Sickle Cell Anemia—Molecular Diagnosis and Prenatal

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ORIGINAL ARTICLE

Sickle Cell AnemiaMolecular Diagnosis and Prenatal

Counseling: SGPGI Experience

Ravindra Kumar & Inusha Panigrahi & Ashwin Dalal &

Sarita Agarwal

Received: 2 November 2010 /Accepted: 15 June 2011 /Published online: 29 June 2011# Dr. K C Chaudhuri Foundation 2011

Abstract

Objective To study the issues and dilemmas in prenataldiagnosis of Sickle cell anemia (SCA) and to evaluate the

role of genetic modifiers in counseling the families.

Methods The authors studied the genotype in 47 individuals

with increased HbS and three representative families were

taken as an example for describing various issues which need

to be sorted out for appropriate counseling.

Results Of 47 individuals 24 were S beta thalassemia, 14

were homozygous sickle cell anemia (SS) and 9 were HbS

trait. In the S beta thalassemia and homozygous SS cases,

anemia was presenting manifestation in all. The transfusion

requirement in these varied from 012 transfusions/ year.

Hepatosplenomegaly was seen in 27 cases (71%) and onlysplenomegaly in 9 cases (23.7%). Jaundice was observed in

34 cases (84.2%). All the 47 subjects (including HbS trait)

were studied by Hb Variant system and underwent DNA

analysis for beta globin gene mutations, alpha globin gene

number and XmnI polymorphism. One or two alpha gene

deletion of 3.7 kb (3.7/ or 3.7/3.7) was found

in 11 out of 47 cases whereas alpha triplication was found

in 2 cases. 28 cases were heterozygous (+/) for XmnI

polymorphism, 9 were homozygous negative (/) and 10

were homozygous positive (+/+). Patients with SCA co-inherited with -thalassemia have less hemolysis as

revealed by lower reticulocyte counts than with normal

alpha genotype. The authors further discuss the issues and

dilemmas faced during prenatal counseling of three families

during this study.

Conclusions The knowledge of the relationship between

genotype and phenotype, effect of the modifier genes has an

important role in genetic counseling and for planning

individualized treatment for sickle cell anemia.

Keywords Genetic counseling . HbS . Modifier genes .

Prenatal diagnosis

Introduction

Sickle cell anemia (SCA) is a common autosomal recessive

blood disorder [1]. It is prevalent in many parts of India,

especially central India [2, 3]. The clinical phenotype of

sickle cell anemia comprises of chronic hemolytic anemia,

microvascular thrombosis, ischemic pain, leg ulcers etc [4].

The sickle-cell gene is common in Africa because the

sickle-cell trait confers some resistance to falciparum

malaria during a critical period of early childhood.

However, inheritance of two abnormal genes leads to

sickle-cell anemia and with malaria is a major cause of

ill-health and death in children with sickle-cell anemia in

Africa. The manifestations of sickle-cell anemia are more

unpredictable and variable than those of thalassemia. Many

affected individuals, however, have a good quality of life,

and in some parts of the world like: Bahrain, India, eastern

Saudi Arabia additional genetic factors (genes) may reduce

the severity of the disorder (WHO report 2006) [5].

R. Kumar: S. Agarwal (*)

Department of Genetics,

Sanjay Gandhi Post Graduate Institute of Medical Sciences,Lucknow 226014, Uttar Pradesh, India

e-mail: [email protected]

I. Panigrahi

Department of Pediatrics,

Post Graduate Institute of Medical Education and Research,

Chandigarh, India

A. Dalal

Diagnostics Divison,

Center for DNA Fingerprinting and Diagnostics,

Hyderabad, India

Indian J Pediatr (January 2012) 79(1):6874

DOI 10.1007/s12098-011-0510-1


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The phenotype of sickle--thalassemia depends on the

type of-thalassemia gene mutation as well as the status of

various genetic modifiers like alpha gene number, Xmn1

polymorphism etc and is not well studied [1]. Mukherjee et

al reported that the clinical presentation is milder in sickle

cell anemia co-inherited with alpha thalassemia [6, 7].

