all cases and was compa-rable between TM and TI patients (p=0.4), and between well-chelated thalassemia patients (SF less than 1500 ng/ml) and poor-chelated patients (SF ≥1500 ng/ml) (p>0.05). Thus, in spite of iron overload in β-thalassemia patients, hepcidin increases were not proportionate to the degree of iron overload. This was in agreement with the previous studies (11, 23, 10, 7, 14, 4).

As the gold standard of assessement of iron overload is the liver iron concentration (LIC) and it wasn’t available for our cases, we tested hep-cidin and hepcidin/ferritin ratio by ROC curve, to determine which is more appropriate in pre-dicting severe iron overload, we found that areas under the curve of hepcidin (0.667) (p=0.032)

while that of hepcidin to ferritin ratio (0.568) (p=0.368) indicating that overall predictability of hepcidin is significantly high when comparedto hepcidin to ferritin ratio. The best cut off of hepcidin was ≥1.3 ng/ml; this makes adoption serum hepcidin as a good predictor of iron over-load more likely.

In conclusion, serum hepcidin is generally el-evated in B-thalassemia patients in the pediatric age group, but this elevation is more evident in TM patients mostly due to the regular transfu-sion therapy. Serum hepcidin can be adopted as an indicator of severe iron overload. However, further large studies are required to confirm thediagnostic and prognostic and possible thera-peutic role of hepcidin in thalassemia, especially in non transfusion dependent β-thalassemia pa-tients.

REFERENCES1. Camberlein E, Zanninelli G, Detivaud L, et al. (2008):

Anemia in β-thalassemia patients targets hepatic hepci-din transcript levels independently of iron metabolism genes controlling hepcidin expression. Haematologica; 93:111.

2. Chauhan R, Sharma S, Chandra J. (2014): What regu-lates hepcidin in poly-transfused β-Thalassemia Ma-jor: Erythroid drive or store drive? Indian J Pathol Microbiol;57:39.

3. Donovan A, Lima CA, Pinkus JL, et al. (2005): The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell Metab.;1:191.

4. El Beshlawy A, Alaraby I, Abdel Kader MSEM, et al , 2012 Study of serum Hepcidin in Hereditary hemolytic anemias. Hemoglobin; 36(6): 555.

5. El-Beshlawy A, Youssry I. (2009): Prevention of he-moglobinopathies in Egypt. Hemoglobin; 33 Suppl 1:S14.

6. Ganz T. (2006): Molecular pathogenesis of anemia of chronic disease. Pediatr Blood Cancer. 46(5):554.

7. Gardenghi S, Marongiu MF, Ramos P, et al. (2007): Ineffective erythropoiesis in beta-thalassemia is charac-terized by increased iron absorption mediated by down-regulation of hepcidin and up-regulation of ferroportin. Blood; 109:5027.

8. Hendy OM, Allam M, Allam A, et al. (2010): Hep-cidin levels and iron status in beta-thalassemia ma-jor patients with hepatitis C virus infection. Egypt J Immunol.;17(2): 33.

9. Kaddah NA, El Gindi HD, Mostafa NO, et al. (2011): Role of Hepcidin in the pathogenesis of iron overload in children with B-thalassemia. IJAR; 3 (4) . I Part 62.

http://www.ncbi.nlm.nih.gov/pubmed?term=Youssry%20I%5BAuthor%5D&cauthor=true&cauthor_uid=20001619

http://www.ncbi.nlm.nih.gov/pubmed?term=Hendy%20OM%5BAuthor%5D&cauthor=true&cauthor_uid=23082485

http://www.ncbi.nlm.nih.gov/pubmed?term=Allam%20M%5BAuthor%5D&cauthor=true&cauthor_uid=23082485

http://www.ncbi.nlm.nih.gov/pubmed?term=Allam%20A%5BAuthor%5D&cauthor=true&cauthor_uid=23082485

http://www.ncbi.nlm.nih.gov/pubmed/23082485


Study of Serum Hepcidin as a Diagnostic Tool of Iron Overload 710. Kattamis A, Papassotiriou I, Palaiologou D, et al.

(2006): The effects of erythropoetic activity and iron burden on hepcidin expression in patients with thalas-semia major. Haematologica ;91:809 .

11. Kearney SL, Nemeeh E, Neufeld EJ, et al (2007): Uri-nary hepcidin in congenital chronic anemias Pediatr. Blood Cancer., 48:57.

12. Kemna E, Tjalsma H, Laarakkers C, et al . (2005): Novel urine hepcidin assay by mass spectrometry. Blood.;106(9):3268.

13. Kemna EHJM, Tjalsma H, Podust VN et al (2007): Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical impli-cations. Clin Chem; 53:620.

14. Kroot JJ, Laarakkers CM, Kemna EH, et al.( 2009): Regulation of serum hepcidin levels in sickle cell dis-ease. Haematologica;94:885.

15. Lin L, Valore EV, Nemeth E, et al (2007): Iron trans-ferrin regulates hepcidin synthesis in primary hepa-tocyte culture through hemojuvelin and BMP2/4. Blood.;110:2182.

16. Lin TJ., Liao LY., Chou JM., et al (2009): Serum pro-hepcidin levels correlate with hepatic iron stores in chronic hepatitis C patients. Hepatogastroenterology. 56(93):1146.

17. Longo F, Zecchina G, Sbaiz L, et al (1999): The influ-ence of hemochromatosis mutations on iron overload of thalassemia major. Haematologica;84:799.

18. Nemeth E (2010): Hepcidin in β-thalassemia. Ann N Y Acad Sci.; 1202: 31.

19. Nemeth E and Ganz T (2006): Hepcidin and iron-load-ing anemias. Haematologica.; 91:727.

20. Nemeth E and Tomas G (2009): The Role of Hepcidin in Iron Metabolism. Acta Haematol. 122(2–3): 78.

21. Nemeth E, Valore EV, Territo M, et al (2003): Hepci-din, a putative mediator of anemia of inflammation, is atype II acute phase protein. Blood;101:2461.

22. Nicolas G, Chauvet C, Viatte L, et al. (2002): The gene encoding the iron regulatory peptide hepcidin is regu-lated by anemia, hypoxia, and inflammation. J Clin In-vest.; 110(7):1037.

23. Origa R, Galanello R, Ganz T, et al (2007): Liver iron concentrations and urinary hepcidin in beta-thalasse-mia. Haematologica.; 92:583.

24. Pak M, Lopez MA, Gabayan V,et al (2006). Suppres-sion of hepcidin during anemia requires erythropoietic activity. Blood.;108:3730.

25. Papanikolaou G, Tzilianos M, Christakis JI, et al.( 2005): Hepcidin in iron overload disorders. Blood;105:4103.

26. Pigeon C, Ilyin G, Courselaud B, et al. (2001): A new mouse liver-specific gene, encoding a protein ho-mologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem ; 276:7811.

27. Sharma V, Panigrahi I, Dutta P, et al. 2007: HFE muta-tion H63D predicts risk of iron overload in thalassemia intermedia irrespective of blood transfusions. Indian J Pathol Microbiol 50:82.

28. Steinberg MH, Forget BG, Higgs DR, et al (2001): Dis-orders of hemoglobin: Genetics, Pathophysiology and Clinical Management. Cambridge, United Kingdom: Cambridge University Press; 2001.

29. Taher A, Isma’eel H, Cappellini MD.( 2006): Thalasse-mia intermedia: revisited. Blood Cells Mol Dis;37:12.

30. Vokurka M, Krijt J, Sulc K, et al (2006): Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis. Physiol Res.;55(6):667.

http://www.jci.org/articles/view/15686



http://www.ncbi.nlm.nih.gov/pubmed?term=Pak%20M%5BAuthor%5D&cauthor=true&cauthor_uid=16882706

http://www.ncbi.nlm.nih.gov/pubmed?term=Lopez%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=16882706

http://www.ncbi.nlm.nih.gov/pubmed?term=Gabayan%20V%5BAuthor%5D&cauthor=true&cauthor_uid=16882706

http://www.ncbi.nlm.nih.gov/pubmed/16497104?dopt=Abstract

Kaddah, A. M. et al.8

المتوسط البحر انيميا في مرضى في الدم الحديد لزيادة كمؤشر استخدامه امكانية و مدى في المصل الهيبسيدين لمستوى دراسةاالطفال المصريين في المتوسطة و الكبرى

رجب - ريهام فرحان صالح - مروة عبد السالم أمينة - ماهر قداح احمد

الكبرى المتوسط البحر انيميا من يعانون الذين األطفال في المصل في الهيبسيدين هرمون مستوى قياس الدراسة هذه من الهدف كانالعالقة لتوضيح وكذلك الزائد الحديد تشخيص في المحتملة وفائدته بالدم الحديد ومستوي الهيبسيدين بين العالقة ودراسة والمتوسطهمستقبلية أجريت في وصفية دراسة هذه كانت للمرضى. والمختبرية السريرية الخصائص ومختلف المصل في بين مستوى الهيبسيدينانيميا من يعانون على٣٠ مريضا هذه الدراسة أجريت وقد القاهرة. جامعة الطب، كلية مستشفى المنيرة، لالطفال، الدم، أمراض عيادةاالصحاء الى ستين طفال من باالضافه (TI) المتوسطه انيميا البحرالمتوسط من يعانون مريضا ٣٠ و (TM) الكبرى المتوسط البحركاملة، صورة دم كامل، اكلينيكي لتقييم الدراسة هذه في األفراد تعرض جميع وقد ضابطة. كمجموعة والجنس العمر فى المتطابقينقياس وكذلك المصل الفيريتين في نسب متوسط تسجيل تم كما مباشر), (المجموع، المصل في الكبد, البيليروبين وظائف اختباراتمل / نانوغرام الى ٠٫٨٩ ٠٫١٩ بين األطفال االصحاء في بالمصل الهيبسيدين مستوى تراوح بالمصل. الهيبسيدين هرمون مستويمستوى الهيبسيدين متوسط كان حيث المتوسط البحر حاالت انيميا مع مقارنة أقل كان المستوى و هذا مل / نانوغرام ٠٫٤ متوسط قيمة معالهيبسيدين بالمصل وكان مستوى .(٠٫٠٠١>TI (p مجموعة في و ١٫٤ نانوغرام/مل TM مجموعة في مل / ٣٫١ نانوغرام بالمصل

مستوى متوسط TI مجموعة اظهرت كما الضابطه. المجموعه و TI مجموعه من غيرها مع بالمقارنة أعلى TM مجموعة فىفي الهيبسيدين متوسط أن وجدنا TM. مجموعة من عليه بما كان مقارنة أقل ولكن الضابطة بالمجموعة بكثير مقارنة أعلى الهيبسيدينمجموع من عدد أكبر لديهم الذين بين أولئك أعلى بكثير الهيبسيدين كان متوسط بينما , TM مجموعة في منه اقل كان TI مجموعةبين عالقة هناك تكن لم الحديد. استحالب بادوية المعالجين المرضى بين بكثير الهيبسيدين أعلى متوسط كان وبالمثل الدم نقل عملياتوتواتر نقل المرض، العمر, ومدة مع بين مستوي الهيبسيدين طردية عالقة وجدنا ولقد الضابطة. المجموعة فى و العمر الهيبسيدين نسبهنسبة كانت ،(٦٠ = المتوسط (ن البحر انيميا بين مرضى المصل الفيريتين في ومجموع البيليروبين، ومستوى للنقل، الكلي الدم، العددمن ١ أقل كانت فقد الهيبسيدين الى الفيريتين لنسبة المجموعة TI وبالنسبة بكثير مقارنة مع أعلى TMمرضى فى المصل في الفيريتينالتنبؤ قادرا على مستوى الهيبسيدين ارتفاع ذلك، كان ومع .TI مرضى TM و مرضى بين احصائيا متماثله وكانت الحاالت، جميع فيانيميا في مرضى عموما يرتفع مستوى الهيبسيدين الخالصة: الفيريتين. الى الهيبسيدين بنسبة مقارنته الزائد عند الحديد العام بحاالتالمتكررة. الدم نقل بسبب عمليات غالبا الكبرى المتوسط البحر انيميا مرضى في اكثر االرتفاع لكن هذا و االطفال المتوسط من البحر

الدم. الحديد في نسبة لزيادة كمؤشر اعتماده يمكن المصل الهيبسيدن في

ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISM OF SPARC GENE WITH INCREASED RISK OF HEPATOCELLULAR

CARCINOMADina Hisham Ahmed Soliman

ABSTRACT Background: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is the third most frequent

cause of cancer-related deaths. Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular glycoprotein involved in many biological processes. SPARC gene polymorphism (rs 2304052) has been shown to be associated with increased risk of hepatocarcinogenesis. Aim of the work: The aim of the present study was to investigate the association of SPARC gene polymorphism (rs 2304052) with the risk of development of HCC and its clinical utility as a genetic marker for HCC. Meth-ods: SPARC gene polymorphism (rs 2304052) was detected in peripheral blood of 50 HCC patients on top of HCV infection, 39patients infected with HCV and 20 healthy control subjects using Taqman Real-time PCR. Results: There was non statistical significant difference was found between the HCC,HCV and control groups regarding frequency of TT, CT and CC genotypesof SPARC gene polymorphism (rs 2304052) (P value = 0.2).However a statistical significant difference was found between the3 groups regarding frequency of T and C alleles, where the frequency of C allele was significantly more frequent in HCC groupthan in HCV and healthy control groups (P value = 0.05).CC and CT genotypes (risky genotypes) were associated with a sta-tistically significant higher values of APRI test that evaluate the degree of fibrosis than TT genotype (P value = 0.04).Also CCand CT genotypes were associated with a statistically significant higher levels of AFP than TT genotype (P value = <0.001). Conclusion: There was an association between C allele (mutant allele) of SPARC gene polymorphism (rs 2304052) and HCC. Also there was an association between CC and CT genotypes (risky genotypes) and increased degree of fibrosis which representa risk factor for HCC. Key words: SPARC gene polymorphism, hepatocellular carcinoma, Real-time PCR.

Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University.


INTRODUCTION Hepatocellular carcinoma (HCC) is the

fifth commonest cancer worldwide and isthe third most common cause of cancer-related deaths(26,18,22). Major risk factors for the development of HCC include chronic infections with HBV, HCV, alcohol consumption, exposure to dietary aflatoxin B1, and liver cirrhosis of anyetiology (7).

Hepatocellular carcinoma has many charac-teristics, such as fast infiltrating growth, metas-tasis in early stage, high grade malignancy, and poor therapeutic efficacy. The most urgent needsin management of HCC are to find sensitivemarkers for diagnosis and monitoring of postop-erative recurrence and to provide adequate treat-ment (25,24).

Currently, imaging techniques including ul-trasonography, computed tomography scanning and magnetic resonance imaging are used for the diagnosis of HCC. However, these techniques cannot adequately differentiate benign hepatic lesions from HCC (5,14).

Serum tumor markers with satisfactory accu-racy could be supplementary to ultrasonography and computed tomography in the diagnosis of HCC(25). Serum AFP has been widely used for screening and diagnosis of HCC. However de-velopment of false-negative tests with AFP was as high as 40% for patients with early stage He-patocellular carcinoma (24).Thus the identificationof novel biochemical markers for HCC remains an important goal around the world (25).

SPARC/ Osteonectin is a glycoprotein in-volved in extracellular matrix remodeling that appears to regulate cell growth through interac-tions with the extracellular matrix and cytokines. It influences cell migration, proliferation, angio-genesis, matrix cell adhesion and tissue remod-eling (2).

As a regulator of cell-extracellular matrix interactions, SPARC/Osteonectin is thought to represent a major factor in extracellular matrix remodeling occurring during tumor invasion. SPARC is also thought to be involved in hepatic

Soliman, D. H. A. 10fibrogenesis related to chronic injuries, HCC an-giogenesis and tumor progression (18,12).

The role of SPARC is controversial and it seems that the capacity of SPARC to promote or inhibit tumor progression is dependent on the initiating cell type, the tumor stage and the con-text of the tumor microenvironment (2,3). It was evident that SPARC has a tumor suppressive function in the liver, so the disruption of SPARC contributes to the development and progression of HCC (16,4).

SPARC gene polymorphism (rs 2304052) is identified to be associated with HCC suscepti-bility. Although further studies should be done to clarify the role of this polymorphism in the pathogenesis of HCC, it may serve as good marker that can be used to identify a subgroup that has a higher risk of developing HCC (16).

Aim of work

Therefore, the aim of the present study was to investigate the association of SPARC gene polymorphism (rs 2304052) and increased risk of development of HCC and its clinical utility as a genetic marker for HCC.


Subjects:

The present study was conducted in Kasr Al-Ainy Hospital; Cairo University. 119 subjects were included in the present study. All patients included in the present study presented to the Tropical Medicine Department, Kasr Al-Ainy Hospital, Cairo University. The study was per-formed on three groups:

1. Hepatocellular carcinoma (HCC) group: included 50 patients with hepatocellular carci-noma (HCC) on top of HCV infection.

2. Hepatitis C Virus (HCV) group: included 39 patients infected with HCV.

3. Control group: included 20 apparently healthy subjects without any evidence of liver disease.

The patients were subjected to the following:

• Full history taking and complete clinical evaluation including assessment of conscious-

ness level and abdominal examination.

• Laboratory investigations:

1- Routine tests:

• Routine liver function tests including serum total bilirubin, direct bilirubin, Alanine Trans-aminase (ALT), Aspartate Transaminase (AST), Albumin. All were done by the antoanalyzer: Bechman CX5 Ireland.

• Serum Alpha-fetoprotein (AFP) level, by using electro chemiluminesence technique (Bay-er healthcare, USA).

• Hepatitis markers (HCV Ab, HBs Ag), were assayed on Axsym autoanalyser using kits sup-plied by ABBOTT LABORATORIES Diagnos-tic Division,Germany.

2- Molecular study:

• Genomic DNA analysis of SPARC gene polymorphism using Taqman Real-time PCR.

DNA Analysis for SPARC gene polymor-phism by Real-time PCR:

1- Extraction of Genomic DNA from peripheral blood:

This was done using QIA-amp® DNA blood Mini Kit (Qiagen, catalog no.51104)19 and per-formed on fully automated Qiacube.

DNA Amplification and Detection usingApplied Biosystems Step one™ Real-Time PCR:

Real-time PCR was done using TaqMan 5-nuclease probes(9,6). Poison Information Center Mainz, Germany.

Primers: forward primer 5 CGGGAGTTCAGGGTAAGGT-3 reverse primer 5-ACAGG CA CCTGCGATCATC-3

Real-time cycler conditions: 40 number of cycles, PCR initial activation step 10 min at 95oC, denaturation 15 seconds at 92oC and com-bined annealing/extenion 60 seconds at 60oC.

APRI TEST (AST-to-Platelet Ratio Index):

APRI test is a non-invasive method used to identify patients with cirrhosis. Patients are clas-sified into three groups; If the APRI score is lessthan or equal to 0.5, this means no fibrosis orjust a little. If APRI score is 1.5 or above, prob-

Association of SNP SPARC Gene & HCC 11ably there is established cirrhosis. APRI scores between 0.5 and 1.5 are related to progressive fibrosis stages (21).

Also the quantitative result of APRI test was used in comparison between different geno-types. It was calculated by dividing AST by the ULN [(upper limit of normal (42 IU/L)] of AST, this result was then divided by the platelet count (with the last three zeros chopped off), and mul-tiply by 100. APRI test formula = (AST/ULN)/platelets x 100 (21).

Statistical analysis: The results were entered on the computer us-

ing “Microsoft Office Excel Software” program(2010) for windows. Data was then transferred to the Statistical Package of Social Science Soft-ware program, version 21 (SPSS) to be statisti-cally analyzed. Qualitative data were expressed as frequencies and percentages. Quantitative data were expressed as mean and SD and dif-ferences between the 3 groups were compared by Student t-test if data were not normally dis-tributed (parametric). The quantitative data were expressed as median and interquartile range and differences between 3 groups were compared by Mann Whitney test if data were not normally distributed (non-parametric).

• Pearson or Spearman correlation coefficientswere calculated to get the association between quantitative or ordinal variables respectively.

• Logistic (standard) regression model was per-formed to get the significant predictors of HCC.

• P values less than 0.05 were considered statistically significant, and less than 0.01 wereconsidered highly significant.

RESULTS

HCC group included 50 patients they were 40 males (80%) and 10 females (20%) with male

to female ratio 4:1 their age ranging from 45 to 80 years (57.2 ± 8.2). HCV group included 39 patients, they were 25 males (64.1%) and 14 fe-males (35.9%) with their ages ranging from 30 to 64 years (49.9 ± 9.4). Normal control group included 20 subjects; they were 6 males and 14 fe-males with their ages ranging from (33.1 ± 9.2).

Age differed significantly across the groups(P value = 0.001) also there was significant dif-ference in sex distribution between groups (P value = 0.001).

There was a statistical significant differencebetween TT, CT and CC genotypes of SPARC gene (rs 2304052) regarding level of AFP. CC genotype is associated with higher levels of AFP (1761.0ng/ml) than CT (9.8ng/ml) and TT (9.0ng/ml) genotypes (P value = <0.009). SPARC gene (rs 2304052) polymorphic genotypes (TT, CT and CC) showed significant difference regardingquantitative degree of liver fibrosis calculated byAPRI test score (p=0.04) where Values of APRI test were significantly high in CC genotype car-riers [median 2.5 (2.1 – 3.2)] than CT genotype carriers [1.1 (0.4 – 1.7)] and TT genotype carriers [0.9 (0.4 – 2.0)], data are illustrated in table 1.

There was no statistical significance be-tween HCC, HCV and control groups regard-ing frequencies of polymorphic genotypes (TT, CT and CC) of SPARC gene polymorphism (rs 2304052) (P value = 0.2). There was statistical significant difference between HCC, HCV andcontrol groups regarding frequency of allele dis-tribution. Frequency of C allele (mutant allele) is higher in HCC than HCV and control groups (P=0.05).

Odds ratio for developing HCC in subjects with C allele is 4.5 (95% CI = 1.0 – 20.1) (p= 0.04), data are illustrated in table 2.

Table 1: comparison between different SPARC (rs2304052) polymorphism Genotypes,AFP level and APRI test in the three studied groups:

Parameter TT (n=67) CT (n =17) CC (n=5) P-value

AFP (ng/ml) 9.0 (2.0-36.7)a

9.8 (2.1-765.2)a,b

1761.0 (16.7-5430)b < 0.009

APRI test 0.9 (0.4-2.0)a

1.1 (0.4-1.7)a

2.5 (2.1-3.2)b 0.04

‡Data presented as median (25th-75th percentiles) . Groups bearing different initials are statistically sig-nificantly different at p value of 0.05. **p value < 0.01 is highly significant.

Soliman, D. H. A. 12

DISCUSSION

Egypt has the highest prevalence of HCV in the world, with reported average of 14.7% among healthy population. (13). Recent investigations in Egypt have shown the increasing importance of HCV infection in the etiology of HCC, now esti-mated to account for 40-50% of HCC cases (15,8).

It was evident that SPARC gene has a tumor suppressive function in the liver, so the disrup-tion of SPARC contributes to the development and progression of HCC (16,4).

The aim of the present study was to detect the association of SPARC gene polymorphism (rs 2304052) with increased risk of development of HCC and its clinical utility as a non-invasive biomarker in the diagnosis of HCC.

In the present study The frequencies of genotypes TT, CT and CC of SPARC gene (rs 2304052) in HCC group were (68.0%, 26.0% and 6.0% respectively), in HCV group were (84.6%, 10.3% and 5.1% respectively) and in control group were (90.0%, 10.0% and 0.0% re-spectively). However, the frequency of CC and CT genotype (risky genotypes) was higher in HCC group than HCV and control groups, yet the difference was statistically insignificant withP value= 0.2. This may be attributed to the small study number when compared with that of Segat

et al(20).. They applied their study on 230 HCC patients and 230 healthy controls. They reported that the frequencies of genotypes TT, CT and CC of SPARC gene (rs 2304052) in HCC group were (67.0%, 20.0% and 13.0% respectively) and in control group were (80.0%, 44.0% and 1.0% respectively) with P value <0.001.

Also, Motohisa and Masao, (2009)(16), report-ed that the CC genotype at (rs 2304052) SNP of SPARC was associated with the susceptibility of HCC.

In the current study the frequencies of T and C alleles of SPARC gene in HCC group were (81.0% and 19.0% respectively), in HCV group were (89.7% and 10.3% respectively) and that in control group were (95.0% and 5.0% respec-tively). There was statistical significant increasein the frequency of C allele in HCC group when compared with the other 2 groups with P value = 0.05, on the other hand the frequency of T allele was higher in the control group as regards the other 2 groups.

Segat et al., (2009)(20), agreed with these re-sults as they revealed that there were statistical significant differences between HCC patientsand healthy controls regarding the frequencies of T and C alleles of SPARC gene. The frequen-cies of T and C alleles of SPARC gene in HCC

Table 2: Frequencies of polymorphic genotypes of SPARC gene polymorphism (rs 2304052) and allele fre-quency in HCC, HCV and control groups:

HCC (n =50) N,frequency

HCV (n = 39) N,frequency

Control (n =20) N,frequency

p-value O R (95%) CI

TT 34 68.0% 33 84.6% 18 90.0%

0.24.5

(1.0-20.1

CT 13 26.0% 4 10.3% 2 10.0%

CC 3 6.0% 2 5.1% 0 0.0%

T 81 81.0% 70 89.7% 38 95.0%0.05

C 19 19.0% 8 10.3% 2 5.0%‡Data presented as median (25th-75th percentiles). Groups having different initials are statistically signifi-

cantly different at p value of 0.05. *p value < 0.05 is significant

Association of SNP SPARC Gene & HCC 13group were (77.0% and 90.0% respectively) and that in control group were (90.0% and 10.0% re-spectively). The C allele was significantly morefrequent in HCC patients than in healthy con-trols with P value = < 0.001. Motohisa and Ma-sao, (2009)(16), also agreed with these results and reported that the C allele at (rs 2304052) SNP of SPARC was associated with the susceptibility of HCC.

Segat et al., (2009)(20), stated that their results suggest a role of SPARC (rs2304052) in the sus-ceptibility of HCC development independent of the causative agent. The allele C in homozygous or heterozygous conditions could be associated with a modified expression of the SPARC genein HCC hepatic tissues.

Also Motohisa and Masao, (2009)(16), stated that although further analysis should be done to clarify the functional role of SPARC gene poly-morphism (rs 2304052) in the pathogenesis of HCC it may serve as a good marker that can be used to identify subgroup that has a higher risk of developing HCC.

In the current study SPARC gene (rs 2304052) polymorphic genotypes (TT, CT and CC) showed significant difference regarding de-gree of liver fibrosis calculated by APRI test inthe form of quantitative value. Values of APRI test were significantly higher in CC genotypecarriers [median 2.5 (2.1 – 3.2)] than CT geno-type carriers [1.1 (0.4 – 1.7)] and TT genotype carriers, with P value = 0.04.

In 2012, Trombetta-eSilvia and Bradshaw(23) studied the role of SPARC gene in inducing he-patic fibrosis. They conducted a study whereinSPARC expression was evaluated in human liv-er samples from patients with liver cirrhosis and hepatocellular carcinoma. They revealed that SPARC expression is closely tied to increased collagen deposition and is correlated with extent of fibrosis. They also revealed that SPARC playsa major role in key pathogenic events related to the fibrogenic process such as hepatocyte necro-sis, inflammation and recruitment/activation ofmyofibroblast as well as in the induction of theprofibrogenic cytokine TGF-β1 which is a cy-tokine that plays a key role in the activation of

hepatic stellate (HSCs) and in the development of hepatic fibrosis.

In this study, there was statistically signifi-cant higher values of serum AFP level in HCC group (median 139.3 ng/ml) versus HCV group (median 5.3ng/ml) and normal control group (median 1.2 ng/ml) with P value = <0.001.

In the present study, on comparing the three SPARC polymorphic genotypes as regards AFP level, it was observed that there was a statisti-cally significant increase in the level of serumAFP level in CC and CT genotype carriers (risky genotypes) compared to TT genotype carriers with P value = <0.001.

The same finding was reported by, Ali etal., (2004)(1). They found that AFP level was significantly increased in HCC than in chronicliver disease. Also a study done by Hussein et al., (2008)(10) showed a significant difference inAFP levels between patients with HCC (1040.29 ± 2315 ng/ml), and chronic liver disease (8.47±20.035 ng/ml) (P value = < 0.05). AFP was higher in HCC patients when compared to patients with chronic liver disease with signifi-cant difference (P value = < 0.05).

In conclusion, the present study revealed that C allele (mutant allele) of (rs2304052) SNP of SPARC gene was associated with increased risk of development of HCC, also the CC and CT genotypes (risky genotypes) of SPARC gene SNP (rs2304052) were associated with increased degree of fibrosis which represent a major riskfactor for HCC.

REFERENCES1. Ali MA, Koura BA, El-Mashad N, et al (2004). The

Bcl-2 and TGF-beta1 levels in patients with chronic hepatitis C, liver cirrhosis and hepatocellular carcino-ma. Egypt J Immunol; 11: 83.

2. Arnold SA and Brekken RA (2009). SPARC: a matri-cellular regulator of tumorigenesis. J Cell Commun Signal; 3(3-4): 255.

3. Atorrasagasti C, Malvicini M, Aquino JB, et al (2010). Overexpression of SPARC obliterates the in vivo tu-morigenicity of human hepatocellular carcinoma cells. Int. J. Cancer; 126: 2726.

4. Atorrasagasti C, Peixoto E, Aquino JB, et al. (2013). Lack of the Matricellular Protein SPARC (Secreted Protein, Acidic and Rich in Cysteine) Attenuates Liver Fibrogenesis in Mice. PLoS ONE 8(2): e54962.

Soliman, D. H. A. 145. Befeler AS and Di Bisceglie AM (2002). Hepatocellu-

lar carcinoma: diagnosis and treatment. Gastroenterol-ogy; 122: 1609.

6. Bustin SA (October 2000). Absolute quantification ofmRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol; 25 (2): 169.

7. . Carr BI, Guerra V, Giannini EG, et al (2014): for the italian liver cancer (ITA.LI.CA) group Significance ofplatelet and AFP levels and liver function parameters for HCC size and survival int J. Biol Markers. Jan 17:0.

8. Cheng J, Wang W, Zhang Y, et al (2014): Hepatocel-lular carcinoma: systematic review and meta-analysis. Jan, 30; 9 (1): e87011

9. Heid CA, Stevens J, Livak KJ, et al (1996). Real time quantitative PCR. Genome Res; 6: 986.

10. Hussein MM, Ibrahim AA, Abdella HM, et al (2008). Evaluation of serum squamous cell carcinoma antigen as a novel biomarker for diagnosis of Hepatocellular Carcinoma in Egyptian patients. Indian Journal of Can-cer; 45 (4): 167.

11. Kutyavin IV, Afonina IA, Mills A, et al (2000). 3′-Mi-nor groove binder-DNA probes increase sequence spec-ificity at PCR extension temperatures”. Nucleic AcidsRes. 28 (2): 655.

12. Lau CP, Poon RT, Cheung ST, et al (2006). SPARC and Hevin expression correlate with tumour angiogenesis in hepatocellular carcinoma. J Pathol. 210:459.

13. Lehman E, Wilson M (2009). Epidemiology of hepati-tis viruses among hepatocellular carcinoma cases and healthy people in Egypt: A systematic review and meta-analysis. Int. J. Cancer; 124: 690.

14. Libbrecht L, Serveri T, Cassiman D, et al., (2006). Glypican-3 expression distinguishes small Hepatocel-lular Carcinomas from cirrhosis, dysplastic nodules and focal nodular hyperplasia-like nodules. American Jour-nal of a surgical Pathology; 30: 1405.

15. Mohamoud YA, Mumtaz GR , Riome S, et al (2013). The epidemiology of hepatitis C virus in Egypt: a sys-

tematic review and data synthesis. BMC Infectious Dis-eases; 13:288.

16. Motohisa Tada and Masao Omata (2009). Another key molecule for pathogenesis of hepatocellular carcinoma. Journal of Gastroenterology and Hepatology; 24:1803.

17. Nakatani K, Seki S, Kawada N (2002). Expression of SPARC by activated hepatic stellate cells and its corre-lation with the stages of fibrogenesis in human chronichepatitis. Virchows Arch. 441(5): 466.

18. Nishikawa H, Kimura T, Kita R, et al (2013). Treatment for Hepatocellular Carcinoma in Elderly Patients: A Literature Review. J Cancer; 4(8):635.

19. QIAamp® DNA Mini Kit Handbook (2012): for DNA extraction from human whole blood.

20. Segat L, Milanese M, Pirulli D (2009). Secreted pro-tein acidic and rich in cysteine (SPARC) gene poly-morphism association with hepatocellular carcinoma in Italian patients. J. Gastroenterol. Hepatol. 24 (12): 1840.

21. Snyder N, Gajula L, Xiao SY, et al (2006). APRI: an easy and validated predictor of hepatic fibrosis inchronic hepatitis C. J Clin Gastroenterol; 40(6):535.

22. Sun J, Lu H, Wang X, et al (2013). MicroRNAs in Hepatocellular Carcinoma: Regulation, Function, and Clinical Implications. The Scientific World Journal Vol-ume 2013, Article ID 924206, 14 pages.

23. Trombetta-eSilva J and Bradshaw AD (2012). The Function of SPARC as a Mediator of Fibrosis. Open Rheumatol J; 6:146.

24. Yao DF, Dong ZZ and Yao M (2007). Specific molecu-lar markers in Hepatocellular Carcinoma. Hepatobili-ary Pancreat Dis Int; 6: 241.

25. Zhou L, Liu J and Luo F (2006). Serum tumor mark-ers for detection of Hepatocellular Carcinoma. World Journal of Gastroenterology; 12 (8): 1175.

26. Zhu A.X., Sahani D.V., Duda D.G., et al. (2009). Ef-ficacy, safety, and potential biomarkers of sunitinibmonotherapy in advanced hepatocellular carcinoma: a phase II study. J Clin Oncol; 27: 3027.

Association of SNP SPARC Gene & HCC 15

الخبيثه الخلويه الكبد بامراض نسبه االصابه " وزياده لجين "اسيارك النيكليوتايد الواحد أشكال تعدد بين العالقهسليمان احمد هشام دينا

من أكثر مصر تعتبر الوفاة. و إلحداث مسببا أكثرها و فى العالم إنتشارا السرطانيه األورام أكثر من الكبد أورام تعتبرالمبكر لهذه يعتبرالتشخص لذلك و سرطان الكبد. و تليف الى يؤدى الذي ”سى“ و المزمن الكبدى بالفيروس الدول اصابةوظائف له الذى ”إسبارك“ بروتين نتج ”إسبارك“ جين ان الفعال. العالج فى البدء لسرعة الهامة األدوات من اإلصاباتأورام حدوث و الكبد ف وي فى تل ح دور له أن الى باالضافة تشكيلها و انقسام الخاليا تنظيم فى هام له دور و متعددة حيويهز و ”إسبارك“ لجين الواحد النيكليوتايد أشكال تعدد العالقة بين بحث هو الدراسة هذه من الرئيسى و الهدف الخبيثة., الكبداشتملت قد و هذا الكبد. سرطان تشخص فى لألورام كدالالت استخدامه من الفائدة و الخبيثة الكبد بأمراض االصابة نسبة ادة

روس الف و الكبد بسرطان مصابا يضا 50 مر عدد علي اشتملت قد و ثالثة مجموعات: المجموعة األولى على الدراسةأما ”سى“, المزمن الكبدى بالفيروس مصابا 49 مريضا عدد على .اشتملت وقد ة الثان المجموعة و ”سى“, المزمن الكبدىاشتملت الدراسة و قد هذا الضابطة. المجموعة بمثابة ل كونوا األصحاء من شخصا 50 عدد المجموعة الثالثة فاشتملت علىألفا الجنينى البروتين الى باإلضافة كاملة الكبد كل من :وظائف صورة فى وذلك المرضى لجميع االتية التحاليل على عملاستخدام بواسطة الدم فى ”إسبارك“ لجين الجينى التركيب دراسة على الدراسة اشتملت قد و (AFP).هذا ( بروتين فيتوإرتفاعا الدراسة أظهرت قد و هذا الضابطة. المجموعة للبوليميريز لجميع المرضى و التسلسلي للتفاعل الكمى القياس جهازذلك الكبد و بسرطان المصابين المرضى مجموعة ما بين allele C ف ال حاملى عدد فى إحصائية داللة و ذو ملحوظامجموعه بين فيما احصائيه دالله وذو ملحوظا اختالفا وجود عدم الدراسه اثبتت و الثالثة.كما الثانية المجموعة بالمقارنة مععدد في والمجموعه الضابطه "سي" الكبدي بالفيروس المصابين المرضي ومجموعه الكبد المصابين بسرطان المرضياختالفا ملحوظا وجد انه )‘.كما TT,CT,CC) بمختلف اشكاله (rs 2304052 ) " اسبارك " لجين الشكلي التعدد حامليحاملي ان وجد انه حيث الكبدي. التليف ونسبه (rs2304052 ) " اسبارك " لجين الشكلي التعدد بين احصائيه وذو داللهالجيني. النوع من حاملي اكثر الكبدى التليف نسبه زباده مع احصائيه دالله ذو ارتباطا مرتبطان CC و CT الجيني النوععند فيتو بروتين االلفا الجنيني البروتين مستوي احصائيه في دالله ملحوظا وذو وجد ارتفاعا ماسبق فقد الي TT وباالضافه

:TT الجيني النوع حاملي من CT اكثر CCو الجيني النوع حاملي

Soliman, D. H. A. 16

ASSOCIATION OF MONOCYTE CHEMOATTRACTANT PROTEIN 1 (MCP-1) GENE POLYMORPHISM WITH LUPUS NEPHRITIS IN

EGYPTIAN PATIENTSLamiaa A. Mohammad*, Dina M. Atef* and Amany Mustafa Abul-Saoud**

ABSTRACTBackground and study aim: Monocyte chemoattractant protein-1 (MCP-1) is a member of CC chemokine that plays an

important role in the recruitment of monocytes/macrophages into renal tubulointerstitium. A biallelic A/G polymorphism at position -2518 in the MCP-1 gene was found to regulate MCP-1 expression. MCP-1 and its A/G gene polymorphism have been implicated in the pathogenesis of some renal diseases. The aim of the study was to investigate the role of the MCP-1 gene polymorphism as early predictors of the development of glomerulonephropathy in SLE patients. We also aimed to measure the serum and urinary levels of MCP-1 in patients with SLE, to find out its relation to clinical disease activity. Methods: 140 SLEpatients (100 with nephritis and 40 without nephritis) and 80 controls were included in this study. MCP-1 gene polymorphism was analyzed by polymerase chain reaction. Serum and urine MCP-1 level were measured using high-sensitivity enzyme-linked immunosorbent assay. Results: The A/A genotype was more common in controls than in SLE patients, whereas both the A/G (P< 0.000) and G/G (P< 0.000) genotypes were more frequent in SLE patients. Carriers of G allele of the MCP-1 -2518 polymorphism had more than 7 fold increased risk to develop glomerulo-nephropathy in patients with SLE. High MCP-1 circulating levels production from patients with A/G and G/G genotypes was significantly higher than in A/A genotype. Inaddition there were significant differences in the mean levels of serum MCP-1 (P<0.001) and urinary MCP-1 (P<0.001)between patients and controls. Conclusion: The present study provides a new evidence that the presence of MCP-1 A (-2518) G gene polymorphism and high circulating MCP-1 levels can play an important role in the development of SLE and nephropathy in Egyptians. Key words: MCP-1, -2518 gene polymorphism, SLE, Lupus nephritis.

Departments of Clinical Pathology* and Rheumatology & Rehabilitation**, Faculty of Medicine,Zagazig University, Egypt

INTRODUCTION

Systemic lupus erythematosus (SLE) is the multi-systemic autoimmune disorder, in which immune complexes play an essential role. The main characteristic of this disease is an immu-nological inflammation with infiltration andsequestration by immune cells of many tissues and organs (such as the skin, joints, kidneys, and Heart) concomitant with their injury and damage(11). SLE has a negative impact on qual-ity of life and is associated with high health-care costs and significant productivity loss(20).

Early renal involvement in SLE is common and serious, and it requires proper evaluation and management. Lupus nephritis (LN) is asso-ciated with increased mortality and morbidity(14). Despite encouraging results for kidney trans-plantation, once end-stage renal disease is es-tablished, the prognosis is relatively poor and no improvement in preventing its development is achieved(6).

Conventional parameters are not sensitive or specific enough for detecting ongoing disease

activity in the SLE. Cytokines and chemokines play a principal role in the pathogenesis of this chronic disease(27). Recently, multiplex based technologies allows to profile a large numberof cytokines, chemokines and cellular growth factors in a wide range of human samples while consuming a very limited amount of sample(23).

The monocyte chemoattractant protein 1 (MCP-1), belongs to the family of chemotactic cytokines. MCP-1 functions as a potent agonist for monocytes, memory T cells, and basophils. Evidence in animal and human studies suggests a significant role of MCP-1 in the progressionof glomerular and tubulo-interstitial injuries and glomerulonephritis in patients with SLE(3).

It has been demonstrated that the MCP-1 –2518 A/G transition in the promoter region may modulate the levels of MCP-1 expression. The MCP-1 –2518 G allele, compared to the –2518 A allele, is linked with increased production of both MCP-1 transcript and protein(17). Involvement of the MCP-1 –2518 A/G promoter polymorphism in SLE development and its contribution to some


Mohammad, L. A., et al.18

clinical manifestations of SLE remains contro-versial (2,22).

In this study, we measured the MCP-1 lev-els in plasma and urine samples as potential biomarkers of disease activity in patients with SLE and analyzed the distribution of functional MCP-1 -2518 A/G polymorphic variants, with the aim to investigate whether the A (-2518) G polymorphism of the gene encoding MCP-1, is associated with Lupus Nephritis, MCP-1 concen-tration, laboratory and clinical manifestations in these patients. Early detection of disease activity in SLE could translate into earlier instauration of the treatment, potentially limiting the flare con-sequences.


To achieve this aim, the study was carried out on 220 individuals; they were classified into 140patients with systemic lupus erythematosus who fulfilled the revised criteria for classification ofSLE by the American College of Rheumatology (ACR), 100 with LN and 40 without LN and 80 apparently healthy individuals serving as a con-trol group matched by sex and age with no signs of active inflammatory or infectious diseases.The patients were selected from those referring to Rheumatology and Rehabilitation Depart-ment, Inpatient and Outpatient Clinics, Zagazig University Hospitals. Laboratory investigations were carried out at The Clinical Pathology De-partment, Zagazig University Hospitals during the period from March 2012/ July 2013. All pa-tients provided a written informed consent.

All subjects were subjected to:

Clinical assessment: careful history taking and complete general examination with stress upon symptoms and signs of SLE and nephritis and detailed locomotor system examination. The total joints tenderness was counted for patients with arthritis considering tenderness (T) score to be equal to zero, if there is no tenderness, and 1, 2, and 3 if there is mild, moderate, or severe tenderness, respectively. Also, the number of swollen joints was counted. For each patient, SLE disease activity was evaluated at the time of sampling; using SLE disease Index (SLEDAI) (1) and the Systemic Lupus International Collabo-

rating Clinics/American College of Rheumatol-ogy Damage Index score(8). Nephropathy was diagnosed clinically by the presence of persis-tent micro-albuminuria [albumin excretion rate (AER) 20–200µg/min] and elevated blood pres-sure (systolic blood pressure >135 mmHg and/or diastolic blood pressure >85 mmHg)(9). The renal SLEDAI (r SLEDAI) score consisted of 4 items of the total SLEDAI: hematuria, pyuria, proteinuria and urinary casts/HPF. The presence of each one of the 4 parameters takes a score of 4 points, thus the r SLEDAI score ranged from 0 to a maximum score of 16(1). The patients group had no history for Diabetes Mellitus.

Sample collection:

• Early morning urine samples were taken from every patient and control for urine analysis and U-MCP-1 concentration which were placed at 4°C and transported directly to the laboratory, where they were centrifuged to remove sediment and frozen in aliquots at -80°C for later cytokine analysis, and 24h urine were collected for detec-tion of proteinuria.

• venous blood samples were taken from ev-ery patient and control under sterile conditions and divided for the following:

1. Three ml were added to three EDTA tubes:

a. Tube for complete blood count.

b. Tube for determination of MCP-1 geno-types (as EDTA reduces DNA degeneration).

c. Tube for MCP-1 concentration: the plas-ma was separated by centrifugation for 15 min-utes at 1000 x g within 30 minutes of collection and then transferred to a plastic tube and stored at -80○C for long term storage.

2. 1.6 ml of blood was added to labeled tube containing 0.4 ml trisodium citrate 32 g/l for ESR measurement.

3. Six ml of blood were placed in plain va-cutainer tubes and the collected serum was used for C-reactive protein, liver enzymes, kidney function tests, ANA, anti-dsDNA, ANCA, and serum C3.

MCP-1 Gene Polymorphism and Lupus Nephritis 19

a. Complete blood count: using automated hematology analyzer sysmex KX-21N (Sysmex, America).

b. ESR measurement: using Westergren method.

c. Liver enzymes (ALT & AST) and kid-

ney function tests (BUN & creatinine) were performed using automated clinical chemistry analyzer Cobas Integra 400 plus (Roche Diag

-

nostics, Deutschland).

d. Serum CRP was measured using au-

tomated nephlometry BN ProSpec (Siemens, UK).

e. Urine analysis for hematuria, pyuria and casts (cellular, granular or tubular) and 24 hour urine collection for protein.

Specific laboratory investigation:

a. Determination of autoantibodies such as; ANA, anti-dsDNA and ANCA by indirect im

-

munoflourescence technique. If the sample ispositive, specific antibodies in the diluted serumsample attach to the antigens coupled to a solid phase. In a second step, the attached antibodies are stained with fluorescein-labeled anti-humanantibodies and visualized with the fluorescencemicroscope.

b. Serum C3 was measured using automat-

ed nephlometry BN ProSpec (Siemens, UK).

c. Measurement of plasma and urine MCP-1 levels by ELISA (Enzyme-linked Immunosor

-

bant assay) technique using Quantikine human CCL2/MCP-1 immunoassay catalog number DCP00 from R & D Systems, Minneapolis, MN, USA.

Principles of the test: A monoclonal anti-body specific for human MCP-1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any MCP-1 pres

-

ent is bound by the immobilized antibody. Af-

ter washing away any unbound substances, an enzyme-linked polyclonal antibody specific forhuman MCP-1 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of MCP-1bound in the initial step. The reaction

is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is pre-pared from seven MCP-1 standard dilutions and MCP-1 sample concentration is determined. The detection limits of this ELISA system were 30 pg/ ml for human MCP-1. Urinary MCP-1 levels were standardized by the amount of creatinine in the urine.

Genotyping:

For determination of MCP-1 A (-2518) G gene polymorphism by polymerase chain reac-tion (PCR) followed by restriction fragment length polymorphism (RFLP) and agarose gel electrophoresis, four steps were performed:

1. Extraction of genomic DNA: DNA was purified from peripheral blood samples usingthe E.Z.N.A. Blood DNA Kit (Omega – biotek. Inc).

2. Amplification of genomic DNA by poly-merase chain reaction (PCR) using thermal cy-cler (Gene Amp, PCR system 9700).

3. Digestion of amplified products with re-striction endonuclease enzyme PVU II.

4. Identification of MCP-1 genotypes bygel electrophoresis (Scie – plas).

Genotyping of the MCP-1 ~2518 polymor-phism was performed as described by Rovin et al(12). The distal regulatory region was amplifiedusing the following primers: Forward primer 5’-CCGAGATGTTCCCAGCACAG-3’. Reverse primer 5’-CTGCTTTGCTTGTGCCTCTT-3’. DNA was amplified using puReTaq Ready- to-Go PCR Beads which are desingned and manu-factured by Amersham Biosciences. Cycle con-ditions used were 94°C for 5 minutes, followed by 40 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1.5 minutes, with a finalextension of 72°C for 7 minutes. The polymerase chain reaction product was digested with 5U of PvuII endonuclease restriction enzyme (supplied by Jena Bioscience). The DNA fragments were subjected to electrophoresis in a 2% agarose gel stained with ethidium bromide.

The restriction digestion resulted in 708- and 222-bp fragments for G/G homozygous, a 930-bp fragment for A/A homozygous, and 930-,

Routine laboratory investigations:

Mohammad, L. A., et al.20708-, and 222-bp fragments for the A/G hetero-zygous.

Statistical data analysis

The data were coded, entered and analyzed using Statistical Package for Special Sciences (SPSS) software computer program version “19.0, IBM Corp., USA”. Statistical tests were applied to determine the relationships between MCP-1 A (-2518) G genotypes,plasma and urine MCP-1 concentrations, clinical and laboratory parameters in Egyptian patients with SLE and controls.

Quantitative data were presented as mean ± standard deviation. Comparison of quantitative variables between the study groups was done using the Student (t) test. Qualitative data were expressed as frequency and percentage. Associa-tion between qualitative data was done using the chi-square test. Differences with (P) values less than 0.05 were considered significant. Odds ratioand 95% confidence interval were calculated toestimate the strength of the associations.

RESULTS

Basic characteristics of the studied popula-tion: As regards (SLEDAI) of patients groups; the mean value was 13.1± 5.2 with range of 2-26, 28 patients were inactive (SLEDAI<6) and

132 patients were active (SLEDAI≥6).

There were 140 patients (22 male and 118 fe-male) and 80 subjects (28 male and 52 female) in control group. The mean age was 31.2 ± 9.5 years in patients group and 30.3 ± 5.5 years in control group. Laboratory characteristics of patients and controls were summarized in Table (1). There was no significant difference regarding age, sex,WBCs and platelets counts (P>0.05) between the two groups. There was a significant increase inthe mean values of systolic and diastolic blood pressure, Hemoglobin level, ESR in 1st hour, se-rum ALT, AST, BUN and creatinine in patients compared with control group. ANA was positive in 105 patients (75%), anti ds-DNA in 119 pa-tients (85%), ANCA in 21 patients (15%), serum C3 was low in 42 patients (30%), proteinuria was found in 95 patients (67.8%), hematuria in 35 patients (25%), pyuria in 32 patients (22.8%) and urinary casts in 38 patients (27.1%).

In the present study, as regards the serum MCP-1 concentration, it was statistically signif-icantly higher in patients (187.9± 93.6 pg/ml) than in controls (67.8± 17.1 pg/ml); P<0.001. Regarding the urinary MCP-1 level, it was statis-tically significantly higher in SLE patients thanthe control group 89.3± 42.5 and 33.8± 19.6 pg/ml respectively; P<0.001.

Table (1): Laboratory characteristics of patients and controls.

Characteristic Patients (140) Control (80) P Sex (Male/Female) 22/118 28/52 >0.05

Age 31.2±9.5 30.3±5.5 >0.05Duration 11.2±2.8 -

SBP (mmHg) 150±8 116±3 <0.001*DBP (mmHg) 90±4 78±3 <0.001*hematuria Sex 25% 0%

Pyuria 22.8% 0%Urinary casts 27.1% 0%Proteinuria 67.8% 0%

ANA 75% 0%ANCA 15% 0%

Anti-ds DNA 85% 0%Low serum C3 30% 0%

CRP POSITIVE % 45% 0%Hemoglobin (g/dl) 11.1±1.7 13.1±1.2 <0.001*

WBCs (x 103/ mm3) 6.7± 2.4 8.1± 2.2 >0.05Platelets count(x 103/ mm3) 232.7± 88.9 247.1± 55.7 >0.05

ESR 1st hour (mm/ hr) 47.3± 24.7 9.8± 4.2 <0.001*ALT (U/L) 48.1± 43 22.1± 7.86 <0.001*AST (U/L) 41.5± 51.6 24± 6.7 <0.05

BUN (mg/dl) 47.1± 9.7 13.2 ± 3.4 <0.05Creatinine (mg/dl) 1. 5 ± 0.7 0.74± 0.2 <0.001*S. MCP-1 (pg/ml) 187.9±93.6 67.8±17.1 <0.001*U. MCP-1 (pg/ml) 89.3 ± 42.5 33.8 ± 19.6 <0.001*

*Significant if (P < 0.05).

MCP-1 Gene Polymorphism and Lupus Nephritis 21

MCP-1 A (-2518) G genotypes frequencies are mentioned in table (2). The distribution of wild (AA) and mutant (AG/GG) genotypes was 80% and 20% in control group with low frequen-cy of GG genotype (5%) and G allele (12.5%). The exact test of population distribution indi-cated that the distributions of MCP-1G allele frequencies were significantly higher in SLE-pa-tients (51.8%) compared to healthy control sub-jects (12.5%, P<0.0000).

The MCP-1(-2518) GG genotype frequencies were significantly higher in the patients groupcompared with the controls (P = 0.000) and were associated with increased risk for SLE develop-

ment (odds ratio, 17.57; 95% confidence inter-val, 5.97 to 51.68; P = 0.000) (Table 2).

The genotype frequency showed significantincrease in mutant genotypes (80% vs. 35%, P<0.0001) with the predominance of homozy-gote mutant (GG) genotype (58% vs. 10%, P < 0.0001, OR =18.85; 95% confidence interval,5.85 to 60.67) in lupus nephritis patients group compared with non-lupus nephritis patients group. Moreover, the higher OR in homozygotes (18.85) than in heterozygotes (2.86) in com-paring LN cases with non LN group suggests a dominant mode of action of the homozygote MCP-1 (-2518) polymorphism in the develop-ment of GN (Table 3).

