GENETIC TESTING Volume 12, Number 3, 2008 ª Mary Ann Liebert, Inc. Pp. 461–464 DOI: 10.1089=gte.2008.0053
CTLA4 Gene Polymorphisms in Children and Adolescents with Autoimmune Thyroid Diseases ¨ zgu¨r Erkal,2 Ediz Yes¸ilkaya,1 Altug˘ Koc¸,2 Aysun Bideci,1 Orhun C ¸ amurdan,1 Mehmet Boyraz,1 O 2 1 Mehmet Ali Ergun, and Peyami Cinaz
Two common forms of autoimmune thyroid diseases are Graves’ disease and Hashimoto’s thyroiditis. Cytotoxic T lymphocyte antigen 4 (CTLA4) encoded by the CTLA4 gene on chromosome 2q33 plays a role in susceptibility to Graves’ disease and is probably important also for Hashimoto’s thyroiditis as well as for the other endocrine autoimmune disorders. The CTLA4 locus is the only nonhuman leukocyte antigen locus that has been found in association with Graves’ disease repeatedly. Particularly, association of three polymorphic markers of CTLA4 gene, namely, C(318)T, A49G, and (AT)n dinucleotide repeat, with Graves’ disease was demonstrated in most of the population-based investigations. On the other hand, there are few studies to reveal the association of these markers with Hashimoto’s thyroiditis. A49G polymorphism was proposed to be associated with Hashimoto’s thyroiditis, and C(318)T was suggested to be not associated. The patient groups consisted of 88 patients (10 males and 78 females; mean age: 14.5 3.2 years [4.6–21.0 years]) with a previous diagnosis of Hashimoto’s thyroiditis and 112 euthyroid volunteers (51 males and 61 females; mean age: 14.1 2.9 years [5.2–18 years]). The frequency of A=G (A49G) genotype was high and statistically significant in patients with Hashimoto’s thyroiditis in comparison with the control group. Although the frequency of C=T [C(318)T] genotype is not significantly high in children with Hashimoto’s thyroiditis according to the control group, the risk of Hashimoto’s thyroiditis in A=G genotype group was 4.66 times greater than the group with A=A genotype. In this study, we documented that the A49G polymorphism might increase the susceptibility for Hashimoto’s thyroiditis.
Introduction
A
utoimmune thyroid disease (AITD) is the most common cause of thyroid disorders in children and adolescents; also, it is the most common cause of acquired hypothyroidism with or without goiter. Besides, it is regarded as the most common human autoimmune disease, affecting up to 5% of the general population (Wang and Crapo, 1997; Wang et al., 2006; Setian, 2007). The AITDs most likely develop as a consequence of a complex interaction between genetic susceptibility and environmental effects (Chistiakov and Turakulov, 2003). There are several known genetic loci responsible for endocrine autoimmune disorders other than the human leukocyte antigen (HLA) system; one of them is the cytotoxic T lymphocyte antigen 4 (CTLA4) gene, which is related with susceptibility to Graves’ disease (Wang and Crapo, 1997; Chistiakov and Turakulov, 2003; Zhernakova et al., 2005). Also, CTLA4 gene polymorphism studies revealed that the locus might display susceptibility for Hashimoto’s thyroid-
itis, type 1 diabetes mellitus, Celiac, and Addison’s diseases (OMIM 123890). CTLA4 is a member of the immunoglobulin superfamily and is a costimulatory molecule expressed by activated T cells (Chambers and Allison, 1997). Three polymorphic markers of the CTLA4 gene were studied in AITDs. The first one is C(318)T polymorphism of the promoter region; the second one is A49G transition of first exon; the last one is (AT)n dinucleotide repeat polymorphism in 30 UTR of exon 4 (Chistiakov and Turakulov, 2003; Bugeon and Dallman, 2000). It has been shown that A49G polymorphism is strongly related with Graves’ disease (Kinjo et al., 2002). Although there are several reports regarding the association of CTLA4 polymorphic markers with Graves’ disease, there are limited number of studies considering Hashimoto’s thyroiditis (Donner et al., 1997; Heward et al., 1999; Kinjo et al., 2002; Chistiakov and Turakulov, 2003; Zhernakova et al., 2005). The aim of our study was to search for A49G and C(318)T polymorphisms of CTLA4 gene in patients with Hashimoto’s thyroiditis and to compare these polymorphisms between euthyroid and hypothyroid patients.
