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Original Paper

Horm Res Paediatr 2012;78:67–72 DOI: 10.1159/000338997

Association between the Polymorphism A/G at Position 49 of Exon 1 of the CTLA-4 Gene and Antithyroid Antibody Production in Children with Hashimoto’s Thyroiditis

Anna M. Kucharska a Alicja Wisniewska a Katarzyna Popko b Urszula Demkow b

Departments of a Pediatrics and Endocrinology and b Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw , Poland

polymorphism and the susceptibility to HT in Polish children and confirms the existence of a link between (+49)A/G poly-morphism and anti-Tg Ab level.

Copyright © 2012 S. Karger AG, Basel

Introduction

Hashimoto’s thyroiditis (HT) together with Graves’ disease (GD) are the most prevalent autoimmune disor-ders, affecting up to 5% of the general population [1] . Au-toimmune thyroid diseases (AITD) are relatively rare in children. Both HT and GD are characterized by the pres-ence of autoantibodies against thyroid antigens, the hy-poechogenic structure of thyroid gland on ultrasound ex-amination, and subsequent development of various de-grees of thyroid dysfunction. The production of antithyroid antibodies (Abs) and the development of AITD result from the coexistence of multiple genetic and environmental factors [2] . The genetic predisposition to antithyroid antibody (Ab) production was suggested over 50 years ago [3] . This hypothesis was confirmed by the frequent presence of thyroid autoantibodies in family members of AITD patients independently on their ethnic origin [4, 5] . Some authors [6, 7] suggested an autosomal dominant mendelian trait, but this idea has now been re-

Key Words

CTLA-4 gene polymorphism � Antithyroid antibodies � Children

Abstract

CTLA-4 gene is considered to be one of the strongest factors determining the predisposition to antithyroid antibody (Ab) production. The aim of the study was to evaluate the asso-ciation of the polymorphism A/G of exon 1 of CTLA-4 gene and antithyroid Ab level in children with Hashimoto’s thy-roiditis (HT). Material and Methods: 45 children with HT (aged 14.9 8 2, range 8.1–7.9) and 55 healthy controls (aged 14.8 8 2.34, range 8.0–17.4) were enrolled. Controls were eu-thyroid and free from any autoimmune disease. CTLA-4 gene (+49)A/G polymorphism was evaluated by a single-strand conformation polymorphism method and restriction frag-ment-length polymorphism. Results: The frequency of GG genotype in HT children was significantly higher than in con-trols: 31 vs. 14.5% respectively (p ! 0.04, OR = 2.65, CI = 0.99–7.06). Anti-Tg Ab titers were higher in patients homozygous for G allele than with AA genotype. The GG genotype seemed to be protective from hypothyroidism at the moment of HT diagnosis, but this observation was not statistically con-firmed. Conclusions: Our study provides the evidence sup-porting the association between CTLA-4 gene (+49)A/G

Received: November 15, 2011 Accepted: April 23, 2012 Published online: August 21, 2012

HORMONERESEARCH IN PÆDIATRICS

Anna M. Kucharska Klinika Pediatrii I Endokrynologii Str. Marszalkowska 24PL–00-576 Warszawa (Poland) Tel. +48 225 227 307, E-Mail anna.kucharska   @   litewska.edu.pl

© 2012 S. Karger AG, Basel1663–2818/12/0782–0067$38.00/0

Accessible online at:www.karger.com/hrp

Kucharska   /Wisniewska   /Popko   /Demkow  

Horm Res Paediatr 2012;78:67–7268

placed by the hypothesis of multifactoral inheritance. Several genes have been proposed to be linked and/or as-sociated with AITDs, but the results of these studies have been variable. The two genetic regions as loci strongly involved in the susceptibility to AITD are HLA and the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) gene [8] . The first evidence for the association of the CTLA-4 locus and antithyroid Abs was reported by Tomer et al. [9, 10] , and then confirmed by Ban et al. [11] , Zaletel et al. [12, 13] and Sahin et al. [14] in patients with AITD. A limited number of scientists thoroughly analyzed the risk factors for the development of thyroid Abs in chil-dren and to date no study has been undertaken in order to associate antithyroid Abs with CTLA-4 gene mutations in a pediatric population.