Table (2): Distribution of 2518 A/G genotype frequency of MCP-1 gene in studied groups

Polymorphism SLE case(n= 140)

Control(n=80 ) OR 95% Cl P

MCP-1

AA n (%) 51 (36.4) 64 (80)

AG n (%) 33 (23.6) 12 (15) 3.45 1.62-7.35 0.001

GG n (%) 56 (40.0) 4 (5) 17.57 5.97-51.68 0.000

G allele n (%) 145 (51.8) 20 (12.5) 7.52 4.45-12.69 0.000Abbreviations: MCP-1= Monocyte chemoattractant protein-1; SLE =systemic lupus erythematosus;

OR = odds ratios; 95% CI = 95% confidence interval. Significant if (P < 0.05).

Figure (1): MCP-1 gene shown on an electrophoresis gel stained with ethidium bromide. Lanes from left: DNA ladder (1), AA genotypes (Lane No 2,3,4,6,9,10,11), AG genotype ( 5,7,8) and GG genotype (12,13).

Table (3): Distribution of MCP-1 genotype in SLE patients with and without nephritis

PolymorphismSLE

with nephritis(n= 100)

SLE without nephritis(n=40) OR 95% Cl P

MCP-1

AA n (%) 20 (20) 26 (65)AG n (%) 22 (22) 10 (25) 2.86 1.11-7.38 0.02GG n (%) 58 (58) 4 (10) 18.85 5.85-60.67 0.000

G allele n (%) 138 (69) 18 (22.5) 7.66 4.19-14.03 0.000 Abbreviations: MCP-1= Monocyte chemoattractant protein-1; SLE =systemic lupus erythematosus;

OR = odds ratios; 95% CI = 95% confidence interval. Significant if (P < 0.05).

Mohammad, L. A., et al.22

Analyzing MCP-1 levels in SLE patients within different genotypes reveals positive as-sociation between high MCP-1 level in both se-rum and urine and MCP-1 genetic mutation in LN and non-LN patients. In hetero- and homo-zygote individuals, sMCP-1 and uMCP-1 levels were higher in SLE patients with nephritis than these without nephritis. Significant differenceswere shown between the levels of MCP-1 in both serum and urine samples in mutant genotypes in SLE with nephritis were detected on comparing with non nephritis cases (P = 0.000) (Table 4).

DISCUSSION

MCP-1 is a low molecular weight CC-che-mokine (13 KDa) linked to the innate immunity that could easily filter into the urine. MCP-1attracts monocytes, T cells, NK cells, and ba-sophils in acute inflammatory conditions, andalso can act as an important mediator in chronic inflammation(5).

In the present work, we demonstrated that the production of MCP-1 has been found to be deeply deregulated in SLE patients and is prob-ably involved in the development of GN. This conception is based on the significantly elevatedMCP-1 level in both the serum and urine sam-ples in LN patients compared to the SLE patients without LN and healthy controls.

The role of circulating MCP-1 in renal dam-age in different glomerular diseases, such as SLE(22), IgA nephropathy(26), DM(21) and polycys-tic kidney(28) has been intensively investigated. Although many studies have focused on the as-sociation between circulating or urinary MCP-1 level and such renal disorders, only few of

them were concerned with its role in nephropa-thy which seems to be controversial. This is in agreement with the study of Chiarelli et al.(4), who focused on a role of circulating MCP-1 in the development of early nephropathy in patients with type 1 DM. This is explained by the piv-otal role played by increased MCP- 1 production due to stimulation by cytokines(7) and exposure to urinary proteins in the progressive tubule-interstitial damage(24). So, we suggest that this marker reflects the progression of nephropathyand could be a predictor of the development of GN (irrespective of the underlying etiology).

However, Kiyici et al.(10) reported that serum MCP-1 levels were significantly higher in type 1diabetic patients than healthy normal group but not correlated with development of nephropa-thy. Morii et al.(16) demonstrated that urine lev-els, but not serum levels, of MCP-1 correlates with the development of diabetic nephropathy. Interestingly, Qiu-yue and Fen-qin verified thatboth serum and urinary MCP-1 had an impact on kidney affection a superior role of urinary MCP-1 as early predictor before appearance of proteinuria(18).

Notwithstanding the convincing evidence that MCP-1 polymorphism plays an important role in susceptibility to SLE, contradictory result has been reported among different populations. The role of MCP-1 polymorphisms in SLE has been studied by other investigators, but has led to differing conclusions. Several studies have observed a significant association of SLE withMCP-1 gene polymorphisms(15,22,25). While on the contrary, other studies showed a lack of asso-ciation with that genetic variation(12.13.17,19),. Rea-

Table (4): Serum and urine level of MCP-1SLE

with nephritis(n= 100)

SLE without nephritis(n=40) P

Plasma MCP-1 (pg/ml)AA 155.6±6.1 99.1±4.8 0.000

AG+ GG 201.7±79.8 138.6±11.5 0.000

Urine MCP-1 (pg/ml)AA 87.6±10.5 41.6±5.2 0.000

AG+ GG 115.4±16.4 69.3±9.5 0.000Abbreviations: MCP-1= Monocyte chemoattractant protein-1; SLE =systemic lupus erythematosus; Sig-

nificant if (P < 0.05).

23sons for the variability in associations are still ambiguous. Thus, investigating the frequencies and distribution of MCP-1 gene across popula-tions is essential for understanding disease asso-ciation and discovery of population differences, especially for Egyptian as they have a much high SLE prevalence.

So that, another finding in this study was that,patients with SLE who have a particular MCP-1 polymorphism have a greater likelihood of hav-ing renal disease. Our results indicate that SLE patients with an A/G or G/G MCP-1 -2518 geno-type have a higher risk of developing LN than those with an A/A genotype. G allele frequency was significantly higher in GN group than othertwo groups. This is in agreement with the study done by Ye DQ et al.(25) and Moon et al.(15) who found that the -2518 G frequency is obviously over-represented in Asian, Mexican, Korean and Chinese populations,

Our results demonstrated that SLE patients who were G allele carriers especially the homo-zygotes were at highly risk for GN with positive association between MCP-1 mutant genotype and G allele and high serum and urinary MCP-1 levels. These results may be suggested that G allele carriage can be responsible for over pro-duction of circulating MCP-1 in SLE patients with renal manifestations and predisposes to the development of nephropathy rather than the oc-currence of the disease itself.

Moon et al.(15) added that carriage of -2518 A allele in the MCP-1 gene was associated with the susceptibility of kidney failure in Korean pa-tients with type 2 DM. Tucci et al.(22) also report-ed that, the A/G and G/G MCP-1 polymorphisms are associated with a greater likelihood of renal disease in patients with SLE. Patients with these genotypes have increased MCP-1 production both peripherally and locally within the kidney. Furthermore Piotrowski et al. (17) observed that the MCP-1 -2518 G variant can be associated with some clinical findings in patients with SLEsuch as renal manifestations and thrombocyto-penia.

However, Liu et al.(13) had not detected a sig-nificant association between MCP-1 promoter -

2518 A/G polymorphism and SLE. Kim et al.(9) also reported that urinary excretion of MCP-1 was greater in AA homozygotes than in other genotypes in Korean patients with lupus nephri-tis. Although, Kouyama et al.(12) failed to detect an association between MCP-1A/G polymor-phism and type 2 diabetes, they noticed that the -2518 AA genotype was associated with higher serum MCP- 1 concentration.

These inconsistent results might be popula-tion-dependent and/or phenotype specific.

In conclusion, the present study implicates the role of circulatory MCP-1 and its -2518 G gene polymorphism as a predictor of the devel-opment of GN in Egyptian SLE patients. Urinary levels of MCP-1 measurement may be a useful tool both for the early detection of LN and for monitoring the response to therapy.

Acknowledgements

The authors are grateful to the study partici-pants. Institutional review board approval was obtained for all sample collections.

The authors declare that they have no conflictof interest.

REFERENCES1. Bombardier C, Gladman DD, Urowitz MB, et,al. 1992

Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis Rheum.; 35:630.

2. Brown KS, Nackos E, Morthala S, et al. 2007 monocyte chemoattractant protein-1: plasma concentration and A (-2518) G promotor polymorphism of its gene in sys-temic lupus erythematosus. J Rheumatol.; 34(4):740.

3. Charo IF and Taubman MB. 2004 Chemokines in the pathogenesis of vascular disease. Circ Res ; 95: 858.

4. Chiarelli F, Cipollone F, Mohn A, et al. 2002 Circulat-ing monocyte chemoattractant protein-1 and early de-velopment of nephropathy in type 1 diabetes. Diabetes Care. ; 25:1829.

5. Chong BF and Mohan Ch. 2009 Targeting the CXCR4/CXCL12 axis in systemic lupus erythematosus. Expert Opin Ther Targets ; 13:1147.

6. Croca SC, Rodrigues T, Isenberg DA. 2011 Assessment of a lupus nephritis cohort over a 30-year period. Rheu-matology (Oxford) ; 50:1424.

7. Gerritsma JS, VanKooten C, Gerritsen AF, et al. 1998 Transforming growth factor-beta 1 regulates chemo-kines and complement production by human proximal tubular epithelial cells. Kidney Int. ; 53:609.

MCP-1 Gene Polymorphism and Lupus Nephritis

Mohammad, L. A., et al.248. Gladman D, Ginzler E, Goldsmith C, et al. 1996 The

development and initial validation of the Systemic Lu-pus International Collaborating Clinics/American Col-lege of Rheumatology damage index for systemic lupus erythematosus. Arthritis Rheum. ; 39:363.

9. Kim HL, Lee DS, Yang SH, et al. 2002 The Polymor-phism of Monocyte Chemoattractant Protein-1 Is As-sociated With the Renal Disease of SLE. Am J Kidney Dis ; 40: 1146.

10. Kiyici S, Erturk E, Budak F, et al. 2006 Serum mono-cyte chemoattractant protein-1 and monocyte adhesion molecules in type 1 diabetic patients with nephropathy. Arch Med Res. ; 37:998.

11. Kong KO, Tan AW, Thong BYH, et al. 2009 Enhanced expression of interferon-inducible protein -10 corre-lates with disease activity and clinical manifestations in systemic lupus erythematosus. Clin Exp Immunol ; 156:134.

12. Kouyama K, Miyake K, Zenibayashi M, et al. 2007 As-sociation of serum MCP-1 concentration and MCP-1 polymorphism with insulin resistance in Japanese indi-viduals with obese type 2 diabetes. Kobe J Med Sci. ; 53:345.

13. Liu T, Zhai JX, Wang HY, et al. 2012 Ye DQ. Meta-analysis of MCP-1 promoter 22518 A/G polymorphism and SLE susceptibility. Mol Biol Rep. ; 39:8475.

14. Mahmoud SS, Bazaraa HM, Lotfy HM, et al. 2012 Renal involvement in childhood-onset systemic lupus erythematosus in Egypt. Rheumatol Int ; Jan; 32(1):47.

15. Moon JY, Jeong L, Lee S, et al. 2007 Association of polymorphisms in monocyte chemoattractant protein-1 promoter with diabetic kidney failure in Korean pa-tients with type 2 diabetes mellitus. J Korean Med Sci.; 22: 810.

16. Morii T, Fujita H, Narita T, et al. 2003 Association of monocyte chemoattractant protein-1 with renal tubular damage in diabetic nephropathy. J Diabetes Complica-tions. ; 17:11.

17. Piotrowski P, Lianeri M, Gasik R, et al. 2010 Monocyte Chemoattractant Protein-1 −2518 A/G single nucleotide polymorphism might be associated with renal disease and thrombocytopenia of SLE. Journal of Biomedicine and Biotechnology ; Article ID 130265, 6 pages.

18. Qiu-yue W and Fen-qin C. 2009 Clinical significanceand different levels of urinary monocyte chemoattrac-tant protein-1 in type 2 diabetes mellitus. Diabetes Res

Clin Practice. ; 83:215.

19. Sanchez E, Sabio JM, Callejas JL, et al. 2006 Associa-tion study of genetic variants of pro inflammatory che-mokine and cytokine genes in systemic lupus erythema-tosus. BMC Med Genet. ; 7:48.

20. Slawsky KA, Fernandes AW, Fusfeld L, et l, 2011 A structured literature review of the direct costs of adult systemic lupus erythematosus in the US. Arthritis Care Res (Hoboken) ; 63:1224.

21. Takebayashi KT, Matsumoto S, Aso Y, et l, 2006 Asso-ciation between circulating monocyte chemoattractant protein-1 and urinary albumin excretion in non obese type 2 diabetic patients. J Diabetes Complications ; 20:98.

22. Tucci M, Barnes EV, Sobel ES, et al. 2004; Strong as-sociation of a functional polymorphism in the mono-cyte chemoattractant protein 1 promoter gene with lu-pus nephritis. Arthritis Rheum 50:1842.

23. Vega L, Barbado J, Almansa R, et al. 2010 Prolonged standard treatment for systemic lupus erythematosus fails to normalize the secretion of innate immunity-re-lated chemokines. Eur Cytokine Netw ; 21:71.

24. Wang Y, Rangan GK, Tay YC, et al. 1999 Induction of monocyte chemoattractant protein1 by albumin is me-diated by nuclear factor B in proximal tubule cells. J Am Soc Nephrol. ; 10:1204.

25. Ye DQ, Hu YS, Li XP, et al. 2005 The correlation be-tween monocyte chemoattractant protein-1 and the ar-thritis of systemic lupus erythematosus among Chinese. Arch Dermatol Res. ; 296:366.

26. Yokoyama H, Wada T, Furuichi K, Segawa C, Shimizu M, Kobayashi K, et al. Urinary levels chemo-kines (MCAF/MCP-1, IL-8) reflect distinct disease activitiesand phases of human IgA nephropathy. J Leukoc Biol. 1998; 63:493–499.

27. Yu SL, Kuan WP, Wong CK, et al. 2012 Immunopatho-logical roles of cytokines, chemokines, signaling mol-ecules, and pattern-recognition receptors in systemic lupus erythematosus. Clin Dev Immunol ; 715190.

28. Zheng D, Wolfe M, Cowley BD, et al. 2003; Grantham JJ. Urinary excretion of monocyte chemoattractant pro-tein-1 in autosomal dominant polycystic kidney dis-ease. J Am Soc Nephrol. 14:2588.

25

و سي بي-١) (ام الوحيدة للخلية الكيميائي بالجاذب الخاص للجين الشكلي التنوع بين اإلرتباط مدىالعربية مصر بجمهورية الحمراء الذئبة مرضى فى الكلوى األلتهاب

السعود أبو أمانى مصطفى - عاطف محمد دينا - الوهاب محمد عبد لمياء

عائلتين الى الكيموكين مجموعة وتنقسم لتركيزه .. معين تبعا اتجاه البيضاء في لخاليا الدم محركة جزيئات هم الكيموكين مجموعةاالكبر لتاثيره الحاد نظرا في االلتهاب مهم دور له سي اكس سي عائلة من انترليوكين-٨ اكس سي). (سي و ( (سي سي وهم اساسيتينمختلفة و منشطة لخاليا جاذبة فهي سي بي-١/سي سي ال-٢) (ام ضمنها التي من و سي سي ال عائلة اما متعادلة الصبغة. الخاليا علىااللتهابات في فلهم دورهام ولذلك القلوية الصبغة و الحمضية ،الخاليا الليمفاوية ،الخاليا االكلة الكبيرة الخاليا ، الوحيدة الخاليا تشملالحمراء الذئبة حدوث مرض بزيادة احتمالية مصحوب يكون بي1-) سي للجين (ام ج (٢٥١٨–) أ ان التنوع الشكلي قد وجد المزمنة.التعرف هو من هذا البحث الهدف المرض. لهذا المصاحبين التهاب الكلى و الدموية االوعية التهاب حدوث خطر من يزيد انه .كمامرض الذئبة فى الكلى في حدوث ألتهاب يؤثر الظهور كعامل مبكر سي ال1-) (سي ال –٢٥١٨)ج لجين أ( الشكلي التنوع دور علىالمرض بنشاط عالقتهما مدى معرفة و المرضى هؤالء في و البول المصل من كال فى (١– بي مستوى (ام سي قياس و كذلك الحمراء ،ضابطة. كمجموعة ظاهريا صحيح و 80 شخص الحمراء الذئبة بمرض مصابا 140 مريضا على الدراسة هذه إجراء األكلينيكى.تممستوى قياس و البلمرة المتسلسل تفاعل طريق بي-١( عن سي (ام لجين الجيني أ( –٢٥١٨)ج الطراز اختالف عن الكشف تم قد ومرضى فى شيوعا األكثر هم (ج/ج) و الجيني (أ/ج) الطراز أن وقد لوحظ االليزا. بطريقة المصل و البول فى بي 1- ) ( ام سي الحمراء الذئبة فى مرضى الكلى و خطورة اإلصابة بأمراض القابضة. المجموعة األكثر شيوعا فى هو الطراز (أ/أ) بينما الذئبة الحمراءالبول و المصل بي-١ في مستوى ام سي ارتفاع لوحظ كما فى تركيبهم الجينى. (ج) األليل الحاملين المرضى فى أضعاف 7 من أكثروجود التعدد الجينى ل أن على دليال جديدا الدراسة تقدم هذه وبذلك الضابطة . المجموعة عن المرضى بالذئبة الحمراء مجموعة فيالكلى لدى الذئبة الحمراء وأمراض مرض في تطور دورا هاما يلعب بى1-يمكن أن سى أم مستوي وأرتفاع ج (-2518) أم سى بى1-

المصريين.

MCP-1 Gene Polymorphism and Lupus Nephritis

STEADY-STATE SERUM LEVELS OF VASCULAR CELL ADHESION MOLECULE-1 IN CHILDREN WITH SICKLE CELL DISEASE:

RELEVANCE TO STROKE RISK AND DISEASE SEVERITY.Mona Hassan El-Tagui*, Mona El- Ghamrawy*, Foad Abd Allah ** , Fadwa Said***

and Ahmed Ibrahim*

ABSTRACTBackground:Vascular cell adhesion molecule-1 (VCAM-1) has been implicated as being important in the pathophysiol-

ogy of vaso-oclusive crisis (VOC)of sickle cell disease (SCD). The impact of chronic transfusion therapy and hydroxyurea (Hu) treatement on serum VCAM-1 level is still controversial.Transcranial doppler has been implicated as important tool in prediction of VOC. Patients and Methods : Soluble VCAM-1 (sVCAM-1) levels were measured in plasma using an ELISA as-say (R&D systems) in 85 patients with SCD( HbSS & HbS/beta thalassemia)between the age of 2-18 years).All patients with Hemoglobin level above 7g/dL were subjected to Transcranial Doppler (TCD) Results: Patients with severe VOC had higher sVCAM-1 levels compared to subjects with moderate and mild crisis (P = 0.007,0.039,respectively).A positive correlation be-tween frequency of blood transfusion and VCAM level in whole group was detected (P= 0.001).Among SS patients, VCAM level was significantly lower in patients receiving Hydroxyura(HU) compared to those not on HU( P=0.049).A positive correlationbetween the Middle cerebral artery (MCA) time – averaged mean of the maximum velocity (TAMMvel) and sVCAM1 level was recorded (P =0.041). Conclusion: Our findings of increased sVCAM-1 level during VOC offer a rationale for therapeutic useof cytokine and other VCAM-1 modulators.The reduction of sVCAM-1 levels observed in patients receiving transfusion and Hu treatement offers insight about their protective effect against the VOC.TCD can serve as a tool for prediction of VOC. Keywords: Sickle cell Disease - Vaso-occlusive-crisis - VCAM-1 – Blood transfusion - Hydroxyurea

Departments of Pediatric Hematology*, Neurology** and Clinical & Chemical Pathology***, Faculty of Medicine, Cairo University, Cairo, Egypt

INTRODUCTION

Sickle cell disease (SCD) is one of the com-monest genetic disorders world-wide and is the most common inherited hematological disease affecting humans (17).

SCD has its cardinal features of chronic he-molytic anemia and recurrent painful episodes. These and all other elements of the disease are the result of mutant sickle cell hemoglobin (Hb S) within the red blood cells (19).

Approximately half the individuals with ho-mozygous HbS disease experience vaso-occlu-sive crises (VOC). The frequency of crises is extremely variable. Some individuals have as many as 6 or more episodes annually, whereas others may have fewer or no episodes (12).

The red cell adheres to the endothelium through a series of mechanisms, either directly via exposed red cell membrane phosphatidyl-serine or sulfated glycans, or by using soluble adhesion molecules (e.g., integrins, thrombos-pondin, high-molecular-weight von Willebrand factor and/or vascular cell adhesive molecule–1 (VCAM-1)) as a bridge (5).

VCAM-1 which plays a very important role in the pathophysiology of VOC and stroke is an immunoglobulin-like adhesion molecule ex-pressed on activated endothelial cells and par-ticipates in neointima formation after vascular wall injury, because it facilitates monocyte in-filtration into injured arteries or/and directly en-hances smooth muscle cell proliferation (10).

As stroke -which has devastating conse-quences on children- affects approximately 11% of SCD patients younger than 20 years of age(6), detection of high risk patients and those liable for strokes is very critical in management of those patients (1).

Careful screening with trans-cranial Dop-pler (TCD) ultrasonography and treatment with chronic blood transfusion will likely reduce the number of strokes in children with sickle cell anemia (4). The stratification of stroke risk byTCD served as the basis for the development and testing of a therapeutic strategy using blood transfusion for the primary prevention of stroke in a randomized multicenter trial initiated in 1995 called the Stroke Prevention Trial in Sickle Cell Anemia (STOP)(2).


El-Tagui, M. H., et al.Risk stratification of stroke by TCD was based

on the time-averaged mean of the maximum ve-locity (TAMMvel) in the distal internal carotid artery or proximal middle cerebral artery. Study concluded that children with elevated velocities (≥ 200 cm/sec) have an astonishingly high rate of stroke: ~ 10,000/100,000 patient-years and interpreted the TAMMvel as follows: “normal,” all mean velocities less than 170 cm/sec; “con-ditional,” at least 1 mean velocity of 170 to 199 cm/sec with none greater than or equal to 200 cm/sec; “abnormal (high risk)” at least one mean ve-locity of 200 cm/sec or higher; or “inadequate,” when no readings from the right- or left-middle cerebral artery and internal carotid artery could be obtained. During the transfusion period, most of the TCD studies revert to or toward normal; unfortunately, the risk of stroke reappears once the transfusion regimen is stopped (2).

The role of chronic blood transfusion is to suppress endogenous red cell production and re-duce HbS to less than 30% (5).

Hydroxyurea (HU) is a tremendously impor-tant drug in the management of patients with SCD who have severe clinical manifestation due to its ability to stimulate production of HbF (11). It also decreases the white blood cells, platelets, and reticulocytes, improves in red cell hydration, and decreases in red cell adhesiveness to the vascular endothelium (13).

Our aim is to estimate serum levels of VCAM-1 in children with Sickle cell anemia and correlate it with the severity of vasocclu-sive crisis, findings of trans-cranial Doppler, fre-quency of blood transfusion and treatment with hydroxyurea.

PATIENTS AND METHODS

The study patients evaluated in this cross-sectional study represent a cohort followed at the Pediatric Hematology Clinic of El- Mounira Children’s Hospital, Faculty of Medicine, Cairo University .

85 patients of SCD (HbSS &HbS/beta Thal-assemia) between the age 2-18 years both males and females seen in the clinic for routine follow-up, and who agreed to participate were enrolled.

Patients were ineligible if they were in pain

or had a vaso-occlusive crises (defined as anepisode of acute pain in the abdomen and/or ex-tremities not otherwise explained) (26) at the time of their visit to the clinic or over the last 4 weeks. Patients with previous cerebrovascular accidents (e.g. stroke) or associated with any other neu-rological disorders (e.g. cerebral palsy or brain atrophy) were also excluded.

The study protocol was approved by Research Committee of Pediatric Departement , Cairo uni-versity and informed consent was obtained from the parents or the patients’guardians. Laboratory investigations were done at El-Mounira Hospital Lab, CairoUniversity.

Methods

All patients were subjected to full history tak-ing and review of medical records with special emphasis on age, duration of disease, frequency and severity of vasoocclusive crises, disease re-lated complications (e.g. acute chest syndrome, pulmonary hypertensions, leg ulcers, priapism etc…),occurrence of neurological symptoms (e.g. headache, cranial nerves affection, TIA etc…).

Duration and frequency of blood transfusion had been recorded. A detailed history of treat-ment given including dose and duration also had been obtained. The diagnosis of Sß thalassemia and SS was based on the complete blood count by Cell Dyn 3700, hemoglobin pattern by HPLC [using the VARIANT II Beta-Thalassemia Short Program, Bio-Rad Laboratories] .

VCAM-1 assay:

The levels of VCAM-1 had been measured using (ELISA) technique using (Quantikine Hu-man sVCAM-1 Immunoassay) kit manufactured BY: R&D Systems, Inc. USA (Ref. No. 892718) , with normal reference value ranged of (349 – 991) with mean 557 and SD of139 ng/ml (14)

TCD:

For all recruited patients with their hemo-globin level above 7g/dL, TCD had been con-ducted at Neurovascular Unit of Neurology De-partment, Cairo University, using Multi-Dop-T. device of DWL Technologies to measure aver-age mean peak velocity across Middle cerebral,

28

VCAM-1 in Children With SCD and Stroke Risk Anterior cerebral, Posterior cerebral, Basilar and intracranial part of Internal carotid arteries on both sides, using Stroke Prevention Trial (STOP) Protocol Values’ as reference (Velocity ≥ 200 classified as high risk, between 199-170 condi-tional and ≤ 170 classified as Normal(2).

Statistical analysis:

All data had been collected and tabulated, VCAM-1 levels had been compared with find-ings of transcranial Doppler and clinical data us-ing t-tests, Pearson Chi-square tests and McNe-mar test using SPSS statistical program version 16 IBM Software program to detect the sensitiv-ity and specifity of VCAM-1 as predictor of highrisk patients and stroke in children with sickle cell disease and to identify any association, if present, between serum VCAM-1 levels and dis-ease severity, transfusion therapy and treatment given.

RESULTS

Characteristics of sickle cell disease pa-tients are demonstrated in table (1). The VCAM level among studied patients ranged from 392 to 5336 ng/ml with mean level of 1438.12 ± 848.2 ng/dl. Classification of Patients accord-ing VCAM-1 level in table (2).Patients hav-ing severe vasooclusive crisis had significantlyhigher VCAM1 level compared to subjects with moderate and mild crisis; p-value=0.007 and 0.039, respectively table (3). Analysis to corre-late serum sVCAM1 level with RT&LT MCA TAMMvel using Pearson Correlation test are de-picted in table (4). There was positive correla-tion between frequency of blood transfusion and VCAM level in whole group (P = 0.001). Among SS patients, VCAM level was significantly lowerin patients receiving HU compared to those not on HU (mean level of 1049±495.6 ng/ml versus 1368.5±599.9 ng/ml with P= 0.041). The levels was much lower in patient receiving higher dos-es of HU (P = 0.049 ) .

Table (1) : Characteristics of SCD patient

Parameter SCD patients (n=85)Demographic and phenotype

Age (years) Male sex

HbSS HbS/β thalassemia

10.19±4.6 (3-18)47 (55.3)57 (67.1)28 (32.9)

Medical history VOC severe VOC mild VOC/ year

Hydroxyurea therapy Hydroxyurea dose (mg/kg/day)

Transfusion frequency/year

59 (69.5)26 (30.5)2.7±1.251 (60)

18.3±4.32.6±1.2

VCAM-1 (ng/ml) 1438.12±848.2 (392-5336)

Table (2): Classification of Patients according sVCAM-1 level

VCAM level* Number of patients Percentage

Normal 25 29.5%

High 60 70.5%*NB: According to R&D VCAM Quintikine Assay Kit’s provided normal Values - Normal: within the nor-

mal mean±SD; High: Higher than the upper normal range.

29

El-Tagui, M. H., et al

DISCUSSION

Adhesion of sickle erythrocytes to vascular endothelium plays a central role in sickle cell disease complications such as stroke and acute painful crises. Adherence of sickled erythrocytes to the endothelial cell surface results in their delayed passage. This delay enhances sickling, which ultimately leads to tissue hypoxia and infarction (7). It is believed that these manifesta-tions are mediated in part by vascular cell ad-hesion molecule-1 (VCAM-1) upregulation in vascular endothelial cells and this process is not counterbalanced by the release of cyto-protec-tive mediators that normally inhibit this endo-thelial VCAM-1 upregulation (15).

In sickle cell disease (SCD), stroke affects approximately 11% of patients younger than 20 years of age (6). Thus, detection of high risk patients is very critical in management of those patients(1). In our study the mean VCAM level among involved patients was 1438.12 ± 848.2ng/ml which is much higher than the standard refer-ence level (557±139.6 ng/ml). Our data showed that sixty patients (70%) had high abnormal lev-els of VCAM while 25 (30%) showed normal levels . Patients having severe vasooclusive cri-sis had significantly higher VCAM1 level com-pared to subjects with moderate and mild cisis p-value=0.007 and 0.039, respectively.

This is in accordance with previous studies suggesting that VCAM-1 presents in high levels

and plays a very important role in the pathophys-iology of VOC, hemolysis and stroke in patients with SCD (8, 18, 14, 9 ,6) . A study including 52 patients in 2010 showed comparable results with level for patients with history of mild VOC was 1157.25 ± 486.54 and 2024.00 ± 1366.35 for patients with history of severe VOC with P Value of 0.018( 6) . So our findings along with thesestudies support that VCAM seemed to correlate with crisis evolution.

In our study correlation of serum VCAM1 level with the Middle cerebral Artery (MCA) time-averaged mean of the maximum velocity (TAMMvel) showed significant positive correla-tion (P value of 0.041).

This is in accordance with Armstrong-Wells et al , who stated that Trans - cranial Doppler (TCD) is a well-established tool that has been used for prediction of future cerebrovascular symptoms and that careful screening TCD ul-trasonography and treatment with chronic blood transfusion will likely reduce the number of strokes in children with sickle cell anemia (3)

Interestingly, our study showed higher lev-els of sVCAM1 in patients needed more blood transfusion with positive correlation of 0.562 and P Value of 0.001. Because of high prevalence of HCV among our population, we follow very restrictive regimen for blood transfusion with regular transfusions being generally not recom-mended. In another study (n=61) the impact of

Table (3): Levels of sVCAM-1 in relation to severity of VOC

Table (4): Correlation between MCA time-averaged mean of the maximum velocity (TAMMvel) and sVCAM-1 level

Mean VCAM

RT MCA 125.97±41.35Pearson Correlation 0.225

Sig. (2-tailed) 0.041*

LT MCA 115.6±38.3Pearson Correlation .066

Sig. (2-tailed) 0.555

30

VCAM-1 in Children With SCD and Stroke Risk

chronic transfusion on sVCAM1 levels showed that chronically transfused patients had signifi-cantly lower levels of sVCAM1as it reduces the percentage of sickle cells and leads to dimin-ished expression of sVCAM1 reducing the ten-dency toward initiation of pathologic events(14). This reflects that the higher the sVCAM1 levelthe more the need for blood transfusion.

As many studies recognized the role of HU in controlling the severity of VOC in SCD (16,

20, 11, 13), we tried to correlate the effect of HU on VCAM levels to get better understanding of the mechanism of action of HU. Our study also supported the protective value of HU treatment in controlling the vaso-occlusive process in pa-tients with SCD as in SS subgroup, VCAM level was significantly lower in patients receiving HUcompared to those not on HU with P Value of 0.041. Moreover, the levels was much lower in patient receiving higher doses of HU (P Value 0.049).These findings support that HU may de-crease the severity of disease in patients with SCD through controlling erythrocyte adhesion rather than increasing in hemoglobin-F levels.

The present study demonstrates that the VOC in sickle cell disease patients is related to the level sVCAM-1 level which may give clue for the importance of therapeutic modulators of sV-CAM-1 and other cytokines. Blood transfusion and Hu play an important role in crisis preven-tion.The TCD can serve in early crisis detection in Egyptian population .

REFERENCES1. Adams RJ 2001: Stroke prevention and treatment in

sickle cell disease. ARCH Neurol; 58:101:103.

2. Adams RJ, McKie V, Hsu L, et al. 1998: Prevention of a first stroke by transfusion in children with abnormalresults of transcranial Doppler ultrasonography. N Engl J Med. ;339:5.

3. Armstrong-Wells J, Grimes B, Collombatti S, et al,. 2009: Utilization of TCD screening for primary stroke prevention in children with sickle cell disease. Neurology; 72:1316.

4. Bruce W, 2007: Reduce Stroke Risk in Sickle Cell Ane-mia Patients: clin rounds J Iss. 202 March 2007 P58.

5. Corrina McMahon 2006: Sickle Cell Anemia. In: Pedi-atric Hematology, 3rd Ed. Eds. Robert J. Arceci, Ian M. Hann, Owen P. Smith, Copyright © 2006 by Blackwell

Publishing Ltd. ISBN-13: 978-1-4051-3400-2 Ch. 10 p213

6. Dworkis DA, Klings ES, Solovieff N, et al, 2011:Severe sickle cell anemia is associated with increased plasma levels of TNF-R1 and VCAM-1 Am J Hematol. 2011; 86:220.

7. Ernest, 2010: Disorders of Hemoglobin Structure: Sick-

le Cell Anemia and Related Abnormalities. By Ernest Beutler In: Williams Hematology, Eighth Edition. Eds.: (Kenneth Kaushansky, Ernest Beutler, Uri Seligsohn et al.) The McGraw-Hill Companies, Inc. ISBN 978-0-07-162144-1 Ch 48. 2010

8. Kaddah N et al 2003: Cellular Adhesion In Children With Sickle Cell Disease. Nourmeen kaddah, emad E. Salama, Naglaa O. Moustafa, Nahed A.R. Emara. Kasr el Aini medical journal Vol. 9, NO. 5.

9. Kato GJ, Martyr S, Blackwelder WC, et al. 2005: Lev-

els of soluble endothelium derived adhesion molecules in patients with sickle cell disease are associated with pulmonary hypertension, organ dysfunction, and mor

-

tality. Br J Haematol;130:943.

10. Klaus Ley & Yuqing Huo 2001: VCAM-1 is critical in atherosclerosis J Clin Invest.;107(10):1209.

11. Lal Ashutosh & Elliott P Vichinsky 2005: Sickle cell anemia. In: Postgraduate Haematology 5th ed. Eds. A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tud

-

denham by Blackwell Publishing Massachusetts USA. ch7 p 92:102

12. Maakaron E Joseph 2011: Sickle Cell Anemia. A med-

scape article No. 205926 Last updated: Dec 16, 2011.

13. Platt OS 2008: Hydroxyurea for the treatment of sickle cell anemia. N Engl J Med 358:1362. [PMID: 18367739]

14. Sakhalkar V S, Rao S P, weedon J, Miller S T 2004. Elevated Plasma sVCAM-1 Levels in Children With Sickle Cell Disease: Impact of Chronic Transfusion Therapy. American Journal of Hematology ; 76:57.

15. .Shiu Y, Udden MM, McIntire LV. 2000: Perfusion with sickle erythrocytes up-regulates ICAM-1 and VCAM-1 gene expression in cultured human endothelial cells. Blood;95:3232.

16. Steinberg MH, Franca Barton; Oswaldo Castro, et al., 2003: Effect of Hydroxyurea on Mortality and Morbid

-

ity in Adult Sickle Cell Anemia. Risks and Benefits Upto 9 Years of Treatment. JAMA, Vol 289, No. 13.

17. Steinberg MH. 2001: Compound heterozygous and oth-

er sickle hemoglobinopathies. In: Disorders of Hemo-

globin: Genetics, Pathophysiology, Clinical Manage-

ment. Eds. Forget BG, Higgs DR, et al., Cambridge, UK: Cambridge University Press, . P 598

18. Walmet PS, Eckman JR, Wick TM. 2003: Inflamma-

tory mediators promote strong sickle cell adherence to endothelium under venular flow conditions. Am J

31

El-Tagui, M. H., et alHematol;73:215.

19. Yogen Saunthararajah, Elliott P. Vichinsky, Stephen H. Embury 2005: Sickle Cell Disease, In: HEMATOLOGY Basic Principles and Practice 4th Ed. Ronald Hoffman, Edward J. Benz, Sanford J. Shattil, Bruce Furie (eds).

Copyright © 2005, Elsevier Inc. Ch. 37 P605:636.

20. Zimmerman SA, William HS, Shelly Burgett, et al,2004: Hydroxyurea therapy lowers transcranial Do- ppler flow velocities in children with sickle cell anemia. Blood 2007 110: 1043

االطفال المرض في وشدة المخيه الجلطات بحدوث عالقته و الدم في 1 رقم االلتصاقي الخلوي الجزء مستوىالمنجلية الخاليا بانيميا المرضى

ابراهيم امين - احمد العظيم عبد سعيد – فدوى عبد اهللا فؤاد الغمراوي– التاجي – منى حسن منىالموجات الصوتية فحوصات بنتائج مقارنته الدم و 1 في رقم االلتصاقي الجزيء الخلوي مستوى قياس الرسالة الى هذه لقد هدفتاالطفال لدى عليه العالج الطبي و نقل الدم تأثير مدى تحديد و المرض شدة و المخية بحدوث الجلطات عالقته لتحديد دوبلر) ) المخيةاالطفال لدى المخية الجلطة تسبب التي الخطورة نسبة عوامل تحديد الى الرسالة أيضا هدفت كما المنجلية. الخاليا المرضى بأنيمياعلى والمترددين المنجلية الخاليا بأنيميا المصابين األطفال من 85 على الرسالة اجريت هذه قد و هذا المنجلية. الخاليا بانيميا المصابيناالناث من 38 من بينهم كان يونيو 2013. بين يونيو2011 الى ما الفترة في الجديد. الجامعي االطفال بمستشفى الدم عيادة امراضفي الدم أعلى واحد رقم االلتصاقي الخلوي الجزئ مستوى ان النتائج اظهرت قد و سنة. بين 3 الى 18 اعمارهم تتراوح 47 الذكور. والجزئ من عالية مستويات لديهم المرضى %70.5 من ظهر ان حيث الطبيعين االشخاص من نظرائهم المنجليةعن الخاليا مرضى بينمستوياته ان اظهرت الدراسة ايضا و قد . هذا الدم في المعدل الطبيعي الجزيء في مستوى % 29.5 يقع في الدم االلتصاقي الخلوي الخلوي االلتصاقي الجزئ يلعبه الذي المحوري الدور يؤكد لنقل الدم مما المريض احتياج و المرضية االعراض ازدياد شدة تزداد معتوفى بعد ان 83 طفل المخية ل الصوتية الموجات عمل تم المنجلية الخاليا انيميا مرضى المرضية في حدوث االعراض و تفاعل فيفي الدم رقم واحد االلتصاقي الخلوي الجزئ مستوى بين عالقة طردية النتائج اظهرت و قد المخية الصوتية الموجات عمل قبل طفالنفي فعال تأثير الدم و عقار الهيدروكسييوريا لهم من نقل كال ان االحصائيات قد اظهرت هذا و المخية الشرايين في الدم تدفق وسرعة

الجلطات المخية. الحماية من

32

ASSOCIATION BETWEEN MDM2-SNP309 POLYMORPHISM AND TUMOUR SIZE, SITE AND NUMBER IN HEPATOCELLULAR

CARCINOMA Noha Kamal Abdel Moamen, Hany Hussien El Sied, Marianne Fathy Ishak, Moataz Syam,

Maha Hasaballa, Afaf Ahmed Abdelhadi and Hebat Allah Mohamed Sharaf El Deen

ABSTRACTBackground: The murine double minute 2 (MDM2), an oncogene, is an important regulator of tumor development. MDM2,

an E3 ubiquitin ligase, regulates p53 by controlling both the stability of the p53 protein and the activity of p53 that a transcrip-tion factor. Aim of the work: This study aims to evaluate the association of the MDM2 promoter SNP309 and development of hepatocellular carcinoma among Egyptian patients with chronic viral hepatitis. Subjects and Methods: MDM2 SNP309 (T/G) genotyping was assayed in 67 HCC patients on top of HCV infection and 36 chronic viral hepatitis patients. MDM2 SNP309 genotyping was assayed using realtime polymerase chain reaction (PCR) Results: The frequency of the distribution pattern of the MDM2 SNP309 TT, TG, and GG genotypes in HCC group were 38.8%, 50.7% and 10.4% respectively and that in the con-trol group were 38.9%, 50% and 11.1% respectively. There was with no significant difference between the two studied groupswith [p value 0.994]. There were no significant difference in the distribution pattern of the SNP309 (TT+TG versus GG orTG+GG versus TT genotypes between the studied two groups with P value 0.917 and 0.993 respectively. There was no signifi-cant difference between the MDM2 SNP309 genotypes and regarding the tumour site, size or number. The male:female ratio in HCC group was 4.2 and that in the control group was 1.4. In the control group, the distribution of the genotype (TT, GG and TG) and the prescence of cirrhosis were (85.7%, 100% and 88.9%) with no significant difference [P value 0.725]. Conclusion:Current data suggest that the association between MDM2-SNP309 genotype and HCC in the cohort study in Egyptian popula-tion is not significant. Key words: MDM2 SNP309, chronic hepatitis and hepatocellular carcinoma

Departements of Clinical Chemistry, Theodor Bilharz Research Institute (TBRI), Egypt

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers worldwide. In Egypt, the disease is usually detected in an advanced stage at which no treatment may be effective in-cluding surgery. Early detection of the disease is thus an important goal allowing the patient to be treated before the enlargement of the tumor or its metastasis to distant organs (12).

Hepatitis B virus (HBV) and hepatitis C vi-rus (HCV) are the known primary risk factors for HCC (29).

More than 170 million patients worldwide are chronically infected with hepatitis C virus (HCV). Prevalence rates range from 0.5% in Northern European countries to 28% in some areas of Egypt (3). Egypt has the highest preva-lence of chronic hepatitis C virus (HCV) infec-tion worldwide and has rising rates of hepatocel-lular carcinoma (HCC) development (16,17).

Hepatocellular carcinoma exhibits numerous genetic abnormalities (including chromosomal deletions, rearrangements, aneuploidy, gene am-

plifications, and mutations), as well as epigen-etic alterations (including modulation of DNA methylation). The combination of genetic and epigenetic alterations activates positive media-tors of cellular proliferation (including cellular protooncogenes and their mitogenic signalling pathways) and inactivates negative mediators of cellular proliferation (including tumor suppres-sor genes), resulting in cells with autonomous growth potential. The current knowledge of mo-lecular signatures of early HCC is preliminary and further studies are required to elucidate he-patocarcinogenesis in concordance with histo-pathological classification of early HCC (31).

The murine double minute 2 (MDM2), an oncogene, is an important regulator of tumor development. MDM2, an E3 ubiquitin ligase, regulates p53 by controlling both the stability of the p53 protein and the activity of p53 that a transcription factor(20). The importance of the p53 tumor suppressor pathway for the preven-tion of transformation has long been recognized, as inactivation of the pathway is a frequent step in the development of the majority of human and murine cancers (15,30).


Abdel Moamen, N. K., et al.34SUBJECTS AND METHOD

This study included 103 subjects. They were divided into 2 groups according to the radiologi-cal findings into (Group 1) which Included 67patients with hepatocellular carcinoma on top of chronic viral hepatitis patients. The two groups were recruited from those admitted in the Gastro-enterology Department of Theodor Bilharz Re-search Institute (TBRI). Written informed con-sent were obtained from all participants, and the study protocol as well as the suggested informed consent were submitted to the Institutional Re-view Board (IRB) of TBRI before start of en-rolling participants. HCC patients were diag-nosed by clinical examination, typical findingson abdominal ultrasound, abdominal triphasic CT scan and (Group 2) included 36 patients with chronic viral hepatitis for more than 10 years serve as control they were diagnosed by clinical examination, biochemical and ultrasonographic criteria.

DNA was extracted from whole blood using QIAamp DNA Mini Kit catalog number 51304. Max-Volmer-Strabe 4-40724-Hilden-Germany.

MDM2 SNP309 genotyping using realtime SNP Genotyping Using the Type-it Fast SNP Probe polymerase chain reaction (PCR) Master Mix were designated as follows: Forward primer 5/-CGGGAGTTCAGGGTAAAGGT- 3/ and Reverse primer 5/-ACAGGCACCTGCGAT-CATC-3/, and with VIC-labeled probe 5/-CTCCCGCGCCGAAG-3/, FAM-labeled probe 5/-TCCCGCGCCGCAG-3/. The probes were MGB probes designed specifically for TaqmanAllelic Discrimination. Taqman universal PCR master mix, forward and reverse primers (900 Amol/L each), 200 nmol/L VIC-labeled probe, and 200 nmol/L FAM-labeled probe. Ther-mal cycle conditions were 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 92°C for 15 seconds and 60°C for 1 minute. Completed PCR plates were read on an ABI PRISM 7000 sequence detector and analyzed usingthe Allelic Discrimination Sequence Detection Software (Applied Biosystems).

Statistical analysis

The results were analyzed using the SPSS computer software package, version 13 (Chicago,

USA).Quantitative data were presented as mean ±SD for normally distributed data and as medi-ans and percentiles for skewed data. Qualitative data were presented in the form of frequencies and percentiles. Differences among groups were computed using one-way analysis of variance (ANOVA) with post-hoc test and Kruskal-Wal-lis analysis of variance, and/or Mann Whitney test for normally distributed and skewed data re-spectively. Pearson Chi square test and/or Fish-er’s exact test were used to detect associations between two variables. For correlations between variables, Pearson’s &/or Spearman Correlation Coefficients were calculated. All tests were twotailed and considered statistically significant atP <0.05.

RESULTS

The characters of the studied groups are shown in table (1) at which the male:female ra-tio in HCC group was 4.2 and that in the control group was 1.4

there was no significant difference in thefrequency of the distribution pattern of the SNP309 (TT, TG and GG) between the studied two groups [p value 0.994]. Also there were no significant difference in the distribution patternof the SNP309 (T/T+T/G versus GG or TG+GG versus TT) genotypes between the studied two groups [p value 0.917 and 0.993 respectively] as shown in table (2).

Frequency distribution of sex, tumour mass number, tumour site, and presence of cirrhosis tumour number among genotypes of studied group 1(HCC) are shown in table(2) in which, the number of tumour in different MDM2 genotype were as following, TT genotype was 80.8% single tumour mass and 19.2% double tumour masses while that in GG genotype was 100% single tumour mass and that in TG geno-type 64.7% was single tumour mass, 20.6% was douple tumour masses and 14.7% was multiple tumour masses, showing no significant differ-ence [P value 0.114]. Regarding the tumour site, MDM2 TT genotype were 76.9% in the right hepatic lobe and 23.1% in the left hepatic lobe while that in GG genotype were 85.7% in the right hepatic lobe and 14.3% in the left hepatic lobe and that of TG genotype were 73.5% in the

Association between MDM2-SNP309 polymorphism and HCC 35right hepatic lobe, 23.5% in the left hepatic lobe and in both lobes were 2.9%, showing no sig-nificant difference between different genotype[P value 0.85]. In the control group, the preva-lence of cirrhosis in different genotype TT, GG and TG were 85.7%, 100% and 88.9% with no significant difference [P value 0.725] as shownin table (3).

Table (3) represents the association between the genotype and age in the group 1(HCC) and

in the control group and the median values of tumor size among genotypes in HCC group.

The age of HCC group were 58.8±7.4 years for TT genotype, 54±8.9 years for GG genotype and 57±9.6 years for TG genotype showing no significant difference [p value 0.403].Consider-ing the tumour size in the genotype TT, GG and TG were 2.8(2-6), 3(2-4) and 3(2.5-4) cm, re-spectively and this showed no significant differ-ence [P value 0.646].

Table (1) Age and sex distribution among studied groups

Age results were normally distributed and presented as mean ±SD,P value calculated by ANOVA test.P value not significant >0.05

Table (2) Frequency of genotype and allelic distribution among the studied subjects:

Age results were normally distributed and presented as mean ±SD, P value calculated by ANOVA test.P value not significant >0.05 Groups having the same letter were not statistically significant*Versus GG genotype ** Versus TT genotype

Abdel Moamen, N. K., et al.36

Table (3) The frequency between the genotype and the tumour number, site and size and the presence of cirrhosis among the studied groups:

P value calculated by ANOVA test. P value not significant >0.05 Groups having the same letter were notstatistically significant *Versus GG genotype ** Versus TT genotype

Association between MDM2-SNP309 polymorphism and HCC 37

Table (4) The association between the genotype and age in the group 1(HCC) and group 2(control) and Frequency of sex in the studied subjects

P value is significant <0.05 *Versus GG genotype ** Versus TT genotype

Figu1(H

1

ure (1): FrHCC) (n=67

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

00%

equency di7).

TT

istribution of tumour

GG

site amongg genotype

TG

es of studiedd group

Both

Lt�lobe

Rt�lobe

Figure (1): Frequency distribution of tumour site among genotypes of studied group 1(HCC) (n=67).

Abdel Moamen, N. K., et al.38DISCUSSION

MDM2, an oncogene, acts as a major regu-lator of the p53 protein. A polymorphism in the MDM2 promoter, SNP309 (T/G), has been shown to alter protein expression and may thus play a role in carcinogenesis. MDM2 is a nucle-ar phosphoprotein that binds to P53 and inhibits P53-dependent transcription. Over-expression of this gene can result in inactivation of P53, di-minishing its tumor suppressor function. Studies investigating the association between MDM2 polymorphism and human cancer risk reported conflicting results. Some studies showed no sig-nificant association between cancer and MDM2SNP309 (35,18), others showed significant associa-tion between MDM2 SNP309 G allele and can-cer (37,14).

In this study there was no significant differ-ence in the frequency of the distribution pattern of the SNP309 (TT, TG and GG) between the studied two groups [p value 0.994]. Also there were no significant difference in the distributionpattern of the SNP309 (T/T+T/G versus GG or TG+GG versus TT) genotypes between the stud-ied two groups [p value 0.917 and 0.993 respec-tively].

This was in agreement with another study performed on Taiwanese population (58 patients with HCC and 138 cancer free healthy adults) showed that proportion of The frequency of MDM2-SNP309 were TT, TG and GG genotype of the SNP309 in patients with HCC were 19%, 63.8% and 17.2% respectively and that in con-trol group were 25.3%, 58% and 16.7% had no significantly difference [P value 1, 0.99 and 0.87respectively]. there were no significant differ-ence between the studied two groups in the dis-tribution pattern of the SNP309 (TG+GG versus TT or TT+TG versus GG) genotypes [p value 0.97 and 0.85 respectively] (18). Another study performed on 93 patients with HCC and 100 can-cer free healthy adults showed that proportion of the frequency of MDM2-SNP309 TT, TG and GG genotype in patients with HCC were 46.4%, 43.5% and 10% respectively and that in control group were 48%, 40% and 12% with no signifi-cant difference [ P value 0.891 ] . The MDM2 SNP-309 genotypes of HCC were not statisti-

cally different from those of 100 controls (38).

On the other hand some studies performed on other types of tumours rather than HCC were in agreement with this study as a meta-analysis study included 1620 colorectal cancer patients showed no association with MDM2 SNP309 and cancer development (35), also in non-melanoma skin cancer, had not observed increases in cancer risk (35,23), moreover there were no evidence that the G/G genotype accelerated breast cancer de-velopment (10,11,21,24).

In contrary to this study, another study re-ported that MDM2 SNP309 was associated with the presence of hepatocellular carcinoma with chronic hepatitis in Japanese patients (12). Another study performed on Japanese patients with HCV infection (n=468) and HCC patients (n=265) reported that MDM2 was significantlyassociated with development and recurrence of HCC (33). This was supported by other studies done on Moroccan and Korean patients, indicat-ed an association between the G genotype and the risk for hepatocellular carcinoma with chron-ic hepatitis B infections (37,14). Another study per-formed on Turkish population showed that the genotypes containing G allele or GG genotype [p value were 0.013 and 0.001 respectively] were associated with significant increased sus-ceptibility to HCC (2). Another study performed on HCC on top of HBV infection (n=350), HBV non-HCC patients (n=230) and healthy controls (n=96) found that MDM2 G/G genotype is as-sociated with increased risk of developing HCC (36). Another study examined the association of the SNP with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) in Han Chinese population which included 310 HBV-related HCC patients, 314 non-HCC subjects with HBV infection and 480 healthy controls and the results suggested that the MDM2 SNP309 might influ-ence the risk of developing HBV-related HCC in Han Chinese population and also revealed that subjects with the G allele had higher HBV-re-lated HCC susceptibility than those with the T allele (34).

The explanation of this discrepancy between the results of this study and the others could be attributed to the difference in methodology and

Association between MDM2-SNP309 polymorphism and HCC 39racial difference. In the current study gene ex-pression was quantitated using TaqMan Real Time PCR while for in other studies, was carried out by reverse transcriptase PCR (37,14), and in another study, MDM2 SNP 309 was genotyped by using a PCR-RFLP method (34). This may be also due to a racial difference between the popu-lation in the different studies.

Difference between the current study results and other studies can be attributed also to our small sample size. Therefore, further studies on larger groups are needed to evaluate the role of MDM2 SNP309 on HCC patients.

Also this discrepancy may reflect differentpathways of evolution in different tumors and the presence of alternative forms of MDM2 SNP309 transcripts not detected by the current methodology.

