Archives of the Balkan Medical UnionCopyright © 2017 Balkan Medical Union

vol. 52, no. 2, pp. 144-151June 2017

RÉSUMÉ

L’effet du polymorphisme des genes APO-1/FAS, CTLA-4 et BCL-2 sur le risque d’apparition des formes nodulaires de goître avec de la thyroidite au-to-immune parmi la population de la Bukovine

Introduction. La thyroïdite auto-immune (TAI) est l’un des problèmes non résolus de l’endocrinologie moderne, car la question de son étiologie et de sa pathogenèse est insuffisante. Les cytokines jouent un rôle important dans la régulation de la réponse im-munitaire et inflammatoire, en tant que gènes qui les codent, sont considérés comme des candidats poten-tiels pour le risque de pathologie thyroïdienne auto-im-mune. Cependant, un certain nombre de problèmes non résolus restent particulièrement liés au rôle des facteurs génétiques dans le développement de l’AIT.

ABSTRACT

Introduction. Autoimmune thyroiditis (AIT) is one of the unsolved problems of modern endocrinology, as the issue of its insufficient etiology and pathogenesis comes now clear. Cytokines play an important role in the regulation of immune and inflammatory response, as the genes that encode them are seen as potential candidates for the risk of autoimmune thyroid patho-logy. However, a number of unsolved issues remains, in particular related to the role of genetic factors in the development of AIT.Objectives. To analyze the frequency of polymor-phic variants of genes BCL-2 (rs17759659), CTLA-4 (rs231775), APO-1 / Fas (rs2234767) in patients with thyroid pathology regarding its forms: nodular goiter with autoimmune thyroiditis (NGAIT) and thyroid adenoma (TA).

ORIGINAL PAPER

EFFECT OF APO-1 / FAS, CTLA-4 AND BCL-2 GENES POLYMORPHISMS ON THE RISK OF GOITER NODULAR FORMS WITH AUTOIMMUNE THYROIDITIS OCCURRENCE AMONG THE BUKOVINIAN POPULATION

Michael Ivanovich Sheremet1, Larysa Petrivna Sydorchuk2, Viktor Oleksandrovich Shidlovskyi3, Anatoly Dmitrovich Bedenyuk3, Ruslan Igorevich Sydorchuk1, Viktor Markiyanovich Batig4, Gennady Sergeevich Kurochkin5, Alexey Viktorovich Levitsky5, Kristina Andreyvna Chympoi6

1 Surgery Department, Bukovinian State Medical University, Ukraine2 Family Medicine Department, Bukovinian State Medical University, Ukraine3 Surgery Department, I.Y. Horbachevsky State Medical University, Ukraine4 Department of Therapeutic Stomatology, Bukovinian State Medical University, Ukraine5 Department of Family Medicine, Genetics Laboratory, State University of Medicine and Pharmacy „Nicolae Testemitanu“, Republic of Moldova6 Department of internal medicine and infectious diseases, Bukovinian State Medical University, Ukraine

Corresponding author: M.I. SheremetSurgery Department No 1 of BSMU, 191 Golovna str, 58018 Chernovtsy, Ukrainee-mail: mihayl71@gmail.comphone – 0956064607

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INTRODUCTION

Nowadays, the question of etiology, patho-genesis, diagnosis and surgical treatment of comor-bidities like nodular goiter and AIT remains valid1-3. An indirect confirmation of the existence of genetic component is based on a high prevalence of family

autoimmune thyroid disease, which can be found in every third case4-6. Various research groups over the years have repeatedly shown that individuals with a family history of thyroid disease have higher risk of this disease. TH Brix et al have shown that autoim-mune thyroid disease, including Graves’ disease and Hashimoto thyroiditis, are caused by the immune

Objectifs. Analyser la fréquence des variantes poly-morphes des gènes BCL-2 (rs17759659), CTLA-4 (rs231775), APO-1 / Fas (rs2234767) chez les patients atteints de pathologie thyroïdienne concernant ses formes: goitre nodulaire avec thyroïdite auto-immune (GNTAI) et l’adénome de la thyroïde (AT).Méthodes. Le polymorphisme des gènes BCL-2 (rs17759659), CTLA-4 (rs231775), Fas (rs2234767) a été étudié par réaction en chaîne en polymère en temps réel chez 95 patients atteints de GNAIT, 30 pa-tients atteints d’AT et 25 personnes en bonne santé. Les changements d’activité fonctionnelle de la glande thyroïde (GT) (activité normale, hypothyroïdie subcli-nique et clinique) et des degrés d’hyperplasie TG (IB, II et III) ont été analysés.Résultats. La mutation des gènes BCL-2 (rs17759659) et CTLA-4 (rs231775) dans l’état homozygote parmi la population adulte des résidents de la Bukovine de Nord se produit avec une fréquence de 3,2-4,0%, aucu-ne différence fiable entre les patients et les sujets sains. Les mutations du gène APO-1 / Fas (rs2234767) dans l’état homozygote ne sont pas observées. La nature de l’ensemble de distribution allélique: rapport de pari-té entre les allèles principaux A et G du gène BCL-2 (52% contre 48% des patients, p> 0,05) et 54% contre 46% – en bonne santé (p> 0,05)). Une prédominance de la mutation de l’allèle sauvage du gène CTLA-4 de l’allèle G 2,57 fois chez les patients (2 = 96,8; p

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response to antigens of own thyroid and “affect“ ap-proximately 2-5% of the general population7.