The aim of the present study was to evaluate the asso-ciation between the polymorphism A/G at position 49 of exon 1 of the CTLA-4 gene and antithyroid Ab level in children with HT.

Material and Methods

Material One hundred Caucasian children from Poland, 45 with HT (40

girls and 5 boys aged 14.9 8 2, range 8.1–17.9) and 55 healthy con-trols (48 girls and 7 boys aged 14.8 8 2.34, range 8.0–17.4) were enrolled. The HT diagnosis was based on the presence of anti-TPO and/or anti-Tg Abs and hypoechogeneity of the thyroid gland on ultrasound. Control children were free from any thyroid dysfunction and antithyroid Abs as well as autoimmune, allergic and hematological disorders. There were no significant differenc-es in age and sex between the HT children and the control group. Written informed consent was obtained from the parents of all patients and controls. The study protocol was approved by the Bioethical Committee of the Medical University of Warsaw.

Methods DNA Isolation and Genotyping of A/G Polymorphism at Po-

sition 49 of Exon 1 in the CTLA-4 Gene. Genomic DNA was obtained from peripheral blood leukocytes with the use of a Genomic Midi AX kit (ASA Biotechnology). An appropriate fragment of the CTLA-4 gene containing a polymorphic placewas amplificated by PCR with the use of primers: forward5 � -GCTCTACTTCCTGAAGACCT-3 � and reverse 5 � -AGTCT-CACTCACCTTTGCAG-3 � >according to the protocol of Harper et al. [15] . The A/G polymorphism was analyzed by a single-strand conformation polymorphism (SSCP) method and by re-striction fragment-length polymorphism (RFLP) simultaneously [16] . In brief: (a) PCR-SSCP: 2 � l of PCR product was mixed with the same volume of formamide and denaturated for 5 min at 95   °   C. The sample was incubated on ice (4   °   C) and electrophoresis was performed in 7.5% polyacrylamide gel at 4   °   C with the use of Fast-System (Biosystem). Electrophoregrams were silver stained. (b) PCR-RFLP: 15 � l of the PCR product was added to 10 � l mix-ture of 1 ! NEBuffer 2 and 1.5 � l restriction enzyme Bbv I (Bio-

labs, New England) and incubated for 24 h at 37   °   C. The digested products were separated by 2% agarose gel electrophoresis at room temperature and detected by ethidium bromide staining.(c) Thyroid function, anti-TPO Abs, anti-Tg Abs: TSH, FT 4 (free thyroxine), and FT 3 (free triiodothyronine) levels were measured using an immunoenzymatic method (microparticle enzyme im-munoassay, AxSYM; Abbott). The normal range for TSH was 0.49–4.67 mIU/l and for FT 4 0.71–1.85 ng/dl. The upper normal limit for anti-Tg Abs was set at 34.0 IU/ml, while that for the anti-TPO Abs at 12 IU/ml. Ultrasonography of the thyroid gland was performed by ATL 3000 HDI, with linear sound 7.5–12.0 MHz. The diagnosis of hypothyroidism was based on increased levels of TSH and a decreased concentration of FT 4 on two or more con-secutive occasions. L -Thyroxine administration was started in all cases of hypothyroidism.

Statistical Analysis The results in different groups were compared by Mann-Whit-

ney U test. To compare the frequency of alleles and genotypes of the CTLA-4 between patients and controls, the Fisher-Snedecor test was used. The median values of anti-TPO and anti-Tg Abs were analyzed by the Kruskal-Wallis ANOVA test and also by the Mann-Whitney U test. The results were processed statistically us-ing Statistica XL7.0 software. Statistical significance was accepted at a level of p ! 0.05. The Hardy-Weinberg equilibrium test was applied to evaluate genotype frequencies.