There is also some evidence that SNP309 accelerates tumor formation in cells with an in-creased steroid receptor status. Possible mecha-nisms of the interactions between SNP309 and estrogen receptor status, environmental stress and other genetic predispositions have been re-viewed by Bond and Levine (6). This may sug-gest that MDM2 SNP309 can affect cancer inassociation with other etiological factors whichshould be studied with the MDM2 frequency incancer. True risk variants may need to be soughtthrough an extended and comprehensive analy-sis of the MDM2 gene.

In this study, the frequency of the MDM2 SNP309 T/T, T/G, and G/G genotypes in HCC group were 38.8%, 50.7% and 10.4% respec-tively and that in the control group were 38.9%, 50% and 11.1% respectively. According to these results, the least distribution of genotype among the Egyptian population in this cohort study was GG genotype, which limits studying the risk of G allele among the Egyptian HCC patients. these results coinside with Acun et al., 2010, who per-formed a study on African population (n=33) 31 patient of them was TT genotype and TG was 2 while GG was 0 patient. On the other hand Acun et al., 2010 studied the distribution of MDM2 genotype of HCC patients living in different geographical regions (TT, TG and GG) geno-type in African patients (n=33) were 31, 2 and 0,

while that in Japanese patients (n=13) were 0, 10 and 3 respectively, and that in Chinese patients (n=21) were 7, 3, and 11 respectively, and that in European patients (n=32) were 11, 16 and 5 respectively while for all HCC cases (90) were 49, 31, and 19 respectively (1).

The genotype distributions of the MDM2 309T>G polymorphism (TG, TG and GG) in a studied Turkish population were (54.04%, 40.09% and 5.85%, respectively), among the controls, which were significantly different fromthose of HCC which were (40.62%, 47.92% and 11.46%, respectively). The GG genotype was more frequent in the HCC cases than in con-trols. Multivariate logistic regression analyses revealed that compared with the SNP 309 TT genotype, the ORs for HCC patients carrying the TG, GG and TG+GG genotypes were 1.59 (95% CI, 0.96–2.64; P = 0.071), 2.60 (95% CI, 1.08–6.28; P = 0.029) and 1.72 (95% CI, 1.06–2.79; P = 0.027), respectively. The frequency of the MDM2 309G allele among control was 0.260(14)

which was significantly higher than thosereported in African-Americans (0.114) (21) but lower than those in Koreans (0.534) (25), Han Chinese of northeast China (0.456) and Cauca-sians (0.358–0.402) (3,19,21). This may suggest that the frequency of TT, GG or TG genotype varies with different geographical regions.

In this study, the prevalence GG genotype was 81.8% among males and 18.2% among fe-male, this was in agreement with Ezzikouri et al., 2009, who found that SNP309 GG was es-pecially more prevalent among males. MDM2 SNP309 GG may synergize with androgens during human liver carcinogenesis (14). MDM2 SNP309 accelerates tumor formation in a gen-der-specific and hormone dependent manner (7,8). The situation is, thus, quite different in HCC with a mildly enhanced male susceptibility. However, it has been shown in a mouse model as well as in tumor cells culture that MDM2 plays a critical role in the growth of hormone-dependent pros-tate cancer (5).

In this study, the age of HCC group were 58.8±7.4 years for TT genotype, 54±8.9 years for GG genotype and 57±9.6 years for TG geno-type showing no significant difference [p val-

Abdel Moamen, N. K., et al.40ue 0.403]. Leu et al., 2009 failed to prove that MDM2-SNP309 could accelerate the develop-ment of HCC (18), although the median age at diagnosis of patients with homozygous MDM2-SNP309 genotype (GG) was 3.5 years lower than that of patients with the common genotype (TT)). This result also agrees with the studies in the Japanese (12) and Moroccan (14) but not in the Korean population (37). In other studies, an asso-ciation between SNP309 GG and lower age at diagnosis of cancer was shown (8, 3,32)

In the control group, the prevalence of cir-rhosis in the different three genotypes were not significant difference and this was in agreementwith Dharel et al., 2006, who found that the ef-fect of SNP309 on the rate of fibrosis progres-sion and presence of cirrhosis and found no sig-nificant difference among the three genotypes ofSNP309 with regard these variables (12).

To the best of our knowledge, this is the firststudy to investigate the Egyptian population to analyze the effects of MDM2-SNP309 genotypes on HCC development. SNP309 alone has little or no effect on the risk of common cancers, but it might modify the time of tumor onset, especially when tumor suppressor pathways are weakened, and it might also modify the survival prognosis. The translation of even promising functional data to the risk of common human cancers ap-pears to be frustrating complex.

We still in need of a long follow up study in those hepatitis patients to examine which geno-type in the future will have a higher incidence in cancer transformation which may help in pre-dicting the risk of HCC development in fibroticpatients.

An intriguing question is why and how MDM2 gene itself is activated. Reports reveal that both genetic and epigenetic mechanisms are likely to be implicated in the process of hepatocarcino-genesis (28). Thus, DNA methylation, functional p53 play an important role in the regulation of the MDM2 gene.

This suggests the need for further genomic analysis of MDM2 in human tumors as well. Whether overexpression of MDM2 is associated with better or worse prognosis appears to vary

depending on the tumor type. It will be impor-tant to revisit this in the future to gain molecular insight into the basis for the varying prognostic outcomes. As MDM2 has been characterized extensively for its interaction with p53, it is not surprising that an emphasis has been placed on its role in tumorigenesis in that context. Never-theless, the increasing numbers of p53-indepen-dent effects that have been ascribed to MDM2 suggest that it has relevance in human cancer on its own. Given the exciting and sometimes con-fusing findings that are emerging from labora-tory studies, placing these recent developments in an integrated context will be critical. This is especially true as MDM2 emerges from the shadow of its relationship with p53.

REFERENCES1. Acun T, Terzioglu-Kara E, Konu O, et al (2010):

Mdm2 Snp309 G allele displays high frequencyand inverse correlation with somatic P53 mutations in hepatocellular carcinoma. Mutat Res. 684 (1-2): 106.

2. Akkiz H, Sumbul AT, Bayram S, et al (2010): MDM2 promoter polymorphism is associated with increased susceptibility to hepatocellular carcinoma in Turkish population. Cancer Epidemiol. Biomarkers Prev. 34 (4):448

.3. Alhopuro P, Ylisaukko-Oja SK, Koskinen WJ, et al(2005): The MDM2 promoter polymorphism SNP309T!G and the risk of uterine leiomyosarcoma, colorectal cancer, and squamous cell carcinoma of the head and neck. J Med Genet. 42: 694.

4. Bartosch B and Dubuisson J (2010): Recent advances in hepatitis C virus cell entry. Viruses. 2(3): 692.

5. Bianco R, Caputo R, Caputo R, et al (2004): Combined targeting of epidermal growth factor receptor and MDM2 by gefitinib and antisense MDM2 cooperative- ly inhibit hormone- independent prostate cancer. Clin Cancer Res. 10: 4858.6. Bond GL and Levine AJ (2007): A single nucleotide

polymorphism in the p53 pathway interacts with gen-der, environmental stresses and tumor genetics to influ-ence cancer in humans. Oncogene. 26: 1317.

7. Bond GL, Hirshfield KM, Kirchhoff T, et al (2006): M DM2 SNP309 accelerates tumor formation in a gend- erspecific and hormone-dependent manner. Cancer Res. 66: 5104.8. Bond GL, Hu W, Bond EE, et al (2004): A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans. Cell. 119: 591.9. Bougeard G, Baert-Desurmont S, Tournier I, et al

(2006): Impact of the MDM2 SNP309 and p53 Arg72Pro polymorphism on age of tumour onset in Li–Fraumeni syndrome. J. Med. Genet. 43: 531.

Association between MDM2-SNP309 polymorphism 41

10. Campbell IG, Eccles DM, Choong DYH (2006): No association of the MDM2 SNP309 polymorphism with

risk of breast or ovarian cancer. Cancer Letts. 240(2)195.

11. Copson ER, White HE, Blaydes JP, et al (2006): In- fluence of the MDM2 single nucleotide polymorphism SNP309 on tumour development in BRCA1 mutation carriers. BMC Cancer. 6: 80

12. Dharel N, Kato N, Muroyama R, et al (2006): MDM2 promoter SNP309 is associated with the risk of hep-

atocellular carcinoma in patients with chronic hepatitis C. Clin. Cancer Res. 12: 4867.13. El-Tayeh SF, TD Hussein, El-Houseini ME, et al (2012): Serological biomarkers of hepatocellular carcinoma in Eg- yptian pa tients. Dis Markers. 32(4): 255.

14. Ezzikouri S, El Feydi AE, Afifi R, et al (2009): MDM2 SNP309T>G polymorphism and risk of hepatocellular carcinoma: a case–control analy sis in a Moroccan population. Cancer Detect. Prev. 32: 380.15. Hollstein M, Rice K, Greenblatt MS, et al (1994): Dat- abase of p53 gene somatic mutations in human tumors and cell lines. Nucleic Ac- ids Res. 22: 3551.16. Lehman E and Wilson M (2009a): Epidemiology of

hepatitis viruses among hepatocellular carcinoma cases and healthy people in Egypt: a systematic review and meta-analysis. Int J Cancer. 124(3): 690.

17. Lehman E and Wilson M (2009b):Epidemic hepatitis C virus infection in Egypt: estimates of past incidence and future morbidity and mortality.J Viral Hepa.

18. Leu JD, Lin IF, Sun YF, et al (2009): Association between MDM2-SNP309 and he

-

patocellular carcinoma in Taiwanese population. World J Gastroenterol. 15(44): 5592.

19. Lind H, Zienolddiny S, Ekstrom PO, et al (2006): Ass- ociation of a functional polymorphism in the promoter of the MDM2 gene with risk of nonsmall cell lung cancer. Int J Cancer. 119: 718.20. Marine JC, Francoz S, Maetens M, et al (2006): Kee- ping p53 in check: essential and synergistic functions of Mdm2 and Mdm4. Cell Death. Differ. 13: 927.

21. Millikan RC, Heard K, Winkel S, et al (2006): No As- sociation Between the MDM2 –309 T/G Promoter Poly

-

morphism and Breast Cancer in African-Americans or Whites. Cancer Epidemiol. Biomarkers Prev. 15: 175 .

26. Petenkaya A, Bozkurt B, Akilli-Ozturk O, et al (2006): Lack of association between the MDM2-SNP309 poly-

morphism and breast cancer risk. Anticancer Res. 26: 4975.

27. Reifenberger G, Liu L, Ichimura K, et al (1993): Am- plification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 m- utations. Cancer Res. 53: 236.

28. Sadikovic B, Al-Romaih K, Squire JA et al (2008): Cause and Consequences of Genetic and Epigenetic Alterations in Human Cancer. Curr Genomics. 9 (6): 394.

29. Schiefelbein E, Zekri AR, Newton DW, et al (2012): Hepatitis C virus and other risk factors in hepatocellular carcinoma. Acta Virol. 56(3): 235.30. Sherr CJ (2006): Divorcing ARF and p53: an unsettled

case. Nat Rev Cancer. 6:663.

31. Suriawinata A and SN Thung (2010): Molecular Sig-nature of Early Hepatocellular Carcinoma. Oncology. 78(1): 36.

32. Swinney RM, Hsu SC, Hirschman BA, et al (2005): MDM2 promoter variation and age of diagnosis of acute lymphoblastic leukemia. Leukemia. 19: 1996.33. Tomoda T, Nouso K, Sakai A, et al (2012): Genetic Risk of Hepatocellular Carcinoma in Patients With Hepatitis C Virus. J Gastroenterol Hepatol. 27(4): 797.

22. Momand J, Jung D, Wilezynski S et al (1998): The MD M2 gene amplification database. Nucleic Acids Res. 26: 3453.

23. Nan H, Qureshi AA, Hunter DJ et al (2009): A functional SNP in the MDM2 promoter, pigmentary phenotypes, and risk of skin cancer. Cancer Causes Control. 20: 171.

24. Oliner JD, Pietenpol JA, Thiagalingam S, et al (1993): Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature. 362: 857.

34. Wang X, Zhang X, Qiu B,et al (2012): MDM2 SNP30 9T>G polymorphism increases susceptibility to hepatitis B virus-related hepatocellular carcinoma in a northeast Han Chinese population. Liver Int. 32(7): 1172.

35. Wilkening S, Hemminki K, Rudnai P, et al (2007): No association between MDM2 SNP309 promoter polymorphism and basal cell carcinoma of the skin. Br J Der- matol. 157: 375.36. Yang Y, Xia T, Li N, et al (2013): Combined effects of p53 and MDM2 polymorphisms on susceptibility and surgical prognosis in hepatitis B virus-related hepatocellular

carcinoma. Protein Cell. 4(1):71.37. Yoon YJ, Chang HY, Ahn SH, et al (2008): MDM2

and p53 polymorphisms are associated with the development of hepatocellular carcinoma in patients with chronic hepatitis B virus infection. Ca-

rcinogenesis. 29: 1192.38. Zhou X, Bloomston M, Shirley LA, et al (2013): The p53 Negative Regulator, MDM4 but Not MD M2, Is Frequently Activated in Hepatocellular Ca- rcinoma. Poster Session VI # 123. 1721.

25. Park SH, Choi JE, Kim EJ, et al (2006): MDM2 309T.G poly-morphism and risk of lung cancer in a Korean popu-

la tion. Lung Cancer. 54: 19.

Association between MDM2-SNP309 polymorphism ��

10. Campbell IG, Eccles DM, Choong DYH (200�): No association of the MDM2 SNP309 polymorphism with

��risk of breast or ovarian cancer. Cancer Letts. ��.

11. Copson ER, White HE, Blaydes JP, ��(2006):�In��fluence of the�MDM2 single nucleotide polymorphism ��SNP309 on tumour development in BRCA1 mutation ��carriers. BMC Cancer. 6: 80

12. Dharel N, Kato N, Muroyama R, ��(2006): MDM2 ��promoter SNP309 is associated with the risk of hep��atocellular carcinoma in patients with chronic hepatitis ��C. Clin. Cancer Res. 12: 4867.13. El-Tayeh SF, TD Hussein, El-Houseini ME, ��(2012): ��Serological biomarkers of hepatocellular carcinoma in Eg��yptian pa tients. Dis Markers. 32(4): 255.

14. Ezzikouri S, El Feydi AE, Afifi R,��(2009): MDM2 ��SNP309T>G polymorphism and �risk of hepatocellular ��carcinoma: a case–control analy sis in a Moroccan popu��lation. Cancer Detect. Prev. 32: 380.15. Hollstein M, Rice K, Greenblatt MS, ��(1994): Dat��abase of p53 gene somatic mutations in human tumors ��and cell lines. Nucleic Ac-��ids Res. 22: 3551.16. Lehman E and Wilson M (2009a): Epidemiology of



18. Leu JD, Lin IF, Sun YF, ��(2009): Association bet��ween MDM2-SNP309 and he

-��

patocellular carcinoma in�� Taiwanese population. World �J Gastroenterol. 15(44): ��5592.

19. Lind H, Zienolddiny S, Ekstrom PO, ��(2006): Ass��ociation of a functional polymorphism in the promoter ��of the MDM2 gene with risk of nonsmall cell lung can��cer. Int J Cancer. 119: 718.20. Marine JC, Francoz S, Maetens M, ��(2006): Kee��ping p53 in check: essential and synergistic functions ��of Mdm2 and Mdm4. Cell Death. Differ. 13: 927.

21. Millikan RC, Heard K, Winkel S, ��(2006): No As��sociation Between the MDM2 –309 T/G Promoter Poly

-

��morphism and Breast Cancer in African-Americans or ��Whites. Cancer Epidemiol. Biomarkers Prev. 15: 175 .

��

26. Petenkaya A, Bozkurt B, Akilli-Ozturk O, ��(2006):�� Lack of association between the MDM2-SNP309 poly��morphism and breast cancer risk. Anticancer Res. 26:

��4975.27. Reifenberger G, Liu L, Ichimura K, ��(1993): Am��plification and overexpression�of the MDM2 gene in�� a subset of human malignant gliomas without p53 m��utations. Cancer Res. 53: 236.

28. Sadikovic B, Al-Romaih K, Squire JA ��(2008): ��Cause and Consequences of �Genetic and �Epigenetic ��Alterations in� Human Cancer. Curr Genomics. 9�(6): ��394.

29. Schiefelbein E, Zekri AR, Newton DW, �� (2012): ��Hepatitis C virus and other risk factors in hepatocellu��lar carcinoma. Acta Virol. 56(3): 235.30. Sherr CJ (2006): Divorcing ARF and p53: an unsettled



32. Swinney RM, Hsu SC, Hirschman BA, ��(2005): ��MDM2 promoter variation and age of diagnosis of�� acute lymphoblastic leukemia. Leukemia. 19: 1996.33. Tomoda T, Nouso K, Sakai A, �� (2012): Genetic ��Risk of Hepatocellular Carcinoma in Patients With ��Hepatitis C Virus. J Gastroenterol Hepatol. 27(4):��797.

22. Momand J, Jung D, Wilezynski S �� (1998): The MD��M2 gene amplification database. Nucleic Acids Res. 26:�� 3453.

23. Nan H, Qureshi AA, Hunter DJ �� (2009): A functio��nal SNP in the MDM2 promoter, pigmentary phenot��ypes, and risk of skin cancer. Cancer Causes Control.�� 20: 171.

24. Oliner JD, Pietenpol JA, Thiagalingam S, ��(1993): ��Oncoprotein MDM2 conceals the activation domain of ��tumour suppressor p53. Nature. 362: 857.

34. Wang X, Zhang X, Qiu B,��(2012): MDM2 SNP30��9T>G polymorphism increases susceptibility to hepatit��is B virus-related hepatocellular carcinoma in a northe��ast Han Chinese population. Liver Int. 32(7): 1172.

35. Wilkening S, Hemminki K, Rudnai P, �� (2007): No ��association between MDM2 SNP309 promoter polymo��rphism and basal cell carcinoma of the skin. Br J Der��matol. 157: 375.36. Yang Y, Xia T, Li N, ��(2013): Combined effects of�� p53 and MDM2 polymorphisms on susceptibility and su��rgical prognosis in hepatitis B virus-related hepatocellular

carcinoma. Protein Cell. 4(1):71.37. Yoon YJ, Chang HY, Ahn SH, ��

and p53 polymorphisms are associated with the development of hepatocellular carcinoma in pat��ients with chronic hepatitis B virus infection. Ca�

��rcinogenesis. 29: 1192.38. Zhou X, Bloomston M, Shirley LA, �� (2013): �� The p53 Negative Regulator, MDM4 but Not MD��M2, Is Frequently Activated in Hepatocellular Ca��rcinoma. Poster Session VI # 123. 1721.

25. Park SH, Choi JE, Kim EJ, ��(2006): MDM2 309T.G ��poly-morphism and risk of lung cancer in a Korean popu�

��la tion. Lung Cancer. 54: 19.










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Abdel Moamen, N. K., et al.42

--

أهم العوامل و من السرطان شيوعا أكثر أنواع وأصبحت األخيرة، السنوات في مصر في بسرطان الكبد معدالت األصابة زادت لقدأهم من يعتبر حيث سى. الفيروسى الكبدى و التهاب بى الفيروسى الكبدى باأللتهاب هى األصابة الكبد سرطان الى المؤدية المعروفةاألورام أنواع من أعنف الكبد يعد سرطان و الحاالت. من فى 5-20٪ تقدريبا 5 سنوات خالل الكبد سرطان و الكبدى التليف مضاعفاتهذلك في بما فعال عالج وجود عدم مع متأخرة مرحلة في المرض عن الكشف يتم ما عادة مصر في العالم. أنحاء جميع في السرطانيةالعديد تستنتج أو أنتشاره. الورم زيادة حجم قبل المريض بمعاجة يسمح ضرورى, المرض المبكر عن يعتبر الكشف وبالتالي الجراحة.العوامل على أيضا نفسه ولكن الورم تحدث في التي الجسدية الطفرات على فقط يعتمد ال الكبد سرطان تطور في التباين الدراسات أن منتؤثر ((MDM2 SNP309 T/G الجينية الطفرة للبروتين P53 ان رئيسي MDM2 كمنظم لألورام المسبب الجين يعمل الوراثية.من مع P53 ويثبط نووي) فسفورى (بروتين MDM2 جين يتفاعل التسرطن. في دورا تلعب قد وبالتالي البروتينى التعبير علىلألورام. الهدف الكابتة فاعليته مقلال من P53 يؤدي إلى تثبيط الجين فى هذا الزيادة على ذلك فان P53 و على المعتمدة النسخ عمليةالمرضى المصريين فى سرطان الكبد و MDM2 SNP309 الجينية الطفرة بين وجود عالقة إمكانية التحقق من الدراسة هو هذه منإشتملت األولى المجموعه : مجموعتين إلى تقسيمهم تم شخصا ثالثة مائة و على الدراسة هذه المزمن. اشتملت الكبدى باأللتهاب المصابينثالثين وستة و ”بى“ و ”سى“ المزمن الكبدي الفيروسى بااللتهاب نتيجة إصابتهم الكبد بسرطان مصابين مريضا ستين و سبعة علىالجيني التنميط عن الكشف يتم الضابطة. المجموعة ليكونوا 10 أعوام من ألكثر المزمن الفيروسى الكبدي بااللتهاب مصابين مريضاأن الدراسة أظهرت قد و .(Realtime PCR) البوليميراز النزيم التسلسلي للتفاعل الفعلى الوقت باستخدام MDM2 SNP309 للالكبدي بااللتهاب المصابين المرضى مجموعة فى و 4.2 الكبد بسرطان المصابين المرضى مجموعة في الذكور و اإلناث النسبة بينأن النمط اإلناث في حين من ٪ و 22.5 الذكور TT هو ٪77.5 من النمط الجيني نسبة ان الدراسة هذه من قد تبين و .1.4 الفيروسىلكن و من اإلناث ٪ و 34.6 ذكور الجيني TG كان ٪65.4 من للنمط وبالنسبة اإلناث من ٪ و 18.2 ذكور من GG كان 81.8٪ الجينيمجموعة في MDM2 SNP309 GG, TG, TT الجينية األنماط توزيع أن الدراسة هذه تبين من قد و إحصائية. له داللة يكن لم ذلكالفيروسى الكبدي التهاب مرضى مجموعة فى أما و التوالي ٪50.7 و ٪10.4 على ،38.8٪ هى الكبد بسرطان المصابين المرضىلم أن الدراسة أظهرت المورثات توزيع إحصائية. داللة له يكن لم لكن ذلك على التوالي و التوالي 11.1٪ ٪50 على ،38.9٪ كانتهناك داللة يكن لم كما بين المجموعتين (GG مقابل TT+ TG ) الجينية األنماط توزيع نمط في إحصائية داللة ذات عالقة هناك يكنالجيني TT كان النمط توزيع أن الدراسة أظهرت المجموعتين. بين ) TT مقابل TG) + GG الجينية األنماط توزيع نمط إحصائية فيكان GG 100٪ في الجيني النمط , أما توزيع المتعدد الورم حالة و ٪64.7 فى الثنائى الواحد و ٪100 الورم الورم ٪80.8 في حالةالورم حالة و ٪14.7 فى الثنائى ، ٪20.6 الورم الواحد الورم حالة ٪64.7 في TGكان الجيني النمط ، أما توزيع الواحد الورم حالة(TT + TG مقابل GG) 71.7٪ في الجيني توزيع النمط أن أظهرت الدراسة إحصائية داللة ذات عالقة له يكن ذلك لم و ، المتعددكان (TT مقابل TG + GG) توزيع النمط الجيني في وأنه حالة الورم المتعدد في و 8.3٪ الثنائى الورم ٪20 في و الواحد الورم حالةو إحصائية. داللة ذات وجود عالقة مع عدم المتعدد الورم حالة و ٪12.2 في الثنائى الورم ، ٪17.1 في الواحد الورم حالة ٪70.7 فينسبته فى بينما األيسر الكبد فص في وكان 23.1٪ ، األيمن فص الكبد في TT كان 76.9٪ النمط الجيني توزيع أن الدراسة تبين من قدو في األيسر الكبد فص في 14.3٪ ، األيمن للكبد الفص في النمط الجيني GG كان 85.7٪ نسبة حين أن ٪0 في كانت معا الفصينو 2.9٪ األيسر الكبد فص في وكان 23.5٪ ، للكبد األيمن الفص في كان 73.5٪ TGالجيني النمط في وأنه 0٪ كانت و الفصين كال GG) 75٪ مقابل TT + TG) الجيني توزيع النمط أن الدراسة أظهرت إحصائية. ذات داللة عالقة عدم وجود مع معا، الفصين فىالنمط توزيع أيضا كان إحصائية. داللة ذات وجود عالقة مع عدم الفصين معا ، في و 1.7٪ الفص األيسر في 23.3٪ الفص األيمن، فيعالقة الفصين معا مع عدم وجود في و 2.4٪ الفص األيسر في و 22٪ األيمن، الفص في TT) 75.5٪ مقابل TG + GG) الجيني (2-4)3 كان 2.8(2-6), ( TG و TT , GG) الجيني النمط أن توزيع الدراسة من تبين قد الورم, لحجم و بالنسبة إحصائية. داللة ذاتكان (GG مقابل TT + TG) النمط الجيني و أما توزيع داللة إحصائية. ذات عالقة عدم وجود مع التوالي، على سم، 2 (2.5-4)3 و(TT مقابل TG + GG) الجيني النمط وأنه مع إحصائية عالقة ذات داللة وجود مع عدم التوالي على سم (2-4)3 و (2.1-4.4)3

داللة إحصائية. ذات عالقة وجود عدم التوالي مع على سم (2-6)2.8 و كان1.9(2.5-4)

في سرطان الكبد الورم وعدد وموقع وحجم SNP٣٠٩- MDMالجينية ٢ الطفره بين العالقه- اهللا حسب مها - صيام معتز - اسحق ماريان فتحي السيد - حسين هاني - المؤمن عبد كمال نهي

الدين شرف محمد اهللا هبه - الهادي أحمد عبد عفاف

MDM2 PROMOTER POLYMORPHISM AND ITS ASSOCIATION WITH HEPATOCELLULAR CARCINOMA IN EGYPTIAN POPULATION

WITH CHRONIC VIRAL HEPATITIS Noha Kamal Abdel Moamen, Hany Hussien El Sied, Marianne Fathy Ishak, Moataz Syam,

Maha Hasaballa, Nihal moustafa Safwat El Assaly, Afaf Ahmed Abdelhadi and Hebat Allah Mohamed Sharaf El Deen

ABSTRACT Background: Hepatocellular carcinoma (HCC) ranks among the 10 most common cancers worldwide. The main risk fac-

tors for its development are hepatitis B and C virus infections. The murine double minute 2 (MdM2), an oncogene, negatively regulates the stability and activity of p53 protein, by binding to p53, leading to its degradation. It is likely that damaged cells escape from the cell-cycle checkpoint control and become carcinogenic, by the attenuation of p53 function, when MDM2 is overexpressed. A single nucleotide polymorphism (SNP) of the MDM2 gene (T309G) was found in the P53-responsive intronic promoter region. A SNP309 in the promoter region of MDM2 has been shown to be associated with both hereditary and spo-radic cancers in humans. Objective: To investigate the association between potentially functional MDM2 oncogene, E3 ubiq-uitin protein ligase (MDM2) T309G polymorphism and susceptibility to hepatocellular carcinoma among Egyptian patients infected by hepatitis B and C virus. Subjects and Methods: We analyzed MDM2-SNP309 genotypes from 67 HCC patients on top of HCV infection and 36 chronic viral hepatitis patients. MDM2 SNP309 genotyping was conducted by realtime polymerase chain reaction (PCR). Results: There was no significant difference between the MDM2 SNP309 genotypes and the type of viralinfection whether it was HCV or HBV. In HCC group, the prevalence of HCV in TT, GG and TG genotype were 92.3%, 100% and 94.1%, respectively [P value 0.747] while in the control group, were 85.7%, 85.7% and 100% [P value 0.189]. In HCC group, the prevalence of HBV in TT, GG and TG genotype were 7.7%, 0% and 5.9%, respectively [P value 0.747] while in the control group, were 14.3%, 0% and 0%, respectively [P value 0.189]. There was no significant difference in the distributionpattern of the MDM2 SNP309 TT, TG, and GG genotypes between the studied two groups with [p value 0.994]. AFP level was highly significantly increased in HCC group [40(7.6-164ng/dl)] than that of the control group [5(2.7-7.7ng/dl)] with P value of0.001. Conclusion: Current data suggest that the association between MDM2-SNP309 genotype and HCC in the cohort study in Egyptian population is not significant. Key words: MDM2 SNP309, chronic hepatitis and hepatocellular carcinoma

Departement of Clinical Chemistry, Theodor Bilharz Research Institute (TBRI), Egypt

INTRODUCTION

Hepatocellular carcinoma (HCC) increased in Egypt in the past years, becoming the most common cancer among men. Hepatitis B vi-rus (HBV) and hepatitis C virus (HCV) are the known primary risk factors for HCC (29).

HCV transmission is ongoing in Egypt, and incidence rates have been estimated at 2.4 per 1,000 person-years (165,000 new infections annually) (22). HCV is a small, single-stranded RNA virus classified in the Flaviviridae family,remains a major cause of hepatic cirrhosis and HCC worldwide (3). The virus is able to estab-lish persistence in approximately two-thirds of infected subjects and has thus become a major cause of chronic liver inflammation that canprogress to liver cirrhosis or even HCC (15).

HBV is the prototype virus of the hepadnavir-idae, a name that signifies the hepatotropism and

DNA nature of the genome of its members(28). HBV causes acute and chronic liver cell injury and inflammation and is strongly associated withliver cirrhosis and HCC (14).

Identification of HCC in high-risk patients,who are at a potentially curable stage, is still an unsolved clinical problem. At present, assess-ment of AFP levels in conjunction with ultra-sound is the “standard procedure” for screening of high-risk patients (35). As elevated AFP is found only in 40%-75% of patients with HCC(7,30,11) and the reported diagnostic value of AFP for detect-ing HCC by using the most commonly reported cutoff value (AFP level > 20 µg/L), its sensitiv-ity is 41% to 65% and the specificity is 80% to94% (12), there is still up till now a strong need to find a complementary or supplementary markerfor prediction and screening of HCC in high-risk patients.


Abdel Moamen, N. K. et al.44

Many studies infer that the variations in HCC development not only depend on somatic muta-tions occurring in the tumor itself but also on host genetic factors (37,10,1).

Although the p53-independent tumorigenic-ity of MDM2 is not fully understood, MDM2 binds to a number of proteins with various func-tions (38).

A polymorphism in the MDM2 promoter, single nucleotide polymorphism at nucleotide 309 (SNP309) (T/G), has been shown to alter protein expression and may thus play a role in carcinogenesis (1).

SUBJECTS AND METHOD

The two groups were recruited from those admitted in the Gastroenterology Department of Theodor Bilharz Research Institute (TBRI). This study included 103 subjects. They were divided into 2 groups according to the radiological find-ings into (Group 1) which Included 67 patients with hepatocellular carcinoma on top of chronic viral hepatitis patients. HCC patients were diag-nosed by clinical examination, serum AFP level and typical findings on abdominal ultrasound,abdominal triphasic CT scan and (Group 2) in-cluded 36 patients with chronic viral hepatitis for more than 10 years serve as control. they were diagnosed by clinical examination, bio-chemical and ultrasonographic criteria. Written informed consent were obtained from all par-ticipants, and the study protocol as well as the suggested informed consent were submitted to

The murine double minute 2 (MDM2), an oncogene, is an important regulator of tumor de-velopment. MDM2, an E3 ubiquitin ligase, regu-lates p53 by controlling both the stability and the activity of the p53 protein , leading to its degra-dation (20). The importance of the p53 tumor sup-pressor pathway for the prevention of transfor-mation has long been recognized(13,31). A single nucleotide polymorphism (SNP) of the MDM2 gene (T309G) was found in the P53-responsive intronic promoter region, and it was revealed that the G allele could increase the affinity forbinding the transcription factor Sp1, subsequent-ly leading to elevated MdM2 protein levels (6).

the Institutional Review Board (IRB) of TBRI before start of enrolling participants.

All patients and controls in this study were subjected to the following serum albumin(8). which was assayed by the autoanalyzer: Bechman CX5. (BeckmanCoulter Ireland Inc. MervueBusi-ness Park, mervue, Gateway) HBVs antigen, HCV anti-body were assayed. Serum AFP was assayed by using the electro-chemiluminescence technique which purchased from Bayer healthcare LLC (Bayer healthcare LLC, Tarry town, NY 10591, 5097 USA)

DNA was extracted from whole blood us-ing QIAamp DNA Mini Kit catalog number 51304 (Max-Volmer-Strabe 4-40724-Hilden-Germany). MDM2 SNP309 genotyping using realtime SNP Genotyping Using the Type-it Fast SNP Probe polymerase chain reaction (PCR) Mas-ter Mix were designated as follows: Forward primer 5'-CGGGAGTTCAGGGTAAAGGT- 3' and Reverse primer 5' -ACAGGCACCTGC-GATCATC-3', and with VIC-labeled probe 5'-CTCCCGCGCCGAAG-3', FAM-labeled probe 5'-TCCCGCGCCGCAG-3'. The probes were MGB probes designed specifically for TaqmanAllelic Discrimination. Taqman universal PCR master mix, forward and reverse primers (900 Amol/L each), 200 nmol/L VIC-labeled probe, and 200 nmol/L FAM-labeled probe. Ther-mal cycle conditions were 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 92°C for 15 seconds and 60°C for 1 minute. Completed PCR plates were read on an ABI PRISM 7000 sequence detector and analyzed using the Allelic Discrimination Sequence Detection Software (Applied Biosystems).Statistical analysis

The results were analyzed using the SPSS computer software package, version 13 (Chicago, USA).Quantitative data were presented as mean ±SD for normally distributed data and as medi-ans and percentiles for skewed data. Qualitative data were presented in the form of frequencies and percentiles. Differences among groups were computed using one-way analysis of variance (ANOVA) with post-hoc test and Kruskal-Wallis analysis of variance, and/or Mann Whitney test for normally distributed and skewed data respec-tively. Pearson Chi square test and/or Fisher’s exact test were used to detect associations be-tween two variables. For correlations between variables, Pearson’s &/or Spearman Correlation

MDM2 Polymorphism and its Association with HCC 45Coefficients were calculated. All tests were twotailed and considered statistically significant atP <0.05.

RESULTS

The characteristics of the study are shown in table (1) as which alpha fetoprotein (AFP) levels in HCC group was significantly high-er 40 (7.6-164 ng/dl) than that in the control group 5(2.7-7.7ng/dl) [P value of 0.001]. In this study there was no significant difference inthe frequency of the distribution pattern of the SNP309 (TT, TG and GG) between the studied two groups [p value 0.994]. Also there were no significant difference in the distribution patternof the SNP309 (T/T+T/G versus GG or TG+GG

versus TT) genotypes between the studied two groups [p value 0.917 and 0.993 respectively]. In this study, the frequency of the MDM2 SNP309 T/T, T/G, and G/G genotypes in HCC group were 38.8%, 50.7% and 10.4% respectively and that in the control group were 38.9%, 50% and 11.1% respectively.

As shown in table (2) and (3), there was no significant difference between the MDM2SNP309 genotypes and the type of viral infection whether it was HCV or HBV. In HCC group, the prevalence of HBV in TT, GG and TG genotype were 7.7%, 0% and 5.9%, respectively) [P value 0.747], while in the control group, were 14.3%, 0% and 0%, respectively [P value 0.189].

Table 1 Characteristic of HCC and the control group

Albumin and alpha fetoprotein (AFP) results were abnormally distributed and presented as median (25th percentile- 75th percentile), p value calculated by Kruskal-Wallis test.

†P value is significant <0.05††P value is highly significant <0.001*Versus GG genotype** Versus TT genotypeP value is significant <0.05Groups having the same letter were not statistically significant.

Abdel Moamen, N. K. et al.46

Table 2 Frequency distribution of HCV and HBV infection among genotypes of studied group 1 (HCC) (n=67):

P value is significant <0.05 Groups having the same letter were not statistically significant *Versus GGgenotype ** Versus TT genotype

Table 3 Frequency distribution of HCV and HBV infection among genotypes of studied group 2 (control) (n=36):

P value is significant <0.05 Groups having the same letter were not statistically significant*Versus GG genotype** Versus TT genotype

MDM2 Polymorphism and its Association with HCC

Figure (1): Frequency distribution of HBV and HCV infection among genotypes of studied group 1 (HCC).

Figure (2): Frequency distribution of HBV and HCV infection among genotypes of studied group 2 (control) (n=36).

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

TT GG TG

HBV +ve Ag

HCV +ve Ab

Figure (2): Frequency distribution of HBV and HCV infection among genotypes of studied group 2 (con-trol) (n=36).

DISCUSSION

A SNP309 in the promoter region of MDM2 has been shown to be associated with both hered-itary and sporadic cancers in humans (4). Several groups have reported an association between the MDM2 SNP309 G variant and increased risk for epithelial cancer, including gastric cancer (24,26,17), breast (19), lung (18) and bladder cancers (25). In other studies, however, no association between MDM2 309T>G polymorphism and cancer risk was found (27,21,5). It is possible that some of these studies did not find a correlation between thepolymorphism and cancer because they did not

control for ethnic differences in allele frequen-cy in unaffected individuals. It is also possible, however, that relevant environmental factors at-tenuate or increase the impact of MDM2 poly-morphism on cancer development (10).

In this study there was no significant differ-ence between the MDM2 SNP309 genotypes and the type of viral infection whether it was HCV or HBV. In HCC group, the prevalence of HBV in TT, GG and TG genotype were 7.7%, 0% and 5.9%, respectively) [P value 0.747] , while in the control group, were 14.3%, 0% and 0%, respec-tively [P value 0.189]. In HCC group, the preva-

Abdel Moamen, N. K. et al.48lence of HCV in TT, GG and TG genotype were 92.3%, 100% and 94.1%, respectively [P value 0.747] while in the control group, were 85.7%, 85.7% and 100% [P value 0.189]. In another study, a multivariate analysis performed on Tai-wanese population included (58 HCC patients and 138 cancer free healthy adults) showed that the association between MDM2- SNP309 (TT, TG and GG) and HCC in the hepatitis patients was not significant [P value 1.000, 0.59 and 0.63respectively] (16). On the other hand, Ezzikouri et al., 2009 showed that SNP309 GG was es-pecially more prevalent among HCV-infected patients. Stratification based on HCV infectionstatus showed that patients with HCV are more likely to be carriers of the GG genotype (10).

Another study performed on HCC on top of HBV infection (n=350), HBV non-HCC patients (n=230) and healthy controls (n=96) found that MDM2 G/G genotype is associated with in-creased risk of developing HCC (36). Wang et al., 2012 examined the association of the SNP with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) in Han Chinese population which included 310 HBV-related HCC patients, 314 non-HCC subjects with HBV infection and 480 healthy controls and the results suggested that the MDM2 SNP309 might influence the riskof developing HBV-related HCC in Han Chinese population and also revealed that subjects with the G allele had higher HBV-related HCC sus-ceptibility than those with the T allele (33).

In this study there was no significant differ-ence in the frequency of the distribution pattern of the SNP309 (TT, TG and GG) between the studied two groups [p value 0.994]. Also there were no significant difference in the distribu-tion pattern of the SNP309 (T/T+T/G versus GG or TG+GG versus TT) genotypes between the studied two groups [p value 0.917 and 0.993 re-spectively]. This was in agreement with another study performed on Taiwanese population (58 patients with HCC and 138 cancer free healthy adults) showed no significant difference betweenthe studied two groups in the distribution pattern of the SNP309 (TT,TG,GG) with [P value 1, 0.99 and 0.87 respectively] (16).

Another study performed on 93 patients with HCC and 100 cancer free healthy adults showed that proportion of The frequency of MDM2-SNP309 TT, TG and GG genotype in patients with HCC were 46.4%, 43.5% and 10% respec-tively and that in control group were 48%, 40% and 12% with no significantly difference [P value0.891]. The MDM2 SNP-309 genotypes of HCC were not statistically different from those of 100 controls (39).

On the other hand some studies performed on other types of tumours rather than HCC were in agreement with this study as a meta-analysis study included 1620 colorectal cancer patients showed no association with MDM2 SNP309 and cancer development (34), also in non-melanoma skin cancer, had not observed increases in cancer risk (34,23), Another study reported that MDM2 SNP309 was associated with the presence of hepatocellular carcinoma with chronic hepatitis in Japanese patients (6). A study performed on Japanese patients with HCV infection (n=468) and HCC patients (n=265) reported that MDM2 was significantly associated with developmentand recurrence of HCC (32). This was supported by other studies done on Moroccan and Korean patients, indicated an association between the G genotype and the risk for hepatocellular carci-noma with chronic hepatitis B infections (37,10). Another study performed on Turkish population showed that the genotypes containing G allele or GG genotype [p value were 0.013 and 0.001 respectively] were associated with significant in-creased susceptibility to HCC (2).

There was a significant difference in albuminlevel between the HCC group [2.9(2.6-3.4 gm/dl)] and the control group [2.3(2.0-3.4 gm/dl)] [ P value 0.03 ] as shown in table (1) and this was in agreement with Dharel et al., 2006 who conducted a study on HCC Japanese patients on top of HCV (187) and chronic HCV infect-ed patients (248), who found that albumin was significant different in HCC group [3.6 (2.3-4.7gm/dl)] than that in non-HCC group [4.1 (2.3-5.0 gm/dl)] [P value <0.0001] .

Alpha fetoprotein (AFP) levels in HCC group was significantly higher 40 (7.6-164 ng/dl) thanthat in the control group 5(2.7-7.7ng/dl) [P value

49of 0.001] as shown in table (1), and this was in agreement with other study included HCC pa-tients (n=144), chronic hepatitis (n=47) and cir-rhosis (n=49), AFP level was significantly higherin patients with HCC than in those without HCC (P < or = 0.0001) (9). Also Youn et al., 2008 per-formed a study on Korean patients showing a significantly higher AFP levels in HCC group [Pvalue < 0.001] (37). AFP level was significantlyhigher in patients with HCC than in those without HCC (P < or = 0.0001) (9). Also Youn et al., 2008 performed a study on Korean patients showing a significantly higher AFP levels in HCC group [Pvalue < 0.001] (37).

This study concludes that there was no sig-nificant difference in the distribution pattern ofthe MDM2 SNP309T/G between the HCC and the control group. There was no significant dif-ference between the MDM2 SNP309 genotypes and the type of viral infection whether it was HCV or HBV.

The recommendation of this study were long term follow up is needed in hepatitis patients to examine which genotype in the future will have a higher incidence in cancer transformation which may help in predicting the risk of HCC development in fibrotic patients. Further studieson larger groups are needed to evaluate the role of MDM2 SNP309 on HCC Egyptian patients and patients with different types of cancer. Also we need to incorporate normal healthy subjects to identify the distribution pattern of MDM2 SNP309T/G among the Egyptian population. MDM2 SNP309 can affect cancer in associa-tion with other etiological factors which should be studied with the MDM2 frequency in cancer. True risk variants may need to be sought through an extended and comprehensive analysis of the MDM2 gene. It is necessary to incorporate the mutation status of p53 when the effects of MDM2 SNP309 on tumors are explored.

REFERENCES1. Acun T, Terzioglu-Kara E, Konu O, et al (2010): Mdm2

Snp309 G allele displays high frequency and inverse correlation with somatic P53 mutations in hepatocel-lular carcinoma. Mutat Res. 684(1-2): 106.

2. Akkiz H, Sumbul AT, Bayram S, et al (2010): MDM2 promoter polymorphism is associated with increased susceptibility to hepatocellular carcinoma in Turkish population. Cancer Epidemiol. Biomarkers Prev. 34(4): 448.

3. Alter MJ, Seeff LB, Bacon BR, et al (2004): Testing for hepatitis C virus infection should be routine for per-sons at increased risk for infection. Ann Intern Med. 141:715.

4. Bond GL, Hu W, Bond EE, et al (2004): A single nu-cleotide polymorphism in the MDM2 promoter attenu-ates the p53 tumor suppressor pathway and accelerates tumor formation in humans. Cell. 119: 591.

5. Campbell IG, Eccles DM, Choong DYH (2006): No association of the MDM2 SNP309 polymorphism with risk of breast or ovarian cancer. Cancer Letts. 240 (2): 195.

6. Dharel N, Kato N, Muroyama R, et al (2006): MDM2 promoter SNP309 is associated with the risk of hepato-cellular carcinoma in patients with chronic hepatitis C. Clin. Cancer Res. 12: 4867.

7. DiBisceglie A, Rustgi V, Hoofnagle J, Dusheiko G, et al (1988): NIH conference: hepatocellular carcinoma. Ann Intern Med. 108: 390.

8. Doumas B (1975): Standards for total serum protein as-say a collaborative study. Clin. Chem. 21: 1159 - 1166.

9. Durazo FA, Blatt LM, Corey WG, et al (2008): Des-gamma-carboxyprothrombin, alpha-fetoprotein and AFP-L3 in patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma. J Gastroenterol Hepatol. 23(10): 1541.

10. Ezzikouri S, El Feydi AE, Afifi R, et al (2009): MDM2SNP309T>G polymorphism and risk of hepatocellular carcinoma: a case–control analysis in a Moroccan pop-ulation. Cancer Detect. Prev. 32: 380.

11. Gebo K, Chander G, Jenckes M, et al (2002): Screen-ing tests for hepatocellular carcinoma in patients with chronic hepatitis C: A systematic review. Hepatol. 36: S84.

12. Gupta S, Bent S and Kohlwes J (2003): Test character-istics of alpha -fetoprotein for detecting hepatocellular carcinoma in patients with hepatitis C. A systematic re-view and critical analysis. 139: 46.

13. Hollstein M, Rice K, Greenblatt MS, et al (1994): Da-tabase of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res. 22: 3551.

14. Lau JYN and Wright TL (2000): Molecular virology and pathogenesis of hepatitis B. Lancet. 342: 1335–1340.

15. Lauer GM and Walker BD (2001): Hepatitis C virus in-fection. N Engl J Med. 345: 41.

16. Leu JD, Lin IF, Sun YF, et al (2009): Association be-tween MDM2-SNP309 and hepatocellular carcinoma in Taiwanese population. World J Gastroenterol. 15(44): 5592.

17. Li G, Zhai X, Zhang Z, et al (2006): MDM2 gene pro-moter polymorphisms and risk of lung cancer: a case–control analysis. Carcinogenesis. 27: 2028.


Mohsen, M. , et al.5018. Lind H, Zienolddiny S, Ekstrom PO, et al (2006): Asso-

ciation of a functional polymorphism in the promoter of the MDM2 gene with risk of nonsmall cell lung cancer. Int J Cancer. 119: 718.

19. Ma H, Hu Z, Zhai X, et al (2006): Polymorphisms in the MDM2 promoter and risk of breast cancer: a case-control analysis in a Chinese population. Cancer Lett. 240(2): 261.

20. Marine JC, Francoz S, Maetens M, et al (2006): Keep-ing p53 in check: essential and synergistic functions of Mdm2 and Mdm4. Cell Death. Differ. 13: 927.

21. Millikan RC, Heard K, Winkel S, et al (2006): No Asso-ciation Between the MDM2 –309 T/G Promoter Poly-morphism and Breast Cancer in African-Americans or Whites. Cancer Epidemiol. Biomarkers Prev. 15: 175.

22. Mostafa A, Taylor S, El-Daly M, et al (2010): Is the hepatitis C virus epidemic over in Egypt? Incidence and risk factors of new hepatitis C virus infections. Liver Int. 31:560.

23. Nan H, Qureshi AA, Hunter DJ et al (2009): A func-tional SNP in the MDM2 promoter, pigmentary pheno-types, and risk of skin cancer. Cancer Causes Control. 20: 171.

24. Ohmiya N, Taguchi A, Mabuchi N, et al (2006): MDM2 promoter polymorphism is associated with both an in-creased susceptibility to gastric carcinoma and poor prognosis. J. Clin. Oncol. 24: 4434.

25. Onat OE, Tez M, Ozcelik T et al (2006): MDM2 T309Gpolymorphism is associated with bladder cancer. Anticancer Res. 26:3473.

26. Park SH, Choi JE, Kim EJ, et al (2006): MDM2 309T.G polymorphism and risk of lung cancer in a Korean population. Lung Cancer. 54: 19.

27. Petenkaya A, Bozkurt B, Akilli-Ozturk O, et al (2006): Lack of association between the MDM2-SNP309 poly-morphism and breast cancer risk. Anticancer Res. 26: 4975.

28. Schafer S, Tolle T, Lottmann S et al (1998). Animal models and experimental systems in hepatitis B virus research. In Hepatitis B Virus: Molecular Mechanisms in Disease and Novel Strategies for Therapy (Koshy, R. C. & Caselmann, W. H., Eds), pp. 51. Imperial College Press, London, UK.

29. Schiefelbein E, Zekri AR, Newton DW, et al (2012): Hepatitis C virus and other risk factors in hepatocellular carcinoma. Acta Virol. 56(3): 235.

30. Sherman M, Peltekian K and Lee C (1995): Screening for hepatocellular carcinoma in chronic carriers of hep-atitis B virus: incidence and prevalence of hepatocel-lular carcinoma in a North American urban population. Hepatol. 22: 432.

31. Sherr CJ (2006): Divorcing ARF and p53: an unsettled case. Nat Rev Cancer. 6:663.

32. Tomoda T, Nouso K, Sakai A, et al (2012): Genetic Risk of Hepatocellular Carcinoma in Patients With Hepatitis C Virus. J Gastroenterol Hepatol. 27(4):797.

33. Wang X, Zhang X, Qiu B, et al (2012): MDM2 SNP309T>G polymorphism increases susceptibility to hepatitis B virus-related hepatocellular carcinoma in a northeast Han Chinese population. Liver Int. 32(7): 1172.

34. Wilkening S, Hemminki K, Rudnai P, et al (2007): No association between MDM2 SNP309 promoter poly-morphism and basal cell carcinoma of the skin. Br J Dermatol. 157: 375.

35. Yagmur E, Rizk M, Stanzel S, et al (2007): Elevation of endoglin (CD105) concentrations in serum of patients with liver cirrhosis and carcinoma. Eur J Gastroenterol Hepatol. 9: 755.

36. Yang Y, Xia T, Li N, et al (2013): Combined effects of p53 and MDM2 polymorphisms on susceptibility and surgical prognosis in hepatitis B virus-related hepato-cellular carcinoma. Protein Cell. 4(1):71.

37. Yoon YJ, Chang HY, Ahn SH, et al (2008): MDM2 and p53 polymorphisms are associated with the devel-opment of hepatocellular carcinoma in patients with chronic hepatitis B virus infection. Carcinogenesis. 29: 1192.

38. Zhang Z and Zhang R (2005): p53-independent activi-ties of MDM2 and their relevance to cancer therapy. Curr. Cancer Drug Targets. 5: 9.

39. Zhou X, Bloomston M, Shirley LA, et al (2013): The p53 Negative Regulator, MDM4 but Not MDM2, Is Frequently Activated in Hepatocellular Carcinoma. Poster Session VI # 123. 1721.