Polymorphism of genes – inhibitors of the ac-tivation of T-lymphocytes or CTLA-4 (cytotoxic T-lymphocyte antigen-4, associated with 4 mol-ecules) and PTPN22 (Protein tyrosine phosphatase, non-receptor type 22) play an important role in the development of NGAIT8-15. Other genes mutations are described that affect the function of the thyroid gland lymphoid tissue or apoptosis, including that of thyrocytes: the gene of apoptosis regulator BCL-2 (B-cell lymphoma 2) (rs17759659), APO-1 / Fas (apop-tosis antigen 1/cluster of differentiation 95 (CD95)) (rs2234767), loci associated with NGAIT available on the chromosomes 2 (2q33), 6 (6p21), 8 (8q24), 12 (12q22) and 13 (13q32), etc16-18.

The role of genetic factors is also evident by the association of NGAIT and TA with antigens of HLA system: it is combined with genes HLA-B8, DR3-HLA and HLA-DR5; hypertrophic form of NGAIT – with genes HLA-DR5, the atrophic one – with the genes HLA-DR3 and HLA-B8. HLA DQw7 is a marker of the most significant hereditary risk factor of NGAIT development. Specific clones of T-lymphocytes capa-ble of interacting with their own thyroid antigens might be inherited, too19-22.

In Ukraine, the polymorphism of genes associ-ated with apoptosis of thyrocytes, lymphoid cells, or regulation of T cell activation (Bcl-2, Fas (APO-1),

CTLA-4) in NGAIT and TA had not been studied previously, while there may be several national and local peculiarities.

MATERIAL AND METHODS

Ninety-five women with NGAIT underwent exa-mination during 2013 to 2016, in Chernovtsy Regional Hospital, Ukraine. The age of patients ranged from 23 to 72 years. Diagnosis was established based on clinical and laboratory examinations (thyroid peroxidase anti-bodies (ATPO) – 60-250 U/ml thyroglobulin antibody (ATTH) – 60-500 U/ml; thyroid-stimulating hormone (TSH) – 4.10 IU/l), and thyroid sonography and histo-logic confirmation after surgery.

The group of 30 women with thyroid adenoma was selected based on ultrasound, fine needle aspira-tion puncture biopsy (TAPB) and thyroid histologi-cal conclusion after surgery. We have identified this group because this pathology is one of the most com-mon forms of nodular goiter. Control group included 25 healthy donors.

Genetic studies were performed in the genetics laboratory of the State University of Medicine and Pharmacy “Nicolae Testemițanu“, Chisinau (Republic of Moldova). DNA was extracted from whole venous blood lymphocytes. Venous blood was stored in test tubes, stabilized with K2-EDTA. Isolation and purification of DNA from the material obtained

Figure 1. CTLA4 gene polymorphism (rs 231775) alleles discrimination

Figure 2. BCL-2 gene polymorphism (17759659) alleles discrimination

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was performed according to methodological guid-ance of Thermo Scientific GeneJET Genomic DNA Purification kit (#K0721, Thermo Fisher Scientific).

Polymerase chain reaction (PCR) was performed in real-time (RT-PCR), using Taq-DNA polymerase and specific primers on QuantStudio 6 equipment, Applied Biosystems (USA), which allowed us to ob-tain amplicons in order to determine their number in “real time“ and reduce the likelihood of diagnostic error. Analysis of the data was performed using the QuantStudio RealTime Software (Fig. 1-3).

Statistical analysis was performed using Statistica 7.0 (StatSoft Inc, USA) software. Nominal data were presented in the form of quantitative analy-sis and percentages. For the genotypes distribution comparison, we used 2-Pearson criterion. Analysis of qualitative data (categorical variables), risk of thyroid pathology development was assessed with a binary lo-gistic regression model, using the relative risk (RelR), risk ratio (RR) and odds ratio (OR) with 95% confi-dence interval [95% CI], chi-square test (2) (df=1). The difference was considered significant at p 0.05

2p

2 =76.78p

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Table 2. The distribution of polymorphic variants of BCL-2 (rs17759659), CTLA-4 (rs231775) and APO-1/Fas (rs2234767) genes in the patients depending on the thyroid pathology.

The genes under study, n (%) Control, n=25 (%)

Study group, n (%)2 p

Total,n=150

(%) TA, n=30 NGAIT, n=95

BCL-2 (A/G), n (%)

AA 3 (12.0) 1 (3.33) 9 (9.47) p>0.05 13 (8.67)

GA 21 (84.0) 29 (96.67) 81 (85.26) p>0.05 131 (87.33)

GG 1 (4.0) 0 5 (5.26) - 6 (4.0)

2p

2 =64,80p

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and APO-1 / Fas (rs2234767) did not differ signifi-cantly in the experimental groups (table 1).