Results

Association of CTLA-4 (+49)A/G Polymorphism with HT. Genotype frequency in the examined population was determined by Hardy-Weinberg equilibrium. The GG genotype was significantly more common in children with HT compared to the control group (p ! 0.04, OR = 2.65, CI = 0.99–7.06) ( table  1 ). AG heterozygotes were more frequent in the control group than in HT children (p ! 0.03), but the OR was too low to consider this finding as being significant (OR = 0.38, CI = 0.17–0.86). The fre-quency of A allele did not differ between controls and HT patients as well as the frequency of G allele: 49 vs. 54% and 51 vs. 46%, respectively ( table 1 ). The number of pa-tients carrying at least one A allele (AA homozygotes + AG heterozygotes) was similar in HT children and in the control group (31/45 (69%) and 47/55 (85%), respectively). The number of patients carrying at least one G allele (GG homozygotes and AG heterozygotes together) did not dif-fer in HT and controls either (32/45 (71%) and 42/55 (78%), respectively). The overt hypothyroidism at the time of HT diagnosis was observed only in carriers of A allele: in 3 of 13 patients (23%) of AA homozygotes and in 2 of 18 patients (11%) of AG heterozygotes. All patients with GG genotype were in euthyroid state ( table 2 ). The difference was not statistically significant. Homozygous

(+49)A/G Polymorphism of CTLA-4 and Antithyroid Antibodies in Children

Horm Res Paediatr 2012;78:67–72 69

GG carriers statistically significantly more often had a family history of AITD than patients with AA genotype: 42.8 vs. 7%, respectively (p = 0.02) ( table 2 ). Patients with GG, AG and AA genotypes were similar with respect to age, gender, and age at the time of the diagnosis.

Association between CTLA-4 (+49)A/G Polymorphism and Thyroid Autoantibodies. The comparison of anti-TPO Ab and anti-Tg Ab concentration in patients homo-zygous for AA vs. heterozygous AG vs. homozygous GG by Kruskal-Wallis test and ANOVA did not show any sta-tistical difference, nevertheless the analysis by Mann-Whitney U test revealed a statistically significant differ-ence between the anti-Tg Ab values in AA homozygous patients (144.25 8 111.64 IU/ml) compared to GG homo-zygous patients (mean 523.63 8 693.52 IU/ml) ( table 2 ). In GG carriers the titers of anti-Tg Abs were statistically significantly higher than in patients carrying AA geno-type (p = 0.03) and also titers of anti-Tg Abs were statisti-cally significantly higher in patients carrying at least one G allele (GG homozygotes + AG heterozygotes) than in patients homozygous for A allele (p = 0.02) ( fig. 1 ). In pa-tients homozygous for G allele in comparison to patients

Table 1. Frequency of genotypes at position +49 of exon 1 of the CTLA-4 gene in the group examined

Genotype HT patients Controls p value OR CI

A, % 13/45 (29%) 12/55 (21.8%) ns 1.46 0.59–3.61GG, % 14/45 (31%)* 8/55 (14.5%)* <0.04* 2.65* 0.99–7.06AG, % 18/45 (40%) 35/55 (64%) <0.03 0.38 0.17–0.86A allele, % 44/90 (49%) 59/110 (54%) ns 0.83 0.47–1.44G allele, % 46/90 (51%) 51/110 (46%) ns 1.21 0.69–2.11AA+AG, % 31/45 (69%) 47/55 (85%) ns 0.38 0.14–1.00GG+AG, % 32/45 (71%) 42/55 (78%) ns 1.01 0.42–2.51

* Statistically significant value. ns = Not significant.