51المصري الشعب في المزمن الفيروسي الكبدي بااللتهاب عالقته و MDM2-SNP309 الجينية الطفرة

حسب اهللا مها - معتز صيام فتحى اسحق - ماريان - هانى حسين السيد عبد المؤمن - نهى كمال

الدين شرف محمد اهللا هبه عبد الهادى - عفاف أحمد العسالى - صفوت مصطفى نهال

مرحلة الكشف عن المرض في يتم ما عادة مصر في العالم. أنحاء جميع في السرطانية األورام أعنف أنواع من الكبد سرطان يعدقبل المريض ضرورى, يسمح بمعالجة المرض المبكر عن الكشف يعتبر وبالتالي ذلك الجراحة. في بما عالج فعال وجود عدم مع متأخرةالسرطان أنواع أكثر وأصبحت األخيرة، السنوات في مصر في الكبد بسرطان األصابة معدالت زادت لقد أنتشاره. أو الورم زيادة حجمالفيروسى الكبدى التهاب و بى الفيروسى الكبدى باأللتهاب األصابة هى الكبد الى سرطان المؤدية المعروفة أهم العوامل و من شيوعاالعديد من تستنتج الحاالت. من فى 5-20٪ تقدريبا 5 سنوات خالل الكبد و سرطان الكبدى التليف مضاعفاته من أهم يعتبر حيث سى.على العوامل أيضا في الورم نفسه ولكن تحدث التي الجسدية الطفرات على فقط يعتمد ال الكبد سرطان تطور في أن التباين الدراسات(MDM2 SNP309) T/G الجينية الطفرة ان P53 للبروتين رئيسي MDM2 كمنظم لألورام المسبب الجين يعمل الوراثية.من مع P53 ويثبط نووي) فسفورى (بروتين MDM2 جين التسرطن. يتفاعل في دورا تلعب قد وبالتالي البروتينى على التعبير تؤثرلألورام. الهدف الكابتة فاعليته من مقلال P53 يؤدي إلى تثبيط الجين فى هذا الزيادة على ذلك فان P53 و على المعتمدة النسخ عمليةالمرضى المصريين فى سرطان الكبد و MDM2 SNP309 الجينية الطفرة بين وجود عالقة إمكانية التحقق من الدراسة هو هذه منإشتملت األولى المجموعه : مجموعتين إلى تقسيمهم تم شخصا ثالثة مائة و على الدراسة هذه المزمن. اشتملت الكبدى باأللتهاب المصابينثالثين وستة و ”بى“ و ”سى“ المزمن الكبدي الفيروسى بااللتهاب نتيجة إصابتهم الكبد بسرطان مصابين مريضا ستين و سبعة علىالجيني التنميط عن الكشف يتم الضابطة. المجموعة ليكونوا 10 أعوام من ألكثر المزمن الفيروسى الكبدي بااللتهاب مصابين مريضاوجود الدراسة أظهرت .(Realtime PCR) البوليميراز النزيم التسلسلي للتفاعل الفعلى الوقت باستخدام MDM2 SNP309 للمجموعة عن الكبد، بسرطان المصابين المرضى مجموعة في الدم مصل فى (albumin) الزالل مستوى فى إحصائية داللة ذات زيادة(AFP) فى ألفا الجنيني البروتين فى مستوى إحصائية داللة وجود زيادة ذات الدراسة أظهرت الفيروسى الكبدي األلتهاب مرضىهذه الدراسة أن تبين من قد و الفيروسى. الكبدي التهاب مرضى مجموعة عن الكبد، بسرطان المصابين في مجموعة المرضى مصل الدم 50.7٪ هى 38.8٪، الكبد بسرطان المصابين المرضى مجموعة في MDM2 SNP309 GG, TG, TT الجينية األنماط توزيعو التوالي على التوالي 11.1٪ ٪50 على كانت 38.9٪، التهاب الكبدي الفيروسى مرضى مجموعة فى و أما التوالي ٪10.4 على وتوزيع األنماط نمط في إحصائية ذات داللة عالقة هناك يكن لم أن الدراسة أظهرت المورثات: توزيع إحصائية. داللة له يكن لم ذلك لكن GG +TG ) الجينية األنماط في نمط توزيع داللة إحصائية هناك يكن لم المجموعتين كما بين (GG مقابل TT+ TG ) الجينيةالعدوى ونوع MDM2 SNP309 الجينية األنماط داللة إحصائية بين عالقة ذات يكن هناك لم أيضا TT) بين المجموعتين. و مقابل

سى. أو بى الكبدى الفيروس كان إذا ا الفيروسية


EVALUATION OF OSTEOPONTIN AND ALPHA L-FUCOSIDASE AS DIAGNOSTIC MARKERS OF HEPATOCELLULAR CARCINOMA

Hanaa H. Elsaid*, Lamiaa Mamoud Kamel*, Abd el Motaleb Mohamed**and Reham Ahmed**

ABSTRACTIntroduction: Alpha fetoprotein (AFP) is not secreted in all cases of hepatocellular carcinoma (HCC), it may be normal in

40% of patients. Alpha-L-fucosidase (AFU) and osteopontin (OPN) have been suggested as tumor markers of HCC. This study aimed to evaluate the diagnostic values of AFU & OPN as tumor markers of HCC in hepatitis C virus patients. Methods: This case-control study was conducted on 60 patients (25 patients with HCC, 20 patients with chronic hepatitis and 20 patients with cirrhosis) in addition to 20 apparently healthy individuals who served as a healthy control group. Results: AFP,OPN and AFU levels were significantly higher in the HCC group compared to chronic hepatitis , liver cirrhosis and healthy control groups(p < 0.001 in each) and in liver cirrhosis compared to the control group (p< 0.01,p<0.001,p<0.001) respectively. There was statistically significant increased in AFU level in the liver cirrhosis compared to chronic hepatitis group (p< 0.001) ,while therewas no significant difference between both groups as regards to AFP and OPN ( p >0.05). In patients with HCC AFP level wassignificantly higher in TNM stage III than TNM stage II (P <0.001) . The AFP sensitivity was 75 %, OPN sensitivity was75% and AFU sensitivity was 70%. Combined use of OPN, AFP produced 80% sensitivity while combined use of AFU, AFP produced 90% sensitivity. Combined use of OPN, AFP and AFU produced 100% sensitivity. Conclusion: Combined detection of OPN and AFU activity may be used as diagnostic markers for the diagnosis of HCC .Combined detection of AFU, OPN and AFP could improve early diagnosis of HCC and differentiate it from chronic liver disease. Keywords: Hepatocellular carci-noma, Alpha-L-fucosidase, osteopontin Corresponding author: Hanaa H. Elsaid

Departments of Clinical Pathology* and Medical Oncology** , Faculty of Medicine, Zagazig University, Egypt.

INTRODUCTION

Hepatocellular carcinoma (HCC) is consid-ered the fifth commonest cancer in the world,and the third oncological cause of death. HCC is the major cause of mortality in patients with chronic liver diseases(31). In Egypt HCC repre-sents 14.8% of all cancer mortality, with a high-er incidence in males (17.3%) than in females (11.5%). Chronic hepatitis usually leads to the sequential occurrence of liver fibrosis and cirrho-sis with a high risk of development of HCC(3,9). It has been estimated that 20% of HCV-infected patients develop liver cirrhosis and approxi-mately 40% of these develop HCC within 10-15 years (8). Alpha-fetoprotein (AFP) is widely used as a screening test for HCC among patients with cirrhosis, despite its limited performance, par-ticularly in early-stage HCC(10). Other markers (e.g., lectinbound AFP [AFP-L3], des-gamma carboxyprothrombin (DCP) and glypican-3 have been proposed for HCC detection(32,36). How-ever, neither DCP nor AFP-L3 presented better performance characteristics than AFP for the diagnosis of early-stage HCC. American hepa-topathologists treat every tumor larger than one cm in diameter in cirrhotic liver as HCC, conse-

quently ignoring AFP serology (very frequently false negative)(24).Alpha-L-fucosidase (AFU) is a sort of enzyme to hydrolyze fucoseglyco-sidic linkages of glycoprotein and glycolipids. Its activity increases obviously in the serum of HCC patients compared with that in the serum of healthy adults, patients with cirrhosis and patients with chronic hepatitis (23). Osteopontin (OPN) is an integrin-binding glycophosphopro-tein that is expressed by several cell types espe-cially by transformed malignant epithelial cells and believed to be involved in many physiologi-cal cellular functions especially in regulation of migration, invasion and thus metastasis as well as survival of tumor cells(4). OPN overexpres-sion indicates a poor prognosis for patients with HCC, it may also have predictive role for HCC invasion and metastasis (4). In our study we aimed to evaluate AFP, AFU and OPN together in order to improve sensitivity of HCC detec-tion.


This study has been conducted on 60 patients who attended clinics and admitted to the Internal-Medicine and Oncology Departments of Zagazig University Hospitals between February 2013 and


Elsaid, H. H. et al.54

May 2014. The plan for secondary data analysis was approved by the Institutional Review Board (IRB) of the Faculty of Medicine, Zagazig Uni-versity, and all the patients gave their informed consent prior to their inclusion in the study. Inclusion criteria, patients with HCC, liver cir-rhosis and chronic hepatitis were included in the study; all patients were HCV positive .Exclusion criteria were patients with HBV infection, car-diovascular, chest or renal diseases or patients receiving chemotherapy or radiotherapy.

SubjectsStudied subjects were divided into three

groups; Control group: included 20 healthy vol-unteers (14 males and 6 females) with age range from 39 to 58 years with mean value ± SD of 50.56±7.91years.Chronic hepatitis group: in-cluded 20 patients(13 males and 7 females) with age range from 39 to 57 years with mean value ± SD of 48.25 ± 9.07years.Chronic cirrhosis group: included20 patients (14 males and 6 females) with age range from 39 to 59 years with mean value ± SD 49.5±10.5years,Hepatocellular car-cinoma group: included 25 patients (16 males and 9 females) with age range from 43 to 57 years with mean value ± SD of 54.0±8.56years.According to American Joint Committee on Cancer (AJCC) Staging System this group was subdivided to TNM stage II and TNM stage III (tumor, node, and metastasis) (22). Abdomi-nal ultrasonography, computerized tomography (CT)and laboratory investigations(liver func-tions, CBC and AFP) were done for all patients to prove the presence of chronic liver diseases or cirrhosis. Clinical assessment of liver disease severity was done by Child Pugh score based on serum albumin, bilirubin, prothrombin time, the presence of ascites and encephalopathy (28). HCC diagnosis was based on a clinical examination, triphasic abdominal computed tomography and the serum AFP level. HCV infection was diag-nosed by real time PCR on Cobas® AmpliPrep/ Cobas® TaqMan® (Roche diagnostics- Swit-zerland) .Peripheral blood was collected from each participant. Sera and plasma were stored at -80°C until measurements of alpha L. fucosi-dase and OPN. The following laboratory tests were performed, liver function tests were done on Cobas 6000Roche diagnostics- Switzerland and while AFP level was measured on Cobas E411 immunoassay Roche Diagnostics, USA.

Complete blood count was performed on Sys-mex-Kx-21 (Sysmex Corporation- Japan) and INR was performed on Sysmex CA-1500 in-struments (Sysmex Corporation - Japan). The AFU assay was done on serum samples by a kit from Bio Supply UK, based on the enzymatic cleavage of the synthetic substrate 2-chloro-4-nitrophenyl-α-L-fucopyranoside to α-L-fucoside and 2-chloro-4-nitrophenol, which was quanti-fied by measuring the absorbance at 405 nm ina kinetic fashion. One unit of AFU is defined asthe amount of AFU that cleaves one μ mole of 2-chloro-4-nitrophenyl-α-L-fucoside per min at 37 °C with Intra assay CV% < 5.1%, Inter assay CV% < 6.2%(44). Plasma concentrations of OPN were measured by quantitative sandwich en-zyme immunoassay technique (ELISA) accord-ing to the protocol provided by the manufacturer (R&D Systems, Minneapolis, USA). The optical density was measured at 450 nm using a micro plate reader. With an intra-assay CV 2.9% and inter-assay CV 5.4%.Statistical analysis

Mean and standard deviation were used for descriptive statistics. Analysis of variance (ANOVA) and Least Significant Difference(LSD) were conducted for a comparison of pa-rameters concentration levels among the differ-ent groups of subjects included. A comparison between two groups was performed using the student t-test. Receiver operating characteristics (ROC) analysis was used to evaluate the diag-nostic value of OPN, AFU and AFP to identify the cutoff values, sensitivity, specificity, positiveand negative predictive values of OPN, AFU and AFP. Calculations were done with the Statisti-cal Package for the Social Sciences version 19 (SPSS, Inc., Chicago, IL, USA).

RESULTSTable 1: Showed that, there were significant

differences between all studied groups as regards liver function tests. Liver function tests were sig-nificantly different in the HCC group comparedto chronic hepatitis , liver cirrhosis and healthy control groups (p < 0.001 in each) except ALP & bilirubin there was no difference between HCC and cirrhotic groups (p> 0.05) while there was no significant difference between HCC and con-trol groups as regards INR(P>0.05). There was significant differences between chronic hepa-titis and cirrhotic groups (p< 0.001) except AST and ANR (p>0.05).

Osteopontin and Alpha L-Fucosidase in HCC 55 111Table 2: Presents the comparison of AFP,

AFU, and OPN among the studied groups. There were statistically high significant difference inthe mean values of AFP (ng/ml) (p<0.0001), AFU (U/l) (p<0.001) and OPN. (ng/dl) (p<0.001). AFP, AFU and OPN levels were significantlyhigher in the HCC group compared to chronic hepatitis , liver cirrhosis and healthy control groups (p < 0.001 in each) and in liver cirrhosis group compared to the control group (p< 0.01, p<0.001,p < 0.001) respectively. There was sig-nificant increase in AFU level in the liver cir-rhosis compared to chronic hepatitis group (p< 0.001) ,while there was no significant differencebetween both groups as regards to AFP and OPN ( p >0.05 in each ). AFP level was significantlyhigher in the chronic hepatitis group compared to control group (p < 0.01) while there was no significant difference between both groups as re-

gards to AFU and OPN (p >0.05 in each).Table 3: AFP was significantly elevated in

TNM stage III than TNM stage II of HCC (t=4. 6, p<0.001).while there was no significant dif-ference between stage III and stage II as re-gards AFU and OPN (t= 0.69, p>0.05), (t= 0.91, p > 0.05) respectively.

Table 4: Presents the diagnostic performance of AFP,APO and AFU in diagnosis of HCC , At cutoff value 200 ng/ml, AFP had 65%sensitivity, 80% specificity, this mean 35% of HCC patientsgave false negative, However 100% sensitivity and 93.8% specificity were obtained in combi-nation between AFU, OPN and AFP. Receiver operator characteristic (ROC) curves showed that the area under the curve (AUC) for OPN and AFU was 0.8 5with 95% confidence interval(CI) (0.75 - 0.95) and 0.87: 95% CI (0.56 -1), respectively (Figure 1, 2).

5

Table 4:Presents the diagnostic performance of AFP,APO and AFU in diagnosis of HCC

, At cutoff value 200 ng/ml, AFP had 65%sensitivity, 80% specificity, this mean 35% of

HCC patients gave false negative ,However 100% sensitivity and 93.8% specificity were

obtained in combination between AFU, OPN and AFP. Receiver operator characteristic

(ROC) curves showed that the area under the curve (AUC) for OPN and AFU was 0.8

5with 95% confidence interval (CI) (0.75 - 0.95) and 0.87: 95% CI (0.56 -1), respectively.

(Figure 1, 2).

Table 1: Comparison between studied groups as regards biochemical parameters

Parameters Control n=20 X±SD

Chronichepatitis n=20 X±SD

Liver Cirrhosis n=20 X±SD

HCC n=25 X±SD

P. Value

Bilirubin (mg/dL) 0.56±0.17 1.18±0.72 2.52±0.85 2.57±1.4 <0.001 Albumin(g/dL) 4.43±0.49 3.91±0.39 2.8±0.54 3.11±0.54 <0.001 AST (IU/L) 19.1±7.09 90.1±19.9 79.7±14.2 105.1±33.5 <0.001ALT (IU/L) 20.03±5.88 108.5±19.9 62.4±17.9 164.6±17.9 <0.001ALP (IU/L) 196.4±31.1 199.8±45.8 265.3±55.4 255.1±79 <0.001 INR 1.05±0.1 1.77±0.36 1.79±0.4 1.14±0.14 >0.05

LSD test was carried out between two groups, HCC group versus control group: Bilirubin (P < 0.001), albumin (P < 0.01), ALT (P <0.001), AST (P<0.001) ALP (P<0.001), INR (P >0.05) .HCC vs. chronic hepatitis group: Bil. (P < 0.001), albumin (P < 0.01), ALT (P <0.001), AST (P<0.01), ALP (P<0.001), INR (P <0.01).HCC vs. cirrhotic group: Bil. (P >0.05), albumin (P < 0.01), ALT (P < 0.001), AST (P < 0.01), ALP (P >0.05), INR (P < 0.01). Chronic hepatitis vs. control: Bil. (P > 0.05), albumin (P < 0.01), ALT (P <0.001), AST (P <0.01), ALP (P >0.05), INR (P <0.01).Cirrhosisvs. Control: Bil. (P <0.01), albumin (P < 0.01), ALT (P <0.001), AST (P <0.01), ALP (P <0.01), INR (P <0.01).Cirrhosis vs. chronic hepatitis: Bil. (P <0.01), albumin (P < 0.01), ALT (P < 0.001), AST (P > 0.05), ALP (P <0.001), INR (P > 0.05).

Table 2: Comparison of AFP, OPN, and AFU among studied groups

Parameters Control (n = 20)

chronic hepatitis (n = 20)

Cirrhosis (n = 20)

HCC (n = 25) P. value

AFP (ng/ml)X±SD 4.48±0.29 49.05±9.3 68.95±18.6 456.3±153 <0.0001

OPN (ng/dl)X±SD 85.4±32 91.4±31.2 116.6±40.4 540.9±87.5 <0.001

AFU (U/L)X±SD 16.4±5.7 22.7±7.6 40.6±11.4 83.9±31 <0.001

AFP: Alpha fetoprotein, OPN: Osteopontin, AFU: Alpha-L-fucosidase. LSD test was carried out between two groups, HCC group versus control group: AFP (P = 000), AFU (P = 0.000), OPN (P = 0.000).HCCvs. cirrhosis: AFP (P = 000), AFU (P< 0.001), OPN (P = 0.000). HCC vs. chronic hepatitis group: AFP (P = 000), AFU (P < 0.001), OPN (P = 0.000).Cirrhosis vs. controlgroup: AFP (P <0.01), AFU (P < 0.001), OPN (P <0.01). Cirrhosis vs. chronic hepatitis: AFP (P >0.05), AFU (P < 0.001), OPN (P >0.05) .Chronic hepatitisvs. Control group: AFP (P <0.01), AFU (P>0.05), OPN (p>0.05).


LSD test was carried out between two groups, HCC group versus control group: Bilirubin (P < 0.001), albumin (P < 0.01), ALT (P <0.001), AST (P<0.001) ALP (P<0.001), INR (P >0.05) .HCC vs. chronic hepatitis group: Bil. (P < 0.001), albu-min (P < 0.01), ALT (P <0.001), AST (P<0.01), ALP (P<0.001), INR (P <0.01).HCC vs. cirrhotic group: Bil. (P >0.05), albumin (P < 0.01), ALT (P < 0.001), AST (P < 0.01), ALP (P >0.05), INR (P < 0.01). Chronic hepatitis vs. control: Bil. (P > 0.05), albumin (P < 0.01), ALT (P <0.001), AST (P <0.01), ALP (P >0.05), INR (P <0.01).Cirrhosisvs. Control: Bil. (P <0.01), albumin (P < 0.01), ALT (P <0.001), AST (P <0.01), ALP (P <0.01), INR (P <0.01).Cirrhosis vs. chronic hepa-titis: Bil. (P <0.01), albumin (P < 0.01), ALT (P < 0.001), AST (P > 0.05), ALP (P <0.001), INR (P > 0.05).

5

Table 4:Presents the diagnostic performance of AFP,APO and AFU in diagnosis of HCC

, At cutoff value 200 ng/ml, AFP had 65%sensitivity, 80% specificity, this mean 35% of

HCC patients gave false negative ,However 100% sensitivity and 93.8% specificity were

obtained in combination between AFU, OPN and AFP. Receiver operator characteristic

(ROC) curves showed that the area under the curve (AUC) for OPN and AFU was 0.8

5with 95% confidence interval (CI) (0.75 - 0.95) and 0.87: 95% CI (0.56 -1), respectively.

(Figure 1, 2).


Parameters Control n=20 X±SD

Chronichepatitis n=20 X±SD

Liver Cirrhosis n=20 X±SD

HCC n=25 X±SD

P. Value

Bilirubin (mg/dL) 0.56±0.17 1.18±0.72 2.52±0.85 2.57±1.4 <0.001 Albumin(g/dL) 4.43±0.49 3.91±0.39 2.8±0.54 3.11±0.54 <0.001 AST (IU/L) 19.1±7.09 90.1±19.9 79.7±14.2 105.1±33.5 <0.001ALT (IU/L) 20.03±5.88 108.5±19.9 62.4±17.9 164.6±17.9 <0.001ALP (IU/L) 196.4±31.1 199.8±45.8 265.3±55.4 255.1±79 <0.001 INR 1.05±0.1 1.77±0.36 1.79±0.4 1.14±0.14 >0.05

LSD test was carried out between two groups, HCC group versus control group: Bilirubin (P < 0.001), albumin (P < 0.01), ALT (P <0.001), AST (P<0.001) ALP (P<0.001), INR (P >0.05) .HCC vs. chronic hepatitis group: Bil. (P < 0.001), albumin (P < 0.01), ALT (P <0.001), AST (P<0.01), ALP (P<0.001), INR (P <0.01).HCC vs. cirrhotic group: Bil. (P >0.05), albumin (P < 0.01), ALT (P < 0.001), AST (P < 0.01), ALP (P >0.05), INR (P < 0.01). Chronic hepatitis vs. control: Bil. (P > 0.05), albumin (P < 0.01), ALT (P <0.001), AST (P <0.01), ALP (P >0.05), INR (P <0.01).Cirrhosisvs. Control: Bil. (P <0.01), albumin (P < 0.01), ALT (P <0.001), AST (P <0.01), ALP (P <0.01), INR (P <0.01).Cirrhosis vs. chronic hepatitis: Bil. (P <0.01), albumin (P < 0.01), ALT (P < 0.001), AST (P > 0.05), ALP (P <0.001), INR (P > 0.05).


Parameters Control (n = 20)

chronic hepatitis (n = 20)

Cirrhosis (n = 20)

HCC (n = 25) P. value

AFP (ng/ml)X±SD 4.48±0.29 49.05±9.3 68.95±18.6 456.3±153 <0.0001

OPN (ng/dl)X±SD 85.4±32 91.4±31.2 116.6±40.4 540.9±87.5 <0.001

AFU (U/L)X±SD 16.4±5.7 22.7±7.6 40.6±11.4 83.9±31 <0.001




Elsaid, H. H. et al.56

6

Table 3: Relation between TNM classification, AFP, AFU, and OPN within HCC group.

Parameters TNM (II) n=15

TNM (III) n=10 t P

AFP(ng/ml)X±SD 368.33 ±122.83 588.25 ±79.68 4.46 <0.001

OPN (ng/dl)X±SD 557.1±89.4 521.1±86.1 0.91 >0.05

AFU (U/L) X±SD 87.6±25.3 79.3±21.2 0.69 >0.05

TNM: (tumor, node, metastases), AFP: Alpha fetoprotein, OPN: Osteopontin, AFU: Alpha-L-fucosidase

Table 4: Diagnostic Performance of AFP, OPN and AFU in diagnosis of HCC. Variables Cut-Off Sensitivity Specificity PPV NPV Accuracy

AFP(ng/ml) 200 65% 80% 100% 90.3% 91.8%OPN(ng/dl) 170 75% 73.8% 47.9% 90.6% 95.3%AFU (U/L) 64 70% 86.2% 60.9% 90.3% 82.4%

Combined AFP&AFU AFP=200AFU=64 90% 80% 58.6% 96.3% 82.4%

Combined AFP& OPN AFP=200OPN=170 80% 80% 55.2% 92.9% 80%

Combined OPN& AFU AFU=64 OPN=170 100% 93.8% 83.3% 100% 95.3%

Combined AFP, OPN& AFU

AFU=64OPN =170AFP=200

100% 93.8% 83.3% 100% 95.3%

PPV: Positive predictive value, NPV: Negative predictive value, AFP: Alpha fetoprotein, OPN: Osteopontin, AFU: Alpha-L-fucosidase,



6


Parameters TNM (II) n=15

TNM (III) n=10 t P

AFP(ng/ml)X±SD 368.33 ±122.83 588.25 ±79.68 4.46 <0.001

OPN (ng/dl)X±SD 557.1±89.4 521.1±86.1 0.91 >0.05

AFU (U/L) X±SD 87.6±25.3 79.3±21.2 0.69 >0.05


Table 4: Diagnostic Performance of AFP, OPN and AFU in diagnosis of HCC. Variables Cut-Off Sensitivity Specificity PPV NPV Accuracy

AFP(ng/ml) 200 65% 80% 100% 90.3% 91.8%OPN(ng/dl) 170 75% 73.8% 47.9% 90.6% 95.3%AFU (U/L) 64 70% 86.2% 60.9% 90.3% 82.4%

Combined AFP&AFU AFP=200AFU=64 90% 80% 58.6% 96.3% 82.4%

Combined AFP& OPN AFP=200OPN=170 80% 80% 55.2% 92.9% 80%

Combined OPN& AFU AFU=64 OPN=170 100% 93.8% 83.3% 100% 95.3%

Combined AFP, OPN& AFU

AFU=64OPN =170AFP=200

100% 93.8% 83.3% 100% 95.3%


Table 4: Diagnostic Performance of AFP, OPN and AFU in diagnosis of HCC.


7

Figure 1: ROC curve of OPN. The curves show cut-off value for OPN of 170 ng/dl, AUC was 0.8: 95%CI(0.75 - 0.95)

Figure 2: ROC curve of AFU. The curves show cut-off value for AFU of 64 U/L, AUC was0.87: 95% CI (0.56 -1)

7





57DISCUSSION

Patients with chronic liver disease and cirrho-sis should be carefully monitored for the early detection of HCC. AFP is the main tumor marker of HCC, Even in patients with advanced HCC, the AFP levels may remain normal in 15–30% of the patients. Therefore, it is of maximum im-portance to identify sensitive biomarkers that allow the prediction of HCC development at an early stage and at the same time are easily mea-surable and minimally invasive (7,32). Our study investigated the concept of a combined detection using several markers in order to support the de-tection of HCC using AFP. In this study serum levels of ALT, AST and Bilirubin were higher in HCC group than other groups , that was in agreement with Bruix and Sherman (30) who showed that the serum level of ALT and AST were elevated in HCC especially the advanced cases; and the difference becomes greater as the disease progresses. Hepatocellular carcinoma can cause either obstructive jaundice or hepato-cellular jaundice(30). In current study there were elevation of AFP levels in HCC patients com-pared to cirrhotic and chronic hepatitis patients, these results were supported by Nagwa et al.(27) who found that the mean concentration of AFP was significantly high in untreated patients withHCC as compared to patients with chronic liver disease. Marrero et al., (24) found that elevation of AFP levels in patients with cirrhosis or ex-acerbations of chronic hepatitis while it may be normal in up to 40% of patients with HCC, par-ticularly during the early stages , this disagree-ment may be due to discrepancy between cases number and stages of liver disease. In our study there were elevation of OPN levels in cirrhotic patients compared to chronic hepatitis patients and healthy subjects. Urtasun et al ( 35) reported that the OPN levels were significantly higherin HCV-cirrhotic patients compared to healthy individuals. OPN expression increases in tumor genesis, angiogenesis and in response to inflam-mation, cellular stress and injury (12). However OPN were statistically elevated in patients with HCC compared to patients with cirrhosis or chronic hepatitis, these results were in accordance with Bessa et al.(5) who found that OPN level was significantly higher in HCC patients group than

than the pathological and healthy controls. The mean value of AFU in patients of HCC in this study was significantly higher than those found inpatients of cirrhosis, chronic hepatitis and control, this was in agreement with 35. Tangkijvanich et al and Zahran et al(34,38). One possible explanation for the increase of AFU is an increased synthesis of proteins by tumor with a consequent increase in fucose turnover. Grizzi et al.(15) and Ishizuka et al.(18) demonstrated the activity of AFU was increased in the sera of HCC patients compared with chronic liver disease and healthy individu-als. In this study we showed that HCC stage III had significantly high AFP more than HCC stageII these results supported by Shian-Yang et al(31). they reported that HCCs with high AFP had more frequent large size (> 5 cm) and high-grade tu-mors as compared to those with lower AFP. In our study there were no significant relation betweenAFU levels and tumor stages these may be due to most of the HCC patients had an early stage tu-mor (60%, TNM stage II). In agreement with our results, El-Houseiniet al(11). showed no significantrelation between AFU and tumor size. The liver contains various cell types that produce cytokines and chemokines(21), which up regulate AFU in the later stages of inflammation, this process is con-sistent with activation of a natural regulatory loop, resulting in a gradual reduction of the potential of blood-borne leukocytes to enter the endothelium at the sites of current inflammation(2). This anti-inflammatory immune-suppressive response maystimulate HCC metastases. In the present study there were no significant difference between tu-mor stages and plasma OPN levels this may be due to small number of cases specially advanced HCC, this was not in accordance with Lee et al.(20) who found that plasma OPN levels increased sig-nificantly in patients with large and multiple tu-mors and distant metastasis. In addition, plasma OPN levels were increased significantly withadvanced tumor stages and with tumor recur-rence. These studies suggested that plasma OPN levels could be used as a prognostic and predic-tive marker for HCC(19,39, 41). OPN regulates the transformation of normal cells to malignant cells by induction of phosphorylation and activation of phosphoinositide 3-kinase.This induces DNA binding and activation of various transcription

Osteopontin and Alpha L-Fucosidase in HCC

Elsaid, H. H. et al.58factors, including nuclear factor kappa-beta. The latter helps “switching on” of genes expressing anti-apoptotic proteins. The end-result is anti-apoptosis, tumor cell growth, motility and inva-sion (25). In the current study for HCC diagnosis, AFP had sensitivity 65% at cut off value 200ng/ml this means that 35% of the studied patients with HCC are negative for AFP.Our findings arein concordance with reports by Bruix J (8) and Spangenberg et al.(32) who showed AFP levels do not discriminate between benign liver disease and HCC. Additionally, they have poor sensi-tivity and specificity and vary with the etiologyof liver disease, treatment and tumor stage.We found that the sensitivity, specificity and diag-nostic accuracy of AFU at the cut-off value of 64 U/L were 70, 86.2 and 82.4%, respectively. Montaser et al.(25) reported that 87.5 % sensitiv-ity and 98 % specificity of AFU at 25 U/L cut-off value , at a cut-off value of 2.3005 ìmol −1 min−1, AFU yielded a sensitivity and specificityof 90% and 97.5%, respectively(26) .In our study a relatively different cut off values of AFU compared to other studies mostly due to the difference in cases number . AFU has been recommended as a serum biomarker for HCC in some studies. The present study shows that the concentration of AFU is significantly higher in patients withHCC than patients with benign liver diseases and control subjects; In addition, AFU recorded 70% sensitivity and 86.2% specificity showingits validity as a diagnostic biomarker of HCC. Therefore, AFU activity may be regarded as a biomarker for the diagnosis of HCC. Zhao et al. (42) showed that, AFU may be a valuable supple-mentary marker in HCC detection. However, the specificity of AFU is relatively poor, and is alsooverexpressed in diabetes, pancreatitis and hypo-thyroidism patients. The activity of AFU is also susceptible to ethnicity. Therefore, the clinical value of AFU requires additional investigation.

We have been reported that the combined de-tection of AFP and AFU can improve the sen-sitivity of HCC detection to 80%, while speci-ficity slightly not changed; this finding camein agreement with BO Tao et al. (6). and Huang Xingang et al. (17) they demonstrated that com-bined use of AFU and AFP will improve the sensitivity and accuracy of diagnosis of primary

hepatic carcinoma. The combined use of AFP and AFU can improve the sensitivity, specific-ity and diagnostic accuracy of AFP from 70, 85 and 79.7%, respectively, to 95, 100 and 99.1%, respectively (26). The combined detection of AFU with other tumor markers should be commonly used in clinical practice to improve the diagnos-tic sensitivity of AFU (43). In our study, plasma OPN sensitivity, specificity and diagnostic ac-curacy were 75, 73.8 and 95.3%, respectively at a cut off value 170 ng/dl. Abu El Makarem et al.(1) who studied the diagnostic performance of OPN level for discrimination of the HCC from chronic liver disease and healthy subjects found that the sensitivity, specificity, PPV and NPVwere 97.7%, 100%, 100%, 97.6% respectively at a cut off value 300ng /ml. In the current study the combined detection of AFP and OPN can improve the sensitivity and specificity of HCCdetection to 80 % and 80%, respectively. Hafez et al.(16) found that diagnostic performance of OPN for discrimination of the HCC patients was 100% in all aspects of diagnostic performance at a cut off value 2000pg/ml.

In this study OPN and AFU levels are ele-vated in HCC patients than other liver diseases, these results suggest that OPN and AFU together can use as diagnostic markers for HCC. In ad-dition combined use of AFU and OPN can im-prove the sensitivity (100 %) and specificity(93.8%) of HCC detection, the AFP does not add any value to diagnosis when combined with them. Although has to be considered AFP the gold standard for HCC tumor markers and serve as an important tool in the monitoring of HCC patients, its performance in early stage of HCC is deficient(34,36). OPN is expressed by several cell types especially by transformed malignant cells(4), AFU activity is increased in another diseases rather than liver diseases as diabetes (42). Consequently, in order to improve the diagnostic performance for HCC, the combination of AFP with other serum tumor markers is recommended in the diagnosis of HCC (12,41). Previous studies have proven that a combination of serum tumor markers had a better diagnostic performance for HCC (12, 14, 41), to avoid misdiagnosis especially in early stage HCC could use the combination of AFP, OPN and AFU if they are available to decr-

59ease the false negative rate of AFP level when it is used alone.

Conclusion: Combined detection of OPN and AFU activity may be used as diagnostic markers for the diagnosis of HCC. Combined detection of AFU, OPN and AFP could improve early diag-nosis of HCC and differentiate it from chronic liver disease.

REFERENCES1. Abu El MakaremMA, Abdel-Aleem A, Ali A, et al.,

(2011): Diagnostic significance of plasmaosteopontinin hepatitis C virus-related hepatocellular carcinoma. Annals of hepatology, 10(3): 296.

2. Ali S, Jenkins Y, Kirkley M, et al., (2008): Leukocyte extravasation: an immunoregulatory role for alpha-L-fucosidase? J Immunol. 181 (4): 2407.

3. Anwar WA, Khaled HM, Amra HA, et al., (2008): Changing pattern of hepatocellular carcinoma (HCC) and its risk factors in Egypt: possibilities for prevention .Mutat Res 659: 176.

4. Behrend EI, Craig AM, Wilson SM, et al., (1994): Re-duced malignancy of ras- transformed NIH3T3 cells expressing antisense OPN RNA. Cancer Res; 54:832.

5. Bessa SS, Elwan NM, Suliman GA. et al. (2010): Clini-cal significance of plasma osteopontinlevel: in Egyp-tian patients with hepatitis C virus- related hepatocel-lular carcinoma. Arch. Med -Res., 41(7): 541.

6. BO Tao, Huanghua, Zhangbao-zhen. (2013): measured analysis of serum AFP, AFU, GGT in patient of primary hepatic carcinoma. Guide of China Medicine, 2013.

7. BruixJ, Sherman M. (2011): American Association for the Study of Liver Diseases. Management of hepatocel-lular carcinoma: an update. Hepatology; 53(3):1020.

8. Bruix J, Sherman M. (2005): Management of hepato-cellular carcinoma. Hepatology; 42:1208.

9. Bugianesi E. (2007): Non-alcoholic steatohepatitis and cancer .Clin Liver Dis. Feb; 11(1):191.

10. Daniele B, Bencivenga A, Megna AS, et al. (2004): Alpha-fetoprotein and ultrasonography screening for hepatocellular carcinoma. Gastroenterology; 127: 108.

11. El-Houseini EM, Mohammed SM, Elshemey MW, et al., (2005): Enhanced Detection of Hepatocellular Car-cinoma, Cancer Control, Vol. 12, No. 4, 248.

12. El-Tanani MK, Campbell FC, Kurisetty V, et al., (2006): The regulation and role of osteopontin in malignant transformation and cancer .Cytokine Growth Factor Rev; 17:463.

13. Ertle J.M., Heider D., Wichert M., Keller B., Kueper R., Hilgard P.(2013): A combination of alpha-fetopro-tein and des-gamma-carboxyprothrombin is superior in detection of hepatocellular carcinoma. Digestion; 87: 121.

14. Ge T., Shen Q., Wang N., Zhang Y., Ge Z., Chu W.(2015): Diagnostic values of alpha-fetoprotein, dick-kopf-1, and osteopontin for hepatocellular carcinoma. Med. Oncol; 32: 59.

15. Grizzi F, Franceschini B, Hamrick C, et al., (2007): Usefulness of cancer-testis antigens as biomarkers for the diagnosis and treatment of hepatocellular carcino-ma. J Transl Med. 5:3.

16. Hafez SS, Abd AL Aziz MM, Mahmoud MA et al., (2013): Alfa-Fetoprotein L3 Subfraction and Osteopon-tin: Novel Markers for the Diagnosis of Hepatocellular carcinoma. Journal of American Science; 9(3) 322.

17. Huang Xingang, SHIXiaobo, GUOXinrong (2012): Clinical evaluation of combined AFU and AFP detec-tion in diagnosis for patients with primary hepatic car-cinoma .Laboratory Medicine and Clinic.100.

18. Ishizuka H, Nakayama T, Matsuoka S, et al., (1999): Prediction of the development of hepatocellular carci-noma in patients with liver cirrhosis by the serial de-terminationsof serum alphaL-fucosidase activity. Intern Med 38:927.

19. Kim J, Ki SS, Lee SD, et al., (2006): Elevated plasma osteopontin levels in patients with hepatocellular carci-noma. Am J Gastroenterol;101: 2051.

20. Lee HJ, Yeon JY, Suh SJ, et al. (2014): Clinical Utility of Plasma Glypican-3 and Osteopontin as Biomarkers of Hepatocellular Carcinoma, Gut Liver. Mar; 8(2):177.

21. Leonardi GC, Candido S, Cervello M, et al. (2012): The tumor microenvironment in hepatocellular carcinoma (review). Int. J. Onco. l40 (6):1733.

22. Liver. In: Edge SB, Byrd DR, Compton CC et al., (2010): AJCC Cancer Staging Manual. 7th ed. New York, NY: Springer, 2, pp 191.

23. Lok AS, Sterling RK, Everhart JE, et al., (2010): Des-gamma-carboxyprothrombin and alpha-fetoprotein as biomarkers for the early detection of hepatocellular carcinoma. Gastroenterology; 138: 493.

24. Marrero JA, Feng Z, Wang Y, et al. (2009): Alpha-feto-protein, des-gamma carboxypro-thrombin, and lectin-bound alpha-fetoprotein in early hepatocellular carci-noma. Gastro-enterology;137:110.

25. Montaser FM, SakrAM ,Khalifa OM. (2012): Alpha-L-fucosidase as a tumor marker of hepatocellular carci-noma. Arab J Gastroenterol.13:9.

26. Mossad NA, Mahmoud HE, Osman EA, et al., (2014): Evaluation of squamous cell carcinoma antigen-im-munoglobulin M complex (SCCA-IGM) and alpha-l -fucosidase (AFU) as novel diagnostic biomarkers for hepatocellular carcinoma. Tumor Biology, Volume 35, Issue 11, pp 11559.

27. Nagwa AG, Wafaa IR, TahanyR, et al., (2008): Prog-nostic Significance of Interleukins Determination inLiver Diseases Research Journal of Medicine and Med-ical Sciences, 3(2):124. .


Elsaid, H. H. et al.6028. Pugh RNH, Murray-Lyon IM, Dawson JL, et al., (1973):

Transection of the esophagus for bleeding esophageal varices. Brit. J. Surg. 60: 646.

29. Rangaswami H. (2004): Nuclear factor inducing kinase plays a crucial role in osteopontin induced MAPK/IkBa kinase dependent nuclear factor kB mediated proma-trixmetalloproteinase-9 activation. J. Biol. Chem., 279: 38921.

30. Sherlock S, Dooly J.(2002): Diseases of the Liver and Biliary System, 11th Edition. Oxford Blackwell scien-tific publication Ch 3,10,19,28.

31. Shian-Yang Peng , Wei J. Chen , Po-Lin Lai ,et al ., (2004): High α-fetoprotein level correlates with high stage, early recurrence and poor prognosis of hepato-cellular carcinoma: Significance of hepatitis virus in-fection, age, p53 and β-catenin mutations, International Journal of Cancer, vol. 112, Issue 1, 44.

32. Spangenberg HC, Thimme R, Blum HE. (2006): Se-rum markers of hepatocellular carcinoma. Semin Liver Dis; 26:385.

33. Stefaniuk P, Cianciara J, Wiercinska-Drapalo A. (2010): Present and future possibilities for early diagnosis of he-patocellular carcinoma. World J Gastroenterol. World J Gastroenterol. Jan 28; 16(4): 418.

34. Tangkijvanich P, Tosukhowong P, Bunyongyod P, et al., (1999): Alpha-L-fucosidase as a serum marker of he-patocellular carcinoma in Thailand. Southeast Asian J Trop Med Public Health; 30: 110.

35. Urtasun R, Lopategi A, George J, et al.(2012) : Osteo-pontin, an oxidant stress sensitive cytokine, up-regu-lates collagen-I via integrin α(V) β(3) engagement and PI3K/pAkt/NFκB signaling. Hepatology; 55:594–608.

36. Villanueva A, Minguez B, Forner A, et al. (2010): He-patocellular carcinoma: novel molecular approaches

for diagnosis, prognosis, and therapy. Annu Rev Med; 61:317.

37. Wang L, Yao M, Dong Z, et al., (2014): Circulating spe-cific biomarkers in diagnosis of hepatocellular carcino-ma and its metastasis monitoring. Tumour Biol; 35:9.

38. Zahran F, Hussein YM, Mohey El-Deen I, et al., (2014): Cyclooxygenase-2 single-nucleotide polymorphisms and hepatocellular carcinoma in Egypt, MolBiol Rep 41:1461.

39. Zhang CH, Xu GL, Jia WD, et al. (2012): Prognostic significance of osteopontin in hepatocellular carcino-ma: a meta-analysis. Int J Cancer; 130:2685.

40. Zhang G., Ha S.A., Kim H.K.,et al (2012): Combined analysis of AFP and HCCR-1 as an useful serological marker for small hepato-cellular carcinoma: a prosp ective cohort study. Dis. Markers;32:265.

41. Zhang H, Ye QH, Ren N, et al., (2006): The prognostic significance of preoperative plasma levels of osteopon-tin in patients with hepatocellular carcinoma. J Cancer Res Clin Oncol; 132:709.

42. Zhao YJ, Qiang JU, Li GH. (2013): Tumor markers for hepatocellular carcinoma. MolClin Oncol. Jul; 1(4): 593.

43. Zhu J, Jiang F, Ni HB, et al., (2013): Combined analy-sis of serum γ-glutamy-ltransferaseisoenzyme II, α-L-fucosidase and α-fetoprotein detected using a commer-cial kit in the diagnosis of hepatocellular carcinoma. Experimental and TherapeuticMedicine journal, Vol-ume 5 Issue 1, Pages: 89.

44. Zielke K, Okada S, O’Brien JS. (1972): Fucosidosis: diagnosis by serum assay of α-l-fucosidase. J. Lab. clin. Med. 79: pp. 164.

61

الكبدية الخاليا سرطان تشخيص الفافيوكيديز و االوستيوبونتين في انزيم اداء تقيمريهام احمد – عبدالمطلب محمد - كامل محمود السيد - لمياء هناء حسني

فى يساعد الفا قد الجنينى مستوى البروتين فى ارتفاع انتشارا السرطانات أكثر من واحدا الكبديه سرطان الخاليا المقدمة: يعتبرالفا البروتين الجنينى مستوى يكون الكبدية الخاليا سرطان مرضى %40 من حوالي ان وجد ولكن الكبدية الخاليا تشخيص سرطانوالفيروسى.وقد لوحظ المزمن الكبدى مثل االلتهاب الكبد لمرضى االخري الحاالت فى المستوي الموجود نفس الى يقترب او طبيعىأداءانزيم تقيم البحث: من الغرض الكبدية. الخاليا سرطان فى مرضى مستوى االوستيوبونتين وزيادة الفافيوكيديز انزيم نشاط زيادةشملت وقد البحث: طريقة (سي). الفيروسى الكبدي االلتهاب عن الناتج الكبدية الخاليا سرطان الفافيوكيديزواالوستيوبونتين فى تشخيصناتج بالكبد مزمن إلتهاب من مريضا يعانون الكبد,عشرين فى (س) ويعانون باورام بفيروس مصابون مريضا وعشرين خمسة الدراسة هذهاالشخاص عشرين من اإلصابه بفيروس (سي) باالضافة الى ناتج عن تشمع الكبد من يعانون مريضا (سي) ,عشرين بفيروس اإلصابه عنبالدم سيولة كاملة، نسبة دم صورة كبد، وظائف التالية: المعملية الفحوصات عمل مع شامل إكلينيكي فحص عمل تم وقد ظاهريا االصحاءقياس االوستيوبونتين و مستوى الفا وتقدير الجنينى البروتين مثل األورام ودالالت ”س“) الكبدي (الفيروس الكبدية الفيروسات دالئل وو الفا الجنينى البروتين لمستوى إحصائية إرتفاع ذو داللة • النتائج: الدراسة. المدرجين فى هذة االشخاص لكل الفافيوكيديز انزيم نشاطوالمجموعة الكبد تشمع و الكبد المزمن إلتهاب بمرضى اورام الكبد مقارنة حاالت الفافيوكيديزفى انزيم وزيادة نشاط االوستيوبونتينفارق وجود عدم المزمن مع الكبد بإلتهاب تشمع الكبد مقارنة نشاط انزيم الفافيوكيديزفى لزيادة إحصائية داللة ذو إرتفاع • . الضابطةلمستوى إحصائية داللة ذو إرتفاع • االوستيوبونتين و الفا الجنينى البروتين لمستوى من بالنسبة لكال المجموعتين واضح بينإرتفاع • الضابطة المجموعة مقارنة تشمع الكبد حاالت الفافيوكيديزفى انزيم نشاط و االوستيوبونتين وزيادة الفا الجنينى البروتينفي تشخيص والحساسية الدقة زيادة • الفا. الجنينى الفافيوكيديزمقارنة بالبروتين انزيم نشاط وزيادة الوستيوبونتين التشخيصية القيمةعلى منهما كل بإستخدام مقارنة الفافيوكيديز) انزيم نشاط ،وقياس الوستيوبونتين ) معا الداللتين عند إستخدام كال الكبد سرطان مرضجيدة الفافيوكيديزمعا كداللة تشخيصية قياس نشاط انزيم و االوستيوبونتين استخدام يمكن انه نستخلص النتائج من هذه : اإلستنتاج حدة.المبكرللمرض. االكتشاف على القدرة اليهما لتحسين الفا البروتين الجنينى اضافة سابقة يفضل دراسات على واعتمادا الكبد. لسرطان


SERUM MATRIX METALLOPROTEINASE-9 (MMP-9), TISSUE INHIBITOR OF METALLOPROTEINASE-1

(TIMP-1) AND TRANSFORMING GROWTH FACTOR-β1 (TGF-β1) IN BRONCHIAL ASTHMA

Neveen Abd El-Moneum Hassan*, Aliaё Abd-Rabou Mohamed-Hussein**, Ebtsam F. Mohammed*, Omnia Abd El-Moneum Mohamed*

and Manal Mohamed Tammam Mahmoud*.

ABSTRACTRationale: Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a

role. Remodeling is assumed to result in persistent airflow limitation and airway hyper-responsiveness. Assessment of airwayremodeling by analysis of blood and sputum remodeling markers has been developed that is minimally invasive and bypass tissue biopsy. Aim of the work: To evaluate the usefulness of estimation of serum MMP-9, TIMP-1and TGF-β1 as noninvasive markers of airway remodeling in patients with bronchial asthma. Patients and Methods: The study included 68 adult asth-matic patients and 20 apparently healthy subjects as controls. The patients group was classified according to different levelsof asthma control in the last three months into controlled asthma 29 patients, and uncontrolled asthma 39 patients. According to asthma severity into mild 14 patients, moderate 25 patients and severe 29 patients and according to disease duration, less than 5 years 34 patients and more than 5 years 34 patients. Blood samples were taken to estimate the serum levels ofMMP-9, TIMP-1and TGF-β1 for all studied groups. Pulmonary function tests were done for patients only. Results: The serum levels of MMP-9, TIMP-1, MMP-9/TIMP-1 ratio and TGF- β1 were significantly higher in the patients group than controls and inpatients with uncontrolled asthma compared to patients with controlled asthma (P <0.001 for each). Results also revealed that MMP-9, TIMP-1, MMP-9/TIMP-1 ratio and TGF-β1 show significantly increased levels with increasing degree of asthmaseverity (P <0.001, P <0.001, P <0.05 and P >0.001 respectively). Concerning the relation between duration of asthma and remodeling parameters our results revealed that serum levels of MMP-9, TIMP-1 and TGF-β1 were significantly higher inpatients with disease duration more than 5 years than in those with disease duration less than 5 years (P<0.001 for each). Pul-monary function tests, forced vital capacity (FVC%), forced expiratory volume in the first second (FEV1%) and FEV1/FVC%were significantly lower in patients with uncontrolled asthma than in patients with controlled asthma (P <0.01 for each) and inpatients with asthma duration more than 5 years than with asthma duration less than 5 years (P <0.001 for each). Correlation studies revealed significant positive correlation between studied parameters with each other (P <0.001 for each). Also, signifi-cant negative correlation between studied parameters and pulmonary function tests showed (P <0.001 for each). Conclusion: Serum MMP-9, TIMP-1, MMP-9/TIMP-1 ratio and TGF-β1 could be considered as non-invasive markers of airway remodel-ing that can bypass biopsy sampling. Also serum sample is easily handled and not subjected to technical error as sputum or bronchoalveolar lavage fluid.


INTRODUCTION

Asthma and related atopic syndromes have emerged as major public health concerns, and studies from around the world suggest that the incidence and prevalence of asthma began to rise more than two decades ago with no signs that these disturbing trends may be reversing. Asth-ma is characterized by episodic dyspnea, lung inflammation, and in some patients, progressiveirreversible airway dysfunction (23,61).

Airway remodeling encompasses the struc-tural alterations in asthmatic compared with nor-

mal airways. Airway remodeling in asthmatic pa-tients involves a wide array of pathophysiologic features, including epithelial changes, increased smooth muscle mass, increased numbers of ac-tivated fibroblasts, myofibroblasts, subepithelialfibrosis, and vascular changes. Multiple cyto-kines, chemokines, and growth factors released from both inflammatory and structural cells inthe airway tissue create a complex signaling envi-ronment that drives these structural changes(1,61). However, investigations have changed our un-derstanding of asthma from a purely inflamma-tory disease to a disease in which both inflam-

Departments of Clinical pathology*, Chest**, Faculty of Medicine, Assiut University

Hassan, N. A., et al.64

matory and structural components are equally involved (1).

Matrix metalloproteinases (MMPs) and their inhibitors, �ssue inhibitors of metalloproteinases (TIMPs) are thought to contribute to the pathogen-esis of asthma via their influence on the functionand migration of inflammatory cells as well asmatrix deposition and degradation. TIMPs inac-tivate MMPs by binding them in a 1:1 fashion. Thus, an increase in the molar ratio of MMP/TIMP may favor tissue injury, while the reverse could be associated with increased fibrosis (18,15). It is possible that the increased levels of TIMP-1 observed in asthmatics may represent an endog-enous protective mechanism to down-regulate the proteolytic activity of MMPs in lung paren-chyma or that an excess of TIMP-1 could lead to airway fibrosis(43). MMP-9 is the predominant MMP in asthma, and its expression is enhanced when patients have spontaneous exacerbations or in response to local instillation of allergen in the airway. After acute inflammation had resolved,MMP-9 levels returned toward normal(43,15).

Transforming growth factor-β (TGF-β), a pro-fibrotic cytokine, plays an important rolein promoting the structural changes of airway remodeling. TGF-β effects on the proliferation, differentiation and extracellular matrix (ECM) metabolism of airway structural cells. Further-more, it decreases synthesis of enzymes that degrade the ECM, namely matrix metallopro-teinase (MMPs) and increases the production of proteins that inhibit enzymes that degrade the ECM (40,47,50). Clinicians should consider airway remodeling in all subjects with asthma. Fixed airflow obstruction is regarded to be a late andirreversible manifestation of airway remodeling. For this reason, even without reliable and easily available tools to confirm the presence of remod-eling, clinicians should adjust allergic airway therapy to prevent development or worsening of airway and tissue remodeling (9,40). Assessment of airway remodeling by analysis of blood and spu-tum remodeling markers (MMP-9, TIMP-1 and TGF-β1) has been developed that is minimally invasive and bypass tissue biopsy (9).

Aim of the work

1- Determination of serum levels of MMP-

9, TIMP-1 and TGF-β1 in asthmatic patients with different degree of asthma severity, control and disease duration.

2- Study the correlation between serum levels of these parameters and pulmonary func-tion tests (FVC, FEV1 and FEV1/FVC ratio).

3- Evaluate the usefulness of these param-eters as noninvasive markers of airway remodel-ing in bronchial asthma.

PATIENTS AND METHODS

This study included 68 asthmatic patients at-tending Assiut University Hospital, Chest Outpa-tient Clinic, their ages ranged from (18-64 years) and 20 apparently healthy subjects, age and sex matched as control group. Patients were diag-nosed as bronchial asthma according to the Glob-al Initiative for Asthma (GINA) criteria and also they were classified according to different levelsof asthma control in the last three months(25) into controlled asthma (day time symptoms < twice per week, no nocturnal symptoms, no limita-tions of activities and need to reliever < twice per week) 29 patients, and uncontrolled asthma (day time symptoms > twice per week, nocturnal symptoms, limitations of activities and need to reliever > twice per week) 39 patients. Accord-ing to asthma severity, the patients were divided into mild (FEV1 > 65%) 14 patients, moderate (FEV1 50-65 %) 25 patients and severe (FEV1 <50) 29 patients (7), and according to disease du-ration into less than 5 years 34 patients and more than 5 years 34 patients.

The following are considered as exclusion criteria postoperative period, acute coronary ar-tery disease, collagen diseases, cancers, chronic renal failure, chronic liver disease, pregnancy and smoking. Patients who received any system-ic corticosteroid therapy in the last three months were also excluded.

All patients and controls included in this study were subjected to the following:

• Careful history taking and clinical ex-amination.

• Chest X-Ray and pulmonary function tests as forced vital capacity (FVC), forced ex-piratory volume in the first second (FEV1) and

http://ukpmc.ac.uk/abstract/MED/12476081/?whatizit_url_gene_protein=http://beta.uniprot.org/uniprot/?query=key:P01033+OR+key:P12032

http://ukpmc.ac.uk/abstract/MED/12476081/?whatizit_url_gene_protein=http://beta.uniprot.org/uniprot/?query=key:P01033

http://ukpmc.ac.uk/abstract/MED/12476081/?whatizit_url_go_term=http://www.ebi.ac.uk/ego/GTerm?id=GO:0009405

http://ukpmc.ac.uk/abstract/MED/12476081/?whatizit_url_go_term=http://www.ebi.ac.uk/ego/GTerm?id=GO:0009405

http://ukpmc.ac.uk/abstract/MED/12476081/?whatizit_url_gene_protein=http://beta.uniprot.org/uniprot/?query=key:P01033

http://ukpmc.ac.uk/abstract/MED/12476081/?whatizit_url_gene_protein=http://beta.uniprot.org/uniprot/?query=key:O18733+OR+key:P14780+OR+key:P41245+OR+key:P41246+OR+key:P50282+OR+key:P52176

http://ukpmc.ac.uk/abstract/MED/12476081/?whatizit_url_gene_protein=http://beta.uniprot.org/uniprot/?query=key:O18733+OR+key:P14780+OR+key:P41245+OR+key:P41246+OR+key:P50282+OR+key:P52176

MMP-9, TIMP- 1& TGF- β1 in Bronchial Asthma 65FEV1/FVC ratio were done for patients only.