Whereas, inheritance of alpha triplication may worsen the

clinical picture due to excess alpha chains [1]. The XmnIsite (CT) polymorphism at position 158 of promoter

region of G gene leads to enhanced gamma chain

production, mainly G chains under condition of erythro-

poietic stress, partially compensating for beta chain synthe-

sis and reducing the free alpha chains, thereby consequent

amelioration of globin chain imbalance and of the clinical

phenotype [1]. Other than these known risk factors some

poly morphism have been noted in many genes that

plausibly affect the phenotype of sickle cell anemia [8].

Some studies are available which describe the clinical

characteristics and hematological parameters of patients

with sickle cell anemia in India [2, 3, 916]. But there isvery little literature reported from India regarding the role

of genetic modifiers on phenotype of various sickle

syndromes. However, the presence of genetic modifiers

poses a significant problem in counseling the families after

prenatal diagnosis. In this article , auth ors report 47

consecutive patients with increased HbS; their hematolog-

ical characteristics, genetic constitution of alpha globin

gene, beta globin gene and Xmn1 polymorphism. Further,

the authors discuss the issues and dilemmas in counseling

after prenatal diagnosis using examples of three represen-

tative families.

Material and Methods

This study was conducted in 47 consecutive individuals

with increased HbS analysed by HPLC (Hb Variant system

-Bio-Rad Laboratories, Hercules, CA). The demographic

parameters and clinical details were noted. Red cell indices

were performed by automated blood cell counter (Sysmex

KX-21-Japan). Percentages of HbA2 and HbF were

determined by HPLC. Structural hemoglobin variants were

determined by cello-gel electrophoresis also in tris- glycine

buffer (pH 8.5) using standard technique. DNA was

extracted from the peripheral blood by the method of

Poncz et al comprising of proteinase K digestion and

phenol-chloroform extraction [15]. For prenatal diagnosis,

DNA was extracted from chorionic villi using DNA

extraction kit (Qiagen, USA).

-globin gene mutation was characterized by Amplifi-

cation Refractory Mutation System (ARMS) PCR[17].

GAP-PCR technique was used for analysis of alpha gene

number as reported in the authors previous publication

[18]. The XmnI polymorphic site was studied by PCR-

RFLP Method [19].

Results

There were a total of 47 individuals comprising of 24: S beta

thalassemia, 14: homozygous sickle cell anemia (SS) and 9:HbS trait (Table 1). Of 38 patients with S beta thalassemia,

and homozygous sickle cell anemia; anemia was presenting

feature in 100% cases. The age of onset varied from 7 months

to15 years age, with a median of 4 years. Transfusion

requirement in the patients varied from 012 transfusions/

year; a median of 3 transfusions per year. Episodes of vaso-

occlusive crises were seen in 22 cases (57.9%) in form of

joint pains or dactylitis or bony pains or chest pain. Jaundice

was seen in 34 cases (84.2%). Hepatosplenomegaly was seen

in 27 cases (71%) and only splenomegaly in 9 cases (23.7%).

Spleen was not palpable in 2 cases (5.3%). Growth

retardation was seen in 32 cases (92.1%). 28 cases (73.7%)were receiving hydroxyurea therapy. Splenectomy had been

done in only 8 patients. Overall presentation was similar in

both S beta thalassemia, and homozygous SS patients. The

confirmation was mainly on parental studies and DNA

analysis. The 9 HbS trait patients were asymptomatic.