Table 2. Clinical characteristics of HT patients according to genotypes

Characteristics AA homozygotes (n = 13)

GG homozygotes (n = 14)

AG heterozygotes (n = 18)

Whole A group (n = 45)

Male/female 2:11 2:12 1:17 5:40Family history of AITD, n 1/13 (7%) 6/14 (42.8%) 4/18 (22.2%) 11/45 (24%)Hypothyroid patients, n 3/13 (23%) 0/14 (0%) 2/18 (11%) 5/45 (11%)Mean value of TSH (median), mIU/l 35.6879.2 (2.6) 2.681.0 (2.4) 6.6812.5 (2.3) 13.2843.1 (2.4) Mean age at the time of diagnosis, years 14.4981.96 15.8481.51 15.2481.61 14.982.25Mean value of anti-Tg Abs, IU/ml 144.28111.6 523.68693.5 412.08659.4 707.382519Mean value of anti-TPO Abs, IU/ml 518.88601.2 460.98375.6 94781034 736.98906.3

4.5

4.0

3.5

3.0

2.5

An

ti-T

g A

b [l

og10

]

2.0

1.5

1.0GG GG+AGAA AA+AG

Fig. 1. Anti-Tg Ab concentrations (in logarithmic scale) in pa-tients accordingly to genotype at position +49 of exon 1 of the CTLA-4 gene.

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carrying at least one A allele (AA homozygotes + AG het-erozygotes), the titers of anti-Tg Abs did not differ sig-nificantly (p 1 0.05) ( fig. 1 ). The anti-TPO Ab values did not differ statistically significantly between patients car-rying various genotypes.

Discussion

CTLA-4 is an immune regulatory molecule expressed on the surface of activated T cells and a key inhibitor of T-cell activation and is important for the maturation and function of suppressive T-regulatory (T reg ) cells [17, 18] . CTLA-4 knockout mice develop an autoimmune lym-phoproliferative disorder leading to death within neona-tal age [19, 20] . Human CTLA-4 gene consists of four ex-ons and three introns [21] . Many studies conducted over the last decade have claimed an association of AITD with an adenine to guanine transition at position 49 of exon 1 (A/G49) and CT60 , but several other polymorphisms of the same gene have also been evaluated [22, 23] . The re-sults have not always been consistent. A detailed genom-ic analysis of CTLA-4 in GD, HT, and type 1 diabetes mellitus involving 108 single nucleotide polymorphisms was published several years ago [22] . The G allele of CT60 polymorphism showed a very strong association to GD. Other SNPs in addition to CT60 , such as JO31 , JO30 , and JO27_1 , were also highly associated and found to be in strong linkage disequilibrium, making it very difficult to map disease susceptibility to a single polymorphism. The GG genotype of A/G49 CTLA-4 gene polymorphism was associated with weaker inhibitory function of T reg cells, and the G allele was associated with lower CTLA-4 ex-pression on the surface of T cells [24] . CTLA-4 is an im-portant antigen in the maintenance of the autotolerance, therefore it is not surprising that the association of CTLA-4 gene with thyroid autoantibodies was found in the whole genome screening [10] . The association was report-ed in patients with GD and HT [10–14] . In the present study we have shown that the frequency of the polymor-phic GG genotype at position 49 of exon 1 of the CTLA-4 gene is significantly increased in Polish HT children compared to the control group. These results confirm the association between A/G49 CTLA-4 gene polymorphism and AITD reported previously in the Polish population by other authors [25, 26] . In the present study the mean concentration of anti-Tg Abs was significantly higher in sera from GG homozygous patients compared to wild AA homozygotes. This finding suggests the association be-tween the A/G49 CTLA-4 gene polymorphism and anti-