• Blood samples were taken and serum was separated for all patients and controls and subjected to the following investigations:

Routine investigations

• Peripheral haemogram on micros 60.

• Serum glucose level, liver and kid-ney function tests on Hitachi 911- Boehringer Mannheim.

Special investigations

• Serum levels of MMP-9, TIMP-1 and TGF-β1 were determined by sandwich enzyme-linked immunosorbent assay (ELISA) by using R&D system quantikine kit catalog no. DMP 900. And DTM 100 for MMP-9, TIMP-1 re-spectively, purchased from R & D system USA, and by using DRG TGF-β1 ELISA kit catalog no. EIA 1864, purchased from DRG diagnostic, Germany.


Data entry and analysis were done by using SPSS software v.17 (Chicago USA).

Continuous values were described by mean and standard deviation. Univariate analy-sis for determining the difference of laboratory variables between studied groups was performed using student’s T test for continuous variables.

The Analysis Of Variance (ANOVA) was used in determining the difference of laboratory variables in cases where there are more than two groups.

Correlations among the studied variables were tested by Spearman’s correlation coeffi-cient. Difference was statistically significant ifP-value was less than 0.05.

RESULTS

Table (1), Figure (1) show that the serum lev-els (mean ±SD) of MMP-9, TIMP-1, MMP-9/TIMP-1 ratio and TGF- β1 in the patients group were (1181±394ng/ml, 569±126ng/ml, 2.04±0.32 and 26.69±3.35 pg/ml respectively) and were significantly higher than in controls(538 ± 155ng/ml, 345±71ng/ml, 1.56±0.35 and 14.45±3.36 pg/ml respectively) with P <0.001 for each.

Table (2), Figure (2) show that the serum levels (mean ±SD) of MMP-9, TIMP-1, MMP-9/TIMP-1 ratio and TGF-β1 in uncontrolled asthma patients (1425±309ng/ml, 635±88 ng/ml, 2.23±0.28 and 28.29±2.69pg/ml respec-tively) were significantly higher than controlledasthma group (853±219ng/ml, 480±114ng/ml, 1.78±0.15 and 24.53±2.94 pg/ml respectively) with P <0.001 for each.

Table(3), Figure(3) show that the mean ±SD of

Fig.(1): Serum levels of MMP-9, TIMP-1and TGF-β1 in patients and control groups.


pulmonary function tests were significantly lowerin patients with uncontrolled asthma FVC%, FEV1% and FEV1/FVC % (55.62±31.13 , 42.46±25.19 and 58.33±33.57 respectively) than in patients with controlled asthma (75.38±7.69, 59.97±11.91 and 78.79 ±9.26 respectively)(P <0.01 for each) .

Table (4), Figure(4) show that the serum

levels (mean ±SD) of MMP-9, TIMP-1, MMP-9/TIMP-1 ratio and TGF-β1 showed significantlyincreased levels with increasing degree of asthma severity, mild asthma were (800±318 ng/ml , 418 ± 97 ng/ml , 1.86 ± 0.28 and 22.93 ± 2.59 pg/ml respectively) moderate asthma were (1229±330 ng/ml, 568 ±107 ng/ml , 2.03 ±0.32 and 27.04 ±2.43) and severe asthma were (1323 ±367 ng/

Table (1) Serum levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of matrix metalloprotein-ase-1 (TIMP-1), (MMP-9/TIMP-1) ratio and transforming growth factor-β1 (TGF- β1) in control and patients groups.

Items Control group(n= 20)

Patients group(n= 68) P-value

MMP-9 (ng/ ml)

<0.001*** Range 320 – 890 550 – 2000

Mean ± SD 538 ± 155 1181 ± 394

TIMP-1 (ng/ ml)

<0.001*** Range 210 – 470 320 – 810

Mean ± SD 345 ± 71 569 ± 126

MIMP-9 / TIMP-1Ratio

<0.001*** Range 1.1 - 2.3 1.5 - 2.6

Mean ± SD 1.56 ± 0.35 2.04 ± 0.32

TGF- β1 (pg/ ml)

<0.001*** Range 9.0 - 20.5 20.5 - 33.5

Mean ± SD 14.45 ± 3.36 26.69 ± 3.35

Independent samples t-test * Statistical significant differences (P < 0.05)** Statistical significant differences (P <0.01) *** Statistical significant differences (P < 0.001)

Table (2): Serum levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of matrix metalloprotein-ase-1 (TIMP-1), (MMP-9/ TIMP-1) ratio and transforming growth factor- β1 (TGF- β1) in patients with controlled asthma and uncontrolled asthma groups.

Items controlled asthma (n= 29) uncontrolled asthma (n= 39) P-value

MMP-9 (ng/ ml) <0.001***Range 550 – 1190 900 – 2000

Mean ± SD 853 ± 219 1425 ± 309TIMP-1 (ng/ ml)

<0.001***Range 320 – 670 450 – 810Mean ± SD 480 ± 114 635 ± 88

MMP-9 / TIMP-1 Ratio<0.001***Range 1.5 - 2.2 1.6 - 2.6

Mean ± SD 1.78 ± 0.15 2.23 ± 0.28TGF- β1 (pg/ ml)

<0.001***Range 20.5 - 29.0 24.5 - 33.5Mean ± SD 24.53 ± 2.94 28.29 ± 2.69

Independent samples t-test

MMP-9, TIMP- 1& TGF- β1 in Bronchial Asthma 67

Fig.(2): Serum levels of MMP-9, TIMP-1 and TGF-β 1 in patients with controlled asthma and uncontrolled asthma groups.

Table (3): Forced vital capacity (FVC %), forced expiratory volume in one second (FEV1%) and (FEV1/FVC %) in patients with controlled asthma and uncontrolled asthma groups.

Items Controlled asthma (n= 29)

Uncontrolled asthma (n= 39) P-value

FVC:%<0.01**Range 65 – 88 0 – 80

Mean ± SD 75.38 ± 7.69 55.62 ± 31.13FEV1:%

<0.01**Range 40-80 0-68Mean ± SD 59.97±11.91 42.46±25.19

FEV1/ FVC:%<0.01**Range 61 – 91 0 – 89

Mean ± SD 78.79 ± 9.26 58.33 ± 33.57Independent samples t-test

Fig. (3): Pulmonary function tests [FVC%, FEV1% and FEV1/FVC%] in patients with controlled and uncontrolled asthma groups.

Hassan, N. A., et al.68Table (4): Serum levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of matrix metalloprotein-

ase-1 (TIMP-1), (MMP-9/TIMP-1) ratio and transforming growth factor- β1 (TGF- β1) in patients group according to disease severity.

Items FEV1% (Disease severity) P-value

Mild Moderate Severe> 65%(n= 14)

50 – 65%(n= 25)

< 50%(n= 29)

MMP-9 (ng/ ml) <0.001***Range 570 – 1450 550 – 1970 870 – 2000

Mean ± SD 800 ± 318 1229 ± 330 1323 ± 367TIMP-1 (ng/ ml)

<0.001***Range 320 – 610 320 – 750 530 – 810Mean ± SD 418 ± 97 568 ± 107 643 ± 81

MMP-9 / TIMP-1 Ratio< 0.05*Range 1.51 - 2.4 1.55 - 2.53 1.57 - 2.64

Mean ± SD 1.86 ± 0.28 2.03 ± 0.32 2.15 ± 0.32TGF- β1 (pg/ ml)

< 0.001***Range 20.5 – 28 20.5 - 31.5 23.5 - 33.5Mean ± SD 22.93 ± 2.59 27.04 ± 2.43 28.21 ± 3.02

ANOVA test

Fig. (4): Serum levels of MMP-9, TIMP-1 and TGF-β1 in patients with mild, moderate and severe asthma groups.

Table (5) Serum levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), (MMP-9/TIMP-1) ratio and transforming growth factor-β1 (TGF- β1) in patients groups with asthma duration (<5yrs.) and (>5yrs.).

ItemsDuration

P-value< 5 years(n= 34)

>5 years(n= 34)

MMP-9 (ng/ ml) <0.001***Range 550 – 1750 870 – 2000Mean ± SD 1016 ± 356 1346 ± 363

<0.001***TIMP-1 (ng/ ml)Range 320 – 720 51 0- 810Mean ± SD 497 ± 121 641 ± 82

> 0.05MMP-9 / TIMP-1 RatioRange 1.51 - 2.64 1.55 - 2.63Mean ± SD 2.00 ± 0.32 2.07 ± 0.33

<0.001***TGF- β1 (pg/ ml)Range 20.5 - 30.5 23.5 - 33.5Mean ± SD 25.09 ± 3.04 28.29 ± 2.87

Independent samples t-test


ml, 643 ±81ng/ml , 2.15 ±0.32 and 28.21 ±3.02 pg/ml respectively ( P < 0.001, P <0.001, P < 0.05 and P < 0.001 respectively).

Table (5), Figure (5) show that the serum lev-els (mean ±SD) of MMP-9, TIMP-1 and TGF-β1 in patients with disease duration (>5yrs) (1346 ±363 ng/ml, 641 ±82 ng/ml and 28.29 ±2.87 pg/ml) were significantly higher than in patientswith disease duration (<5yrs) (1016 ±356 ng/ml, 497 ±121 and 25.09 ±3.04 pg/ml) (P <0.001 for

each).

Table (6), Figure (6) show that (mean ±SD) of pulmonary function tests (FVC%, FEV1% and FEV1/FVC %) were significantly lower inpatients with asthma duration (>5yrs) (50.18% ± 30.70, 34.35% ± 21.68 and 50.24% ± 31.19 respectively) than in patients with asthma du-ration (<5yrs) (77.91% ± 4.90, 65.50% ± 5.99 and 83.88% ± 4.99 respectively) (P<0.001 for each).

Fig. (5): Serum levels of MMP-9, TIMP-1 and TGF-β 1 in patients with asthma duration (<5yrs.) and (>5yrs.).

Table (6): Forced vital capacity (FVC%), forced expiratory volume in one second (FEV1%) and (FEV1/FVC%) in patients with asthma duration (<5 years) and (> 5 years).

ItemsDuration

P-value<5 years(n= 34)

> 5 years(n= 34)

FVC:% <0.001***Range 70 – 88 0 – 76Mean ± SD 77.91 ± 4.90 50.18±30.70

<0.001***FEV1:%Range 43 – 80 0 – 64Mean ± SD 65.50 ± 5.99 34.35±21.68

<0.001***FEV1 / FVC %:

Range 61 – 91 0 – 86Mean ± SD 83.88 ± 4.99 50.24±31.19


Fig (6): FVC%, FEV1% and FEV1/FVC% in patients groups with asthma duration (<5yrs.) and (>5yrs.).

Correlation Study

Correlation of the studied parameters with each other showed

Positive correlation between MMP-9 with TIMP-1 and TGF-β1 (r=0.934, P <0.001 and r=0.850, P <0.001 respectively) (Figure 7-8) as well as between TIMP-1 and TGF-β1 (r=0.863,

P <0.001) (Figure 9).

Correlation of the studied parameters with pulmonary function test showed

Negative correlation between FEV1% with MMP-9, TIMP-1 and TGF-β1 (r= - 0.604, P <0.001, r= -0.742, P <0.001, and r= -0.692, P <0.001 respectively) (Figure 10-12).

Fig. (7): Positive correlation between MMP-9 and TIMP-1

Fig.(8): Positive correlation be-tween MMP-9 and TGF-β1.


Fig. (9): Positive correlation between TIMP-1 and TGF-β1.

Fig.(10) Negative correlation between (FEV1%) and MMP-9.

Fig.(11): Negative correlation between FEV1% and TIMP-1.

Fig. (12): Negative correlation between FEV1% and TGF-β1

Hassan, N. A., et al.72DISCUSSION

Asthma is a chronic inflammatory disorder ofthe airways. The chronic inflammation is associ-ated with airway hyper-responsiveness that leads to recurrent episodes of wheezing, dyspnea, breathlessness, chest tightness and coughing, particularly at night or in the early morning (4,26). These episodes are usually associated with wide-spread, but variable, airflow obstruction withinthe lung that is often reversible either spontane-ously or with treatment (25,61). Airway remodeling is clinically defined as persistent airflow obstruc-tion despite aggressive anti-inflammatory thera-pies. It also leads to a decrease in lung function, and airway hyper-responsiveness (1,61).

In this study the serum levels of matrix me-talloproteinase-9 (MMP-9), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), matrix metalloproteinase-9/tissue inhibitor of matrix metalloproteinase-1 (MMP-9/TIMP-1) ratio and transforming growth factor-β1 (TGF-β1) were estimated in asthmatic patients with varying de-gree of disease severity, asthma control and dis-ease duration as markers of airway remodeling.

The current study revealed that the serum levels of MMP-9, TIMP-1, and MMP-9/TIMP-1 ratio were significantly higher in the patientsgroup compared to that of controls.

In asthma, the potentially most important members of remodeling markers are MMP-9 and TIMP-1. MMP-9 is an important MMPs found commonly in asthma. It contributes to altered extracellular matrix turnover, influx ofproinflammatory cells, epithelial repair, andangiogenesis(37). Basement membrane degenera-tion by MMP-9 and other proteinases may oc-cur in the early phase of asthma exacerbation (58). Over-expression of TIMP-1 causes deposition of ECM and thickening of basement membrane by inhibiting degradation of ECM (53,3).

In agreement with our results (Cataldo,et al.,(16) , Simpson et al.,(52), Mohamed et al.,(43)reported that serum levels of MMP-9 were in-creased in asthma compared with those seen in healthy control subjects. Dogu et al., Hong et al., Zhang et al.,( 21, 31,61) reported that serum MMP-9 levels of asthmatic children were significantly

higher than those of healthy controls. These find-ings suggest that an increase in the serum MMP-9 level may indicate a defect in ECM homeosta-sis even in stable asthmatic children and perhaps propose MMP-9 as a noninvasive marker of in-flammation and remodeling in asthma. Slightlyincreased TIMP-1 levels may indicate homeo-stasis of a repair process (21).

Bronchial tissue expression of MMP-9 and BAL level were higher in asthmatic patients and it was significantly increased with increased se-verity also MMP-9 tissue expression was strong-ly inversely correlated with spirometric param-eters (22).

In accordance with our results Higashimoto et al., Gujin et al., Mohamed et al. (30,28, 43) showed that serum TIMP-1 concentrations of asthmatic patients were significantly higher than those ofthe control subjects.

Bergeron and Boulet(8) reported that MMP-9 levels were significantly higher in the sputumof asthmatic patients compared with control subjects, but TIMP-1 levels were similar to those found in healthy control subjects. Kim et al., Karakoc et al.,(36,33) reported that levels of MMP-9 and TIMP-1 in BAL fluid and exhaledbreath condensate were significantly higher inasthmatic patients than control group.

In consistent with our study Mohamed et al.,(43) reported that MMP-9/TIMP-1 ratio was increased in asthmatic patients compared to con-trol subjects. On contrary Kelly and Jarjour Matsumoto et al.,(34,41) reported that the ratio of MMP-9/TIMP-1 in asthmatics was lower than in control subjects and correlates with the degree of airway obstruction and increased airway wall thickness, suggesting that lower MMP-9/TIMP-1 ratios may be associated with a profibroticenvironment with reduced extracellular matrix turnover leading to airway remodeling.

To study the effect of asthma exacerbation on the studied parameters our results revealed that the serum levels of MMP-9, TIMP-1, and MMP-9/TIMP-1 ratio were significantly higher in pa-tients with uncontrolled asthma than in patients with controlled asthma.

In agreement with our results Mohamed et

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MMP-9, TIMP- 1& TGF- β1 in Bronchial Asthma 73al.,Castano et al., Felsen et al.(43,15, 23) reported that serum MMP-9 level was increased in asth-ma exacerbation and after allergenic challenge and decreased by corticosteroids. Also Rivera et al., (49) reported that the circulating and spu-tum MMP-9 concentrations were increased in patients with asthma exacerbations compared with patients with stable asthma. This increased activity may be related to exaggerated airway inflammation and airway remodeling. Circulat-ing MMP-9 levels may therefore reflect a “spillover” of MMP-9 produced in the airways. In this context, MMP-9 may be a potential target for the management of exacerbations of asthma. In con-trast, Boulay etal.,Mattos etal.,Rivera etal.,(11,42,49) reported that there were no significant differences in the TIMP-1 concentrations, suggest-ing that MMP-9 plays a role in the pathophysiol-ogy of asthma exacerbations.

Tanaka et al., Kelly et al.,(54, 35) found that an imbalance between MMPs and TIMPs was pres-ent in acute asthma or following allergen chal-lenge with an excess of MMP-9 implying a high ratio of MMP-9/TIMP-1 on exacerbation before treatment, meaning that airway tissue destruction and, over time with corticosteroid treatment, the TIMP-1 levels rose, dropping the ratio of MMP-9/TIMP-1. It was suggested that overproduction of MMP-9 and TIMP-1 in acute asthma might contribute to airway tissue remodeling and that TIMP-1 production might not be suppressed by glucocorticosteroids.

This study revealed that serum levels of MMP-9, TIMP-1 and MMP-9/ TIMP-1 ratio were significantly increased with increasingasthma severity.

In consistent with our results, Dar et al., Ventura et al (20,57) reported that serum levels of MMP-9 and TIMP-1 increased significantly withincreasing disease severity in asthmatic patients. In contrast Belleguic et al.,(5) found that no dif-ference was found in TIMP-1 levels in plasma.

MMP-9 levels in sputum from patients with severe asthma, particularly those with irrevers-ible airway obstruction, were even higher when compared with either healthy subjects or patients with mild asthma. However no significant dif-ference was seen between normal subjects and

patients with mild asthma. These findings mayindirectly indicate the involvement of MMP-9 in airway remodeling process in patients with se-vere asthma who have an irreversible component of airway narrowing. Therefore, MMP-9 level measurements could be proposed as a useful tool to determine one of the aspects of disease sever-ity (57).

Furthermore, Gueders et al.,(27) speculated that the MMP-9/TIMP-1 ratio in sputum of acute severe asthmatics may be higher than in the oth-er groups.

Concerning the relation between duration of asthma and studied remodeling parameters our results revealed that serum MMP-9 and TIMP-1 were significantly higher in patients with diseaseduration > 5 years than in those with disease du-ration < 5 years .

It is generally accepted that chronic inflamma-tion leads to airway remodeling in patients with asthma and this may contribute to irreversible airflow obstruction in affected individuals(12,45).

Children with persistent asthma had signifi-cantly increased serum MMP-9 levels compared to children who were diagnosed with asthma for the first time (19).

Bergeron et al., (10) reported that the duration of asthma has been associated with reduced lung function, increased airway hyper-responsive-ness (AHR) and asthma symptoms, as well as greater use of medications. The remodeling pro-cess has been proposed to explain these features. The Childhood Asthma Management Program (CAMP) study demonstrated an association be-tween asthma duration and reduced lung func-tion, higher AHR; greater asthma symptomatol-ogy. These observations may be related to air-way remodeling.

Asthma is a chronic inflammatory diseaseof the airway. Pathological repair of chronic in-flammation leads to airway remodeling. Trans-forming growth factor-β (TGF-β), a profibroticcytokine, plays an important role in promoting the structural changes of airway remodeling. TGF-β effects on the proliferation, differentia-tion and extracellular matrix (ECM) metabolism of airway structural cells (40,47).

Hassan, N. A., et al.74In this study the serum level of TGF-β1 was

significantly higher in the patients group than inthe control group. In agreement with our results Joseph et al., Ozyilmaz et al.,(32,46) found that TGF-β 1 was significantly higher in asthmatic pa-tients compared with controls. Also Manuyakorn et al.,(40) had demonstrated that atopic asthmatic patients had significantly higher levels of serumTGF-β1 compared to controls. TGF-β1 is re-leased in higher amounts in basal and allergen-challenged sites in the BAL fluid of asthmaticpatients than in controls. TGF-β, which is ex-pressed in the airway in asthma, has the potential to induce peribronchial fibrosis through stimula-tion of fibroblasts to produce extracellular ma-trix proteins (collagen, fibronectin)(50)

Yang et al.,(60) found that anti-TGF-β1 anti-body was effective in inhibiting pulmonary fi-brosis and significantly reduced collagen deposi-tion, smooth muscle cell proliferation, and goblet cell mucus production in an asthma model.

Kun et al.,(39) reported that serum and BAL levels of TGF-β1 were significantly increased inchildren with asthma compared to controls.

Concerning the effects of asthma exacerba-tion our results revealed that the serum level of TGF-β1 was significantly higher in patients withuncontrolled asthma than in patients with con-trolled asthma. Parallel with our results Balzar et al.,(2) reported that the serum level of TGF-β1 was significantly higher in the uncontrolled pa-tients than in the controlled patients.Ozyilmaz et al., (46) reported that there was positive correlation between uncontrolled asthma pulmonary func-tion tests and plasma TGF-β-1 levels so plasma TGF-β1 level may be considered as a systemic marker of asthma control.

On the other hand asthmatic patients with de-creased expression of TGF-β1 had better condi-tion as well as longer disease free period than other ones with increased expression of TGF-β1(24).

Regarding the effects of asthma severity on TGF-β1 levels our results revealed that its serum level was significantly increased with increasingdegree of asthma severity.

In agreement with our results Kun et al., (39)

reported that serum TGF-β1 levels were signifi-cantly increased in patients with moderate and severe asthma compared to mild asthma. Also, Boxall et al., Todorova et al., Brown et al.,(13,55,14) reported that increased TGF-β1 levels in the airways of asthmatic patients correlate with the severity of asthma and with the thickness of sub-epithelial basement membrane.

To study the effect of asthma duration on TGF-β1 level our study revealed that it was sig-nificantly higher in cases with disease duration> 5 years than in cases with disease duration < 5 years. Chung and Kimb (19) found that plasma level of TGF-β1 in children with persistent asth-ma was significantly higher than in the childrenwho were diagnosed with asthma for the firsttime and controls.

The secretion of TGF-β1 after an allergic disorder takes part in fibrosis and the irrevers-ible changes associated with airway remodeling in chronic asthma. Furthermore, it has been re-ported that elevated level of plasma TGF-β1 was a predictor of lung fibrosis (40).

With regard to the correlations between the studied parameters our results revealed that there were significant positive correlations be-tween serum levels of MMP-9, TIMP-1, MMP-9/TIMP-1 ratio, and TGF-β1 with each other in the patients group.

Loops of reciprocal control exist between TGF-β and MMPs since MMP-9 activates latent TGF-β, this latter, once activated acts directly on MMP-9 expression and plays a pivotal role in-creasing its production and secretion in a mouse model of remodeling (44,51). On contrary Kucich et al., (38) found that TGF-β depresses the syn-thesis of matrix-degrading proteases and at the same time up-regulates the production of pro-tease inhibitors, in particular tissue inhibitor of metalloproteinase-1 (TIMP-1).

Also Tomkowicz et al.,(56) reported that col-lagen I, III and tissue inhibitor of matrix metal-loproteinase 1 (TIMP-1), both of which were increased in asthma, were strongly induced by TGF-β. Hetzel et al.,(29) reported that TIMP-1 levels in BAL fluid were correlated stronglywith TGF-β1 level.

MMP-9, TIMP- 1& TGF- β1 in Bronchial Asthma 75The present study involved the performance

of some pulmonary function tests includes FVC%, FEV1% and FEV1/FVC %.

Pulmonary function tests were significantlylower in patients with uncontrolled asthma than in patients with controlled asthma, this is in agreement with GINA 2014(25)..

Also, these parameters were significantlylower in patients with asthma duration more than 5 years than in patients with duration less than 5 years. Rivera et al.,(49) showed a close relation-ship between the duration of disease and loss of lung function, supporting the concept of asthma as a slow, progressive disease at least among those patients with a mild to moderate degree of airflow limitation.

To study the correlation between performed pulmonary functions and the studied parameters our results revealed that there were significantnegative correlations between MMP-9,TIMP-1, MMP-9/TIMP-1 ratio and TGF-β1 with pulmo-nary function tests FVC % , FEV1 % and FEV1/FVC %.

In consistent with our results Mohamed et al.,(43) reported significant negative correlationbetween serum MMP-9 levels and both the max-imum percent fall in forced expiratory volume in first second (FEV1%) during the late response.Also Cataldo et al., Wenzel et al., (17, 59) showed that elevated sputum MMP-9 level was associ-ated with a fall in FEV1 after allergen challenge and was linked to asthma severity. Sputum levels of MMP-9 measured after bronchial challenge with common allergens were significantly posi-tively correlated with the maximal fall in FEV-1 observed during the asthmatic reaction (15).

Barbaro et al., (3) reported positive correla-tion between MMP-9 level in exhaled breath and the percentage fall of FEV1 in severe asth-matics. Higashimoto et al., (30) reported that the serum TIMP-1 concentration was negatively correlated with the FEV1/FVC % in asthmatic patients. Moreover Pham et al.,(48) showed that BAL MMP-9 levels positively correlated with the overnight fall in FEV1 and MMP-9/TIMP-1 ratio negatively correlated with the FEV1%. However Benayoun et al., (6) reported that FEV1

was positively correlated with the serum MMP-9/TIMP-1 ratio, but weakly positively correlated with the MMP-9 level.

Conclusion

Serum levels of MMP-9, TIMP-1, MMP-9/TIMP-1ratio and TGF-β1 are increased in asth-matic patients and are associated with increased asthma severity, asthma exacerbation and pro-longed disease duration and negatively correlate with pulmonary function tests, so these param-eters could be used as prognostic tools for fol-low up of asthmatic patients and as non invasive markers of airway remodeling that can bypass biopsy sampling. Also serum sample is easily handled and not subjected to technical error as sputum or bronchoalveolar lavage fluid.

REFERENCES1. Al-Muhsen S M, Johnson J R, and Hamid Q. 2011 Re-

modeling in asthma. J Allergy and Clin Immunol;128(3): 451.

2. Balzar S, Wei Chu H, Silkoff P, et al, 2005 Increased TGF-b2 in severe asthma with eosinophilia. J Allergy and Clin Immunol;115: 110.

3. Barbaro M P F, Spanevello A, Palladino GP, et al, 2014 Exhaled matrix metalloproteinase-9 (MMP-9) in differ-ent biological phenotypes of asthma. European Journal of Internal Medicine ;25: 92.

4. Barrios RJ, Kheradmand F, Batts L, et al, 2014: pa-thology and pathophysiology. Arch Pathol Lab Med 2006;130: 447.

5. Belleguic C, Corbel M, Germain N, et al, 2002 In-creased release of matrix metalloproteinase-9 in the plasma of acute severe asthmatic patients. Clin Exp Al-lergy ; 32: 217.

6. Benayoun L, Druilhe A, Dombret MC, et al, 2003 Air-way structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med ;167: 1360.

7. Benditt J. A. 2008 Primer on Reading, Pulmonary Function Tests. Clinical Respiratory Diseases ;35: 213.

8. Bergeron C and Boulet L. 2006 Structural changes in airway diseases characteristics, mechanisms, conse-quences, and pharmacologic modulation. Chest ;129 (4): 1068.

9. Bergeron C, Tulic MK, Hamid Q. 2007 Tools used to measure airway remodeling in research. Eur Respir J ;29: 596.

10. Bergeron C, Tulic M, and Hamid Q. 2010 Airway re-modeling in asthma: From bench side to clinical prac-tice .Can Respir J ;17(4): e85.

11. Boulay ME, Prince P, Deschesnes F, et al, 2004 Me-

http://chestjournal.chestpubs.org/search?author1=C%C3%A9line+Bergeron&sortspec=date&submit=Submit

http://chestjournal.chestpubs.org/search?author1=Louis-Philippe+Boulet&sortspec=date&submit=Submit

Hassan, N. A., et al.76talloproteinase-9 in induced sputum correlates with the severity of the late allergen-induced asthmatic response. Respiration ;71(3): 216.

12. Bousquet J, Jeffery PK, Busse WW, et al, 2000 : From bronchoconstriction to airways inflammation and re-modeling. Am J Respir Crit Care Med ;161: 1720.

13. Boxall C, Holgate ST, Davies DE. 2006 The contribu-tion of transforming growth factor-beta and epidermal growth factor signaling to airway remodeling in chronic asthma. Eur Respir J ;27: 208.

14. Brown S D, Baxter KM, Stephenson ST, et al, 2012 Airway TGFβ1 and oxidant stress in children with se-vere asthma. Association with airflow limitation. J Al-lergy Clin Immunol 2012;129(2):

15. Castano R, Miedinger D, Maghni K, et al, 2013 Ma-trix metalloproteinase-9 increases in the sputum from allergic occupational asthma patients after specific in-halation challenge. Int Arch Allergy Immunol. ;160(2): 161.

16. Cataldo D, Munaut C, Noel A, et al, 2000 MMP-2- and MMP-9-linked gelatinolytic activity in the sputum from patients with asthma and chronic obstructive pul-monary disease. Int Arch Allergy Immunol ;123: 259.

17. Cataldo DD, Bettiol J, Noel A, et al, 2002. Matrix me-talloproteinase-9, but not tissue inhibitor of matrix me-talloproteinase-1 increase in the sputum from allergic asthmatic patients after allergen challenge. Chest ;122: 1553.

18. Chaudhuri R, McSharry C, Brady J Donnelly I, et al, 2012 Sputum matrix metalloproteinase-12 in patients with chronic obstructive pulmonary disease and asth-ma: Relationship to disease severity. The Journal of Al-lergy and Clinical Immunology;129(3): 655.

19. Chung H and Kimb S. 2004 Increased release of ma-trix metalloproteinase-1 and transforming growth fac-tor-β1 in the plasma of children with persistent asthma. Journal of Allergy and Clinical Immunology ;113(2): Supplement, S195.

20. Dar KA, Shahid M, Mubeen A, et al, 2012 The role of non invasive methods in assessing airway inflammationand structural changes in asthma and COPD. Monaldi Arch Chest Dis. ;77(1): 8.

21. Doğu F, Yildiran A, Loğlu D, et al,.2008 Serum Trans-forming Growth Factor-β1 (TGβ1), MatrixMetallopro-teinase-2), MatrixMetalloproteinase-9 (MMP-9) and Tissue Inhibitors of Metalloproteinase (TIMP-1) levels in Childhood Asthma. Turk J Med Sci. ;38(5): 415.

22. Farhat A A, Mohamad A S, Shareef M M, et al, 2014 Asthma remodeling the pathogenic role of matrix metal-loroteirase-9. Egyptians Journal of Chest Diseases and tuberculosis ;63,issue 4: 755

23. Felsen C N, Savariar EN, 2014 Whitney M and Tsien RY. Detection and monitoring of localized matrix me-

talloproteinase upregulation in a murine model of asth-ma. Am J Physiol Lung Cell Mol Physiol. ; 306: L764.

24. Ghanei M, Ghalejooghi NA, Nourani MR, et al, 2010 Effect of TGFβ1 and TIMP2 on Disease Activity in Asthma and COPD. Iran J Allergy Asthma Immunol. ; 9(2): 79.

25. GINA 2014. Global Strategy for Asthma Management and Prevention 2014 (Update).

26. Greenlee K J, Werb Z and Kheradmand F. 2007 Matrix Metalloproteinases in Lung: Multiple, Multifarious, and Multifaceted. Physiol Rev. ;87(1): 69.

27. Gueders M M, Foidart J, Noel A, Cataldo D. 2006 Ma-trix metalloproteinases (MMPs) and tissue inhibitors of MMPs in the respiratory tract: Potential implications in asthma and other lung diseases. European Journal of Pharmacology ;533(1): 133.

28. Gujin C. 2011 Clinical significance of determinationthe changes of serum CGRP, MMP-9 and T1MP-1 lev-el both before and after treatment in pediatric patients with bronchial asthma. Journal of radioimmunology;24(6)Dec. 621.

29. Hetzel M, Walcher D, Grub M, et al, 2003 Inhibition of MMP-9 expression by PPAR gamma activators in human bronchial epithelial cells. Thorax ;58: 778.

30. Higashimoto Y, Yamagata Y, Iwata T, et al, 2005 In-creased serum concentrations of tissue inhibitor of me-talloproteinase-1 in COPD patients. ERJ ;25(5): 885.

31. Hong Z, Lin YM, Qin X, et al, 2012 Serum MMP-9 is elevated in children with asthma. Mol Med Rep. ;5(2): 462.

32. Joseph J, Benedict S, Badrinath P, et al, 2003 Elevation of plasma transforming growth factor beta1 levels in stable nonatopic asthma. Ann Allergy Asthma Immu-nol. ;91(5): 472.

33. Karakoc GB, Yukselen A, Yilmaz M, et al, 2012 Ex-haled breath condensate MMP-9 level and its relation-ship with asthma severity and interleukin 4/10 levels in children. Ann Allergy Asthma Immunol. ;108(5): 300.

34. Kelly E and Jarjour N. 2003 Role of matrix metallopro-teinases in asthma. Current ,9(1): 28.

35. Kelly, EA, Busse, WW, Jarjour, NN. et al, 2000 In-creased matrix metallopro-teinase-9 in the airway after allergen challenge. Am. J. Respir. Crit. Care Med. ;162: 1157.

36. Kim JS, Kang JY, Ha JH, et al, 2013 Expression of nerve growth factor and matrix metallopeptidase-9/tis-sue inhibitor of metalloproteinase-1 in asthmatic pa-tients. J Asthma ;50 (7): 712.

37. Koloze MT, Anderson J, Bonfield L et al, 2010 TheRole Of matrix metalloproteinases (MMPs) and their proteolytic degradation of chemokines in the lung sub-mitted in partial fulfillment of the requirements for the

http://www.ncbi.nlm.nih.gov/pubmed/?term=Brown%20SD%5Bauth%5D

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http://www.ncbi.nlm.nih.gov/pubmed/?term=Stephenson%20ST%5Bauth%5D

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http://www.ncbi.nlm.nih.gov/pubmed?term=Castano%20R%5BAuthor%5D&cauthor=true&cauthor_uid=23018543

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http://www.ncbi.nlm.nih.gov/pubmed?term=%22Badrinath%20P%22%5BAuthor%5D



http://www.ncbi.nlm.nih.gov/pubmed?term=Karakoc%20GB%5BAuthor%5D&cauthor=true&cauthor_uid=22541398

http://www.ncbi.nlm.nih.gov/pubmed?term=Yukselen%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22541398

http://www.ncbi.nlm.nih.gov/pubmed?term=Yilmaz%20M%5BAuthor%5D&cauthor=true&cauthor_uid=22541398


http://www.ncbi.nlm.nih.gov/pubmed?term=Kim%20JS%5BAuthor%5D&cauthor=true&cauthor_uid=23713676

http://www.ncbi.nlm.nih.gov/pubmed?term=Kang%20JY%5BAuthor%5D&cauthor=true&cauthor_uid=23713676

http://www.ncbi.nlm.nih.gov/pubmed?term=Ha%20JH%5BAuthor%5D&cauthor=true&cauthor_uid=23713676


MMP-9, TIMP- 1& TGF- β1 in Bronchial Asthma 77degree of Master of Science department of pathology. Case Western Reserve University May, .

38. Kucich U, Rosenbloom JC, Herrick DJ, et al, 2001 Signaling events required for transforming growth fac-tor-beta stimulation of connective tissue growth factor expression by cultured human lung fibroblasts. ArchBiochem Biophys ;395: 103.

39. Kun J, He-Bin C, Ying W, et al, 2013 Changes in IL17 and TGF-β1 levels in serum and bronchoalevolarlavage fluid and their clinical significance among children withasthma. CJCP ;15(8): 604.

40. Manuyakorn W, Kamchaisatian W, Atamasirikul K, et al, 2008 Serum TGF-β1 in Atopic Asthma. Asian Pa-cific Journal of Allergy and Immunology ;26: 185.

41. Matsumoto H, Niimi A, Takemura M, et al. 2005: Re-lationship of airway wall thickening to an imbalance between matrix metalloproteinase-9 and its inhibitor in asthma. Thorax ;60(4): 277.

42. Mattos W , Sam Lim, Russell R, et al. 2002 Matrix metalloproteinase-9 expression in asthma effect of asthma severity, allergen challenge, and inhaled corti-costeroids. Chest ;122(5): 1543.

43. Mohamed G M, Mohamed Farres N, Mahmoud H. 2012 Interplay between matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in acute asthma exacerbation and airway remodeling. Egyptian Journal of Chest Diseases and Tuberculosis ;61(3): 35.

44. Ohbayashi H. Matrix 2002 metalloproteinases in lung diseases. Curr. Protein Pept. Sci. ;3: 409.

45. Oshita Y, Koga T, Kamimura T, et al (2003) Increased circulating 92 kDa matrix metalloproteinase (MMP-9) activity in exacerbations of asthma. Thorax ; 58: 757.

46. Ozyilmaz E, Canbakan S, Capan N, et al. 2009 Corre-lation of plasma transforming growth factor beta 1 with asthma control test. Allergy Asthma Proc. ;30(1): 35.

47. Panek M, Pietras T, Szemraj J, et al. 2013 Identificationand association of TGFβ-1 expression in patients with asthma in a Polish population - Lodz metropolitan area study. Int J Biochem Mol Biol. ;4(1): 67.

48. Pham D N, Hong Wei Chu, Richard J. Martin, 2003 Monica Kraft. Increased matrix metalloproteinase-9 with elastolysis in nocturnal asthma. Annals of Allergy, Asthma & Immunology ;90(1): 72.

49. Rivera JM, Alvarez-Puebla MJ, 2007 Puy Uribe San Martín M, Tallens Armand ML. Duration of Asthma and Lung Function in Life-Long Nonsmoking Adults. J Investig Allergol Clin Immunol ; 17(4): 236.

50. Rodriguez B T, Ortega E, Segura-Medina P, et al.

2014 TGF-β: An important mediator of allergic disease and a molecule with dual activity in cancer develop-ment. Journal of Immunology Research ;: 1.

51. Santibanez JF, Guerrero J, Quintanilla M, et al 2002 Transforming growth factor-beta1 modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in transformed keratinocytes. Bio-chem Biophys. Res. Commun. ;296: 267.

52. Simpson JL, Scott RJ, Boyle MJ, et al, 2005 Differ-ential proteolytic enzyme activity in eosinophilic and neutrophilic asthma. American Journal of Respiratory and Critical Care Medicine ;172: 559.

53. Tagaya E and Tamaoki J. 2007 Mechanisms of airway remodeling in asthma. Allergology International ;56: 331.

54. Tanaka H, Miyazaki N, Oashi K, et al, 2000 Sputum matrix metalloproteinase-9/tissue inhibitor of metallo-proteinase-1 ratio in acute asthma. J Allergy Clin Im-munol ;105: 900.

55. Todorova L, Bjermer L, Westergren-Thorsson G, Lars-son AM. et al, 2011 TGFβ induced matrix production by bronchial fibroblasts in asthma: Budesonide and for-moterol effects. Respiratory Medicine ;105: 1296.

56. Tomkowicz A, Kraus-Filarska M, Bar J, et al, 2008 Bronchial hyper-responsiveness, subepithelial fibro-sis, and transforming growth factor-β1 expression in patients with long-standing and recently diagnosed asthma. Arch Immunol Ther Exp (Warsz) ; 56(6): 401.

57. Ventura I, Vega A, Chacón P, et al, 2014 Neutrophils from allergic asthma�c pa�ents produce and re-lease metalloproteinase-9 upon direct exposure to allergens. Allergy ;69(7): 898-905.

58. Vignola A M, Mirabella F, Costanzo G,et al.2003; Airway Remodeling in Asthma. Chest 123(3) suppl. 417S .

59. Wenzel SE, Balzar S, Cundall M, et al, 2003 Subepithe-lial basement membrane immunoreactivity for matrix metalloproteinase-9: association with asthma severity, neutrophilic inflammation, and wound repair. J AllergyClin Immunol ;111: 1345.

60. Yang Y C, Zhang N, Van Crombruggen K, et al, 2012 Transforming growth factor-beta1 in inflammatory air-way disease: a key for understanding inflammation andremodeling. Allergy ;67: 1193.

61. Zhang YY, HZ Xu, Bao XE, et al, 2014 Detection and Clinical Significance of a Potential Mediator of AirwayRemodeling in Preschool Wheezy Children. HK J Pedi-atr ;19: 63.

http://chestjournal.chestpubs.org/search?author1=Waldo+Mattos&sortspec=date&submit=Submit

http://chestjournal.chestpubs.org/search?author1=Sam+Lim&sortspec=date&submit=Submit

http://chestjournal.chestpubs.org/search?author1=Richard+Russell&sortspec=date&submit=Submit

http://www.sciencedirect.com/science/article/pii/S0422763812000210





http://www.sciencedirect.com/science/journal/04227638/61/3

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ozyilmaz%20E%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Canbakan%20S%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Capan%20N%22%5BAuthor%5D


http://www.ncbi.nlm.nih.gov/pubmed/?term=Panek%20M%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Pietras%20T%5Bauth%5D

http://www.ncbi.nlm.nih.gov/pubmed/?term=Szemraj%20J%5Bauth%5D



http://www.sciencedirect.com/science?_ob=PublicationURL&_hubEid=1-s2.0-S1081120610X61981&_cid=279877&_pubType=JL&view=c&_auth=y&_acct=C000228598&_version=1&_urlVersion=0&_userid=10&md5=aec212fa1f09676b5ec7fc8960829aca

http://www.hindawi.com/15641538/







http://chestjournal.chestpubs.org/search?author1=Antonio+M.+Vignola&sortspec=date&submit=Submit

http://chestjournal.chestpubs.org/search?author1=Franco+Mirabella&sortspec=date&submit=Submit

http://chestjournal.chestpubs.org/search?author1=Giorgio+Costanzo&sortspec=date&submit=Submit


المعدني1- اإلنزيمي للبروتين النسيجي والمثبط للقالب9- المعدني اإلنزيمي البروتين ,1 المتحول-بيتا النمو عامل مصلالشعبي الربو فى

تمام منال محمد المنعم محمد - عبد - أمنية فاروق محمد - إبتسام ربه محمد عبد علياء - عبد المنعم كامل نيفين

ضابطة. كمجموعه أصحاء شخصا وعشرون الشعبي بالربو مريضا وستون ثماني شخص، وثمانون ثماني على الدراسة اشتملتشديد) متوسط, (خفيف, إلى المرض شدة حسب تقسيمهم وتم للربو الشعبي العالمية المبادرة حسب الشعبي بالربو المرضي تشخيص تمأكثر لفترة من المرض مرضى يعانون المرض إلى حسب فترة مستقر) وأيضا و غير إلى (مستقر استقرار المرض درجة وحسبمن من أي يعاني الدراسة هذه من أي مريض واستثنى سنوات. خمس من أقل المرض لفترة من يعانون ومرضى خمس سنوات منوالسرطان، الذاتية، المناعة وأمراض الحاد، الشريان التاجي وأمراض الجراحة، النقاهة من فترة أو األنسجة إصابات اآلتية: األمراضفي بالحقن كورتيزون أدوية قد تلقوا أي المرضى يكون ال وأن والتدخين. المزمنة، والحمل الكبد وأمراض المزمن، الكلوى والفشلعمل وتم الدراسة هذه الضابطة في المجموعة و الحاالت لجميع السريرى والفحص المرضى أخذ التاريخ الثالثة األخيرة. وتم األشهرهواء حجم ونسبة األولى الثانية في الزفير القسري هواء حجم القسرية، الحيوية القدرة الرئة ( وظائف و الصدر، السينية علي األشعةومستوى كاملة، دم صورة التالية التحليالت المعملية أجريت فقط. للمرضى ( القسرية الحيوية القدرة األولى / الثانية في القسري الزفيرالمعدني اإلنزيم المتحول–بيتا1, النمو عامل ومستوى مصل وظائف الكلى الكبد، واختبارات واختبارات وظائف الدم، في الجلوكوزعالية ٳحصائية داللة ذات زيادة النتائج وأظهرت القالب 1-. لبروتين المحلل المعدني لإلنزيم النسيجي والمثبط -9 القالب لبروتين المحللالمحلل لبروتين المعدني لإلنزيم النسيجي والمثبط -9 القالب لبروتين المحلل المعدني اإلنزيم ,1 بيتا – النمو المتحول عامل لمصلالربو مرضى مجموعة عن المستقر الغير الربو مجموعة مرضى وأيضا في الحاكمة المجموعة عن الشعبي الربو مرضي 1-في القالبالمرضى الذين يعانون وأيضا في المتوسط أو الخفيف الربو مرضي مجموعه عن الشديد الربو مجموعة مرضى المستقر وكذلك فيذو نقص النتائج سنوات. وأظهرت خمس من أقل لفترة المرض يعانون من الذين المرضى عن سنوات من خمس أكثر لفترة المرض منالزفير هواء حجم نسبة و األولى الثانية في القسري الزفير القسرية، حجم هواء الحيوية (القدرة الرئة في وظائف عالية ٳحصائية داللةفي و المستقر الربو مجموعة مرضى الغير المستقر عن مرضى الربو في مجموعة القدرة الحيوية القسرية) األولى / الثانية في القسريكما سنوات. خمس من من المرض لفترة أقل يعانون الذين المرضى عن سنوات خمس من من المرض لفترة أكثر يعانون الذين المرضىاإلنزيم بيتا1, – المتحول النمو عامل مصل مستوي كل من بين عالية ٳحصائية داللة ذات ايجابية ارتباطات البحث هذا أظهرت نتائجذات ارتباطات كذلك المرضى. كل القالب 1- في لبروتين المحلل المعدني لإلنزيم النسيجي والمثبط -9 لبروتين القالب المعدني المحللالقالب 9- والمثبط النسيجي لبروتين المحلل المعدني ,اإلنزيم 1 – بيتا المتحول النمو عامل مصل مستوى بين عالية سلبية داللة ٳحصائيةونسبة األولى الثانية في القسري الزفير حجم هواء القسرية، الحيوية (القدرة الرئة وظائف و -1 لإلنزيم المعدني المحلل لبروتين القالب

.( القسرية الحيوية / القدرة األولى في الثانية القسري الزفير هواء حجم

THE EFFECT OF UGT1A6 POLYMORPHIC GENETIC VARIANTS ON THE CLINICAL RESPONSE TO VALPROIC ACID IN EGYPTIAN

CHILDREN WITH IDIOPATHIC EPILEPSYMohamod el-Sawy*, Osama Ahmed Badary** Manal Zaghloul Mahran*** ,

Sahar Mohamed Ahmed Hassanein* , Manal El Hamamsy**,

Nesrine Aly Mohamed*** and Engi Abdel-Hady** ABSTRACT

Introduction: Valproic acid (VPA) is one of the most common prescribed drugs for the treatment of epilepsy. Interindivid-ual variability in VPA dose and plasma concentration may reflect functional consequences of genetic polymorphisms in genesencoding drug metabolizing enzymes. VPA is mainly eliminated through conjugation by UDP-glucuronosyltransferases (UGT) which are known to be polymorphic. Aim of the study: To investigate the genetic polymorphisms of UGT1A6 at 541A>G and 552A>C loci in a cohort of Egyptian children with idiopathic epilepsy and their potential influence on both VPA serum leveland clinical response in epilepsy. Methods: The genetic polymorphisms of UGT1A6 at 541A>G and 552A>C loci in 48 epilep-tic patients receiving VPA monotherapy were detected by PCR-restriction fragment length polymorphism (PCR-RFLP). Steady state concentrations at trough level of VPA were determined by homogenous enzyme immunoassay technique. All patients were monitored for seizure frequency and seizure severity (SS) using Chalfont SS scale as well as for adverse drug reactions (ADRs). Results: Patients of variant genotype group (AC & CC) had lower concentration dose ratios (CDRs) than those with wild genotype (AA) for UGT1A6 552A>C (p=0.029). Variant allele carriers had significantly lower CDR than wild allele car-riers for both UGT1A6 541A>G and 552A>C (p= 0.047 and p= 0.001, respectively). Higher SS scores on Chalfont scale were associated with (AA) genotype of UGT1A6 552A>C than variant genotypes group (AC & CC) (p=0.020). No significant effectwas detected on seizure control, while fatigue and cognitive adverse effects were significantly higher in wild genotype groupof UGT1A6 552A>C (AA genotype) and variant genotype group of UGT1A6 541 A>G (GG&AG genotypes), respectively. Conclusion: Although there was a limited sample size, the study demonstrated that UGT1A6 polymorphisms may increase VPA metabolism in Egyptian epileptic children affecting both seizure severity as well as susceptibility to ADRs. Keywords: antiepileptic drugs,adverse drug reactions, pharmacogenetics, glucuronidation,serum level.

Departments of Pediatrics*, Faculty of Medicine, Clinical Pharmacy**, Faculty of Pharmacy, Clinical Pathology & Immunology***, Faculty of Medicine, Ain Shams University, Cairo, Egypt.

INTRODUCTION

Valproic acid (VPA) is one of the major an-tiepileptic drugs possessing a broad spectrum of efficacy that encompasses generalized and focalseizure in both adults and children(21).However, therapy with VPA is clinically complicated by high interindividual variability in both pharma-cokinetics and pharmacodynamics giving rise to a wide dosing range and therapeutic plasma level of 50-100 mg/l which necessitates monitoring its plasma level during therapy(10,18). It is estimated that pharmacogenomics is implicated in up to 95 % of variability in drug disposition and effect(19). The principal pathways of VPA elimination in the liver are glucuronidation and β-oxidation. The former reaches up to 50% of the total metab-olism of the initial dose (2). Invitro studies of hu-man liver microsomes and purified recombinantproteins have identified a number of UGT iso-zymes including UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 involved in VPA glucuronidation(2,9).

Among these iso¬zymes, UGT2B7, UGT1A6 and UGT1A9 count for the majority of VPA in-trinsic hepatic clearance(2). UGT1A6 gene was demonstrated to be highly polymorphic and char-acterized by three single nucleotide polymor-phisms (SNPs).These include polymorphisms rs2070959 (541A>G) and rs1105879 (552A>C), rs6759892 (19T>G) in the coding sequence which can lead to both transcriptional and func-tional changes of its encoded enzymes (6, 23). Two missense mutations in exon 1 of UGT1A6 have been identified;they result in threonine to ala-nine change at amino acid 181(an A to G transi-tion at nucleotide 541) and an arginine to serine change at amino acid 184 (an A to C transver-sion at nucleotide 552)(6).Previous studies con-ducted in vitro indicated that UGT1A6 poly-morphism influences the glucuronidation rateof its substrate compounds(6,20). To date, there have been relatively few clinical studies on the effect of UGT1A6 gene polymorphisms on the levels of VPA in epileptic patients(22,29). All of


El-Sawy, M. et al.80

them were carried out in Asian populations of different ethnic backgrounds, while no studies in Caucasians have been carried out so far. The results reported by those studies were not consis-tent. For example, two Chinese studies reported no association between blood level of VPA and patient genotypes at either rs2070959 (541A>G) or at both rs2070959 (541A>G) and rs1105879 (552A>C)(5,31).Children were the target popula-tion in only two of those studies(14,31). None of the aforementioned studies investigated the in-fluence of such genetic polymorphism on drugresponse and treatment outcome. To that end, the current study was sought in an attempt to comprehensively evaluate the effect of UGT1A6 variants at 541A>G and 552A>C sites, on the re-sponsiveness to VPA regarding both serum level and clinical outcome in a cohort of epileptic chil-dren in Egypt.

MATERIALS AND METHODS

• Patients, sampling and drug analysis

This study was conducted prospectively be-tween April 2010 - September 2011 at Pediat-ric Neurology Clinic, Children’s Hospital, Ain-ShamsUniversity, Cairo, Egypt. The study pro-tocol was approved by the Hospital Ethical Med-ical Committee and signed informed consents were obtained prior to enrollment. A total of 50 randomly selected, consecutive patients were en-rolled, but only 48 of them completed the study. Eligible patients were children (age, 2-18 years) diagnosed as idiopathic epilepsy according to International League Against Epilepsy (ILAE) criteria(18) and received VPA monotherapy. Ex-clusion criteria were: (1) history or evidence of hepatitis; (2) mental retardation; (3) unreliable record of seizure frequency; (4) documented noncompliance to antiepileptic drugs (AED); (5) receive concurrent medication known to either induce or inhibit VPA metabolism; and (6) sus-pected presence of neuro-degenerative, neuro-metabolic or neuro-cutaneous syndromes.

Relevant data including age, gender, physical assessment, EEG, biochemical laboratory results, seizure history, and drug dosage were recorded. VPA was started at an initial dose of 5 to 7.5 mg/kg/day with an increase of 5 – 7.5 mg/kg/day ev-ery 3 – 7 days up to a final dosage of 30 mg/kg/

day(19). The BID (twice a day) or TID (three times a day) schedules were standardized to a regimen of every 12 and 8 hours, respectively. The dos-ing regimen was maintained stably for at least 2weeks (>5 half-lives) to ensure that the blood sampling was performed at the steady-state of VPA pharmacokinetics. Venous blood samples (5 mL) for analysis were collected immediately before the morning dose for measuring trough VPA level, when a stable dose at maintenance has been reached. Blood samples were collected into 2 sterile tubes; the first was allowed to clotat room temperature, centrifuged at 3000 rpm for 10 min then stored as serum at -20ºC for drug assay. The second tube (with EDTA) was frozen immediately at -70 ºC for DNA extraction and genotyping.