In the total surveyed population (both patients as a whole and healthy people), GA-genotype of BCL-2 gene dominated, with parity ratio of wild-A-allele and the G-allele (52% vs. 48% – in patients (p>0.05) and 54% vs 46 % – in healthy people (p>0.05)). The analy-sis of allelic state of CTLA-4 gene (rs231775) showed the dominance of the wild A-allele over the muta-tion G-allele: a 2.57-fold in patients (72% vs. 28%, 2=96.8; p0.05

AG 1.05 1.40 0.87-1.26 0.42-4.63 >0.05

GG 1.0 1.0 0.12-8.20 0.11-8.95 >0.05

The thyroid adenoma

AA 0.28 0.26 0.03-2.51 0.02-2.60 >0.05

AG 1.15 5.52 0.96-1.38 0.58-53.05 >0.05

GG – – – – –

NGAIT

AA 0.79 0.77 0.23-2.70 0.19-3.07 >0.05

AG 1.01 1.10 0.84-1.23 0.33-3.70 >0.05

GG 1.32 1.33 0.16-10.76 0.15-11.96 >0.05Note. RR – relative risk; OR – Odds Ratio, CI – confidence interval

Table 7. Polymorphic variants of the APO-1/Fas (rs2234767) gene as the risk factors of the thyroid gland pathology

Genotypes of the APO-1/Fas gene RelR OR 95% CI RR 95% CI OR p

General TG pathologyAG 0.77 0.71 0.35-1.69 0.26-1.99 >0.05

GG 1.07 1.40 0.84-1.36 0.50-3.90 >0.05

TG adenomaAG 0.69 0.63 0.24-2.01 0.17-2.39 >0.05

GG 1.10 1.58 0.84-1.44 0.42-5.96 >0.05

NGAITAG 0.79 0.74 0.35-1.78 0.26-2.12 >0.05

GG 1.07 1.35 0.84-1.36 0.47-3.87 >0.05Note. RR – relative risk OR –Odds Ratio, CI – confidence interval

Table 8. Polymorphic variants of the CTLA-4 (rs231775) gene as risk factors for the thyroid gland pathologyGenotypes of the CTLA-4 gene RelR OR 95% CI RR 95% CI OR p

Thyroid pathology

AA 0.79 0.60 0.54-1.14 0.25-1.43 >0.05

AG 1.38 1.75 0.79-2.39 0.72-4.25 >0.05

GG 0.80 0.79 0.09-6.86 0.08-7.41 >0.05

The thyroid gland adenoma

AA 0.67 0.44 0.39-1.15 0.15-1.31 >0.05

AG 1.67 2.67 0.92-3.03 0.89-7.98 >0.05

GG – – – – –

NGAIT

AA 0.82 0.65 0.56-1.20 0.27-1.60 >0.05

AG 1.29 1.53 0.73-2.27 0.62-3.81 >0.05

GG 1.05 1.05 0.12-9.01 0.11-9.88 >0.05Note. RelR – relative risk; OR –Odds Ratio; CI-, confidence interval, TG- thyroid gland.

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2 degrees of freedom. Analyzing the allelic status of both genes, a mild excess of heterozygosity in all groups was found (F=-0,23-/-0.05 /; p>0.05), which did not influence the expected population balance.

The distribution of polymorphic variants of genes CTLA4 (rs 231,775), BCL-2 (rs17759659) and APO-1 / Fas (rs2234767) in the patients, depending on the type of thyroid pathology (NGAIT, TA), is shown in table 2. The relative incidence of genotypes of the analyzed genes between the patients with NGAIT and TA did not differ significantly.

Population and race analysis (table 3) showed that the incidence of minor G-allele gene BCL-2 in the people that we surveyed did not differ sig-nificantly from Caucasian populations (p>0.05). Instead, the frequency of wild-A allele in our study is much less than that for the equatorial race and some populations of Mongoloid race (P

A=0.52-0.54 vs.

PC=0.65-1.0; p0.05), respectively22.

Racial and population differences of the poly-morphic variants of CTLA-4 gene (rs231775) in the comparative perspective are displayed in table 4. The incidence of the main A-allele of the CTLA-4 gene in Bukovina residents (P

A=0.72-0.78), and the minor

G-allele (PG=0.22-0.28) correspond to that on aver-

age in Caucasian populations (PA=0.61-0.88 and

PG=0.12-0.39; p>0.05), indicating the relative ho-

mogeneity for the polymorphic locus of the studied gene. At the same time, we have found significant differences when compared with some populations of equatorial race, where the incidence of alleles and genotypes had a wide range and divergence, indicat-ing a relatively high heterogeneity (P

A=0.54-0.67 and

PG=0.33-0.46; p

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