Departments of 1Pediatric Endocrinology and 2Medical Genetics, Gazi University Medical School, Ankara, Turkey.
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YES¸ ILKAYA ET AL.
462 Materials and Methods This study was approved by the Research Ethics Committee of the Medical Faculty at Gazi University, and all patients and volunteers signed informed consent forms. Patients We have enrolled the children and the adolescents with Hashimoto’s thyroiditis who had been treated in our outpatient clinic between 2000 and 2007. The patient group consisted of 88 patients (10 males, 78 females; the mean age was 14.5 3.2 years [4.6–21.0 years]) with a previous diagnosis of Hashimoto’s thyroiditis. Hashimoto’s thyroiditis was defined according to the presence of thyroid autoantibodies (antiperoxidase [TPOAb] and=or antithyroglobulin [TgAb]) and the ultrasonographic findings of the thyroid including echogenity compatible with thyroiditis, regardless of the thyroid function. Patients who had Down syndrome, Turner syndrome, or type 1 diabetes were excluded. One hundred and twelve euthyroid volunteers (51 males and 61 females with a mean age of 14.1 2.9 years [5.2–18 years]) were included as a control group. Subjects who had goiter, history of thyroid disease, or any laboratory abnormalities that could be related to thyroid diseases, use of thyroid hormones or antithyroid drugs, or use of systemic corticosteroids or amiodarone during the last 6 months were excluded. If an elevated thyroid-stimulating hormone (TSH) concentration was associated with a decreased serum thyroxine (T4) concentration, the patient was considered to be hypothyroid. The patients with Hashimoto’s thyroiditis were classified as hypothyroid and euthyroid. The hypothyroid patients had been treated with levothyroxine in increasing dosages until TSH levels within the lower part of the normal range were reached. The mean follow-up period of our patients was 1.9 years (0.1–7.6 years). Laboratory analysis Serum concentrations of serum T4, triiodothyronine (T3), and TSH were measured with specific chemiluminescence assays using an Abbott Architect system (Abbott Laboratories, Chicago, IL). The normal ranges for serum T4 and T3 were 7–15.5 mg=dL and 0.7–2.4 ng=mL, respectively. The normal range for serum TSH was 0.5–5.0 mIU=mL. Serum antithyroglobulin antibody (anti-T) and antithyroid peroxidase antibody (anti-TPO) levels were measured by particle agglutination using commercial kits. Genotype Genomic DNA was prepared from peripheral white blood cells using the DNA purification kit (QIAGEN, Hilden, Ger-
many). We analyzed CTLA4 genotypes and allele with PCR. PCR specific for A49G polymorphism was performed with oligonucleotide primers (forward, 5-CTGAACACCGCTCCCATAAA-3; reverse, 5-CCTCCTCCATCTTCATGCTC-3) using premix Taq (Takara, Shiga, Japan). For the C(318)T polymorphism, the forward 5-GGGATTTAGGAGGACCCTTG-3 and reverse 5-GTGCACACACAGAAGGCACT-3 oligonucleotides were used. The common PCR protocol used for both types of polymorphism was performed by initial denaturation for 5 min at 948C, annealing for 15 s at 608C, extension for 15 s at 728C, denaturation for 30 s at 948C (for 30 cycles), and a final extension for 5 min at 728C. The presence of G (A49G) alleles was determined in each subject by PCR amplification of CTLA4, followed by digestion with TseI, which acts on the G variation, but not on the A variation. If a G allele was at position 49, 99=69-bp fragments were obtained. And the presence of C(318)T polymorphism revealed by digestion with MseI, which acts on T variation and giving 119=94-bp fragments. PCR products were detected by electrophoresis in a 3% agarose gel. Statistics SPSS for Windows 11.5 (Chicago, IL) was used for statistical analysis. Results are given as mean standard deviation, median (min–max), and proportion. The groups were compared with independent samples t-test, Mann–Whitney U test, and w2 test where appropriate. A p-value of < 0.05 was considered statistically significant. Results Fifty-five of the patients were hypothyroid and the rest 33 were euthyroid (Table 1). Comparison