-Measurement of valproic acid serum levels

Drug levels were measured by homogenous enzyme immunoassay technique (Cobas®c 311, Roche diagnostics, USA) according to the man-ufacturer’s instructions. The assay was validated for biosample analysis in a linear range of 2.8 – 150 µg/ml. The assay is a homogeneous im-munoassay based on the principle of measuring changes in scattered light or absorbance which result when activated microparticles aggregate. The microparticles are coated with valproic acid and rapidly aggregate in the presence of a val-proic acid antibody solution. When a sample containing valproic acid is introduced, the ag-gregation reaction is partially inhibited, slowing the rate of the aggregation process. Antibody bound to sample drug is no longer available to promote microparticle aggregation, and subse-quent particle lattice formulation is inhibited. Thus, a classic inhibition curve with respect to valproic acid concentration is obtained, with the maximum rate of aggregation at the lowest valproic acid concentration. By monitoring the change in scattered light or absorbance, a con-centration-dependent curve is obtained.

-A concentration to dose ratio (CDR) was calculated for each patient by dividing steady state serum concentrations (Css) of VPA by the daily dose and body weight.

• Detection of genetic polymorphisms of UGT1A6 gene at 541A>G and 552A>C loci

UGT1A6 Genetic Variants and Clinical Response to VPA in Epilepsy 81byPCR-restriction fragment length polymor-phism (PCR-RFLP).

This was evaluated at the Clinical Pathology Department, Ain-Shams University Hospital as follows:

- DNA Extraction

It was done using the QIAamp DNA Mini Kit supplied by Qiagen( Hilden, Germany). DNA in the sample was liberated using protein-ase K solution and lysis buffer. Released DNA was bound exclusively and specifically to theQIAamp membrane in the presence of binding buffer under appropriate salt iron and pH condi-tions. Denatured protein and other contaminants were removed with several washing procedures. The DNA was then eluted from the membrane with elution buffer.

- Polymerase Chain Reaction

For detection of UGT1A6 541A>G and 552A>C polymorphisms whose amplified prod-uct is 992 bp fragment containing both poly-morphisms, amplification was performed usinga thermal cycler Gene Amp PCR system 9700 Applied biosystems(USA). PCR product were carried out in volume of 50 μl containing 5 μl genomic DNA, 25 μl of the ready to use master mix supplied by Qiagen(2x concentrate contains Taq DNA polymerase, PCR buffer with 3 mM MgCl2 and 400 μM of each dTNTP), 2.5 μl (25 pmol) of forward primer, 2.5 μl (25 pmol) of re-verse primer and 15 μl of deionized water. Prim-ers used were forward primer

(5′-GGAAAATACCTAGGAGCCCTGT-GA-3′) and a reverse primer (5′AGGAGCCAAATGAGTGAGGGAG-3′). The test primers were chosen according to van Oijen et al. (30)and pre-pared by Bioneer (USA). PCR conditions were an initial denaturation step at 94°C for 4 minutes and 32 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 65°C for 1minute, with extension at 72°C for 1 minute, then finalextension at 72°C for 6 minutes.

- Restriction Fragment Length Polymor-phism

a) Restriction digestion for UGT1A6 541A>G polymorphism.

Restriction digestion of the amplified productat site 541 was performed using 0.5 μl (10,000U/ml) NsiI restriction enzyme supplied by New England biolabs (Beverly, UK). Site of restric-tion: (5´ATGCG ↓T 3´) (3´ T↑GCGTA 5´). The enzyme was added to 5 μl of PCR amplifiedproduct, 18.5 μl deionized water and 1 μl of 10 x buffer, followed by gentle mix using pipette then centrifugation for few seconds. Then tubes were placed on a heat block for 1 hour at 37°C.The enzyme was inactivated at 80°C for 20 min. As experimental control: no-enzyme “mock” digest was used.

b) Restriction digestion for UGT1A6 552A>C polymorphism.

Restriction digestion of the amplified productat site 552 was performed using 0.5 μl (5,000U/ml) Fnu4HIrestriction enzyme supplied by New England biolabs (Beverly, UK). Site of restric-tion: (5´GC ↓NGC 3´) (3´ CGN ↑CG 5´). The enzyme was added to 5 μl of PCR amplifiedproduct, 18.5 μl deionized water and 1 μl of 10 x buffer, followed by gentle mix using pipette then centrifugation for few seconds. Then tubes were placed on a heat block for 1 hour at 37°C.The enzyme was inactivated at 65°C for 20 min.

- DNA analysis by gel electrophoresis

Amplified product of DNA samples and re-striction fragments were run on 2% agarose gel (Promega, USA) for 30 minutes at 100V, stained with ethidium bromide (Amresco, Germany). 100bp DNA ladder supplied by Fermentas (USA) was also run to identify the site of bands. The gel was examined under an ultraviolet trans-illuminator.

- Genotyptes of UGT1A6 541A>G and 552A>C polymorphisms were defined accordingto Van Oijen et al.(30).

a) For UGT1A6 541A>G polymorphism

- Homozygous AA genotype (homozygous for the common alleles-wild type) if the band was not divided: 992 bp.

- Homozygous GG genotype (homozygous for the variant alleles) if the band is divided into 2 parts 616 and 376 bp.

82- Heterozygous AG genotype if it yields 3

bands 992,616 and 376 bp.

b) For UGT1A6 552A>C polymorphism

- Homozygous AA genotype (homozygous for the common alleles-wild type) if it yields 2 bands 833 and 159 bp.

- Homozygous CC genotype (homozygous for the variant alleles) if it yields 3 bands 464,369 and 159 bp.

- Heterozygous AC genotype if it yields 4 bands 833,464,369 and 159 bp.

• Follow-up

Drug response involving both seizure control and adverse drug reactions (ADRs) was assessed every 1-2 months during therapy till a minimum of six months (for 6 month or till the end of the study, whichever is longer). On each follow up visit, the number of seizures in the preced-ing period was recorded, drug compliance was checked and the most frequent ADRs of VPA were recorded using a prepared checklist (17). An average number of seizures per month was calculated for 6 months of follow up. A good response was defined as having a seizure fre-quency of <2 month for six consecutive months (24). Seizure severity (SS) was rated using the Chalfont SS scale (8).Complete blood count and serum ALT were performed routinely, ev-ery 3-6 months. Additional laboratory tests were performed such as PT and PTT, blood bilirubin levels, blood ammonia, when a severe form of ADRs was suspected.

Statistical Analysis

Analysis of data was performed using the Statistical Package for Social Sciences version 17 (SPSS Inc, IL, USA). Data were expressed as mean±standard deviation (SD) for paramet-ric data, and as median and interquartile range (IQR) for non-parametric data, respectively. Non-parametric data were analyzed using the Mann–Whitney test to compare two independent groups. Comparisons of demographic character-istics including age, body weight, and gender among genotypic groups were done by Mann-Whitney test or Chi-square test. Association be-tween VPA dosages, Css, CDR, Chalfont SS rat-ing score, and number of ADR per patient, with

genetic polymorphisms was examined using Mann-Whitney test. The relationship between seizure control, occurrence of ADR and geno-typic groups was examined by the Chi-square test or the Fisher’s exact test for small sample size. All p-values are two-sided. A p value of less than 0.05 was considered significant.

RESULTS

A total of 48 pediatric patients completed the study. They comprised 20 males (41.7%) and 28 females (58.3%). They had a median age of 7 years (range, 2-18 years) and a median weight of 24.5 kg (range, 12-96 kg). The median duration of therapy with VPA was 2.6 years (range, 0.8-14years). All of the participants were from the Egyptian population. The mean VPA daily dose was 20.6 ± 7.1 mg/kg/day (range, 7-39.6 mg/kg/day). The maximum maintenance dose received averaged 22.2 ± 10 mg/kg/day (range, 7.7-39.6 mg/kg/day). Average Css was 59.96 ±27.18 µg/ml (range, 20-124 µg/ml). Characteristics of the study groups are displayed in Table 1.

The genotype frequencies of UGT1A6 541A>G and 552A>C are shown in table 2. The genotypic distributions were all consistent with Hardy–Weinberg equilibrium proportions.

The distribution of age, weight and gender was not significantly different among genotyp-ic groups (wild-type versus variant genotypes) for both SNPs (p>0.05). For 552A>C polymor-phism, the variant genotype group (AC& CC) had significantly lower VPA serum level (p=0.010) and CDR (p= 0.029) than wild-type group (AA genotype) (Table 3).

Upon performing the same analyses on polymorphic allele carriers, C allele carriers of 552A>C polymorphism had significantly lowerserum concentrations as well as lower CDRs than patients with A allele (p= 0.001), while for 541A>G polymorphism, G allele carriers had only significantly lower CDRs than A allele car-riers (p= 0.047) (Table 4).

Regarding seizure control, no significant as-sociation was revealed between wild-type and variant genotypic groups for both polymorphic loci (p>0.05). As for the Chalfont SS score, vari-ant genotypic group of 552A>C polymorphism

El-Sawy, M. et al.

83had significantly lower score than wild geno-typic group (p=0.02). It was also found that, C allele carriers had significantly lower score thanA allele carriers (p= 0.009)

Table 5 reveals that the variant genotype group (AG &GG) of 541A>G polymorphism

had significantly higher incidence of difficultyin concentration and/poor scholastic achieve-ment as an ADR than wild genotype group(AA) (p=0.047). On the other hand, variant genotype carriers (AC& CC)of 552A>C polymorphism had a significantly lower incidence of fatigue(p=0.025).

� �

Table 1. Demographic and clinical characteristics of epileptic children (n=48).

The genotype frequencies of UGT1A6 541A>G and 552A>C are shown in table 2. The genotypic distribu-tions were all consistent with Hardy–Weinberg equilibrium proportions.

Table 2.Genotype frequencies of UGT1A6 541A>G and 552A>C in epileptic patients of the study population.

UGT1A6 Genetic Variants and Clinical Response to VPA in Epilepsy

84

Table 3.Association of UGT1A6 541A>G and 552A>C genotypes with maximum valproic acid dosage, Css

and CDR, in epileptic children.

* Variant group’s median is significantly lower than wild-type group’s median.Css : steady state concentration of valproic acid; CDR: Concentration to dose ratio.

Table 4.Association of allelic groups of UGT1A6 541A>G and 552A>C polymorphisms with maximum val-proic acid dosage, Css and CDR, in epileptic children.

Css: steady state concentration of valproic acid; CDR: Concentration to dose ratio.

Table 5.Association of UGT1A6 541A>G and 552A>C genotypes with adverse drug reactions to valproic acid, in epileptic children.

*p=0.047 (Chi-square test). §p=0.025 (Fisher exact test). Data are represented as median (IQR)

El-Sawy, M. et al.

UGT1A6 Genetic Variants and Clinical Response to VPA in Epilepsy 85

Figure 1. Fragmentation patterns of PCR products for detection of genetic polymorphisms in UGT1A6. Electrophoresis patterns of PCR fragments after digesting with NsiI for the UGT1A6 A541G poly-morphism (A) and digesting with Fnu4HI for the UGT1A6 A552C polymorphism (B). M, 100 bp marker. Lanes 1, represents unrestricted fragment, 1 band (experimental control: no enzyme “mock” digest). Lanes 2, homozygosity for the common alleles; lanes 3, heterozygosity; lanes 4, ho-mozygosity for the variant alleles.

DISCUSSION

The present study demonstrated an associa-tion between polymorphisms at 541A>G and 552A>C loci of the drug metabolizing enzyme UGT1A6 gene and CDR of VPA as shown by the significantly lower CDR in variant allelecarriers compared to wild-type allele at both loci (p<0.05). Such findings may imply an in-creased rate of VPA glucuronidation in the vari-ant UGT1A6 carriers. These results are also concordant with those obtained from two for-mer studies in Chinese epileptic patients(15,29), while in disagreement with two other studies, performed by Wang et al. and Chu et al.(5,31).The former concluded no association between 541 A>G variants and adjusted serum level in epileptic children, while the latter reported no significant effect of the genotypes of eitherUGT1A6 552A>C or 541A>G polymorphisms on VPA serum concentration, despite the same population ethnicity in these studies. In the study by Chu et al. (5), only 3 patients had the GG and CC genotype (for 541A>G and 552A>C, respec-tively) out of 136 epileptic patient. This could have contributed to under representation of the homozygous variant forms; therefore, this lack of association they found could be attributed to type II error. Additionally, Jain et al(16). who studied UGT1A6 polymorphisms in a group of 80 epileptic children of Indian population, found

that UGT1A6 polymorphisms had no significanteffect on the valproate dose requirements, serum valproate concentrations, and standardized serum valproate concentrations. However, Aphichartp-hunkawee et al(1).observed that the homozygous variants of UGT1A6 541A>G and 552A>C had higher VPA serum level and required lower dose than heterozygous variants in a group of Thai population.

Population trough CDR of antiepileptic drugs have been suggested to be used as a clinical refer¬ence for prescribing the dose under a target concentration(3). Therefore, in the current study, adjusting patient’s VPA serum concentration to the dose received was considered more realistic at describing the variation of serum VPA level with different genotypes. In the current study, neither UGT1A6 552A>C nor 541A>G genetic variants was found to have a significant effect onmaximum dose required to achieve seizure con-trol, such finding is inconsistent with those re-ported by the study of Hung et al. in adult epilep-tic patients(15). According to the authors, variant genotype carriers for both 541A>G and 552A>C required significantly higher dosage than wild-type carriers. They defined VPA maintenancedose as the dosage that had not been changed for at least 1 year under good patient’s compliance and good seizure control. While in the current study, the maximum maintenance dose of VPA

86recorded for the patient was used in the compari-sons in the attempt of pursuing the contribution of genetic polymorphism to the long term dos-age requirements of patients, irrespective of the time of serum drug analysis. Another possible explanation for the inconsistency of results be-tween the current study and that of Hung et al(15)., is the effect of sample size and confounders such as differences in methodology, ethnicity, pheno-type definition such as drug-responsiveness andage of the study population. Sample size limita-tion is a common problem in association stud-ies, including many of the previously mentioned studies(14,16,29,31) , as well as the current study. All genotype-phenotype association studies of VPA pharmacokinetics were performed in Asian populations in Chinese ethnic groups especially Han Chinese. Genotype and allele distributions of UGT1A6 vary to a significant extent in dif-ferent racial populations, especially between Caucasians and Asians making results extrapo-lation of association studies from population to another rather unrealistic. Significant differencesfor genotype distribution ofUGT1A6 541 A>G and 552A>C were identified between our studypopulation and Chinese population based on data from a large Chinese study(32) (p<0.001 and p= 0.043, respectively). In contrast, genotype frequencies for both loci were comparable with a previous report for a Caucasian population(26).There is paucity of reports in Caucasians re-garding polymorphisms affecting VPA phar-macokinetics. Also, two of the previous studies were conducted in heterogeneous population re-garding patient age, where data from pediatric patients were combined with those from older individuals(5,29).

In the current study, no genotypic group was demonstrated to have a significant associationwith seizure control as expressed by the record-ed number of seizures. Although the number of patients having good seizure control tended to be higher in the wild type allele carriers than vari-ant allele carriers in both SNPs, the difference didn’t reach statistical significance (p>0.05). In-creasing the number of patients may have better revealed this difference. A previous study esti-mating pharmacokinetic parameters of VPA and their relationships to seizure control in epileptic

children found that patients in the uncontrolled-seizure group consumed a relatively higher VPA dosage than seizure-controlled group(25). This may indicate that relationship between dose and response is not consistent. In the current study, patients of variant genotype group (AC & CC) for 552A>C polymorphism showed collectively a significantly lower score for their seizures onChalfont SS scale than (AA) carriers (p=0.02). Although, not significantly different from eachother in seizure control assessment, the wild genotype group was shown previously to have higher CDRs (median 3.1, range, 1.4-6.9) than the former, which may be due to a rather non-consistent relationship between concentration and clinical response.

None of the genotype-phenotype association studies to date addressed the influence of geno-typic variation on clinical response to VPA per se. A study by Munisamy et al. (22) evaluated the effects of UGT1A6 (541A>G, 552A>C) on the pharmacokinetics of sodium valproate in epi-leptic patients showing toxicity to therapy and found significant associations with the allelicand genotype frequencies of UGT1A6 (541A>G, 552A>C) variants between toxicity and non-tox-icity groups of VPA in epileptic cases.

In the present study, variant (AG & GG) group had a higher incidence of difficulty inconcentration and poor school achievement, al-though shown previously to have no significant-ly different CDR than wild type. Despite cogni-tive adverse effects (CAEs) is linked with higher drug dosages, some studies report no relationship between CAEs and dose for valproate(13,27). Fur-ther, Brouwer et al.(4) found no correlation be-tween cognitive performance and VPA plasma levels suggesting that, drug levels measurement may not be regarded totally as good predictors of this subtle side effect. Also, a significantlyhigher percentage of patients was found having fatigue as an ADR within the wild type category (75%) compared to (25%) in the variant geno-type category for 552A>C polymorphism. This finding is in agreement with an earlier study byFortscher et al.(11) who reported significantlyhigher VPA levels in patients complaining of tiredness than patients without this side effect.

El-Sawy, M. et al.

UGT1A6 Genetic Variants and Clinical Response to VPA in Epilepsy 87It is worth noting that, patients within the wild genotype group for UGT1A6 552A>C polymor-phism had significantly higher CDRs (median3.1) than the variant genotype group (median 2.4). When collectively comparing the number of ADR experienced per patient between both UGT1A6 polymorphisms, neither 541A>G, nor 552A>C showed significant association be-tween the number of ADR per patient and their genotypes. Conversely, Jakovljevic et al. (17) ob-served a significant correlation between troughserum concentrations of VPA level and number of ADR per patient. The relationship between VPA concentration and effect has been unclear with both the clinical and toxic effects of the drug considered to be poorly correlated with to-tal serum concentrations(28). Genetic differences in the expression of drug metabolizing enzymes contribute to variability in drug exposure and re-sponse. Studying these genetic factors may help understand, and possibly predict, the relation-ship between the drug dose, the resulting drug concentrations and pharmacologic effect which can help guiding VPA personalized therapy in Egyptian epileptic children.

In conclusion, the too small sized of the in-vestigated groups is a shortcoming of this study. Nevertheless, the observed variations demon-strate that UGT1A6 polymorphisms at nucleo-tide positions 541A>G and 552A>C may be associated with increased VPA metabolism in Egyptian children with idiopathic epilepsy. Vari-ant allele carriers had significantly lower CDRswhen compared with wild type group in 541A>G and 552A>C polymorphism. Certain ADRs were significantly associated with certain genotypicgroups while seizures had significantly a higherscore of SS on Chalfont scale in wild genotyp-ic group than variant group regarding 552A>C polymorphism. It should be emphasized that no studies investigated the influence of such geneticpolymorphism on VPA drug response and treat-ment outcome in Egyptian epileptic children have been conducted to date.

Acknowledgments

The authors would like to Dr. InasElattar, professor of Biostatistics, for assisting in con-ducting the statistical analysis and interpretation of data.

REFERENCES1. Aphichartphunkawee S, Chinvarun2 Y, Kijsanayotin

P (2014): Association of genetic variants in UGT1A6 genes and non-genetic variant with valproic acid doses and plasma concentration in Thai epileptic patients. The Thai Journal of Pharmaceutical Sciences. ;38(2):98.

2. Argikar UA , Remmel RP (2009): Effect of aging on glucuronidation of valproic acid in human liver mi-crosomes and the role of UDP-glucuronosyltransfer-ase UGT1A4, UGT1A8, and UGT1A10. Drug Metab Dispos;1:229.

3. Blanca Sanchez M, Herranz JL, Leno C, et al: (2010).UGT2B7-161C>T polymorphism is associated with la-motrigine concentration-to-dose ratio in a multivariate study. Ther Drug Monit;2:177.

4. Brouwer OF, Pieters MS, Edelbroek PM, et al. (1992): Conventional and controlled release valproate in chil-dren with epilepsy: a cross-over study comparing plasma levels and cognitive performances. Epilepsy Res;3:245.

5. Chu XM, Zhang LF, Wang GJ, et al. (2012): Influenceof UDP-glucuronosyltransferase polymorphisms on valproic acid pharmacokinetics in Chinese epilepsy pa-tients. Eur J Clin Pharmacol;10:1395.

6. Ciotti M, Marrone A, Potter C, et al. (1997): Genetic polymorphism in the human UGT1A6 (planar phenol) UDP-glucuronosyltransferase: pharmacological impli-cations. Pharmacogenetics;6:485.

7. Commission on Classification and Terminology of theInternational League Against Epilepsy. Proposal for Revised Classification of Epilepsies and Epileptic Syn-dromes. Epilepsia 1989;4:389.

8. Duncan JS, Sander JW. (1991):The Chalfont Seizure Severity Scale. J Neurol Neurosurg Psychiatry;10:873.

9. Ethell BT, Anderson GD, Burchell B.( 2003):The effect of valproic acid on drug and steroid glucuronidation by expressed human UDP-glucuronosyltransferases. Bio-chem Pharmacol;9:1441.

10. Ferraro TN , Buono RJ .(2005): The relationship be-tween the pharmacology of antiepileptic drugs and human gene variation: an overview. Epilepsy Behav ;1:18.

11. Froscher W, Schulz HU, Gugler R. (1979): Antiepilep-tic therapy with valproinic acid. Correlations of side effects and serum levels of valproinic acid. Fortschr Med;34:1464.

12. Gerstner T, Bell N, Konig S.( 2008): Oral valproic acid for epilepsy-long-term experience in therapy and side effects. Expert Opin Pharmacother;2:285.

13. Gillham RA, Read CL, McKee PJW, et al. (1991): Cog-nitive function in adult epileptic patients on long-term sodium valproate. Journal of Epilepsy;4:205.

14. Guo Y, Hu C, He X, et al. (2012): Effects of UGT1A6, UGT2B7, and CYP2C9 genotypes on plasma concen-

88trations of valproic acid in Chinese children with epi-lepsy. Drug Metab Pharmacokinet;5:536.

15. Hung CC, Ho JL, Chang WL, et al. (2011): Association of genetic variants in six candidate genes with valproic acid therapy optimization. Pharmacogenomics;8:1107.

16. Jain P, Shastri S, Gulati S, et al. (2015): Prevelance of UGT1A6 polymorphisms in children with epilepsy on valproate monotherapy .Neurol India;663(1):35.

17. Jakovljevic MB, Jankovic SM, Jankovic SV, et al. (2008): Inverse correlation of valproic acid serum con-centrations and quality of life in adolescents with epi-lepsy. Epilepsy Res;2-3:180.

18. Jiang DC, Wang L. (2004): Population pharmacokinet-ic model of valproate and prediction of valproate serum concentrations in children with epilepsy. Acta Pharma-col Sin;12:1576.

19. Kalow W, Tang BK, Endrenyi L.(1998): Hypoth-esis: comparisons of inter- and intra-individual varia-tions can substitute for twin studies in drug research. Pharmacogenetics;4:283.

20. Krishnaswamy S, Hao Q, Al-Rohaimi A et al. (2005): UDP glucuronosyltransferase (UGT) 1A6 pharmaco-genetics: II. Functional impact of the three most com-mon nonsynonymous UGT1A6 polymorphisms (S7A, T181A, and R184S). J Pharmacol Exp Ther;3:1340.

21. Loscher W. (2002): Basic pharmacology of valproate: a review after 35 years of clinical use for the treatment of epilepsy. CNS Drugs; 10:669.

22. Munisamy M, Tripathi M, Behari M, et al. (2013): The effect of uridine diphosphate glucuronosyltransferase (UGT)1A6 genetic polymorphism on valproic acid pharmacokinetics in Indian patients with epilepsy: a pharmacogenetic approach. Mol Diagn Ther;5:319.

23. Nagar S, Zalatoris JJ, Blanchard RL. (2004): Human UGT1A6 pharmacogenetics: identification of a novelSNP, characterization of allele frequencies and func-tional analysis of recombinant allozymes in human liver

tissue and in cultured cells. Pharmacogenetics;8:487.

24. Nathan JK, Purandare AS, Parekh ZB, et al. (2009): Ketogenic diet in Indian children with uncontrolled epilepsy. Indian Pediatr;8:669.

25. Panomvana Na Ayudhya D, Suwanmanee J, Visudtib-han A. (2006): Pharmacokinetic parameters of total and unbound valproic acid and their relationships to seizure control in epileptic children. Am J Ther;3:211.

26. Peters WH, te Morsche RH, Roelofs HM. (2003): Com-bined polymorphisms in UDP-glucuronosyltransferases 1A1 and 1A6: implications for patients with Gilbert’s syndrome. J Hepatol;1:3.

27. Ristic AJ, Vojvodic N, Jankovic S, et al. (2006): The frequency of reversible parkinsonism and cogni-tive decline associated with valproate treatment: a study of 364 patients with different types of epilepsy. Epilepsia;12:2183.

28. Serrano BB, Garcia Sanchez MJ, Otero MJ et, al. (1999): Valproate population pharmacokinetics in chil-dren. J Clin Pharm Ther;1:73.

29. Sun YP, Tan L, Wang Y, et al. (2007): Effect of UGT1A6 genetic polymorphisms on the metabolism of sodium valproate. Zhonghua Yi Xue Za Zhi;29:2033.

30. van Oijen MG, Huybers S, Peters WH, et al. (2005): Polymorphisms in genes encoding acetylsalicylic acid metabolizing enzymes are unrelated to upper gastroin-testinal health in cardiovascular patients on acetylsali-cylic acid. Br J Clin Pharmacol;6:623.

31. Wang Y, Gao L, Liu YP ,et al. (2010): Effect of UGTIA6 A541G genetic polymorphism on the metab-olism of valproic acid in Han epileptic children from Henan. Zhongguo Dang Dai Er Ke Za Zhi;6:429.

32. Xing Y, Yang L, Wang L, et al. (2009): Sys-tematic screening for polymorphisms within the UGT1A6 gene in three Chinese populations and function prediction through structural modeling. Pharmacogenomics;5:741.

El-Sawy, M. et al.

Mohsen, M. , et al.41

تأثير التعدد الشكلى الجينى لل UGT1A6 جين علي استجابة االطفال المصريين المصابين بالصرع لعقار الفالبروات

محمد عبد العدل الصاوي - اسامة احمد بداري - منال زغلول مهران - سحرمحمد حسنين - منال حامد الحمامصي نسرين علي محمد - انجي عبد الهادي ابراهيم

UDP - glucuro- أساسا عن طريق االقتران ب ( VPA )المقدمة و الهدف من البحث: يتم التخلص من حمض الفالبرواتnosyltransferases والمعروف بتعدد اشكاله الجينيه. تهدف هذ` الدراسة إلى تقييم شامل لتأثير UGT1A6 متعدداألشكال في موقع النوكليوتيداتA>C 552 وA>G 541 على تركيز حمض فالبرويك فىالمصل واالستجابة االكلينيكية لحمض الفالبروات ، السيطرة علىالنوبات الصرعية والتفاعالت الدوائية الضارة في األطفال المصريين المصابين الصرع. طرق البحث :تشتمل هذ` الدراسة علي 48 مريضا بالصرع ممن يتلقون عقار الفالبروات وقد تم تحديد المتغيرات الجينية لجين (RFLP) .بواسطة تفاعل البلمرة التسلسلى باستخدام تقنية A>G 541و A>C 552في موقع النوكليوتيدات UGT1A6تم قياس مستويات حمض فالبرويك فى المصل بواسطة تقنية متجانسة انزيم المناعه. واجرى رصد لجميع المرضى اكلينيكيا لشدة النوبات باستخدام مقياس تشالفونتالى جانب ذلك ، استخدمت قائمة مرجعية لتسجيل التفاعالت الدوائية الضارة. النتائج :وقد وجد ان نسبة التركيزإلى جرعة للدواء أقل بكثير في المرضي الذين يحملون الساللة الجينية AC,CC للتنوع الجينى (A/C - 552) مقارنة بالمرضي الذين يحملون الساللة الجينيةAA. كما وجد ان حاملي الساللة الجينية AA اظهروا درجة .(A/C - 552) للتنوع الجينى AC,CC أعلى بكثيرعلى مقياس تشالفون تلقياسشدة النوبات مقارنة بحاملي الساللة الجينيةلم نجد أي ارتباط ملموس في هذ` الدراسة بين السيطرة على النوبات الصرعية والمجموعات الوراثية المختلفة . بينما وجدنا A/ - 541)للتنوع الجينى AG,AA و حاملي الساللة الجينية (A/C - 552)للتنوع الجينىAA ان حاملي الساللة الجينية

UGT1A6 اكثرعرضة للتعب واالعياء و اكثر تأثرا في الجانب المعرفي. االستنتاج: يؤثر التعدد الشكلى الجينى لل.(Gجين علي معدل االيض لحمض فالبرويك مما يؤثرعلي شدة نوبات الصرع و القابلية للتفاعالت الدوائية الضارة.

NON-INVASIVE METHOD FOR THE ASSESSMENT OF LIVER FIBROSIS AND PREDICTION OF HEPATOCELLULAR CARCINOMA

IN PATIENTS WITH CHRONIC ACTIVE HEPATITISNevine E. El-Abd*, Sherihan H. El-Kady and Mohamed A. El-Attiq**

ABSTRACTHepatitis C virus (HCV) is a major health problem accounting for 40% of cases with end stage cirrhosis and 60% of cases

with hepatocellular carcinoma (HCC). Globally, hepatitis B virus (HBV) has been associated with 100-fold increase in risk for development of HCC. Recognition of the limitations and cost of liver biopsy, led to an increasing need for non-invasive tests for the assessment of the stage as well as progression of liver fibrosis. The current study aimed to evaluate the value of three serumbiomarkers: fibronectin (FN), pseudocholine esterase (PCHE) and prothrombin activity individually and when combined inan equation, in patients with chronic active hepatitis (CAH) B or C in order to assess their ability to discriminate between advanced fibrosis, cirrhosis and for early prediction of HCC ( Discriminant score = PCHE/60 (U/L) X 0.00011 + fibronectin(µg/ml) X 0.039 + Prothrombin activity (%) X 3.51). The study was conducted on 70 patients, they were selected according to METAVIR scoring system as 20 patients with fibrosis ( F3), 25 patients with cirrhosis (F4) and 25 patients with HCC on top ofchronic active hepatitis B or C. All of the groups were compared to 10 apparently healthy individuals as controls. All subjects were subjected to routine liver function tests, Alpfa fetoprotein (AFP), hepatitis B and C markers, fibronectin, pseudocholineesterase and prothrombin activity. Abdominal ultrasonography, liver biopsy and Metavir scoring were done for patients only. Levels of PCHE, FN and equation score were decreased with the progress of liver disease. As regards fibronectin, there was nosignificant difference between F3 and F4 patients. As regards prothrombin activity and the equation, no significant differencewas found between patient with cirrhosis (F4) and HCC. The equation was found to be a good non-invasive method to discrimi-nate between advanced fibrosis and cirrhosis with sensitivity 80% and specificity 70%. Pseudocholine esterase was found to bea good non-invasive marker for follow up of cirrhotic patients and for early detection of HCC; with sensitivity and specificity of84% and 92% respectively. key words: pseudocholine esterase, fibronectin, prothrombin activity, liver cirrhosis, HCC.


INTRODUCTION

Hepatitis C virus (HCV) is a major health problem with an estimated global prevalence of an average 3% or 150-200 million people of the world population(16). Egypt has the highest prevalence of HCV in the world with an esti-mated prevalence of 14.9% of which 80% are among individuals aged 30 years and above(12). The estimated risk of HCC is 15-20 times higher among HCV patients than it is among healthy subjects(36). Hepatitis B virus (HBV) is also a major health problem, with almost 2 billion in-fected subjects, more than 350 million of whom have chronic hepatitis B infection (CHB). It is estimated that 15–40% of CHB patients devel-op liver cirrhosis or HCC, contributing to more than 1 million deaths every year(8). Furthermore, HCC ranked as the third most frequent cause of cancer-related deaths accounting for 9.2%

of all cancer deaths worldwide(29). According to the American Association for the Study of Liver Disease (AASLD), liver biopsy has a ma-jor role in diagnosis, prognosis and therapeutic decisions(21). However, Cost pressures and the limitations have led to an increasing demand for non-invasive tests to assess the stage of liv-er fibrosis. Serum biomarkers of liver fibrosissuch as fibronectin, pseudocholinesterase andprothrombin concentration have been proposed as indicators of change from chronic hepatitis to cirrhosis(28). The analysis of these serum bio-chemical markers is a rapid, inexpensive and non-invasive leading to high predictive identifi-cation of the progression of the fibrotic processand hence proper management(24). Cholinester-ase is synthesized mainly in hepatocytes , thus serum levels are reduced in liver dysfunction due to decreased synthesis. The predominant

Departments of Clinical & Chemical Pathology, Faculty of Medicine, Cairo University* and Gastroenterology & Hepatology, Tanta Fever Hospital, Tanta**, Egypt

El-Abd, N. E., et al.92

hepatic source of serum cholinesterase, suggests that its value might be a more specific indica-tor of liver dysfunction than the other traditional liver function tests(22). Its value in predicting postoperative complications following hepatic resection of HCC has been also proved to be of great importance(9). In normal liver, fibronectinplays major roles in various cellular functions such as cell adhesion, proliferation and differ-entiation, increased expression of fibronectin isthought to be associated with both physiological and pathological tissue remodeling(1). It was also proved that fibronectin plays an important rolein cancer progression, and is thus hypothesized to be highly associated with HCC progression(17). Prothrombin activity is considered as a universal indicator of the severity of liver disease and is incorporated in almost all the prognostic models and major therapeutic decisions worldwide(5).

The aim of this study was to evaluate the utility of the 3 serum biomarkers: fibronectin,pseudocholinesterase and prothrombin activity, individually and when combined in an equation, in patients with post hepatitis liver fibrosis, cir-rhosis and HCC in order to assess their ability to discriminate between different stages of fibrosisas well as for early prediction of HCC.


This study was conducted on 70 patients with age ranging from 40 to 65 years, selected from Hepatology and Gastroenterology department, Cairo University hospital and Tanta Fever Hos-pital. All subjects were classified into 4 groups,group I included 20 patients with CAH (B or C) with liver fibrosis (F3), group II included 25 pa-tients with CAH (B or C) with liver cirrhosis (F4) and group III included 25 patients with HCC on top of CAH (B or C). The 3 groups of patients were selected and classified according to META-VIR scoring system. They were compared to 10 apparently healthy age and sex matched volun-teers as the control group (group IV). The study protocol was approved by the ethics committee of Cairo University Hospital. Routine liver func-tion tests: Albumin(10), AST(33), ALT(27), GGT(30) and Alkaline phosphatase(34) were performed on Beckman Coulter AU 480 auto analyzer. Serum alpha fetoprotein (AFP) was analyzed

by chemiluminescent microparticle immunoas-say on Architect 2000 Si by Abbott(23), HBsAg and HCV Ab were done by enzyme- linked im-munosorbent assay (ELISA)(35). Determination of Prothrombin activity was carried by man-ual method(7), Fibronectin was determined by quantitative competitive enzyme immunoassay ELISA kit supplied by Assaypro(31)[Assaypro, AssayMax Human Fibronectin 30 Triad South Drive, St. Charles, MO 63304.] and Pseudo-cholinesterase was done by a quantitative kinetic assay, using Butyrylthiocholine (BTC) method by a kit supplied by BEN(26)[BEN Biochemi-cal Enterprise S.r.l - via Toselli, 4 - 20127 Mi-lano - Italy]. All patients underwent abdominal ultra sonographic examination and liver biopsy for assessment of degree of fibrosis by using theMETAVIR scoring system(3) which classify pa-tients according to fibrosis as follows:

Stage (0): no fibrosis, Stage(1): stellateenlargement of portal tracts without septae, Stage(2): enlargement of portal tract with rare septae, Stage(3): numerous septae (fibrosis),without cirrhosis and Stage(4): cirrhosis.

Fortunato et al. in 2001, developed a discrim-inate score to assess the degree of hepatic fibro-sis; [Pseudocholinesterase (µKat/L) X 0.00011 + Fibronectin (mg/L) X 0.039 + Prothrombin activity (%) X 3.51 + ALT (µKat/L) X 0.49 – N-acetyl-β – glucosaminidase [B-NAG] (µKat/L) X 0.51 – Manganese superoxide dismutase [Mn-SOD] (µG/L) X 1.29 – 9.41] (14). In the present study, for simplicity, the original equation was modified as follows: [(Pseudocholinesterase (U/L) / 60) X 0.00011] + [Fibronectin (µg/ml) X 0.039] + [Prothrombin activity (%) X 3.51]. The discrimination score was calculated by computer software (Microsoft Excel) by entering the re-sults of the three studied biomarkers.

Statistical method: All results were pro-cessed by SPSS (Statistical Package of Social Science) 16.0 for Windows XP. ANOVA (anal-ysis of variance) among groups of study were done for quantitative variables, considering the p value < 0.05 as statistically significant. Quanti-tative results were expressed as mean values (x) and standard deviations (SD). ROC curves were drawn for detection of reliability of the studied markers to differentiate stages of liver fibrosisand its value in prediction of HCC.

Liver Fibrosis and Prediction of HCC in CAH 93RESULTS

descriptive statistics of liver function tests and AFP are represented in table (1)

Mean values of PCHE in the HCC group were significantly lower (3931U/L) when com-pared to that of F3, F4 and control groups (P < 0.01). its mean value in F4 group (8300U/L) was significantly lower comparing it to that ofF3 group (11400 U/L), P < 0.01, however, F3 and F4 showed no statistically significant dif-ference when compared to the control group (9136U/L), P>0.05. As for Fibronectin, its mean value in HCC group was significantlylower (228.3 µg/ml) when compared to that of F4 (305.0) (P =0.019), however, no statistically significant difference was found between themean values of F3 and F4 groups (333.6 µg/ml and 305.0 µg/ml respectively), at P > 0.05. Re-garding Prothrombin activity, its mean values in F4 and HCC groups (69.7% and 70.9% respec-tively) were significantly lower comparing themto that of F3 group (78.7%), (P < 0.01 and <0.05 respectively), the mean values of F3, F4 and HCC groups were statistically lower comparing them to the control group (90.7%), (P < 0.01). The mean values of the discrimination score, of F4 group (286.6) was statistically lower than that of F3 group (322.1) at P < 0.05, while, no signifi-cant difference was found between the mean val-

ues of F4 and HCC groups (280.4) at P > 0.05. The control group was significantly the highest(370.2) when compared to all of the other 3 stud-ied groups (P < 0.01)

In the ROC curve analysis (Figures 1 and 2); we found the following:

Pseudocholinesterase: at a cut-off value of 11829 U/L, PCHE was able to discriminate be-tween F3 and F4, the AUC (0.784), a sensitivity of 92 % and specificity of 60 %. Moreover, at acut-off value of 4809 U/L, it could also discrimi-nate between F4 and HCC, AUC (0.899) with a sensitivity of 84 % and a specificity of 92 %.

Fibronectin: at a cut-off value of 362.8 µg/ml, it was hardly able to discriminate between F3 and F4, the AUC (0.573) with a sensitivity and specificity of 72 % and 45 % respectively.Furthermore, at a cut-off value of 244.4 µg/ml, it showed a limited value in discrimination be-tween cirrhosis and HCC with AUC (0.739), a sensitivity of 68 % and a specificity of 80 %.

The discrimination score: at a cut-off of 310; it was able to discriminate between fibrosis(F3) and cirrhosis (F4), the AUC (0.820) with a sensitivity and specificity of 80 % and 70 % re-spectively. However, at a cut-off value of 291, it was bearly able to discriminate between F4 and HCC showing AUC (0.555), with a sensitivity of 68 % and a specificity of 44 %.

Table (1): Descriptive statistics for liver biochemical tests and AFP of the studied groups.

Results are presented by mean ±SD (P < 0.05 is Significant)Cells bearing same letters are not statistically significant

El-Abd, N. E., et al.94

Table (2): comparison between the mean values of Prothrombin activity, Pseudocholinesterase (PCHE), Fibronectin (FN) and the equation in the all of the studied groups

Results are presented by mean ±SD (P < 0.05 is Significant) Cells bearing same letters are not statistically significant

Figure (1): ROC curve for Pseudocholinester-ase, Fibronectin and the equation in F3 and F4 patients.

Figure (2): ROC curve for Pseudocholinesterase, Fibronectin and the equation in F4 patients and HCC patients

Liver Fibrosis and Prediction of HCC in CAH 95DISCUSSION

The prognosis and management of chronic liver disease depend largely on the degree and progression of liver fibrosis as well as the de-velopment of HCC. In the past decade HCC has gone from being an almost universal death sentence to a cancer that can be prevented, de-tected at an early stage, and effectively treated(4). Serum markers of liver fibrosis offer an attrac-tive, less invasive and cost effective alternative to liver biopsy for both patients and clinicians. Moreover, measurements may be performed re-peatedly, thus allowing for a dynamic monitor-ing of fibrosis(38). This study aimed to evaluate three biomarkers; Pseudocholinesterase, Fibro-nectin and Prothrombin activity individually and combined in a specific equation, in correlationwith histopathological findings, in patient withchronic HBV or HCV infection; to detect their value as non-invasive markers to discriminate late stages of fibrosis (F3) and cirrhosis (F4) inCAH patients as well as for early prediction of HCC in cirrhotic patients.

Our study revealed that the mean value of PCHE was the lowest in HCC patients than that in F3, F4 and control groups (p <0.01). Further-more, its value in F4 group, was significantlylower as compared to that of F3 group, reveal-ing that PCHE was negatively related to the degree of hepatic fibrosis, with sensitivity 92%and specificity 60% at a cut off value of 11829U/L. In concordances to our study, a recent re-search studying the serum level of PCHE in 30 patients with CAH or cirrhosis compared to 30 apparently healthy controls, found that the value of PCHE was significantly lower in the patientscompared to control group (<0.05) and they sug-gested a cutoff value of 4850 U/L to diagnose liver disease with 90% sensitivity and 100% specificity(22). Another study concluded that cho-linesterase activity is significantly decreased incirrhosis as compared to chronic hepatitis pa-tients at P<0.01(13), which also comes in agree-ment with our results. Moreover, another study, showed that serum cholinesterase decreased sig-nificantly in liver cirrhosis (P<0.001) and thatcholinesterase is easier and more objective in evaluating the liver reserve function in cirrhot-

ic patients(18). A third study proved that PCHE mean value was significantly decreased in livercirrhosis (P <0.01)(20), which again goes hand in hand with another study, reporting that the me-dian value of PCHE was significantly lower incirrhotic group compared to its values in chronic active hepatitis and control groups(25). Our results supported by previous studies might be contrib-uted to decreased synthetic capability of the liver with the advanced stages of liver fibrosis.

We also found that the mean value of PCHE was significantly lower in HCC group whencompared to F4 group at P 0.001, at a cut off value of 4809 U/L showing a sensitivity of 84% and specificity of 92%. These results were inagreement with a study, demonstrating that lower serum CHE level is a significant predic-tor of poor prognosis and severe liver damage in HCC patients(32). Researchers observed in their study that the PCHE levels were lower (31-49% of normal value) in all patients with differ-ent malignancies(6). PCHE levels reported to be decreased in advanced cancer patients, with or without hepatic involvement: the lowest activity has been found in patients with hepatic metasta-ses, despite the normality of other liver function tests(15). One of the possible mechanisms respon-sible for PCHE activity decrease in cancer pa-tients could be secondary anorexia accompany-ing malignancy(19).

On the contrary, a study found that serum val-ue of cholinesterase in cirrhosis was statistically indifferent comparing it to HCC patients(13).

As for Fibronectin, it showed no statistically significant difference between F3 and F4 group(P>0.05). However, its value was significantlylower in HCC group than that in F4, F3 and con-trol groups (P <0.05, <0.01 and <0.01 respec-tively). These findings were in agreement with astudy which concluded that the determination of plasma fibronectin was insignificant in the eval-uation of the degree of liver fibrosis(11). On the other hand, a study found that the level of fibro-nectin was increased significantly with the pro-gression of fibrosis stages but decreased in cir-rhosis and that fibronectin was the most efficientmarker among other tested markers, P <0.01)(2).

El-Abd, N. E., et al.96Another study, demonstrated that circulating fi-bronectin modulates blood vessel formation and tumor growth by modifying the amount of and the response to Vascular Endothelial Growth Factor (VEGF)(37). Researchers concluded that determination of the FN can serve as a prognos-tic biomarker for breast and prostate cancers and possibly other cancers. However, other research-ers found that HCC patients had significantlyhigher plasma levels of FN comparing them to the control group (P<0.05) and that circulating FN levels were dramatically reduced in patients at late stages of HCC(17). Regarding the mean val-ues of Prothrombin activity, we found that it was significantly lower in F4 group when comparedto that of F3 group (78.7%) (P < 0.01), which was previously concluded by scientists who re-ported that the median values of prothrombin ac-tivity were significantly lower in cirrhotic groupas compared to chronic active hepatitis group (P < 0.01)(25). we also found that the mean values of the discrimination score of equation were sig-nificantly lower in F4 in comparison to its valuein F3 group (P value <0.05). The mean values of F3, F4 and HCC groups were significantly lowercomparing them to the control group, P value< 0.01. The equation score can significantly dis-criminates between fibrosis (F3) and cirrhosis(F4), at a cut-off value of 310.1 with sensitiv-ity 80%, specificity 70%. Close results were ob-tained by other researchers who found that the equation significantly discriminates fibrosis andcirrhosis (P = 0.001) with sensitivity 100% and specificity 60% at cut-off value of 243.28(25). A study which used a discriminant similar score, including fibronectin, pseudocholinesterase andprothrombin activity, established a cut-off value of -0.22 (< -0.22 indicated cirrhosis and > -0.22 indicated chronic active hepatitis)(14). However, there was no statistically significant differencebetween the mean values of the equation score in F4 and HCC groups (286.6 and 280.4 respec-tively), (P > 0.05) and the equation was not able to predict HCC on top of cirrhosis; whereas, at a cut off value of 291.4 , the sensitivity and speci-ficity were 68% and 44% respectively.

In conclusion to our results, the value of PCHE appeared to reflect the progression ofchronic liver disease. The decrease in the level of

serum PCHE from stage F3 to stage F4 to HCC; show its importance in the follow up of chronic advanced liver fibrosis for the detection of cir-rhosis and HCC. Although, the statistics showed decrease in PCHE mean values in groups F3 and F4, yet they were considered to be within the normal range, however, in HCC group, PCHE levels were markedly decreased below the nor-mal range; which reflects its great importancein predicting HCC in cirrhotic patients. FN re-sults showed that it has no significant role inthe detection of the stage of advanced fibrosisor in the prediction of HCC. Although FN has an important role in fibrogenesis and the devel-opment of different cancers and several studies were done on the level of FN in these conditions; some reported a decrease in its levels, while oth-ers showed that it is increased, however, the mechanism remains unknown. It is possible that patients at the cirrhotic stage and late stages of HCC lost their synthetic capability because of liver malfunctions. The discrimination score proved its value for evaluation of the progression of liver fibrosis, as it was able to discriminate be-tween fibrosis and cirrhosis (F3 and F4) whichwas attributed to the significant results found inthis study regarding PCHE and prothrombin ac-tivity between both groups. It has proved to be a good non-invasive method for the follow up of chronic active hepatitis patients and may elimi-nate the need for repeated liver biopsy. However, it showed a limited value for prediction of HCC which might be attributed to the non-significantresults found in this study for prothrombin activ-ity and FN between F4 and HCC groups.

REFERENCES 1- Abdel-Latif MS, Amahal EK, Ragab NM, et al (2014).

Cellular fibronectin expression in hepatitis C virus pa-tients. Scand J Infect.Dis.July, 46 (7):508.

2- Attallah, A. M., Abdallah, S. O., Attallah, A. A., et al (2013). Diagnostic value of fibronectin discriminantscore for predicting liver fibrosis stages in chronic hep-atitis C virus patients. Ann Hepatol, 12, 44.

3- Bedossa, P., & Poynard, T. (1996). An algorithm for the grading of activity in chronic hepatitis C. Hepatology, 24(2), 289.

4- Bruix, J., & Sherman, M. (2011). Management of he-patocellular carcinoma: an update. Hepatology, 53(3), 1020.

Liver Fibrosis and Prediction of HCC in CAH 975- Caraceni, P. and D. H. Van Thiel (1995). “Acute liver

failure.” The Lancet 345(8943): 163.

6- Chougule, A., Hussain, S., & Agarwal, D. P. (2008). Prognostic and diagnostic value of serum pseudocho-linesterase, serum aspartate transaminase, and serum alanine transaminase in malignancies treated by radio-therapy. Journal of cancer research and therapeutics, 4(1), 21.

7- Colman, R. W., Hirsch, J., Marder, V. J., et al (1995). He-mostasis and Thrombosis: Basic: Principles and Clini-cal Practice. Blood Coagulation & Fibrinolysis, 6(2), 147.

8- Datta, S., Chatterjee, S., Veer, V., et al (2012). Molecular Biology of the Hepatitis B Virus for Clinicians. Journal of Clinical and Experimental Hepatology. 2(2):353

9- Donadon, M., Cimino, M., Procopio, F., et al (2013). Potential role of cholinesterases to predict short-term outcome after hepatic resection for hepatocellular car-cinoma. Updates in surgery, 1.

10- Doumas, B. T., Ard Watson, W., & Biggs, H. G. (1971). Albumin standards and the measurement of serum al-bumin with bromcresol green. Clinica chimica acta, 31(1), 87.

11- El-Saeid, M. E., Mousa, M., & Hamdy, M. (2012). Evaluation of Cystatin C, Fibronectin and Alpha-Feto Protein as Biochemical Markers in Patients with Liver Diseases. Life Science Journal, 9(4):5389.

12- El-Zanaty, F., & Way, A. (2012). Egypt demographic and health survey 2008. Cairo, Egypt: Ministry of Health, El-Zanaty and Associates, and Macro Interna-tional; 2009.

13- El Zwahry, E., Ahmed, D. F., El Masry, M. et al (2013). Serum cholinesterase activity helps to distinguish be-tween liver cirrhosis, hepatocellular carcinoma and chronic hepatitis C virus in Egyptian patient. Research-er 2013;5(7)

14- Fortunato, G., Castaldo, G., Oriani, G., et al (2001). Multivariate discriminant function based on six bio-chemical markers in blood can predict the cirrhotic evo-lution of chronic hepatitis. Clinical chemistry, 47(9), 1696.

15- Gu, S. Z., Zhao, X. H., Quan, P., et al (2005). Alterations of serum cholinesterase in patients with gastric cancer. World Journal of Gasteroentrology, 11(29), 4604.

16- Hanafiah Mohd, Groeger J, Flaxman AD et al (2013):Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seropreva-lence. Hepatology; 57 (4): 1333.

17- Lin, C. C., & Yin, M. C. (2012). Clinical significanceof circulating IL-10 and fibronectin levels in hepatocel-lular carcinoma patients with HBV infection. BioMedi-cine, 2(3), 122.

18- Meng, F., Yin, X., Ma, X., et al (2013). Assessment of the value of serum cholinesterase as a liver function test for cirrhotic patients. Biomedical Reports, 1(2), 265.

19- Ogunkeye, O. O., & Roluga, A. I. (2006). Serum cho-linesterase activity helps to distinguish between liver disease and non-liver disease aberration in liver func-tion tests. Pathophysiology, 13(2), 91.

20- Rao, S. V., Kiran, V. R., & Indira, S. (2011). A compara-tive study of pseudocholinesterase and liver function test in cirrhosis of liver, infective hepatitis and obstruc-tive jaundice: a case control study.

21- Rockey, D. C., Caldwell, S. H., Goodman, Z. D., et al (2009). Liver biopsy. Hepatology, 49(3), 1017.

22- Ruchi Gokani, Pranay Jadav, Nadeem Shaikh, et al (2014). Serum cholinesterase as a diagnostic marker of liver disease. International Journal of Biomedical and Advance Research 05(09); 439.

23- Ruoslahti, E., Pihko, H., & Seppälä, M. (1974). Al-pha�fetoprotein: Immunochemical Purification andChemical Properties. Expression in Normal State and in Malignant and non�Malignant Liver Disease. Im-munological Reviews, 20(1), 38.

24- Sabatier, L., Chen, D., Fagotto-Kaufmann, C., et al (2009). Fibrillin assembly requires fibronectin. Mo-lecular biology of the cell, 20(3), 846.

25- Salem, M., El Gayar, D., Kamal, M., et al (2010). Bio-chemical markers in blood can predict the cirrhotic evo-lution of chronic hepatitis. Arab. J. Lab. Med. (2010) Vol.(36), No. 3: 599.

26- Schmidt, E., Gerhardt, W., Henkel, E., et al. (1992). Proposal of standard methods for the determination of enzyme catalytic concentrations in serum and plasma at 37 ºC. Cholinestrase (Acylcholine Acylhydrolase, EC-3118). European Journal of Clinical Chemistry and Clinical Biochemistry, 30 (3), 195.

27- Schumann, G.,Bonora, R., Ceriotti, F., et al (2002). IFCC primary reference procedures for the measure-ment of catalytic activity concentrations of enzymes at 37 ºC. Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase.Clinical Chemistry and Laboratory Medicine, 40 (7), 718.