A detailed hematological and HPLC analysis that have

been performed in 24 individuals of S-beta thalassemia are

shown in Table 2; and 14 individuals of SS are shown in

Table 3. The beta chain mutations identified in the present

series of cases included IVS 15 (G-C), 619 bp deletion,

Co8-9(+G), IVS1-1(G-T) and Cap +1. Out of 47 cases, 34

cases had normal alpha genotype. One alpha gene deletion

of 3.7 kb(/) in 9 cases, 2 alpha gene deletion of

3.7 kb (/) in 2 cases and alpha gene triplication (/

) in 2 cases were found . Patients having alpha gene

deletion had later age of onset (22.19.8 years) than those

patients who had normal alpha genotype (15.212.1 years)

(P Value


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Genetic analysis in a number of single gene disorders

had brightened the prospects of our ability to predict

phenotype based on genotype. All patients with sickle cell

anemia do not have the same frequency and severity of

clinical symptoms. It has already been shown that co-

inheritance of alpha thalassemia with sickle cell anemia

results in significantly higher hemoglobin (Hb), hematocrit

(Hct), red blood cells counts (RBC) and hemoglobin A2

(HbA2) levels and milder clinical presentation with fewer

episodes of painful crisis, chest syndromes, infections,

Fig. 1 Characteristics of Family 1


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requirement of hospitalization and blood transfusion [6, 7].

Similarly, the heterozygous (+/) or homozygous (+/+) state

of Xmn1 polymorphism is associated with higher levels of

HbF, which in turn leads to amelioration of phenotype in

sickle cell anemia [1, 8]. This dependence of phenotype of

single gene disorders on other genetic and environmental

factors has lead to the emergence of concept of modifier

genes. The three families illustrated here highlight the

importance of proper use of genetic testing and pre-and

post-test counseling for optimal benefits. Accurate testing

and counseling is more important in antenatal diagnosis

cases as decision about termination of pregnancy has to be

taken by couples on the basis of the facts presented to them.

There can be diversity in decision-making depending on the

faith and religion and this should be kept in mind while

counseling the families [20]. The preference for male child

in some societies like in some ethnic groups in India can

also affect the familys approach for medical care, as

exemplified by family 3. Also the perception of the severity

can vary depending on cultural and financial factors. PND

and newborn screening are two methods by which the

anemia burden on the families and the health system can be

reduced, and optimal care can be provided. Colah et al

reported PND in 85 families with SCA and raised the issue

of justification of prenatal diagnosis in these families due to

relatively benign clinical course in some tribal groups in

India [21]. The study conducted by Telfer et al demonstrat-

ed early identification of affected infants by neonatal

screening and careful follow-up coupled with relatively

simple interventions, substantially reduced the morbidity

and mortality [22]. Unlike beta thalassemia, the clinical

severity of sickle cell disease in Indians is much less

predictable. Thus, sickle cell study at the present centre has

taken a long period of detailed observation but now it has

been possible clearly to define both mild and severe

phenotypes of Sickle cell anemia and S beta thalassemia

in Indian population on the basis of modifiers.

Although it is clear that these phenotypes are molded by

an extremely complex interaction of genetic and environ-

mental factors, the further analysis of the other factors

which have been identified and coming up regularly will

provide a measure of the degree to which the phenotypic

diversity can be explained. This will provide an extremely

valuable basis for further genetic studies, including genome

searches for further secondary and tertiary modifiers.

Further larger studies are needed addressing different

modifiers, so that more clear data are available for proper

genetic counseling especially in Indian sickle cell families.


Indian J Pediatr (January 2012) 79(1):6874 73


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Acknowledgements Theauthors would like to thank the Uttar Pradesh

Council for Science and Technology (UP-CST) for their financial

assistance; and the Japan International Cooperation Agency (JICA),

Government of Japan, for their contribution towards establishing

laboratory facilities for screening and prenatal diagnosis in their institute.

RK is thankful to CSIR for providing fellowship. The authors duly thank

all the patients and families who participated in this study.

Conflict of Interest None.

Role of Funding Source Uttar Pradesh Council for Science and

Technology (UP-CST) provided funding of laboratory works. Council of

Industrial and Scientific Research-New Delhi gave fellowship of RK.

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Sickle Cell Anemia—Molecular Diagnosis and Prenatal

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