of sex, presence of goiter and nodules, anti-T, and anti-TPO levels was not statistically significant ( p > 0.05) between hypothyroid and euthyroid patients. Dosage of levothyroxine replacement therapy in patients with hypothyroidism was 50 mg=day (25–150 mg=day). The frequency of A=G (A49G) genotype was high and statistically significant in children with Hashimoto’s thyroiditis in comparison with the control group ( p ¼ 0.014). Although the frequency of C=T [C(318)T] genotype was high in patients with Hashimoto’s thyroiditis (15.9%) when compared with the control group (8.9%), however, it was not significant statistically ( p ¼ 0.32) (Table 2). The risk of having Hashimoto’s disease in A=G genotype group was 4.66 times greater than the group with A=A genotype, and the difference was statistically significant (95% GA [1.24–17.48]) ( p ¼ 0.023). Although the A=G genotype was higher among hypothyroid Hashimoto’s thyroiditis patients (14.5%) in contrast to euthyroid Hashimoto’s thyroiditis patients (6.1%), the finding was not statistically significant. Also, we found that the allele frequency differ-
Table 1. The Characteristics of Study Group Female (n ¼ 78) Male (n ¼ 10) Total (n ¼ 88) Hypothyroid Euthyroid
51 (92.7%) 27 (81.8%)
4 (7.3%) 6 (18.2%)
55 (100%) 33 (100%)
Age
Goitre Nodule Anti-T (mIU=mL) Anti-TPO (mIU=mL)
14.8 3.1 14.4 3.2
Anti-T: antithyroglobulin antibody; anti-TPO: antithyroid peroxidase antibody.
26 15
8 2
158 (5–1412) 266 (7–2595)
366 (2–1288) 339 (2–1528)
CTLA4 GENE POLYMORPHISMS IN CHILDREN WITH THYROIDITIS
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Table 2. Frequencies of Genotypes and Alleles of A=G Polymorphism at Position 49 in Exon 1 of CTLA4 Gene in Hashimoto’s Thyroiditis and Controls Hashimoto’s thyroiditis (n ¼ 88) Genotype
Hypothyroid
Euthyroid
A=A A=G C=C C=T
47 8 46 9
31 2 28 5
(85.5%) (14.5%) (83.6%) (16.4%)
Controls (n ¼ 112)
(93.9%) (6.1%) (84.8%) (15.2%)
109 3 102 10
(97.3%) (2.7%) (91.1%) (8.9%)
w2*
p*
8.56
0.014
2.30
0.32
*Values are mean SD or median (min-max).
ence between AA and AG was found to be statistically significant ( p ¼ 0.01595). The participants with C=T genotype had 1.93 times greater risk to have Hashimoto in contrast to participants with C=C genotype, but it was not statistically significant (95% GA [0.81–4.58]) ( p ¼ 0.136). Also C=T genotype ratio was higher among hypothyroid Hashimoto’s thyroiditis patients (16.4%) in contrast to euthyroid Hashimoto’s thyroiditis patients (15.2%), but again it was not statistically significant. Discussion Similar genetic and environmental factors may contribute to the development of Graves’ disease and Hashimoto’s thyroiditis (Lorini et al., 2003). Although these diseases are clinically distinct entities, they share many immunological and histological features. Susceptibility genes for AITDs have been investigated, and only the HLA and CTLA4 loci have been consistently associated with AITDs (Donner et al., 1997; Heward et al., 1999; Chistiakov and Turakulov, 2003; Zhernakova et al., 2005). However, recent data have shown linkage and association of chromosome 8q24 (containing the thyroglobulin gene) to AITDs. Graves’ disease and Hashimoto’s thyroiditis are common complex disorders affecting 2–5% of the western population. They result from a breakdown in the normal mechanisms that maintain tolerance to self-thyroid antigens. Although both genetic and environmental factors are important, the reasons why patients with AITD develop a failure of immune tolerance whereby autoreactive lymphocytes and=or antibodies mount an inflammatory response against the thyroid remain unknown. Consistent associations between DNA variants of the HLA gene region on chromosome 6p21 and the CTLA4 gene region on chromosome 2q33 and AITDs along with preliminary linkage data for a number of chromosomal regions support the genetic basis of this disease and explain in part both twin concordance and familial clustering data (Heward et al., 1999; Chistiakov and Turakulov, 2003; Zhernakova et al., 2005). Kinjo et al. (2002) classified their patients with Graves’ disease into three groups as A, B, and C according to the response of TSH-receptor antibody levels to the antithyroid drug treatment. They found that the frequencies of the GG genotype and the G allele were significantly higher in group C patients than in the other groups ( p < 0.0001), and those cases had the worst response and persistently positive TSHreceptor antibodies over 5 years of antithyroid drug treatment. Also, the group C patients did not have the AA genotype. In addition, the periods of time until remission were