28- Schuppan, D., Schmid, M., Somasundaram, R., et al (1998). Collagens in the liver extracellular matrix bind hepatocyte growth factor. Gastroenterology, 114(1), 139.

29- Schwatz J.M, Carithers R.L, Di Bisceglie, et al (2014). Epidemiology and etiologic associations of carcinoma, Wolters Kluewer health. Viewed 12 August 2014. www.update.com/contents/epidemiology- and-etiologic-as-sociations- of- hepatocellular carcinnoma.

30- Shaw M, Stromme H, London L, et al (1983): Part 4 IFCC method for γ-glutamyltransferase. Clin Chem Clin Biochem; 21(10): 633.

El-Abd, N. E., et al.9831- Snyder, E. L., Luban, N. L., & Sherman, L. A. (1986).

Fibronectin: applications to clinical medicine. Critical Reviews in Clinical Laboratory Sciences, 23(1), 15.

32- Takeda, H., Nishikawa, H., Iguchi, E., et al (2013). Im-pact of pretreatment serum cholinesterase level in un-resectable advanced hepatocellular carcinoma patients treated with sorafenib. Molecular and Clinical Oncol-ogy, 1(2), 241.

33- Thomas L (1998): Alanine aminotransferase (ALT), Aspartate aminotransferase (AST). In: Thomas L (ed.): Clinical laboratory diagnostics: Use and assessment of clinical laboratory results. Frankfurt/Main: TH-Books Verlagsgesellschaft; pp: 55.

34- Tietz NW, Rinker D, Shaw LM (1983): IFCC methods for the measurement of catalytic concentration of en-

zymes Part 5. IFCC method for alkaline phosphatase. J Clin Chem Clin Biochem; 21: 731.

35- Torane VP and Shastri JS (2008): Comparison of ELI-SA and rapid screening tests for the diagnosis of HIV, hepatitis B and hepatitis C among healthy blood donors in a tertiary care hospital in Mumbai. Indian J. Med. Microbiol.; 26: 284.

36- Tsai WL and Chung RT (2010): Viral hepatocarcino-genesis. Oncogene; 29: 2309.

37- von Au, A., Vasel, M., Kraft, S., et al (2013). Circu-lating Fibronectin Controls Tumor Growth. Neoplasia (New York, NY), 15(8), 925.

38- Zhou, K., & Lu, L. G. (2009). Assessment of fibrosisin chronic liver diseases. Journal of digestive diseases, 10(1), 7.

الخلوي الكبدية الخاليا بسرطان التنبؤ و الكبد تليف عن للكشف اجتياحية طريقة غير

الناشط المزمن الكبدي االلتهاب مرضي في

العتيق القاضى - محمد العبد - شيريهان نيفين

تطور التي من عملية التالئم جزء وهو الخلية. المصفوفة خارج من البروتينات وغيره للكوالچين مفرط تراكم هو الكبدي التليفإلى النهاية في مما يؤدي الكبد مورفولوجيا الخلية، يغير خارج المصفوفة تدريجيا للبروتينات المتزايد الكبد. الترسيب اصابة على رداللتليف في المرحلي للتحديد الذهبي المعيار الكبد هو األخيرة، خزعة حتى اآلونة الكبد. الي سرطان ايضا ويؤدي وظائف الكبد، ضعفو أيضا ومؤلم إجتياحي إجراء هو الكبد خزعة المزمنة. الكبد أمراض من وغيرها (سي) و (بي) المزمن الوبائي الكبد حاالت التهابنشاط دمجهما مع عند كولينستيراز الزائف و فيبرونكتين تقييم هو دراستنا من الهدف المريض. تهدد حياة مضاعفات خطر حدوث يحملالوبائي االلتهاب الكبدي من يعانون الذين المرضى في المرضية) النسيجية التغيرات مع (في عالقة معادلة معينة في البروثرومبينالكامل، وتليف الكبد المتقدمة الكبد مرحلة تليف بين للتمييز إجتياحي غير كمؤشر من المعادلة القيمة استخدام يتم أو (سي). (بي) المزمنالكامل. الكبدى فوق التليف الكبد سرطان مرضي بنتائج الكامل، الكبدى التليف مرضى نتائج مقارنة خالل من والتنبؤ بسرطان الكبد

مستشفي الحميات من وايضا القاهرة جامعة بمستشفيات الهضمي والجهاز الكبد امراض قسم من اختيارهم 70 مريضا، تم الدراسة شملتالي 20 METAVIR الدرجات لنظام تبعا تقسيمهم تم أو (سي). المزمن (بي) الوبائي الكبدي بااللتهاب جميعهم مصابون بطنطا،و25 مريضا طنطا حميات مستشفي من الكبدي للتليف األخيرة و الرابعة بالمرحلة و25 مريضا الكبدي بالمرحلة الثالثة للتليف مريضا،التصويرة االشعة نتائج قبل (تم تشخيصها من الكبدية الخاليا سرطان مع معقدة الكبدي للتليف األخيرة الرابعة و بالمرحلة تشخيصهم تملجميع اآلتي عمل تم والعمر. الجنس في مطابقة أصحاء متطوعين عشرة من الضابطة المجموعة اختيرت المختبرية). والمعاملترانسفيراز، األالنين أمين إنزيم الزالل، ذلك في بما مصل الدم في وظائف الكبد إختبارات الروتينية: اإلختبارات المعملية المرضي:البروثرومبين. وقت وتركيز فيتو بروتين. وألفا الفوسفاتيز القلوية غاماغلوتاميل ترانسفيراز، األسبارتات ترانسفيراز، إنزيم أمين إنزيمبروتين (سي). الكبد التهاب لفيروس المضادة واألجسام (بي) التهاب الكبد لفيروس السطحي المستضد الوبائي: الكبدي االلتهاب دالالتالبطن فحص مصل الدم، في الزائف الكولينستراز إنزيم فيبرونكتين في البالزما. التليف: دالالت عن الكشف الدم. مصل في فيتو ألفاطريق عن وااللتهاب التليف تقييم مع والثالثة والثانية األولى المجموعات من كبدية أخذ خزعة الثالثة. للمجموعة التليفزيونية باآلشعةو الكولينستراز الزائف إنزيم مستويات أن الرابعة): وجد (المرحلة الكبدي مرضى التليف في .METAVIR الدرجات نظام استخدامهناك فرق يكن لم وفيما يتعلق بمستويات فبرونيكتين، في الكبد. نسبة التليف تقدم المعادلة المحسوبة تنخفض مع و نشاط البروثرومبينالمعادلة أستخدام الممكن من أن التوصل الرابعة). تم (المرحلة الكامل الكبد تليف و (المرحلة الثالثة) المتقدم الكبدي التليف بين كبيرسرطان مرضي في وتحديدية 70٪. ٪ 80 بحساسة الكامل الكبدي والتليف المتقدم التليف الكبدي بين للتمييز إجتياحية غير جيدة كوسيلة(المرحلة الكبدى التليف مرضى في الكبد سرطان تطور مع أكثر انخفضت فبرونيكتين و الزائف الكولينستراز مستويات أن وجد الكبد:لن المعادلة وجد أن الكبد. وسرطان الكبد الكامل بين تليف كبير فرق هناك يكن المعادلة، لم البروثرومبين و بنشاط وفيما يتعلق الرابعة).الدم مصل في الزائف الكولينستراز أنزيم أن الي التوصل تم الكامل. الكبد تليف مرضي في الكبد لتنبؤتطويرسرطان جيدا مؤشرا تكون

٪84 وتحديد 92٪. حساسة مع سرطان الكبد؛ عن المبكر للكشف الكبدى، التليف مرضى لمتابعة إجتياحي جيد غير اختبار ليكون

HAS PERIODONTAL THERAPY A ROLE ON THE EXPRESSION OF THE RECEPTOR OF ADVANCED GLYCATION END PRODUCTS

(RAGE) IN GINGIVAL TISSUES AMONG PATIENTS WITH TYPE 2 DIABETES MELLITUS?

Nagwa Abdel Hamid Osman*, Mona Salah El Din Darhous**, Ashraf Ismail***Dina M. Rasheed Bahgat*** and Rania Khalifa***

ABSTRACTBackground: An extensive body of literature reports that diabetes is a risk factor for gingivitis and periodontitis, and the

degree of glycemic control is a determining factor in the vulnerability to oral health. The receptor for advanced glycation end-products (RAGE) is strongly expressed in gingival tissues obtained from patients with type II diabetes.This suggests that RAGE plays a central role in periodontitis in Type II Diabetes Mellitus. Subjects and Methods:50 male individuals ranging in age from 35 to 60 years old were included in the study and divided into 3 groups; group I consisted of 20 patients suffering from both type 2 DM and Chronic periodontitis (CP), group II consisted of 20 non-diabetic patients suffering from CP, group III consisted of 10 non-diabetic individuals with healthy periodontium. Blood samples were withdrawn to an EDTA tube for measurement of (HbA1c) in group I using high performance chromatography method before and after periodontal therapy, also biopsies were obtained from the marginal gingiva in areas of gingival enlargement for detection of RAGE also before and after periodontal therapy using semi-quantitative RT-PCR.Similarly all baseline clinical parameters were obtained immedi-ately before biopsy acquisition, these parameters included: Gingival index (GI), probing depth (PD), Clinical attachment level (CAL) before and after periodontal therapy. Results: Expression of RAGE did not differ significantly between groupsI and II,pre-or post-operatively, however they were significantly higher than control group. Levels of RAGE expression before and aftertreatment in group I and II were statistically significant. Hb1Ac differed significantly before and after periodontal therapy ingroup I.There was a positive correlation between levels of RAGE and clinical parameters but it was not statistically significant.No statistically significant differences were observed between the diabetic group and the chronic periodontitisgroup in the peri-odontal pocket depth(PD), clinical attachment level(CAL)and gingival index(GI)before treatment. After peri odontal therapy, as regards PD, it showed a significant reduction in groupI and in groupII, also for CAL, there was a significantr eductioningroupI and in groupII after the rapy. The GI also showed a significant reduction in both groups. A positive correlation between thelevel of RAGE expression and HbAlc, PD,CAL,GI,before and after treatment both in groupIandII was observed, yet it was not statistically significant. Regarding groupI, preoperatively, there was no significant correlation between HbAlc level and PD,CAL,GI, however, postoperatively, there was a statistically significant positive correlation between HbA1c and both PD andCAL. Conclusion: RAGEs expression did not differ in groupsI and IIdenoting that they are related to inflammation and notspecific to the diabetic state. Periodontal therapy resulted in lower HbA1c levels in diabetic patients thus it should be includedas a part of diabetes control measures, hence anti-RAGE therapy may provide a wider therapeutic window for periodontitis and glycemic control in diabetic patients. Key Words: RAGE,DM, HbA1c,RT-PCR

Departments of Oral Medicine and Periodontology*, Faculty of Oral and Dental Medicine, Clinical Pathology***, Kasr Al-Aini, Faculty of Medicine, Cairo University, **National Institute of Diabetes and Endocrinology


INTRODUCTION

The relationship between diabetes mellitus (DM) and periodontal disease has been exten-sively researched. A 1993 review of epidemio-logic studies estimated that one in three diabetics have severe periodontitis.This finding asserts ag-gressive periodontitis as the sixth complication of diabetes(12).

More research is emerging that suggests a bidirectional relationship between both types of diabetes and periodontal disease where DM plays an important etiological role and is consid-ered as a risk factor in periodontal diseases and

on the other side periodontal disease has pow-erful and multiple influences on the occurrence and severity of DM(16).

In hyperglycemic states found in diabetics, a non-enzymatic glycation and oxidation of pro-teins and lipids occurs. As a result, advanced glycation end products (AGEs), accumulate in the plasma and tissues, such as gingiva, of dia-betic subjects. The biologic mechanism currently accepted as a possible explanation for the more serve periodontitis found in diabetic patients is the accumulation of AGEs(17)

.

The effect of AGEs is mediated by the recep-

Osman, N. A. et al.100

tor advanced glycation end products (RAGE).RAGE is a multiligand signal transduction receptor belong-ing to the immunoglobulin superfamily and it is ex-pressed by a variety of cell types including endo-thelial cells, smooth muscle cells, lymphocytes, monocytes and neurons(9)

.

The receptor for advanced glycation end-products (RAGE), is strongly expressed in gin-gival tissues obtained from patients with type 2 diabetes and periodontitis compared with systemically healthy patients with chronic peri-odontitis patients(24).This suggests that RAGE plays a central role in periodontitis in type 2 DM(4)

.

This central role is achieved after interaction between RAGE and its ligands which affect the migration and phagocytosis of polymorphonuclear and mononuclear cells, producing a subgingival florawith a predominance of gram negative anaerobes. This would trigger the secretion of soluble mediators that facilitate connective tissue destruction and bone resorption (14.)

At the same time, the periodontal infection would also induce resistance to insulin in the tissues, and would contribute, in turn, to hyper-glycemia and therefore, to the accumulation of AGEs(14).This viscious circle leads to the progres-sion of both periodontitis and uncontrolled dia-betes.

Hence the aim of the study is to detect the expression of RAGE in the gingival tissues of patients with type 2 DM before and after peri-odontal therapy and its correlation to levels of glycated hemoglobin.


Subjects:The present study was conducted on 50 male individuals ranging in age from 35 to 60 years old. They consisted of 3 groups; group I consisted of 20 patients suffering from both type 2 DM and CP, group II consisted of 20 non-diabetic patients suffering from CP, group III consisted of 10 non-diabetic individuals with healthy priodontium.

Informed consent was obtained from all cases. Ethical approval was obtained from local Ethics committee.

Methods: All periodontal evaluation was

performed by one periodontist according to a uniform classification(3). Inclusion criteria for the study were: (1) Well-controlled type 2 diabetes for group I monitored by HbA1C level within the range for controlled dia-betic patients (i.e.6-8%)(2). 2Fasting plasma glucose level <100 mg/dl in non-diabetic patients (groupsII andIII(1)). (3)Generalized CP consisting of loss of attachment >30% of the existing dentation(only for group I and II)(1)(4) No other significant systemic disease(other than diabetes in group I) or reception of any drugs that may modify the periodon-tal condition in all groups.

1-Measurment of glycated hemoglobin:Blood samples were withdrawn to an EDTA

tube for measurement of (HbA1c) using high performance chromatography method which is a reference method for HbA1c on a specificapparatus (D10 Bio-Rad). A value of 6-8% was considered a controlled case(2)

.

2-Clinical parameters:

For all groups, all baseline clinical param-eters were obtained immediately before biopsy acquisition, these parameters included: Gingival index (GI), probing depth (PD), Clinical attach-ment level (CAL)

3-Biopsy acquisition for PCR detection of RAGE:

Biopsies were obtained from the marginal gingiva in areas of gingival enlargement, areas which needed crown lengthening for construc-tion of a fixed prosthesis and/or extraction sites.

Periodontal therapy (for group I and II):

Patients were subjected to thorough full-mouth supra and sub gingival scaling and root planning (SRP).Treatment was carried out in 2 sessions within 24-36 hours. Sessions were per-formed under local anesthesia using standard periodontal curettes and ultrasonic device. Sys-temic combination antibiotics (amoxicillin and flagyl)were administrated twice daily for two weeks. Instructions for proper plague control included tooth brushing with a soft tooth brush twice daily using horizontal scrub technique and mouse wash daily for 2 weeks. Oral hygiene

Role of Periodontal Therapy on the Expression of RAGE in DM 101

control was reviewed 2 times monthly during the whole study period.

Post-treatment procedures:

At the end of 3 months both group I and II were re-evaluated for:

1-Clinical parameters,2-PCR detection of RAGE,3-Measurment of HbA1c.

PCR examination of RAGE:

The gingival biopsy is kept in-70°C until pro-cessed for reverse transcriptase coupled to poly-merase chain reaction (RT-PCR).

RNA extraction:

RNA was extracted using Total RNA Puri-fication Kit (cat.#17200) (Norgen,Biotek cor-poration, Canada). Five milligrams of sample was homogenized in 350 µl of RLT buffer with 1% b-mercapthoethanol, and then centrifuged at 12,000 xg for 3 min. The supernatant was trans-ferred to a new tube and 350 µl of 70% ethanol was added. The sample was placed in aRNeasy mini column and centrifuged at 8000 x g for 15s.

The column was washed with 400 µl of RW1 buffer and also centrifuged at 8000 x g for 15 s. A solution of 10 ml of DNase I and 70 µl of RDD buffer was placed in each spin column and incubated at room temperature for 15 min. The spin column was then washed with 350µl of RW1 buffer once and with 500 µl of RPE buffer twice. Finally the RNA was eluted by placing 50 µl of RNase-free water directly in the silica gel membrane and centrifuged for 1 min at 8000 x g. The collected RNA was stored at –70°C.

RNA quantification

The quantity of the RNA isolated was deter-mined by measuring the absorbance at 260 nm and the purity was determined by the 260/280 nm absorbance ratio. The ratio should be >1.75 before the RNA can be used for RT-PCR.

RT-PCR

Semi-quantitative RT-PCR was performed using RevertAid first strand cDNA synthe-sis Kit #K1621 (Fermentas, Canada). The RAGE primers used (forward 5’-GACTCT-

TAGCTGGCACTTGGAT- 3’ and the reverse 5’-GGACTTCACAGGTCAGGGTTAC-3’) yielded a 326 bp product. In a 0.5 ml tube, 10 µl AMV/Tfl 5 x reaction buffer, 1 µl 0.2mMdNTP mix, 50 pmol forward primer, 50 pmol reverse primer, 2 µl 25mM MgSO4, 1µl (5 U/ml) AMV reverse transcriptase, 1 ml (5 U/ml) Tfl DNA polymerase, and 1 mg of the isolatedRNA were combined and brought to a volume of 50 µl with RNA-free distilled water. The firststrand of cDNA synthesis was obtained by one cycle of 45 min at 48C, followed by one cycle of 2 min at 92°C. The solution was then sequen-tially thermocycled at 95°C for 12 min, then 35 cycles for 1 min at 94°C, 1 min at 60°C and 1 min at 72°C.

The products of the RT-PCR were then sepa-rated by agarose gel electrophoresis and viewed with ethidium bromide and UV light. The bands were semiquantified using densitometry accord-ing to band thickness and density in relation to a reference.


Data were presented as means and standard deviation (SD) values. One way analysis of Vari-ance {ANOVA) was used to compare between the three groups. Duncan’s post-hoc test was used to determine significant differences between the means when ANOVA test result is significant.Student’s t-test was used to compare between PD, CAL and GI of group I andII. Paired t-test was used to study the changes after treatment in-each group. Pearson’s correlation coefficientwas used to determine significant correlations between the different variables.The significancelevel was set at P≤0.05. Statistical analysis was performed with SPSS 16.0® (Statistical Package for Scientific Studies) for windows.

RESULTS

The present study was conducted on fiftymale individuals ranging in age from 35 to 60 years, who were divided into three groups. The first group included twenty patients who had acontrolled type 2 diabetes mellitus with chronic periodontitis, the second group included twenty non-diabetic patients who had chronic periodon-titis and the third group included ten volunteers


who were systemically free with healthy gingiva and served as controls.

PCR results:

Figures (1), (2) and (3) show the expression of RAGE in the two study groups before and af-ter treatment as well as the healthy controls.Statistical analysis of our results showed:

The mean value was reduced by 0.54 % which

M 1 2 3 4 5 Figure (3): Agarose gel electrophoresis (2%) stained with ethidium bromide showing:• M lane: PCR-DNA marker (100 b.p).• Lanes (1-5): Showing extremely low expression of RAGE in healthy controls.

M 1 2 3 4 5 6 7 8Figure (2): Agarose gel electrophoresis (2%) stained with ethidium bromide showing:• M lane: PCR-DNA marker (100 b.p)• Lanes (1,3,5,7): Expression of RAGE in non-diabetic patients before periodontal therapy.• Lanes (2,4,6,8): Expression of RAGE in non-diabetic patients after periodontal therapy

M1 2 3 4 5 6 7 8Figure (1): Agarose gel electrophoresis (2%) stained with ethidium bromide showing:• M lane: PCR-DNA marker (100 b.p)• Lanes (1, 3, 5, 7): Expression of RAGE in diabetic patients before periodontal therapy.• Lanes (2, 4, 6, 8): Expression of RAGE in diabetic patients after periodontal therapy.

is equal to a 7.5% decrease in HbAlc value.

With respect to the clinical variables there was no statistically significant difference be-tween mean PD of group I and group II pre- and post-operatively with P value 0.737 and 0.063, respectively.The statistical analysis comparing the changes in PD after treatment in both groups showed a highly significant decrease in mean PDafter treatment in the two groups (P<0.001).


(Table 4): Correlation between RAGE expression and HbAlc level, PD, CAL and GI in group I and IIVariables Group I Group II

Pre-operative Post-operative

Pre-operative Post-operative

Correlation coefficient (r)

P-value

Correlation coefficient

(r)

P-value


P-value


P-value

0.389 0.090 0.258 0.272

0.293 0.210 0.347 0.134 0.197 0.406 0.110 0.645

0.176 0.459 0.335 0.149 0.089 0.710 0.441 0.756

0.216 0.360 0.084 0.724 0.211 0.437 0.065 0.917

(Table 5): Correlation between HbAlc level and PD, CAL and GI for group I

VariablesGroup I

Pre-operative Post-operativeCorrelation

coefficient (r)P-

valueCorrelation

coefficient (r)P-

valuePD 0.139 0.558 0.492 0.027*

CAL 0.072 0.763 0.438 0.048*

GI 0.418 0.066 0.288 0.219

*: Significant

(Table 3): Comparison of the changes in HbAlc levels before and after treatment in group ITreatment

Group Pre-operative Post-operativeP-value

Mean SD Mean SD

Group I 7.22 0.4 6.68 0.4 < 0.001**: Highly significant

Table (1): Comparison between mean and SD values of the level of RAGE expression among the three groupsGroup

Treatment

Group I Group II ControlP-value

Mean SD Mean SD Mean SD

Pre-operative 2.42 a 0.6 2.02 a 0.4 0.06 b 0.02 <0.001*

Postoperative 0.78 a 0.4 0.65 a 0.3 <0.001*

* Highly significant, means with different letters shows statistically significantly difference

(Table 2): Comparison between mean and SD values of the level of RAGE expression before and after treat-ment in group I and II

Treatment

Group

Pre-operative PostoperativeP-value

Mean SD Mean SD

Group I 2.42 0.6 0.78 0.4 <0.001*Group II 2.02 0.4 0.65 0.3 <0.001*

* Highly significant


Also in case of CAL There was no statisti-

cally significant difference between mean CAL

in group I and group II pre- and post-operative-

ly with P value 0.528 and 0.907, respectively.

The statistical analysis comparing the changes

in CAL after treatment in both groups showed

a highly significant decrease in mean CAL after

treatment in both groups (P<0.001)

The same was in GI where there was no sta-

tistically significant difference between mean GI

of group I and group II pre- and post-operatively

with P value 1.000 and 0.102 respectively.The

statistical analysis comparing the changes in GI

after treatment in both groups showed a highly

significant decrease in mean GI after treatment

in both groups (P<0.001).

DISCUSSION

Under normal conditions, RAGE is present

at low levels on a number of cells, while its

expression is strikingly increased during diabetes,

inflammation and other perturbed states(6).

This study has been performed to see if

treatment of periodontal disease in controlled

diabetic patients would contribute to glycemic

control management. This has been monitored

by studying the clinical response as well as by

estimating the level of RAGE expression in

gingival tissues before and after periodontal

therapy and its correlation to levels of glycated

hemoglobin in diabetic patients.

The study was carried out on male patients

only to exclude the effect of sex hormones on

the periodontium as elevated levels of estrogen

and progesterone may modify the action of

cells of the immune system, including cytokine

production(21).

Statistical analysis of the current study showed

that 3months after removal of dental plaque, there

was no significant difference between means of

RAGE in groupI (0.78±0.4), andII (0.65±0.3),

while both were significantly higher than that

of the control group (0.06±0.02) (P<0.001). This

denotes that after therapy and control of dental

plaque by the patients, RAGE did not reach the

level of healthy patients although it approached

it. This could be explained by the fact that the

habit of oral hygiene practice in these patients

never reached the extent of a healthy one or that

the host response in these patients differ from

that of healthy subjects.

The recommended HbAlc target value for

people with diabetesis <7% (normalis<6%).

However, achieving this goal was difficult as

Koro et al., 2004(11). noted in a population study,

only 36% of people with type 2 diabetes achieved

a target HbAlc of <7%.

However, here, the glycated hemoglobin of

the selected patients was considered within the

controlled level between 6-8% following the

National Diabetes Institute and depending on

the recommendations of the American Diabetes

Association, 2001.

In the current study, before treatment,

HbAlcl evels for diabetic patients with chronic

periodontitis ranged from (6.3-7.8)% with a mean

of (7.22±0.4), while after treatment it ranged

from (6-7.3)% with a mean of (6.68±0.4). Thus,

there was a 7.5% reduction in HbAlc value,

which is a highly significant decrease in mean

HbAlc after treatment (P<0.001).

The present study is in agreement with

Rodrigues et al.200320, as they performed

scaling and root planning (SRP) in combination

with amoxicillin/clavulanic acid and showed

a 4% reduction in HbAlc level after 3 months.

The slightly superior results in our study could

be due to the use of an antibiotic combination

(amoxicillin and flagyl) which significantly

reduced the systemic bacterial challenge more

than amoxicillin alone.

This result also agreed with O’Connell et

al.2008(18), who showed a 13% reduction in

the HbAlc level after SRP in conjunction with

systemic doxycycline. This could be explained

by the possibility that doxycycline has a non-

enzymatic inhibitory effect on glycation, thus

would yield more superior results than other

antibiotic regimens.

However, these findings were in contrast to

Promsudthiet al 2005(19) who conducted their

study on type2 diabetic subjects who received

scaling and root planning with adjunctive

doxycycline. They showed a significant

improvement in periodontal health but an on


significant reduction in HbAlc values. This

might be due to the fact that, unlike the present

study, they selected poorly controlled diabetic

patients.

With respect to the clinical variables,

no statistically significant differences were

observed between groupI and groupie in

the periodontal pocket depth (PD), clinical

attachment level (CAL) and gingival index (GI)

before treatment.

This agreed with Tsai et al. 2002(23) and Meng

200716 who found no difference in periodontal

parameters between well controlled diabetic

patients and non-diabetics.

However, these findings were in contrast to

Khader et al. 2008(10). who found that periodontal

disease was more severe in type2 diabetic

patients than in non-diabetics, as indicated by

significantly mean higher GI, PD and CAL.

In the present study, improvement in the

clinical parameters was evident at the end of

the study period. After periodontal therapy, as

regards PD, it showed asignificant reduction

by 41.9% in group I and by 36% in group II,

while for CAL, there was a significant reduction

by 33.6% in group Iand 35.5% in group II. The

GI also showed a significant reduction in both

groups; 40% in group I and 52% in group II.

The alterations in PD observed in our study are

in accordance with Rodrigues et al.200320 and

O’Connell et al. 2008(18), who showed reduction

in PD of 25% and 37.6% respectively in diabetic

patients who received SRP and doxycycline. The

reduction observed in our study was 41.9% a

slightly superior result that might be explained

by the fact that, unlike the present study, they

selected poorly controlled diabetic patients for

their study.

The improvement in CAL in the present

study was in agreement with O’Connell et al.

2008(18) who showed a mean CAL gain of 0.9

mm as compared to the gain in our study which

was 1.48mm.

However, these results were in contrast to

Rodrigues et al.2003(20) who found no significant

difference in mean CAL after therapy. This

may be explained by their selection of poorly

controlled diabetic patients with different host

response during healing.

In the present study, no significant difference

was found in the gingival index between diabetic

and non-diabetic patients. This could be explained

by Grisi etal. 2001(7), who stated that, gingival

index does not represent a diagnosis because it

does not distinguish between the various forms

of periodontal disease but it is associated with

them. Moreover, Karjalainen,2000(8), stated that

gingival index in diabetics increased only when

the metabolic control is poor.

The improvement in GI seen in the present

study was inagreement with Rodrigueset al.

2003(20) and O’Connell et al. 2008(18); however,

they showed an improvement in GI of 61%

and 80% respectively as compared to the pres-

ent study which showed a 40% improvement in

GI. This may be due to the fac that diabetic pa-

tients included in their study had a higher GI at

baseline due to their poor glycemic control so

the improvement n GI after therapy was expect-

ed to be higher. Moreover, the antimicrobial as

well as the anti-inflammatory effect of doxycy-

cline may be the cause of the greater results by

O’Connelletal,2008(18).

Our results also showed a positive correlation

between the level of RAGE expression and

HbAlc, PD, CAL, GI, before and after treatment

both in groupI andII, yet it was not statistically

significant. This may be due to the small number

of patients in the present study or because

statistical analysis depends on an average of

different readings of patients with different

degrees of RAGE expression whether before or

after treatment.

Previous studies have suggested a close

relationship between the control of diabetes

and periodontitis. HbAlc level was considered

a reliable marker of diabetic control and has

been shown to be associated with the extent of

periodontitis(5).

In the present study, regarding groupI,

preoperatively, there was no significant

correlation between HbAlc level and PD, CAL,

GI.

This was in accordance with Takeda et

OSMAN, N. A. ET AL.106

al.2006(22), who also found no correlation between HbA1c and periodontal status, taking into consideration that the mean HbAlc level in their study was 9.6%, which was higher than that reported in the present study (7.22%).

It is likely that there is individual patient variability in the degree to which glycemic control influences periodontal status, given the multifactorial nature of periodontal diseases, in which systemic conditions play a modifying role rather than a primary, causative role(14) as some poorly controlled diabetic patients do not develop significant periodontal destruction, just as some do not develop the classic diabetic complications. Conversely, well-controlled diabetes places the person at a lower risk for periodontal disease, similar to the risk of non-diabetic individuals; yet, well-controlled diabetic patients may still develop periodontitis just as non-diabetic individuals do(13).

However, postoperatively, there was a statistically significant positive correlation between HbA1c and both PD and CAL, denoting that’subsidence of inflammation after plaque control and reduction in PD and CAL is also reflected on the improvement of HbAlc.

While regarding GI there was no correlation as change in GI is temporary and can change within the same day.

In conclusion, our report notesthat RAGEs are present in gingival tissues of CP patients in group Ito almost the same extent as those in group II with no significant difference denoting that they are related to inflammation and not specific to the diabetic state. Also After periodontal therapy, RAGE in both study groups decreased much, but never reached the level of healthy controls, moreover the clinical parameters improved after periodontal therapy with no statistically significant difference between the two study groups, however the non-surgical periodontal therapy resulted in lower HbA1c levels in diabetic patients thus it should be included as a part of diabetes control measures. .

From these conclusions, we highly recommend that RAGE should be estimated in systemic diseases that modify tissue response

to periodontal pathogens to see if it could be considered a potential marker for periodontal inflammation. Also further studies are needed to be performed to assess the influence of periodontal therapy on the expression of RAGE in uncontrolled diabetic patients.

REFERENCES1- American Academy of Periodontology (2003): Di-

agnosis of Periodontal diseases (Position paper). J Periodontol; 74:1237.

2- American Diabetes Association (2001): Standards of Medical Care for Patients with Diabetes Mellitus. Dia-betes care, 24(suppl. 1), 33.

3- Armitage, G. C. (1999) Development of a clawdytssifi-cation system for periodontal diseases and conditions. Annals of Periodontology 4,1.

4- Chavakis T, Bierhaus A, Nawroth PP (2004): RAGE (re-ceptor for Advancedglycation end products): a central player in the inflammatory response. Microbes Infect. 6(13): 1219.

5- Collin HL, Uusitupa M, Niskanen L, et al. (1998): Peri-odontal findings in elderly patients with non-insulin de-pendent diabetes mellitus. J Periodontol; 69:962.

6- Crabtree M, Pileggi R, Bhattacharyya I, et al (2008): RAGE mRNA Expression and Its Correlation with Nu-clear Factor Kappa Beta mRNA Expression in Inflamed Human Periradicular Tissues. J Endod; 34:689.

7- Grisi M F de Moraes, Fanganiello CL Silva, and Silva-neto CR (2001): Relationship Between the Presence or Absence of Gingival Bleeding and the Enzymatic BANA Test Braz Dent J, 12(1): 23

8- Karjalainen K (2000): Periodontal diseases, dental caries and saliva in relation to clinical characteristics of type 1 DM. Department of Periodontology and Geriatric Den-tistry. Institute of Dentistry.Cited from: http://herkules.oulu.

9- Katz J, Bhattacharyya I, Farkhondeh-Kish F, et al (2005): Expression of the receptor of advanced glycation end products in gingival tissues of type 2 diabetes patients with chronic periodontal disease: “ a study utilizing un-munohistochemistry and RT-PCR. J ClinPeriodontol; 32: 40

10- Khader YS, Albashaireh ZS, Hammad MM (2008): Periodontal status of type 2 diabetics compared with non-diabetics in north Jordan. East Mediterr Health J.; 14(3):654.

11- Koro CE, Bowlin SJ, Bourgeois N, et al (2004): Gly-cemiccontrol from 1988 to 2000 among U.S. adults diagnosed with type 2 diabetes. A preliminary report. Diabetes Care, 27: 17.

12-Löe H.1993 Periodontal disease.The sixth complication of diabetes mellitus.Diabetes Care. Jan;16(1):329


states, p563.

14- Mealey BL (2006): Periodontal disease and diabetes. A two-way street. J Am Den Assoc, 137, No suppl_2, 26S.

15- Mealey BL and Ocampo GL (2007): Diabe-tes mellitus and periodontal disease. Periodontology 2000, 44: 127.

16- Meng HX(2007): Association between periodontitis and diabetes mellitus. Beijing Da XueXueBao, 39(1):18

17- Monnier VM, Sell DR, Genuth S (2005): Glycation products as a markers and predictors of the progression of the progression of diabetic complications. Ann N Y Acad Sci.; 1043:567

18- O’Connell PA, Taba M, Nomizo A, et al (2008): Effects of periodontal therapy on glycemic control and inflam-matory markers. J Periodontol.; 79(5):774.

19-Promsudthi A, Pimapansri S, Deerochanawong C, et al (2005): The effect of periodontal therapy on uncon-trolled type 2 diabetes mellitus in older subjects. Oral

Dis; 11:293.

20- Rodrigues DC, Taba MJ, Novaes AB, et al (2003): Ef-fect of non-surgical periodontal therapy on glycemic control in patients with type 2 diabetes mellitus.JPeri-odontol., 74(9): 1361.

21- Shu L, Guan SM, Fu SM, et al (2008): Estrogen modu-lates cytokine expression in human periodontal liga-ment cells. J Dent Res.; 87(2): 142.

22- Takeda M, Ojima M, YoshiokaH, Inaba H, et al (2006): Relationship of Serum Advanced Glycation End Prod-ucts With Deterioration of Periodontitis in Type 2 Dia-betes Patients J Periodontol; 77 (1): 15

23- Tsai C, Hayes C, Taylor GW (2002): Glycemic control of type 2 diabetes and severe periodontal disease in the US adult population. Community Dent Oral Epidemiol; 30(3): 182.

24-Yoko Morimoto-Yamashita a, Takashi Ito b, Ko-ichi Kawahara b,c, et al (2012): Periodontal disease and type 2 diabetes mellitus : Is the HMGB1–RAGE axis the missing link ⇑ Medical Hypotheses 79 (2012) 452


النوع2؟ من السكري مرض RAGEفى ال فى مستوى عامل هل لعالج اللثة دورخليفة رانيا بهجت – رشيد دينا – اسماعيل – اشرف درويش الدين صالح منى – عثمان نجوى عبدالحميد

عامل الدم في السكر نسبة مراقبة تعد ، و اللثة اللتهاب خطير عامل السكري مرض بأن تفيد االبحاث مجموعة كبيرة من توجدمن المرضى الذين يعانون من عليها الحصول تم التي في أنسجة اللثة بقوة ( RAGE) عامل ال واألسنان. ويوجد الفم لصحة حاسماستخدام تم قد المرضى. و هؤالء اللثة في التهاب مركزيا في يلعب دورا العامل بأن هذا وهذا يوحي .2 النوع من السكري مرضمن مجموعة 1 وتتألف مجموعات؛ إلى ثالث بتقسيمهم قمنا قد و البحث. هذا عاما فى 35 و 60 بين أعمارهم تتراوح ذكر خمسونمن يعانون الذين 20 مريضا من 2وتتألف مجموعة ،(CP) والتهاب اللثة المزمن من النوع 2 من مرض السكري 20 مريضا يعانونبمرض السكربمرض غير مصابين 10 أفراد من 3تتكون والمجموعة النوع2، من السكري بمرض مصابين غير و المزمن اللثة التهابعالج وبعد قبل األداء عالى اللوني الفصل المجموعة1باستخدام طريقة في (HbA1c) لقياس دم سحب عينات تم و قد صحية لثة عندهم،RT-PCRطريقة ال باستخدام اللثة عالج وبعد قبل ( RAGE ال( عامل لقياس المجموعات كل من اللثة من عينات اخذ تم كما اللثة،العمق ، اللثة فهرس شملت: المعلومات هذه العينة، على الحصول قبل مباشرة عليها الحصول تم اكلينيكية معلومات اخذ تم وبالمثل قدالمجموعات1و2، بين كثيرا يختلف لم (RAGE) ان مستوى ال اللثة. و قد اوضحت النتائج قبل وبعدعالج االكلينيكى التمسك مستوى ،العالج. بعد و قبل حدة على مجموعة كل فى كثيرا ايضا اختلف الضابطة وقد المجموعة من بكثير أعلى كان ولكنة و بعد العالج، قبل(RAGE)ال بين مستويات إيجابي هناك ارتباط وكان في المجموعة1. عالج اللثة وبعد قبل كبير حد إلى Hb1Acال اختلف قد وعلى يدل المجموعة1و2مما بين (RAGE) ال يختلف مستوى لم االستنتاج: احصائى. مدلول ذا لم يكن ولكن االكلينيكية والمعلوماتيجب لذلك السكري مرضى HbA1c في مستويات انخفاض إلى أدى تخص السكري. عالج اللثة وال اللتهاب الصلة ذات تكون أن

على المرض. السيطرة وسائل ادراجة كاحد

EFFECT OF MESENCHYMAL STEM CELLS ON CD4, CD8 EXPRESSION AND IL17 FROM RHEUMATOID ARTHRITIS

PATIENTS IN ALLERGENIC CO-CULTUREMaha Abd El Rafeh ELBassuoni*, Gehan Abd El Fatah*and Heba Ahmed El- Esselly**

ABSTRACTBackground: Mesenchymal stem cells (MSCs) hold immune-modulatory properties as they are immunoprivileged cells, due

to the low expression of class II major histocompatibilty complex (MHC-II). So, MSCs are promising tools for the repair of damaged joint tissue such as cartilage, bone, and tendons. Aim: the present study aimed to prove immune modulation of T- lym-phocytes from rheumatoid arthritis (RA) patients by allergenic MSCs. Subjects and methods: The present study involved 30 pa-tients of RA (age ranged from 20 to 60 years), routine data of the patients was obtained. MSCs were isolated from healthy bone marrow. MSCs co-cultured with MNCs from RA patients and the immune modulatory effect of MSCs on lymphocytes was de-tected by flocytometric expression of CD4 and CD8 and measurement of IL17 in culture supernatant by ELISA. Results: MSCshave significantly suppress the expression of CD4 (from35.5±5.7 to 11.9±3.7, p<0.001), CD8 (from 42.4±10.5 to19.02±2.5,p<0.001) and supernatant IL17 (from 142.9±54.1to 86.4±29.5 (p<0.001). Conclusion: MSCs have immune inhibitory effect on CD4, CD8 and IL17 of patients with rheumatoid arthritis in-vitro allogeneic culture. Key words: MSCs, Rheumatoid Arthritis, immune modulation of T- lymphocytes


INTRODUCTION

Mesenchymal stem cells (MSCs) are charac-terized by self-renewal and multi-directional dif-ferentiation (14).

MSCs have the advantages of easy isolation and cultivation, expansion potential, stable phe-notype, compatibility by different delivery meth-ods and minor side effects after implantation (5).

MSCs are used in regenerative medicine main-ly based on their capacity to differentiate into specific cell types and also as bioreactors of sol-uble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold immune-modulatory properties as they are immunoprivileged cells, due to the low expres-sion of class II Major histocompatibilty Complex (MHC-II) and co-stimulatory molecules in their cells and homing to injured tissues(3).

Given these properties, MSCs could be used in the allogeneic setting without the need for im-munosuppression. That said, MSCs are not inert participants, but rather, express a wide range of receptors and can release a number of cytokines and chemokines (9).

Thus, MSCs represent promising candidates for the prevention and treatment of immune-me-diated diseases (17).

The process of immunologic tolerance is critical in preventing autoimmune disease and maintaining immune system homeostasis. In-creased understanding regarding cytokine net-works has led to the development of neutralizing antibodies against tumor necrosis factor (TNF) and against interleukin-1 (IL-1) and IL-6 signal-ing in the treatment of rheumatoid arthritis (RA). However, there remains an unmet need, given the significant number of patients not achievingremission or whose condition fails to respond to these drugs. MSCs are promising tools for the repair of damaged joint tissue such as cartilage, bone, and tendons (9).

Before contemplating clinical studies with MSCs in patients with rheumatic joint disease, immunomodulatory capacity of MSCs in this set-ting needs to be explored. So, the present study aimed to prove immune modulation of T- lym-phocytes from RA patients by allergenic MSCs.


The present study was conducted at Clini-

Departments of Clinical Pathology*, Rheumatology**, Faculty of Medicine, Menoufia University, Egypt

ELBassuoni, M. A., et al.110

cal Pathology Department College of Medicine – Menoufia University during the period fromMarch 2014 to December 2014.

Subjects

1. Rheumatoid Arthritis Patients:

The present study involved 30 patients of RA (age ranged from 20 to 60 years), routine data of the patients was obtained (age, sex, ESR, CRP,RF,ACCP).

2. Bone Marrow Donors:

The study involved patients whose bone mar-row (B.M) is normocellular and without any infiltration as: Hypersplenism and IdiopathicThrombocytopenic Purpura (ITP). Written con-sent was obtained from all subjects.

Methods

1. Primary culture and isolation of aller-genic BM derived MSCs

Under complete aseptic conditions the bone marrow was diluted with sterile PBS in the ratio of 1:1. The diluted blood was layered on ficoll-hypaque (Biochrome AG)at ratio one volume of ficoll: two volume of blood then, the tubes werecentrifuged for 20 minutes at 1800 rpm. The mononuclear cells (MNCs) fraction was collect-ed, washed twice, count and viability was done using trypan blue (Sigma). The MNCs suspen-sions were plated at a concentration of (1×106 cells/ml) and allowed to adhere to tissue cul-ture plastic flasks 25cm2, incubated at 37 oC 5% CO2 in fresh complete nutrients medium which includes: DMEM-LG with L-glutamine, 10% F.B.S, Penicillin-streptomycin (10,000 U/ml and 10,000 µg/ml), Fungizone (250 µg/ml), all from Lonza. Then the media was changed twice weekly until reaching 70% to 90% confluence.After that, the adherent cells were harvested by trypsin-EDTA 0.25% solution. The harvested MSCs were characterized by flowcytometricanalysis for CD34, CD44 and Oct3/4.

2. Peripheral blood mono nuclear cells (P.B. MNCs) isolation and culture from Rheu-matoid arthritis patients

5cm peripheral blood was obtained from RA patients MNCs were separated using ficoll-hypaque solution, counted and viability was

tested.

3. Co-culture P.B. MNCs with BM mesen-chymal stem cells

Preliminary study was done firstly on7samples at different ratios of MSCs: MNCs 1:1,1:2,1:4,1:10,1:40 ,then the study was com-pleted at ratio 1:4.

MSCs were plated in 24 well culture plates in complete media. After 4 hours, 10µg Mitomy-cin c (Sigma) was added to each well for 1 hour. Cells washed twice with PBS. Then MNCs from RA patients was added then PHA (Sigma) was added at concentration 10µg/ml. Then the plates were incubated for 72 hours at 37°c and 5% co2. The sample groups were divided into the follow-ing groups: Group1, MNCs without PHA (-ve control). Group2 MNCs with PHA at concen-tration 10µg/ml without MSCs. Group3 MNCs with PHA and MSCs at ratio 4:1.

4. Study of CD4 and CD8 on lymphocytes by flowcytometry

Using flowcytometry, we investigated the ex-pression T-lymphocytes markers in the presence or absence of MSCs using the following mono-clonal antibodies FITC CD4 (Immunostep), PECD8 (Immunostep).

5. Supernatant IL17 measurement by ELI-SA.

To assess the effect of MSCs on IL17, the supernatant from co-culture was obtained and stored at -20oC for analysis by ELISA. The ab-sorbance was measured at 450 nm. A standard curve prepared from 7 human IL-17A standard dilutions and human IL-17A sample concentra-tion determined.


Data are presented as means ± standard de-viation. The results were analyzed with SPSS 17.0. The statistical significance was assessedby paired t-test, Wilcoxon signed-rank test and Pearson correlation; p value <0.05 was consid-ered statistically significant.

RESULTS

1. Characterization of isolated B.M MSCs

Effect of MSCs on CD4, CD8, IL17 From RA in Allergenic Co-CultureMSCs were successfully isolated from all

B.M samples with viability 96-100% showing adherence to plastic surface and fibroblastoidmorphology (Fig.1a). Using flowcytometry, thecells were positive for CD Oct3/4, Cd44 and negative or low expression to CD34 (Fig.2).

2. Impact of PHA on MNCs of RA pa-tients

Addition of PHA on MNCs culture signifi-cantly increase the expression CD4 (25.4±4.1 before vs35.5±5.7 after PHA, p <0.001) and CD8 (22.9±2.9 before vs42.4±10.5 after PHA,

p <0.001) table1.

3. Impact of MSCs on CD4 and CD8 ex-pression at different ratios

When MSCs co-cultured with the MNCs (Fig. 1b) at different ratios, MSCs had no effect on expression of both CD4and CD8 at ratio 1:1 (p= 0.45, 0.09) respectively, at ratio 1:2, expression of CD8 was significantly decreased (p= 0.02) butCD4 not affected (p= 0.15). Expression of both CD4 and CD8 were significantly decreased at ra-tios 1:4 (p<0.001, 0.007), 1:10 (p<0.001, 0.003) and 1:40 (p=0.02, 0.007) (Table 2).

Table (1): Effect of PHA on cultured lymphocytes

Total cases=30 Paired t- test P valueLymphocytes without PHA Lymphocytes with PHA

CD4Mean±SD 25.4±4.1 35.5±5.7 11.14 <0.001**

CD8Mean±SD 22.9±2.9 42.4±10.5 15.67 <0.001**

**Highly significant difference

Table 2: Effect of MSCs on cultured T lymphocytes of patients with RA in different ratio

Total cases=7Paired t- test P valueLymphocytes

with PHALymphocytes with MSCs Ratio 1/1

CD4 Mean±SD 38.6±4.9 36.8±3.6 0.82 0.45

CD8 Mean±SD 39.5±11.9 37.2±11.2 1.96 0.09

Lymphocytes with PHA Lymphocytes with MSCs Ratio 1/2CD4

Mean±SD 38.6±4.9 35.04±3.7 1.64 0.15CD8

Mean±SD 39.5±11.9 35.8±10.1 3.34 0.02*Lymphocytes with PHA Lymphocytes with MSCs

Ratio 1/4CD4

Mean±SD 38.6±4.9 14.8±2.8 10.20 <0.001**CD8


Ratio 1/10CD4

Mean±SD 38.6±4.9 16.3±4.5 8.33 <0.001**CD8


Ratio 1/40CD4

Mean±SD 38.6±4.9 14.01±6.8 2.37# 0.02*CD8

Mean±SD 39.5±11.9 19.1±5.3 4.07 0.007*


Figure 1: B.M derived MSCs showed fibroblastoid adherent cells with 80% confluence (1a). MSCs co-cultred with P.B MNCs (1b)

6

Figure 1: B.M derived MSCs showed fibroblastoid adherent cells with 80% confluence (1a). MSCs co-cultred with P.B MNCs (1b)

Figure 2: Mesenchymal stem cell markers by flowcytometry showing positive expression for CD44 and oct 3/4 and negative for CD34

R1

1a 1b

Figure 2: Mesenchymal stem cell markers by flowcytometry showing positive expression for CD44 and oct3/4 and negative for CD34

Effect of MSCs on CD4, CD8, IL17 From RA in Allergenic Co-Culture

4. Impact of MSCs on CD4 and CD8 ex-pression at ratio 1:4

After the preliminary results on 7 samples at different ratios, the study proceeded at ratio 1:4, the expression of CD4 and CD8 were significantlysuppressed (35.5±5.7 without MSCs vs11.9±3.7 with MSCs, p<0.001) and (42.4±10.5 without MSCs vs19.02±2.5 with MSCs, p<0.001) re-spectively figure 3 and table 3.

5. Impact of MSCs on supernatant IL17

The supernatant IL17 when measured with

and without MSCs, it was found that IL17 was significantly reduced from 142.9±54.1to86.4±29.5 (p<0.001) (Table 4).

6. Correlation between MSCs immun-emodulatory effect and RF and ACCP

Using Spearman correlation, there was statis-tically significant negative correlation betweenCD4 expression after addition of MSCs and ACCP (r=-0.479, p= 0.007). However, there was no any correlation between expression of CD4, CD8 or supernatant IL17 and RF or ACCP after addition of MSCs table 5.

Figure 3: Flowcytometric analysis of CD4 and CD8 expression of lymphocytes without MSCs (3a) and with MSCs (3b).

7

Figure 3: Flowcytometric analysis of CD4 and CD8 expression of lymphocytes without MSCs (3a) and with MSCs (3b).

Table (3): Effect of MSCs on cultured T lymphocytes of patients with RA at ratio 1:4

Total cases=30 Paired t- test P value

Lymphocytes without MSCs Lymphocytes with MSCsCD4

Mean±SD 35.5±5.7 11.9±3.7 20.91 <0.001**

CD8Mean±SD 42.4±10.5 19.02±2.5 10.44 <0.001**



DISCUSSION

MSCs represent an important paradigm in regenerative medicine attributed to their biologi-cal properties as the ability to home to sites of inflammation following tissue injury when in-jected intravenously; to differentiate into vari-ous cell types; to secrete multiple bioactive mol-ecules capable of stimulating recovery of injured cells and inhibiting inflammation and to performimmunomodulatory functions (16).

In the present study, MSCs were successfully isolated from all B.M samples with high viabil-ity and this with previous studies who reported that MSCs have the advantages of easy isolation and cultivation, expansion potential and stable phenotype (4,8,13). Previous studies investigated the immune modulatory effect of MSCs on T- cells, in vitro (6, 12).

In the present study, the use of allergenic MSCs to be co-cultured with cells of the pa-tients was chosen to evaluate its ability to affect on the lymphocytes that may be preconditioned in these patients and because of the unique abil-ity of MSCs to escape allo-recognition. In the present study, the use of PHA was effective to stimulate the CD4 and CD8 and this is with other studies(2,17).

The present study showed that MSCs have significantly suppressive effect on expression ofCD4 and CD8 on lymphocytes from RA patients in allergenic co-culture and according to our

knowledge this is the first study to investigatethis approach in RA patients. Only one study re-ported by Calkoen et al., 2013(2) who investigate immune suppressive effect of MSCs in RA but they use MSCs from the same patients instead of allergenic culture and they use juvenile pa-tients than adult RA. The suppressive effect in the present study is with previous studies who investigate this effect on lymphocytes from healthy populations (10,11).

In the present study, we investigated the ef-fect of MSCs at different ratios and there was highly suppressive at ratios 1:4, 1:10 and 1:40 but not at 1:1 and at 1:2 CD8 only showed minor suppression and no effect on CD4 and this indi-cate that the ratio of MSCs to lymphocytes is a critical issue to induce the suppressive effect. IL-17 is produced by the activated T cells, induces cytokines and chemokines expression and may play a role in skeletal tissue destruction and in-flammatory processes in the RA. In arthritis, IL-17 andIL-17F induce significant cartilage matrixrelease, inhibit new cartilage matrix synthesis and directly regulate cartilage matrix turnover(7).So, the present study investigated the immune modulatory effect of MSCs on supernatant IL17 in the allergenic culture and there was signifi-cantly down-regulation of IL17.

Qu et al., 2012 (15) showed that bone marrow–derived MSCs were able to inhibit Th17 cell differentiation via interleukin (IL)-10 secretion. The present study showed significantly negative

Table (4): Effect of MSCs on supernatant IL 17of cultured T lymphocytes of patients with RA

Total cases=30 Wilcoxon Signed Rank test

P value

Lymphocytes without MSCs

Lymphocytes with MSCs

Supernatant IL17Mean±SD 142.9±54.1 86.4±29.5 3.62 <0.001**


Table (5): Spearman correlation between effect of MSCs on T lymphocytes and ACCP and RF

ACCP RFR P value R P value

CD4 -0.479 0.007* -0.051 0.79

CD8 -0.144 0.45 -0.072 0.71IL17 -0.347 0.06 0.006 0.98

Effect of MSCs on CD4, CD8, IL17 From RA in Allergenic Co-Culturecorrelation between CD4 expression after addi-tion of MSCs and ACCP.

In conclusion, MSCs have immune inhibitory effect on CD4, CD8 and IL17 of patients with rheumatoid arthritis in-vitro allergenic culture. Therefore, the results of this study support the use of autologous MSCs in clinical trials of pa-tients with RA.

REFERENCES1. Aggarwal S,Pittenger M. 2005 Human mesenchymal

stem cells modulate allogeneic immune responses. Blood ; 105(4): 1815.

2. Calkoen F, Brinkman D, Vervat C et al., 2013 Mesen-chymal sttromal cells isolated from children with sys-temic juvenile idiopathic arthritis suppress innate and adaptive immune responses. Cytotherapy ; 15: 280.

3. De Miguel MP, Fuentes-Julián S, Blázquez-Martínez A et al., 2012 Immunosuppressive properties of mesen-chymal stem cells: advances and applications.CurrMol Med ; 12(5):574.

4. El-Ansary M, Abdel-Aziz I, Mogawer S et al., 2012 Phase II Trial: Undifferentiated Versus Differentiated Autologous Mesenchymal Stem Cells Transplantation in Egyptian Patients with HCV Induced Liver Cirrho-sis. Stemcell Review ; 8(3): 972.

5. Fang B, Shi M, Liao L, et al. 2004 Systemic infusion of FLK1+mesenchymal stem cells ameliorate carbon tetrachloride-induced liver fibrosis in mice. Transplan-tation ; 78: 83.

6. Hagmann S, Gotterbarm T, Müller T, et al. 2013 The influence of bonemarrow- and synovium-derived mesenchymal stromalcells from osteoarthri�spa-�ents on regulatory T cells in co-culture. ClinExpim-munol ; 173(3):454.

7. Hymowitz SG, Filvaroff EH, Yin Jet al. 2001 IL-17s adopt a cystineknot fold: structure and activity of a novel cytokine, IL-17F, andimplications for receptor binding. EMBO J; 20:5332.

8. Karp J, Leng G. 2009 Mesenchymal stem cell homing:

the devil is in the details. Cell Stem Cell ; 4(3): 206.

9. MacDonald G, Augello A, and De Bari C (2011): Role of Mesenchymal Stem Cells in Reestablishing Immu-nologic Tolerance in Autoimmune Rheumatic Diseases. ARTHRITIS& RHEUMATIS ; 63(9): 2547.

10. Nasef A, Mazurier C, Bouchet S et al. 2007 Leukemia inhibitory factor: role in human mesenchymal stem cells mediated immunosuppression. Cell immunol ; 253(1-2): 16.

11. NasefA,chapel A, Mazurier C et al., 2007 Identificationof Il10 and TGF-beta transcripts involved in the inhi-bition of T- lymphocyte proliferation during cell con-tact with human mesenchymal stem cells. Gene Expr ; 13(4-5): 217.

12. Nemeth K, Leelahavanichkul A, Yuen P et al. 2009 Bone marrow stromal cells attenuate sepsis via pros-taglandin E2-depentent reprogramming of host macro-phages to increase their interleukin-10 production. NA. Med ; 15(1): 42.

13. Otto W, Wright N (2011): Mesenchymal stem cells from experiment to clinic. Fibrogenesis Tissue Repair; 4: 20.

14. Pei XT (2003): Stem cell biology. Beijing: Science; 174.

15. Qu X, Liu X, Cheng K, et al. 2012 Mesenchymal stem cells inhibit Th17 cell differentiation by IL-10 secre-tion. Experimental Hematology ; 40: 761.

16. Wang S, Qu X, Zhao R. 2012 Clinical applications of Mesenchymal Stem cells. Hematology and oncology ; 5:9.

17. Zhao S, Wehner R, Bornhäuser M, et al. 2012 Immu-nomodulatory properties of mesenchymal stromal cells and their therapeutic consequences for immune-medi-ated disorders. Stem Cells Dev; 19: 607–14.Wang S, Qu X, Zhao R. Clinical Applications of Mesenchymal Stem cells. hematology and oncology ; 5: 19.

18. Zhou C, Yang B, Tian Y, et al. 2011 Immunomodula-tory effect of human umbilical cord Wharton’s jelly-de-rived mesenchymal stem cells on lymphocytes. Cellular Immunology ; 272: 33.

http://www.ncbi.nlm.nih.gov/pubmed?term=De%20Miguel%20MP%5BAuthor%5D&cauthor=true&cauthor_uid=22515979

http://www.ncbi.nlm.nih.gov/pubmed?term=Fuentes-Juli%C3%A1n%20S%5BAuthor%5D&cauthor=true&cauthor_uid=22515979

http://www.ncbi.nlm.nih.gov/pubmed?term=Bl%C3%A1zquez-Mart%C3%ADnez%20A%5BAuthor%5D&cauthor=true&cauthor_uid=22515979

http://www.ncbi.nlm.nih.gov/pubmed/?term=Hagmann%20S%5BAuthor%5D&cauthor=true&cauthor_uid=23607395

http://www.ncbi.nlm.nih.gov/pubmed/?term=Gotterbarm%20T%5BAuthor%5D&cauthor=true&cauthor_uid=23607395

http://www.ncbi.nlm.nih.gov/pubmed/?term=M%C3%BCller%20T%5BAuthor%5D&cauthor=true&cauthor_uid=23607395


الروماتيزمي االلتهاب 17لمرضي دي4 وسي دي 8وانترلوكين الليمفاوية سي الخاليا علي الميزينشيمية الخاليا تاثيرللمفاصل

العسيلي أحمد هبة – توفيق الفتاح عبد جيهان – البسيوني الرافع عبد مها

إلى العديد والتباين على النمو قادرة وهى الجدارية العظمى النخاع أيضابخاليا تعرف خاليا جذعية هى المزنشيمية الجذعية الخالياأيضا المزنشيمية و للخاليا الجذعية االساسي المصدر هو العظمى النخاع الدهنية. واألنسجة والغضاريف العظم الخاليا منها من أنماطوبنمط بسهولة العزل والزرع المزنشيمية الجذعية وتتميزالخاليا الدهنية. و األنسجة الحبل السري مثال أخرى أنسجة فصلها من يمكنعلي علي قدرتها األولي يعتمد بالدرجة العالج في المزنشيمية الجذعية الخاليا بعد الزرع. إن إستخدام طفيفة وأثارجانبية وثابت مستقرباإلضافة إعادة بناء األنسجة التالفة دورفعال في لها هذه الخاليا والتي من العوامل بعض إفراز وأيضاعلي خاصة نسيجية خاليا التمايزإليلتثبيط الجهاز يمكن استخدامها فإنه هنا ومن المناعية الوظائف التحويرفي علي يتميزبقدرته الجذعية الخاليا من النوع هذا فإن ذلك الياستخدام نجاح برغم الجهازالمناعي . لمرضي جديد أمل أعطت , ولذا فقد والكيموكايين السيتوكايين من الخاليا تفرزه هذه المناعي لمااستخدامه أن كرون إال وداء السكر مرض من لنوع االول مثال الجهازالمناعى أمراض من فىعالج العديد المزنشيمية الجذعية الخالياالسيتوكايينالزا مضادات استخدام علي الذي يعتمد الحديث العالج بعض إن محل جدال. اليزال الروماتيزمي التهاب المفاصل فىعالجالمزنشيمية الجذعية بالخاليا ولذلك فإن العالج األدوية هذه الي اليستجيب المرضي كبيرمن عدد أن حيث الدراسة مزيد من الي ليحتاجتقييم الضروري من فإنه للمفاصل الروماتيزمي عالج االلتهاب في الخاليا هذه استخدام قبل فإنه ولذلك المرضي لهؤالء األمل يبعثالجذعية استجابة الجهازالمناعى لتأثيرالخاليا الدراسه :تقييم من الهدف هؤالءالمرضي . في الجهازالمناعي التأثيرعلي قدرتها عليمن كافى بعدد تمسح الدراسة هذه وفى : البحث مواد وطرق . للمفاصل االلتهاب الروماتيزمي لمرضي الليمفاويه علىالخاليا المزنشيميةالجذعية تأثيرالخاليا معا وتقييم المزنشيمية وزرعها الجذعية والخاليا النواة أحادية لخاليا وفصال السري أوالحبل العظمى النخاع عينات

لقد البحث: نتائج . للمفاصل الروماتيزمي االلتهاب لمرضي 17 وانترلوكين الخلوي التدفق بجهاز الليمفاوية المرنشيمية علىالخاليا17لمرضي دي8وانترلوكين وسي دي4 الليمفاوية سي الخاليا تثبيط علي للخاليا الجذعية المزنشيمية القدرة العالية الحالية الدراسة أثبتت

للمفاصل الروماتيزمي االلتهاب

HUMAN TELOMERASE REVERSE TRANSCRIPTASE MRNA AS A

NOVEL TUMOR MARKER FOR HEPATOCELLULAR CARCINOMAMadeeha Y. Bakheet, Sherief I. Kamel*, Hanan O. Mohamed, Tahra M. Sherif,

Ebtsam F. Mohamed and Dina A. Mohareb

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Alpha-fetoprotein

(AFP) and Protein induced by vitamin K absence (PIVKA-II) are used for patients with HCC, but their results are controver-

sial. Telomerase is a ribo-nucleoprotein enzyme that synthesizes telomeric DNA and adds this sequence to chromosomal ends.

Thereby, it prevents telomere shortening and consequently protects the chromosomes from DNA degradation, end-to-end fu-

sion, rearrangements, and chromosomal loss. Recently human telomerase reverse transcriptase mRNA has been demonstrated

as a novel serum marker for HCC diagnosis. Aim of the work: The present study was designed to investigate the potential role

of Human telomerase reverse transcriptase mRNA (hTERT-mRNA) as a diagnostic, prognostic, non-invasive marker for HCC

Patients: This study was conducted on 90 individuals they were divided into: Group 1: 10 apparently healthy persons as con-

trols Group 2: 40patients with LC. Group 3: 40 patients with HCC The following specific investigations were done for all the

studied persons:-Serum AFP, serum PIVKA-II and h TERT mRNA in whole blood by real time PCR. Results: There were signifi-

cant elevations in both serum levels of AFP and PIVKA-II in the HCC group compared to both the LC and control groups (P <

0.04*, P < 0.000**).,(P < 0.000**, P < 0.000**) respectively. The mean hTERT mRNA level by relative unit was significantly

higher in both HCC and LC with 180 and 1.5 fold relative to controls (P < 0.001**, P < 0.000**).hTERT-mRNA was positively

correlated with AFP, PIVKA-II, tumor size and number (P < 0.04*, P <0.04*, P <0.002**p<0.001**). The serum AFP cutoff

value (≥ 52 ng/ml) resulted in 75% sensitivity, a 100% specificity, 85% diagnostic accuracy (DA) for HCC prediction. The

serum PIVKA-II cutoff level (≥7.5 ng/ml) resulted in a 85% sensitivity, a 100% specificity; 89% DA for HCC prediction. The

hTERT-mRNA cutoff level (1) showed a 99% sensitivity, a 54% specificity, 97%DA for HCC prediction, While at (≥7.9) showed

a 90% sensitivity, a 100% specificity, 95%DA for HCC prediction. Combined use of hTERT mRNA and AFP in prediction of

HCC increased sensitivity to 98% and DA 97% while combined use of AFP,PIVKA-II and h TERT m RNA increase it to 100%.

and DA 99%. Conclusion: In HCC patients ,hTERT mRNA more specific marker and is related more closely to the tumor size

and number of tumor nodules than AFP and PIVKA-II.


INTRODUCTION

Hepatocellular carcinoma (HCC) represents

an international public health concern as one

of the most common and deadly cancers world-

wide. It is the fifth most common cancers and the

third cancer-related death worldwide. It is rarely

to be detected early and usually fatal within few

years (18). Carcinogenesis of HCC is a multi-fac-

tor, multi-step and complex process, which is as-

sociated with a background of chronic and per-

sistent infection of hepatitis B virus and hepatitis

C virus .Hepatitis B and C virus infections along

with alcohol and aflatoxin B1 intake are widely

recognized etiological agents in HCC (23).

The most urgent needs are to find sensitive

markers for early diagnosis and monitoring of

postoperative recurrence of HCC, and to give

adequate treatment for HCC patients. HCC has

many characteristics, such as fast infiltrating

growth, metastasis in early stage, high-grade

malignancy, and poorly therapeutic efficacy.

HCC prognosis is poor and early detection has

been under research for many years particularly

AFP-negative HCC. These false negative results

limit the universality of AFP application. The

development of molecular biology has led to

the successful exploration and identification of

markers for HCC, which is expected to improve

the early diagnostic rate, treatment effect in ad-

dition to curative satisfaction (8).

PIVKA-II is a biomarker for HCC. Liebman

et al, firstly reported PIVKA-II as a candidate

marker for the diagnosis or prognosis of HCC(15).

When compared with total AFP, PIVKA-II

showed the highest sensitivity in the early de-

tection of small HCC. Due to these promising

results, PIVKA-II is under review as a candidate

marker for HCC by the FDA (17).

Department of clinical pathology, department of tropical medicine and gastroentrology*, Faculty of Medicine,

Assiut university, Egypt.

Bakheet, M. Y., et al.118

The genetic markers of HCC or tumor spe-

cific-protein can monitor carcinogenesis of he-

patocytes and diagnose HCC at early stage (3).

Using the appropriate single tumor marker or

combination of other markers may improve the

effectiveness in screening HCC patients. Many

clinical, tumor genetic and molecular biologi-

cal markers have been used to diagnose HCC

or predict HCC recurrence, the biomarkers that

indicate the existence of malignant cells may be

a useful predictor for HCC extra-hepatic metas-

tasis (7).

Telomerase, an RNA-dependent DNA poly-

merase, can maintain the telomeric length by

acting as reverse transcriptase. Telomerase con-

sists of two essential components; human telom-

erase reverse transcriptase (hTERT) the catalytic

subunit with reverse transcriptase activity and

(hTR) human telomerase hTERT is generally

repressed in normal cells and up regulated in im-

mortal cells. Telomerase activity is closely cor-

related with hTERT expression both in tissues &

peripheral blood (28)

In humans, tumor cells escape programmed

cell senescence through reactivation of telomer-

ase. These immortalized cells can compensate

for telomeric shortening at each cell division,

leading to progressive neoplastic evolution.

Telomerase re-expression was found in 85% of

human malignant tumors(29).

Aim of the work

1- The present study was designed to inves-

tigate the potential role of h TERT m RNA as

a diagnostic, non-invasive marker for HCC at

its early stages and to assess its sensitivity and

specificity in comparison with the usual recom-

mended parameter AFP and PIVKA-II.

2- Study the correlation between AFP, PIV-

KA-II and h TERT mRNA with tumor size and

number of tumor nodules.

3-To detect its possible role as sensitive

markers for early prediction of HCC in cirrhotic

patients.


Subjects

This study was conducted on eighty patients

40 patients with liver cirrhosis (group 2) diag-

nosed on basis of abdominal ultrasound: normal

or reduced sized liver, coarse echo pattern, ir-

regular surface, dilated portal vein and enlarged

spleen. 40 patients with Hepatocellular carcino-

ma (group 3) diagnosed on basis of abdominal

CT: hypodense mass on the pre-contrast, after

intravenous contrast there was a heterogeneous

enhancement in the tumor. Group 3 was fur-

ther sub-grouped according to Tumor size into

Group3- I ( ≤ 2cm) : 13patients .Group3- II (

2-4cm) : 10 patients. Group3- III ( >4cm) : 17

patients. And also according to number of tumor

nodules into Group 3 -1(1 Nodule): 33 patients

Group 3-2 (2 or more Nodules):7 patients. They

were recruited from the Tropical medicine and

gastroenterology department Assiut University

hospital after exclusion of patients with other

malignancies or patients receiving chemother-

apy. Ten apparently healthy individuals were

included in this study as the control group for

comparison (group 1).

Methods

10 ml of venous blood was withdrawn and

was divided into: 1.5 ml on EDTA (separation

tube for RNA) (Zhejiang Kangshi Medical De-

vices Co.,Ltd).1.8 ml immediately mixed with

0.2 ml tri-sodium citrate for prothrombin time

and concentration.2 ml on EDTA for determina-

tion of peripheral haemogram.4.7 ml into was-

serman tube left to clot The separated serum was

immediately divided into aliquots for routine

and special investigations. Alphafetoprotien was

measured by chemiluminescent assay by IM-

MULITE 1000 autoanalyzer a kit supplied by

Siemens (Cat. no. L2KAP2), PIVKAII was done

by a sandwich ELISA kit supplied by Glory Sci-

ence (Cat.no. 95595).

Whole blood h TERT RNA m RNA quantifi-

cation was done by Real time PCR, after RNA

extraction using QIAamp RNA Blood Mini

Kit supplied by QIAGEN®, Germany) (Cat.

no.52304). Complementary DNA (cDNA) was

synthesized using 7500 Fast Real-Time PCR

System (Applied Biosystems®USA) kit supplied

by Thermo Scientific® Germany (cat no.1641) ,

Synthesis Kit is a convenient system optimized

Human TERT mRNA, a Novel Marker for HCC 119

for cDNA synthesis in two step real time quanti-

tative RT-PCR (RT-qPCR) applications. The kit

uses Maxima Reverse Transcriptase, an advanced

enzyme derived in vitro. The enzyme features

high thermostability, robustness and increased

cDNA synthesis rate. The Maxima First Strand

cDNA Synthesis Kit is capable of reproducible

cDNA synthesis from a wide range of starting

total RNA amounts (1 pg - 5 μg) at elevated

temperatures (50-65°C). The synthesis reaction

can be completed in 15-30 minutes. Then RNA

quantization was done using 7500 Fast Real-

Time PCR System (Applied Biosystems®USA)

kit supplied by Thermo Scientific™ Solaris™

qPCR,Germany k#0351. The sequences of the

primers and probe used to amplify and detect

GAPDH (Cat.no.: HS3439098) are :

Forward Primer: 5’-GAAGGTGAAGGTCG-

GAGTC-3’

Reverse Primer: 5’-GAAGATGGTGAT-

GGGATTTC-3’

Probe: VIC-CAAGCTTCCCGTTCT-

CAGCC-TAMRA

The sequences of the primers and probe used to

amplify and detect hTERT(Cat. no.: HR3126958)

are :

Forward primer: 5’-TGACACCTCACCT-

CACCCAC-3’

Reverse Primer: 5’-CACTGTCTTCCG-

CAAGTTCAC -3’

Probe: FAM-ACCCTGGTCCGAGGTGT-

GTCCCTGA -TAMRA

The 7500 Fast Real-Time PCR system soft-

ware mathematically uses the comparative meth-

od (2− ΔΔCt) for calculating relative quantifica-

tion (Delta-Delta method for comparing relative

gene expression) to produce the results and the

result graphs: (Livak and Schmittgen, 2008).

Statistical analysis:

• Data collected and analysed by comput-

er program (SPSS ver 17) Chicago. USA.

§ Data expressed as mean ± SE.

§ ROC curve was ploted to determine the

senestivity and speceficity NPV,PPV and DA for

different cut off values.

§ For statistical evaluation, independant T

test used to determine significant for numeric

variables .

§ Person’s correlation used to evaluate the

correlation between different parameters in each

group.

§ Results were as following:

● Insignificant: P > 0.05

● Significant: P < 0.05*

● Highly significant: P < 0.001**

RESULTS

Serum AFP, PIVKA-II and h TERT mRNA in

the studied groups: (Table 1)

Serum levels of AFP and PIVKA-II showed

significant elevations in LC and HCC groups

(2, 3) compared to controls (group 1) (P <0.000,

0.000)(p<0.03*,<0.000). Also in HCC group

compared to LC group ( p<0.04 and, 0.001).In

group 2, hTERT mRNA mean value by relative

unit (1.04±0.24).In group 3, hTERT mRNA mean

value (179.2±45.31). There was a highly signifi-

cant elevation in hTERT mRNA Level in HCC

(group 3) compared to LC (group 2) (P<0.001).

The hTERT mRNA expression was significantly

increased with 180 fold and 1.5 fold in HCC and

cirrhosis groups respectively relative to the cut

off value of controls (cut-off value was set to 1 )

(p<0.000).

Serum AFP, PIVKA-II and hTERT mRNA

in LC subgroups according to Child-Pugh score:

table (2)

No statistically significant differences were

found in AFP and PIVKA-II levels between the

three groups compared to each others. There

was a statistically significant elevation in hTERT

mRNA level in group 2 B compared to group 2

A (P=0.04).Also there were significant elevations

in hTERT mRNA levels in group 2 C compared to

groups 2 B and 2 A (P<0.03, <0.001).

Serum AFP, PIVKA-II and h TERT mRNA in HCC subgroups according to tumor size: (Table 3)

High significant elevation was found in com-

paring AFP level in group 3 -III to group 3- I

(P< 0.003) .No statistically significant difference

was found in comparing PIVKA-II levels in the

three groups compared to each other. Highly

statistically significant elevations were found in


hTERT mRNA in group3-II and 3-III compared

to group3- I (P< 0.005, <0.001). Also there was a

highly statistically significant elevation in group

3-III compared to group 3-II(P<0.001).

Serum AFP, PIVKA-II and h TERT mRNA in

HCC subgroups according to number of tumor

nodules: table (IV)

High statistically significant elevation was

found in AFP and hTERT mRNA levels in group

3-2 compared to group 3-1(P < 0.000). Statistical-

ly insignificant elevation was found in PIVKA-II

level in group 3-2 compared to group 3-1.

Receiver operating characteristic (ROC) curve

was plotted to show the accuracy of AFP, PIVKA-

II and hTERT mRNA in the diagnosis of HCC:

(Table 5 and Fig.1)

By using ROC curve AFP at cut off value ≥

52 ng/ml revealed sensitivity 75 %, specificity

100% and Diagnostic accuracy 85% .PIVKA-II

at cut off value ≥ 7.5 ng/ml revealed sensitiv-

ity 85 % and specificity 100% and Diagnostic

accuracy 89% and hTERT mRNA at cut off

value ≥7.9 revealed sensitivity 90 %,specific-

ity 100%, and a DA 95%.The combination of

AFP and h TERT m RNA showed 98%sensitvity

100% specificity 100% DA 97%.The combi-

nation of AFP,PIVKA-II and h TERT m RNA

showed 100%sensitvity 100% specificity 100%,

DA 100%. Area under Roc Curve was =0.937,

0.965, 0.988 for AFP, PIVKA-II and hTERT

mRNA respectively.

Groups

Items

Group 1 (controls)

N=10

Group 2

(LC)

N=40

Group 3

(HCC)

N=40

P-value

AFP (ng/ml) 2.35± 0.40 8.93± 1.56 166.0± 17.79

2 vs1< 0.000**

3 vs1< 0.000**

3 vs2< 0.04*

PIVKA-II (ng/ml) 0.79± 0.03 2.43± 0.28 11.9± 0.74

2 vs1< 0.03*

3 vs1<0.000**

3 vs2<0.000**

hTERTmRNA

(relative unit)------ 1.04± 0.24 179.2± 45.31 3 vs2<0.001**

Insignificant: P > 0.05

Table (1): Serum AFP, PIVKA-II and h TERT mRNA in the studied groups (mean±SE).

Insignificant: P > 0.05

Significant: P < 0.05*

Highly significant: P < 0.001**

Groups

Items

Child-Pugh in LC subgroups

P-valueGroup 2 A

(N=8 )

Group 2 B

(N=15)

Group 2 C

(N=17)

AFP (ng/ml) 11.9±5.0 5.4±1.4 10.5±2.4

B vs A =0.133

C vs A=0.792

C vs B =0.092

PIVKA-II(ng/ml) 2.7±0.7 2.6±0.4 2.0±0.4

B vs A =0.932

C vs A =0.429

C vs B =0.376

h TERT mRNA

(relative unit)0.1±0.08 0.6± 0.07 1.8±0.5

B vs A <0.04*

C vs A <0.001**

C vs B <0.03*

Serum AFP, PIVKA-II and h TERT mRNA in HCC subgroups according to tumor size: (table III)

Table (2): Serum AFP, PIVKA-II and h TERT mRNA in LC subgroups according to Child-Pugh score

(mean±SE).

Serum AFP, PIVKA-II and h TERT mRNA in HCC subgroups according to tumor size: (table III)


Groups

Items

Group 3-1

( N=13)

<2cm

Group 3-II

(N=10)

2-4cm

Group 3-II

(N=17)

>4cm

P-value

AFP (ng/ml) 87.4± 28.0 175.7± 25.5 214.1± 21.7

II vs I =0.073

III vs I < 0.003**

III vs II=0.28

PIVKA-II (ng/ml) 11.6± 1.3 11. 3± 1.5 12.1± 1.1

II vs I =0.894

III vs I < 0.771

III vs I1=0.681

h TERT mRNA

( relative unit)82.7± 28.0 193.2± 77.5 243.8± 123.2

II vs I <0.005**

III vs I < 0.001**

III vs I1 <0.001*

Serum AFP, PIVKA-II and h TERT mRNA in HCC subgroups according to number of tumor nodules:

Table (3): Serum AFP, PIVKA-II and h TERT mRNA in HCC subgroups according to tumor size

(mean±SE).

Groups

Items

Group 3-1

( N=33)

(1 Nodule)

Group 3-2

(N=7)

(2 or more)

P-value

AFP (ng/ml)

135.1± 17.0

311.4± 15.0.

< 0.000**

PIVKA-II (ng/ml) 11.3± 0.8 14. 5± 1.6 < 0.591

NS

h TERT mRNA

( relative unit)167.0± 1.02 233.0± 50.8

<0.001**

Table (4): Serum AFP, PIVKA-II and h TERT mRNA in HCC subgroups according to number of tumor

nodules (mean±SE)

AFP PIVKA-II hTERTmRNAAFP+hTERT

mRNA

AFP+PIVKAII+h

TERT mRNA

Sensitivity 75% 85% 90% 98% 100%

Specificity 100% 100% 100% 100% 100%

PPV 100% 100% 100% 100% 100%

NPV 15.0% 52% 82% 76% 65%

Accuracy 85% 89% 95% 97% 99%

Cut off point ≥52ng/ml ≥7.5ng/ml ≥7.9(relative unit)≥52ng/ml+7.9

(relative unit)

≥52ng/ml+

7.5ngl+7.9

(relative unit)

Figure (1) Receiver operating characteristic (ROC)

Table (5): The sensitivity, specificity and diagnostic accuracy of the three markers in HCC prediction


DISCUSSION

The expression of telomerase was associ-

ated with HCC development and its abnormal-

ity in liver tissues or in peripheral blood could

be a useful marker for diagnosis and prognosis

of HCC (20). The present study spots a beam of

light on the role of telomerase in HCC and LC

to find its possible role in prediction of HCC as

compared to the commonly used markers such

as AFP and PIVKA-II.

�

0 200 400 60080 1000� 1200�

hTERT

0

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1

1.5

2

2Child- pugh.5

3

3.5

�

r = 0.461

p<0.003** � �

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0 200 400 600 800� 1000� 1200

h TERT m RNA

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0.5

1

1.5

2

2.5

3

3.5

Tumor size

r = 0.461�p<0.003**�

Fig.2Correlation between h TERT mRNA and Child-

Pugh score in LC group

Figure 1 correlation coefficient between hTERT

m RNA and tumor No.in HCC group

�

0 200 400 6008001000� 1200�

h TERT m RNA�

0

0.5

1

1.5

2

2.5

3

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r = 0.461p<0.003**

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Fig.(3,4,) Correlation between h TERT mRNA and AFP, Tumor No. in HCC group.

Figu

Cor

The

The

Figure (1) Receiver operating characteristic (ROC) curve for AFP, PIVKA-II and h TERT m RNA as a predictor

factor for HCC. Area under Roc curve for AFP =0.937, PIVKA-II=0.965, hTERT mRNA=0.988

Correlation coefficients of h TERT mRNA with tumor size and AFP in HCC group &Correlation coefficient of h

TERT mRNA with Child-Pugh score in patients with LC group.

There were a significant positive correlations between hTERT with the Tumor size, number and AFP (r= 0.461 and

P<0.003),(r= 0.484 and P<0.002 ) ( r= 0.541 and P<0.003 ) respectively in HCC group .

There was significant positives correlation between h TERT mRNA and Child-Pugh score in LC group (r= 0.412

and P<0.005).


The present study showed significant eleva-

tion in AFP level in LC group compared to con-

trol group. AFP in chronic hepatitis or cirrhosis

raises in 20% and 40% of patients, respectively,

and tends to fluctuate in the course of the dis-

ease (27). 16 LC patients (representing40%) have

serum level below the calculated cutoff of the

control (≥4ng/ml). This is to be explained by the

fact reported by Cortez-Gonzalez and Zanetti

that AFP is significantly decreased in cirrhotic

patients receiving interferon therapy for HCV(6).

In this study serum level of PIVKA-II was

found to be significantly higher in LC group

compared to control groups which is in accor-

dance with Nakatsura et al., who reported high

concentrations of PIVKA-II and AFP in the cir-

rhotic compared to control groups. In this study,

significant elevation in hTERT mRNA level in

LC group compared to control group(24). Normal

hepatocytes may express negligible amount of

hTERT mRNA and inflamed hepatocytes still

express more weakly than hepatocellular carci-

noma cells. The expression of the mRNA encod-

ing hTERT closely reflects the telomerase activ-

ity (19).

In LC group classified according to Child-

Pugh score no statistically significant differences

were found in serum AFP and PIVKA-II levels

between the three groups. Also by using correla-

tive studies no statistically significant correlation

could be found between serum AFP and serum

PIVKA-II in relation to Child-Pugh score in cir-

rhotic group. Trevisani et al.,(31) and EKram et al.,

reported that AFP showed no significant correla-

tion to any of the clinicopathological variables

in cirrhotic group. Also Farinati et al., reported

that PIVKA-II was not correlated to any of the

clinico-pathological variables in chronic liver

disease (10).

On the other hand the current work revealed

a highly statistically significant elevation in

hTERT mRNA level in child-Pugh B compared

to group child-Pugh A. Also there were signifi-

cant elevations in hTERT mRNA levels in child-

Pugh C compared to both group B and group A,

and its level was found to be positively corre-

lated with Child-Pugh score in cirrhotic group.

Telomerase was significantly reactivated in liver

cirrhosis but significantly lower than in HCC(21).

In HCC group, the present work showed

highly significant elevation in serum AFP lev-

el in HCC group compared to LC and control

groups. Results are parallel to those reported by

Miura et al .,(19) and Ekram et al.,(21). This eleva-

tion most probably due to liver cancer .As AFP is

a serum glycoprotein that was first recognized as

a marker for HCC and has since been described

to detect preclinical HCC. The fetal yolk sac and

fetal liver generate high levels of AFP, which

decline to 5-10 ng/dl within 300 days of birth.

Serum elevations thereafter suggest underlying

pathology which may be malignant. AFP secre-

tion in HCC resulted from re-expression in the

tumor of fetal origin(19). In this work other causes

of elevation such as cancers were excluded.

Our study showed highly significant eleva-

tion in PIVKA-II level in HCC group compared

to LC and control groups. Chaur et al ., reported

that PIVKA-II act as autologous mitogen for

HCC cell line it is elevated in 50%-80% of pa-

tients with HCC(5) .

In the present study, hTERT mRNA was de-

tected in peripheral blood of 99% in HCC group,

87% in LC. Similar detection rates for telom-

erase expression in HCC and LC was reported

by Wu et al.(32) 83%, 43.2%, respectively and by

Deng Fu et al(7). 85.5%and 45% respectively. In

the present study, although hTERT mRNA ex-

pression level was detectable in HCC as well

as LC, the mean level was significantly higher

in HCC group than LC. The hTERT mRNA ex-

pression was significantly increased with 180

fold and 1.5 fold increase in HCC and cirrhosis

groups respectively when compared to that of

controls Amal Abu El-Fadle et al , reported 699

fold and 33fold increase in HCC and cirrhosis

groups correspondingly when compared to that

of controls (2). Wu et al.,(32) Miura et al., Yao et

al., Satra et al.,(28) , Miura et al.,(20), El-Saeed et

al.,(12) Abdel Had et al.,(1) and Ebid et al.,(9) all

reached the same final results parallel to results

of present study that is; telomerase is significant-

ly increased in HCC compared to LC. This is to

be explained by the striking positive correlation


between high telomerase activity and tumors of

different histological origins and types. That’s

why although telomerase per se is not carcino-

genic, it plays a direct role in oncogenesis by al-

lowing the precancerous cells to proliferate con-

tinuously and become immortal(33).

A significantly increased telomerase ac-

tivity in peripheral blood or peripheral blood

mono¬nuclear cells (PBMCs) has been observed

in patients with hepatocellular carcinoma, which

suggests a close association between the activa-

tion of telomerase and the tumorigenesis of the

liver(6).

In this study five patients were below the cut-

off (≥7.9) , those patients had decompensated

liver cirrhosis. In decompensated liver cirrhosis

patients have higher levels of serum transform-

ing growth factor-ß(TGF-ß) that promotes apop-

tosis of immortalized hepatocytes and in these

cases elevated TGF-ß may stimulate apoptosis ,

resulting in reduction of hTERT mRNA (4).

Nittis et al (25)., suggested that immortal he-

patocytes may acquire an Alternative Lengthen-

ing of Telomerase (ALT) mechanism for main-

tenance of their chromosomal stability. More

than 85-90% of all human cancers express the

catalytic subunit of telomerase. The importance

of telomere maintenance is understood by work

demonstrating that inhibition of hTERT results

in telomere loss and crisis. The remaining 10-

15% of human cancers are completely devoid of

telomerase activity, yet maintain stable telomere

lengths through activation of the ALT pathway.

In this study as regards to the tumor size it

was found that the AFP was significantly higher

in HCC patients with lesion > 4cm compared to

those patients with tumor size < 2cm or2-4 cm.

Also a significant positive correlation was found

between serum AFP in relation to tumor size.

The same result was reported by Gupta et al .,(13)

Francisco et al.,(11) Abid et al.,(9) Who revealed

that AFP, level was positively correlated only to

tumor size. This could be attributed to the fact

that AFP has less performance in early stages

and small size tumor (less than 3 cm) (7).

As regards to PIVKA-II no statistically

significant elevation was found in comparing

PIVKA-II levels in the three groups. Also no

significant correlation could be found between

PIVKA-II in relation to tumor size. Yoshida et

al.,(30) found that PIVKA-II did not increase in

relation to tumor size and have no correlation

with the tumor size. As a result, diagnostic appli-

cation of serum PIVKA-II levels to small liver

tumors is limited.

A highly statistically significant elevations

were found in hTERT mRNA levels in patient

with lesion < 2cm or 2-4 cm or >4cm. This find-

ing was supported by finding a significant posi-

tive correlation between hTERT mRNA and tu-

mor size. This is to be explained by the striking

positive correlation between telomerase and tu-

mors of different sizes and number (22).

As regards to the number of tumor nodules a

high statistically significant elevation was found

in serum AFP in patients with multiple nodules

compared to patients with single nodule. This

could be explained by the fact that AFP increas-

es with the progression of the disease (13). In this

study as regards to the number of tumor nodules

a statistically insignificant elevation was found

in PIVKA-II level in patients with multiple nod-

ules compared to PIVKA-II level in patients

with single nodule.

A high statistically significant elevation was

found in hTERT mRNA level in patients with

multiple nodules compared to hTERT mRNA

level in patient single nodule. This is to be ex-

plained by the fact that hTERT is up-regulated

during the rapid proliferation of tumor during

phases of oncogenesis and differentiation (22).

As regards correlation between the studied

markers and tumor number hTERT mRNA and

PIVKA-II showed significant positive correla-

tion between both and number of tumor nodules.

In agreement with our results Norimasa et al.,(26).

reported positive correlation between both and

tumor of different sizes and number.

In this study the best cutoffs that could dif-

ferentiate between HCC and LC patients were,

>52 ng/ml with a sensitivity of 75%, a specific-

ity 100%, PPV level of 100%, NPV of 15%, di-

agnostic accuracy 70% and >7.5ng/ml with a

sensitivity of 85%, specificity 100%, PPV level

Human TERT mRNA, a Novel Marker for HCC 125of 100%, NPV of 82%, diagnostic accuracy 78% and > 7.9 with a sensitivity of 90%, specificity100%, PPV level of 100%, NPV of 52% and 90% diagnostic accuracy for AFP, PIVKA-II and h TERT mRNA respectively.

Our findings revealed that h TERT m RNAis a better marker than AFP in early stages of HCC as 8 patients had tumor size < 2 cm were negative for AFP at cut off ≥52 ng/ml, mean-while those patients were positive for h TERT m RNA at cutoff value ≥7.9.Wu et al.,(32) suggest that AFP sensitivity is lower in certain (African Americans) . Farinati et al.,(10) reported that the usefulness of AFP testing for HCC diagnosis is questioned because AFP misses many HCC cas-es and arouses suspicion of malignancy in many cirrhotic patients. So it has limited utility in dif-ferentiating HCC from benign disorders for its high false positive and negative rates and also patients with acute viral exacerbation may have elevated AFP.

The combination of AFP and h TERT mRNA showed 98% sensitvity 100% specificity 100%PPV and 76% NPV value DA 97%. Abou El-Fadle et al.(2) reported that combined hTERT mRNA and AFP could increase sensitivity for HCC diagnosis to 90.5%. Deng Fu et al.(7) also re-ported that AFP in combination with telomerase expression in peripheral blood could increase ac-curacy for HCC diagnosis to 92.6%. Abdel Hady et al.,(1) reported that Combination of both AFP and telomerase is increasing sensitivity (90.4%), specificity (82.3%) and accuracy (89.9%). In ourstudy the combination of the three markers in-creases the sensitivity and specificity to 100%, PPV and 65% NPV value DA 99%.

Conclusion

hTERT mRNA is more sensitive, specificmarker for prediction of HCC than AFP and PIVKA-II. Also it is correlated to the size and number of tumor nodule, so it could be used as a prognostic tool. We recommend testing of AFP with telomerase, as in combination they showed higher overall accuracy. Also the follow up of cirrhotic patients with hTERT mRNA, as a pre-dictor of HCC development.

Human telomerase reverse transcriptase mRNA 117level of 100%, NPV of 82%,diagnostic accuracy 78% and> 7.9 with a sensitivity of 90%, speci-ficity 100%, PPV level of 100%, NPV of 52%and 90% diagnostic accuracy for AFP, PIVKA-II and h TERT mRNA respectively.

Our findings revealed that h TERT m RNA isa better marker than AFP in early stages of HCC as 8 patients had tumor size < 2 cm were nega-tive for AFP at cut off ≥52 ng/ml, meanwhile those patients were positive for h TERT m RNA at cutoff value ≥7.9.Wu et al.,(2005) suggest that AFP sensitivity is lower in certain (African Americans) . Farinati et al.,(2006) reported that the usefulness of AFP testing for HCC diagnosis is questioned because AFP misses many HCC cases and arouses suspicion of malignancy in many cirrhotic patients. So it has limited utility in differentiating HCC from benign disorders for its high false positive and negative rates and also patients with acute viral exacerbation may have elevated AFP.

The combination of AFP and h TERT m RNA showed 98%sensitvity 100% specificity100%PPV and76 %NPV value DA 97%. Abou El-Fadle et al. (2011) reported that combined hTERT mRNA and AFP could increase sensi-tivity for HCC diagnosis to 90.5%. Deng Fu et al. (2006) also reported that AFP in combination with telomerase expression in peripheral blood could increase accuracy for HCC diagnosis to 92.6%. Abdel Hady et al.,(2013) reported that Combination of both AFP and telomerase is in-creasing sensitivity (90.4%), specificity (82.3%)and accuracy (89.9%).In our study the combina-tion of the three markers increases the sensitivity and specificity to 100%,PPV and 65 NPV valueDA 99%.

Conclusion

hTERT mRNA is more sensitive, specificmarker for prediction of HCC than AFP and PIVKA-II. Also it is correlated to the size and number of tumor nodule, so it could be used as a prognostic tool. We recommend testing of AFP with telomerase, as in combination they showed higher overall accuracy. Also the follow up of cirrhotic patients with hTERT mRNA, as a pre-dictor of HCC development.

REFERENCES 1. Afaf Abdel Hady, Faten El Shanawany, Mona M. Has-

san, Amgad Anas, et al. : (2010) Evaluation of Human Telomerase Activity as a Novel Tumor Marker for He-patocellular Carcinoma , Life Science Journal 4, 7.

2. Amal Abou El-Fadle, Naglaa Fathy Al Husseini Adel F. Al-Kholy, et al. (2011): Tolemerase Reverse Transcrip-tase Gene Expressions a Tumor Marker for Hepatocel-lular Carcinoma .American Journal of Biochemistry and Biotechnology 7 (2): 55.

3. Bae JS, Park SJ, Park KB (2005): Acute exacerbation of hepatitis in liver cirrhosis with very high levels of alphafetoprotein but no occurrence of hepatocellular carcinoma.Korean J Intern Med20(1):80.

4. Cavin LG, Romieu-Mourez R, Panta GR (2003): Inhi-bition of CK2 activity by TGF-beta1 promotes Ikap-paB-alpha protein stabilization and apoptosis of im-mortalized hepatocytes. Hepatology. 38(6):1540.

5. Chaur-Shine Wang, Chih-Lin Lin, Hsi-Chang Lee, et al. (2005): Usefulness of serum �� prothrombin in detection of hepatocel�� omaWorld J Gastroenterol 11(39): 6115

6. Cortez-Gonzalez X, Zanetti M (2007): Telomerase immunity from bench to bedside: round one. J Transl Med. 5:12.

7. Deng-fu Yao, Zhi-Zhen Dong and Min-Yao (2007): Specific molecular markers in HCC. HepatobiliaryPancreatic Dis. Int; 6(3):15.

8. Dong ZZ, Zou L, Qiu LW, Yao DF (2006): Expressions of hepatoma and circulating TGF-b1-mRNA and its clinical values in diagnosis of patients with liver cancer. J Gastroenterol Hepatol 21(S2):A172.

9. Ekram Y.H Ebid, Emtethal E Elkholy, Mohamed S Mo-stafa, et al. (2013):Telomerase as a novel serum tumor marker for detection of hepatocellular carcinoma Jour-nal of American Science;9(2).

10. Farinati F, Marino D, De Giorgio M (2006): Diagnostic and prognostic role of alpha-fetoprotein in Hepatocel-lular carcinoma: both or neither? Am J Gastroenterol. 101(3):524.

11. Francisco A Durazo, Lawrence M Blatt, William G Co-rey, et al. (2008) : Des-g carboxyprothrombin, afetopro-tein and AFP-L3 in patients with chronichepatitis, cir-rhosis and Hepatocellular carcinoma. Hepatology;10: 1445.

12. Gehan Kamal El-Saeed , Maha A. El-Bassuoni 1, Hala Hany El-Saeed , et al. (2009):Molecular Detection of Human Telomerase mRNA (hTERT-mRNA) in Egyp-tian Patients with Hepatocellular Carcinoma.Hepatitis Monthly 9(2): 114.

13. Gupta S, Bent S and Kohlwes J (2003): Test character-istics of alpha fetoprotein for detecting hepatocellular carcinoma in patients with hepatitis C. A systematic re-

Bakheet, M. Y., et al.126 Bakheet , Y. B., et al.118

view and critical analysis. Ann Intern Med; 139 :46.

14. Jorge A. Marrero, Ziding Feng, Yinghui Wang, et al. (2009) ; Alpha-fetoprotein and DCP in early hepatocel-lular carcinoma, Gastroenterology; 137:110.

15. Liebman HA, Furie BC, Tong MJ, et al. (1984). Des-γ-carboxy (abnormal) prothrombin as a serum marker of primary hepatocellular carcinoma. N Engl J Med 310:1427.

16. Lucia Carulli, Claudia Anzivino (2014): Telomere and telomerase in chronic liver disease and hepatocarcino-ma. World J Gastroenterol 20(20): 6287.

17. Marero JA , Su GL , Wei W (2003). Des-gamma-car-boxy prothrombin can differentiate hepatocellular car-cinoma from non malignant chronic disease in Ameri-can patients . Hepatology . 37:1114.

18. Michael C Kew (2014): Hepatocellular carcinoma: epi-demiology and risk factors. Journal of Hepatocellular Carcinoma .1: 115.

19. Miura N, Maeda Y, Kanbe T, Yazama H, et al. (2005): Serum human telomerase reverse transcriptase messen-ger RNA as a novel tumor marker for hepatocellular carcinoma. Clin Cancer Res; 11 :3205.

20. Miura N, Maruyama S, Oyama K, et al. (2007) :De-velopment of a novel assay to quantify serum human telomerase reverse transcriptase. messenger RNA and its significance as a tumor marker for hepatocellularcarcinoma. Oncology; 72(1) :45.

21. Miura N, Shiota G, Nakagawa T, et al. (2003): Sensitive detection of hTERTmRNA in the serum of patients with hepatocellular carcinoma. Oncology 64:430.

22. Miura, N., Y. Osaki, M. Nagashima, et al. (2010):A novel biomarker TERTmRNA is applicable for early detection of hepatoma. BMC Gastroenterol, 10: 46.

23. Montalto G, Cervello M, Giannitrapani L, et al. (2002): Epidemiology, risk factors, and natural history of hepa-tocellular carcinoma. Ann N Acad Sci 963: 13.

24. Nakatsura T, Yoshitake Y, Senju S, Monji M, Komori H, Motomura Y(2003):Glypican 3, overexpressed specifi-cally in human hepatocellular carcinoma is a novel tu-

mor marker. Biochem Biophys Res Commun 6(1):16.

25. Nittis, T., L. Guittat and S.A. Stewa (2008):Alternative Lengthening of Telomeres (ALT) and chromatin: Is there a connection? Biochem, 90: 5.

26. Norimasa Miura a Shigeo Maruyama b Kenji Oyama b Yutaka Horie b Michimori Kohno c Eijiro Noma d Seigo Sakaguchi (2007): Serum Human Telomerase Reverse Transcriptase Messenger RNA and Its Signifi-cance as a Tumor Marker for Hepatocellular Carcino-ma, Oncol.,72:45.

27. Randeep Kashyap, Ashok Jain, Michael Nalesnik, et al. (2001): Clinical Significance of Elevated α-Fetoproteinin Adults and Children.Digestive Diseases and Scienc-es. 46, 8, 1709.

28. Satra M, Gatselis N, Iliopoulos D, et al. (2007): Real-time quantification of human telomerase reverse tran-scriptase mRNA in liver tissues from patients with he-patocellular cancer and chronic viral hepatitis. J Viral Hepat. 14(1):41.

29. Satyanarayana A, Manns M and Rudolph KL (2004): Telomeres and telomerase: a dual role in hepatocar-cinogenesis. Hepatology .40 :276

30. Shuhei yoshida, kazutaka kurokohchi, keiji arima, et al. (2002); clinical significance of lens culinaris agglutinin-reactive fraction of serum alphafetoprotein in patients with hepatocellular carcinoma international journal of oncology 20: 305.

31. Trevisani F, D’ Intino PE, Grazi GL, et al. (2001): Se-rum alpha fetoprotein for diagnosis of HCC in patients with chronic liver disease. Hepatology; 34 (4): 570.

32. Wu W, Yao DF, Qiu LW (2005):Abnormal expression of hepatomas and circulating telomerase and its clinical values. Hepatobiliary Pancreat Dis Int. 4(4):544-9. 11.

33. Xochtil C and Maurizio Z (2007): Telomerase immuni-ty from bench to bedside: round one. Journal of Trans-lational Medicine; 5 :5.

34. Yao DF, Wu W, Yao M, (2006): Dynamic alteration of telomerase expression and its diagnostic significance inliver or peripheral blood for hepatocellular carcinoma.World J Gastroenterol.12(31):4966.


الحامض الريبوزي الرسول للمنتسخه العكسيه التيلوميريه واستخدامه كدالله جديد$ لسرطان الكبد

مديحه يونس بخيت - شريف ابراهيم كامل - حنان عمر محمد - طاهر$ محمد شريف

ابتسام فاروق محمد - دينا احمد محارب

يعتبر سرطان الخاليا الكبديه من أكثر األروام الخبيثة شيوعا في جميع أنحاء العالم. ويستخدم األلفافيتوبروتين وكذلك البروتين الناتج عن غياب فيتامين ”ك“ كدالالت لهذا المرض ولكن نتا ئجهما مصدر للجدل. التلوميريز هو عبارI عن انزيم من مجموعة الريبونيكلوبروتين والذي يصنع جزيئات ”دي ن أ“ ويضيفها الي أطراف الكروموسومات. وبهذة الطريقة يحمي التيلوميرالموجود في نهاية الكروموسوم من التأكل فيحافظ علي الكروموسومات من التحلل ,األلتصاق ,أعادة الترتيب أو فقدان الكروموسومات. منذ وقت قريب تم اكتشاف التلوميريز“أم أر أن أ“ الناسخ العكسي البشري كواحد من الدالالت الناسخ أ“ أن أر التلوميريز“أم من المتوقع الدور دراسة البحث- من الهدف الكبديه. الخاليا سرطان لتشخيص الجديدة العكسي البشري كعامل جديد لتشخيص ومتابعة سرطان الخاليا الكبدية. - كيفية أجراء البحث اشتملت هذI الدراسه علي ٩٠ فردا تم تقسيمهم ألي ثالث مجموعات: المجموعة األولي تحتوي علي ١٠ أشخاص أصحاء المجموعة الثانية تحتوي علي ٤٠ مريضا بتليف الكبد المجموعة الثالثة تحتوي علي ٤٠ مريضا بسرطان الخاليا الكبدية تم عمل الفحوصات التالية لكل األفراد: الفافيتوبروتين في السيريم , البروتين الناتج عن غياب فيتامين ك II في السيريم وكذلك التلوميريز“أم أر أن أ“ الناسخ العكسي البشري في الدم بأستخدام جهاز ” بي سي أر“ -النتائج: كان هناك ارتفاع ملحوظ في مستوي ألفا فيتوبروتين و البروتين الناتج عن غياب فيتامين ”ك“ في المجموعة الثالثة أذا ما قورنت بالمجموعتيين األولي والثانية (٠٠٤‚٠>P). متوسط التلوميريز“أم أر أن أ“ الناسخ العكسي البشري كان أعلي بدرجة ملحوظة في كل من المجموعة الثالثة

والثانية بمعدل ١٨٠ و ۵‚١ مرة أذا ما قورنوا بالمجموعة األولي (٠٠١‚٠>P). كان هناك أرتباط أيجابي بين التلوميريز“أم أر أن أ“ الناسخ العكسي البشري وألفا فيتوبروتين و البروتين الناتج عن غياب فيتامين ”ك“ وكذلك حجم وعدد أالورام (٠٤‚٠>P , ٠٤‚> P , ٠٠١, >P ‚٠٢‚٠>P). القيمة القطعية لأللفافيتوبروتين في السيريم ( ≥۵٢ نانوجرام/ مل) أدت

ألي ٧٥ ٪ حساسية , ١٠٠٪ خصوصية و ٪٨۵ دقة في التنبؤ بوجود سرطان في الخاليا الكبدية . القيمة القطعية للبروتين الناتج عن غياب فيتامين ”ك“ في السيريم ( ≥ ۵‚٧ نانوجرام/ مل) أدت ألي ٨٥ ٪ حساسية , ١٠٠٪ خصوصية و ٨٩٪ دقة تشخيص في التنبؤ بوجود سرطان في الخاليا الكبدية . القيمة القطعية التلوميريز“أم أر أن أ“ الناسخ العكسي البشري ( ١) أدت ألي ٩٩ ٪ حساسية , ۵٤٪ خصوصية و ٩٧٪ دقة تشخيص في التنبؤ بوجود سرطان في الخاليا الكبدية في حين

أن القيمة القطعية ( ≥٩‚٧) أدت ألي ٩٠ ٪ حساسية , ١٠٠٪ خصوصية و ٪٩۵ دقة في التنبؤ بوجود سرطان في الخاليا الكبدية . أستخدام كال من التلوميريز“أم أر أن أ“ الناسخ العكسي البشري والفافيتوبروتين معا أدي ألي ۵‚٩٨ ٪ حساسية , ٩٧٪ دقة تشخيص في التنبؤ بوجود سرطان في الخاليا الكبدية , أدي أستخدام الثالث دالئل معا ألي زيادة في حساسية ودقة التشخيص ألي ١٠٠٪ و ٩٩ ٪ علي التوالي. قيمة األلفافيتوبروتين في تسعة مرضي بسرطان الخاليا الكبدية (۵‚٢٢ ٪) كانت أقل من القيمة القطعية المحسوبة ولكن كانت قيمة التلوميريز“أم أر أن أ“ الناسخ العكسي البشري في هؤالء المرضي كانت موجبة . - الخالصة: يعتبرالتلوميريز“أم أر أن أ“ الناسخ العكسي البشري أكثر حساسية وخصوصية كدليل لتوقع وجود سرطان الخاليا الكبديه من ألفا فيتوبروتين و البروتين الناتج عن غياب فيتامين ”ك“. أيضا هناك أرتباط بينة وبين حجم وعدد عقيدات الورم, ولذلك يمكن أن يستخدم كأداة للمتابعة. نتيجة لما سبق فأن قياس التلوميريز“أم أر أن أ“ الناسخ

العكسي البشري في الدم ممكن ان يكون دليل مبكر ومألم وبدون تدخل لتشخيص سرطان الخاليا الكبديه.

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Egypt. J. Lab. Med. Vol.(27) No.1&2, February/June , 2015 · 2018-01-24 · Omar Ali Alroubi Nevine...

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Transcript of Egypt. J. Lab. Med. Vol.(27) No.1&2, February/June , 2015 · 2018-01-24 · Omar Ali Alroubi Nevine...