significantly shorter in the AA genotype. Kinjo et al. (2002) suggested that Graves’ patients with the G allele need to continue antithyroid drug treatment for longer periods. In this investigation, we have looked for an association between CTLA4 polymorphisms and Hashimoto’s thyroiditis as in Graves’ disease. We have found that A=G genotype augmented the risk of Hashimoto’s thyroiditis development 4.66 times with respect to A=A genotype. The proportion of females was greater in patient group, but we know that there is no relation with sex. Unfortunately, we were not able to show a clear association between TSH-receptor antibody levels or other possible related factors and the A49G polymorphism. C(318)T polymorphism seemed not to be associated with Hashimoto’s thyroiditis, and the result was compatible with the previous reports (Chistiakov and Turakulov, 2003). Tomer et al. (2001) reported that CTLA4 contributed to genetic susceptibility to thyroid antibodies production, but there was no evidence that it contributed specifically to the causation of Graves’ or Hashimoto’s disease. After these pioneer studies, it has been shown that CTLA4 polymorphisms, particularly A49G, had significant role in genetic susceptibility to both Graves’ disease and Hashimoto’s thyroiditis (Donner et al., 1997; Heward et al., 1999; Tomer et al., 2001; Kinjo et al., 2002). In the light of our study, we may conclude that G allele regarding A49G polymorphism may increase the susceptibility to Hashimoto’s thyroiditis in children and the adolescents in Turkey. Further studies with larger samples are needed to confirm our preliminary findings for this polymorphism. References Bugeon L, Dallman MJ (2000) Costimulation of T cells. Am J Respir Crit Care Med 162:164–168. Chambers CA, Allison JP (1997) Co-stimulation in T cell responses. Curr Opin Immunol 9:396–404. Chistiakov DA, Turakulov RI (2003) CTLA-4 and its role in autoimmune thyroid disease. J Mol Endocrinol 31:21–36. Donner H, Braun J, Seidl C, et al. (1997) Codon 17 polymorphism of the cytotoxic T lymphocyte antigen 4 gene in Hashimoto’s thyroiditis and Addison’s disease. J Clin Endocrinol Metab 82:4130–4132. Heward JM, Allahabadia A, Armitage M, et al. (1999) The development of Graves’ disease and the CTLA-4 gene on chromosome 2q33. J Clin Endocrinol Metab 84:2398–2401. Kinjo Y, Takasu N, Komiya I, et al. (2002) Remission of Graves’ hyperthyroidism and A=G polymorphism at position 49 in exon 1 of cytotoxic T lymphocyte-associated molecule-4 gene. J Clin Endocrinol Metab 87:2593–2596.
464 Lorini R, Gastaldi R, Traggiai C, Perucchin PP (2003) Hashimoto’s thyroiditis. Pediatr Endocrinol Rev 2:205–211. Setian NS (2007) Hypothyroidism in children: diagnosis and treatment. J Pediatr 83:209–216. Tomer Y, Greenberg DA, Barbesino G, et al. (2001) CTLA-4 and not CD28 is a susceptibility gene for thyroid autoantibody production. J Clin Endocrinol Metab 86:1687–1693. Wang C, Crapo LM (1997) The epidemiology of thyroid disease and implications for screening. Endocrinol Metab Clin North Am 26:189–218. Wang SY, Tung YC, Tsai WY, et al. (2006) Long-term outcome of hormonal status in Taiwanese children with Hashimoto’s thyroiditis. Eur J Pediatr 165:481–483.
YES¸ ILKAYA ET AL. Zhernakova A, Eerligh P, Barrera P, et al. (2005) CTLA4 is differentially associated with autoimmune diseases in the Dutch population. Hum Genet 118:58–66.
Address reprint requests to: Ediz Yes¸ ilkaya, M.D. ¨ niversitesi Ty´p Faku¨ltesi Gazi U Pediatrik Endokrinoloji Bilim Daly´ Ankara 06200 Turkey E-mail:
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