thyroid Ab production. On the other hand, in HT chil-dren, GG genotype seemed to be protected from hypo-thyroidism at the time of the diagnosis, however the number of hypothyroid patients was too small to confirm this observation statistically ( table 2 ). Zaletel et al. [13] analyzed the influence of the A/G49 CTLA-4 gene on clinical characteristics of adults with HT. They evaluated the presence of CTLA-4 genotypes with regard to the presence of antithyroid Abs, TSH and FT 3 level, gender and the age at the time of diagnosis. They found that in-dividuals carrying G allele had a higher anti-TPO and anti-Tg Ab level than AA homozygotes. Although in sera from GG homozygotes with HT, only anti-TPO Abs were significantly increased and not anti-Tg Abs. This obser-vation is inconsistent with our findings. The observed discrepancy may be explained by the small number of patients in our group, but also by the age of the popula-tion examined. The animal studies had shown that early onset of the diseases as well as early phase of the disease may be characterized by anti-Tg Ab predominance [27] . Thyroglobulin is the most important thyroid antigen and is considered as a candidate antigen initiating the im-mune response against the thyroid tissue [28] . The initia-tion of the autoimmune process is characterized by den-dritic and T-cell infiltration of the thyroid gland, and the production of anti-Tg Abs, later followed by the occur-rence of Abs against other specific thyroid antigens in-cluding TPO [29] . Anti-TPO Abs are mainly produced by a lymphocytic infiltrate at an advanced stage of AITD and only to a small extent by regional lymph nodes or the bone marrow [30, 31] . During spontaneous evolution of HT in humans, anti-Tg and anti-TPO Abs are detected at the onset of the disease and followed by a selective in-crease of anti-TPO Ab level connected with TSH eleva-tion and hypothyroidism [32, 33] . This finding suggests that anti-TPO Ab might represent a marker of deteriorat-ing thyroid function, in agreement with reports showing the correlation between anti-TPO Ab levels and lympho-cytic infiltration of the gland [34] . The excellent evidence confirming this theory is the study of Mazziotti et al. [35] , who reported the natural evolution of thyroid Ab produc-tion in Sri Lankan schoolchildren after the introduction of mandatory iodination. At the beginning of the study these authors observed an increase of anti-Tg and anti-TPO Abs in children, followed by progressive decrease of anti-Tg Abs and the increase of anti-TPO Abs with time. Although progress has been made in determining the ge-netic predisposition to AITD, much of the work per-formed in HT has been on small datasets of adults where subjects have been grouped together with GD patients. In

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Horm Res Paediatr 2012;78:67–72 71

an attempt to gain a greater understanding of the effects of individual CTLA-4 variants and address some of the published inconsistencies, we focused on HT in a pediat-ric population. Our observations highlight the involve-ment of genetic background in the pathogenesis of HT and the links between genotype, the production of anti-thyroid Abs and possible impairment of thyroid gland function. Further studies are needed to evaluate the as-sociation between the structure of the genome, dysregu-lation of the immune response and clinical presentation as well as natural history of the disease.

Conclusions

In this study, we confirmed that the A/G49 CTLA-4 gene polymorphism is associated with the susceptibility of HT in Polish children and adolescent. Our results sup-port the idea that A/G49 CTLA-4 gene polymorphism significantly influences the anti-Tg Ab production in HT children.

References

1 Szyper-Kravitz M, Marai I, Shoenfeld Y: Co-existence of thyroid autoimmunity with oth-er autoimmune diseases: friend or foe? Ad-ditional aspects on the mosaic of autoimmu-nity. Autoimmunity 2005; 38: 247–255.

2 Zaletel K: Determinants of thyroid autoanti-body production in Hashimoto’s thyroiditis. Expert Rev Clin Immunol 2007; 3: 217–223.

3 Hall R, Owen S, Smart G: Evidence for a ge-netic predisposition to formation of thyroid autoantibodies. Lancet 1960;ii:187–188.

4 Hall R, Stanbury J: Familial studies of auto-immune thyroiditis. Clin Exp Immunol 1967; 2: 719–725.

5 Aho K, Gordin A, Sievers K, Takala J: Thy-roid autoimmunity in siblings: a population study. Acta Endocrinol 1983; 251(suppl):11–15.

6 Phillips D, McLachlan S, Stephenson A, Rob-erts D, Moffitt S, McDonald D, Ad’Hiah A, Stratton A, Young E, Clark F, Beever K, Brad-bury K, Rees-Smith B: Autosomal dominant transmission of autoantibodies to thyro-globulin and thyroid peroxidase. J Clin En-docrinol Metab 1990; 70: 742–746.

7 Phillips D, Prentice L, McLachlan S, Upad-hyaya M, Lunt P, Rees Smith B: Autosomal inheritance of the tendency to develop thy-roid autoantibodies. Exp Clin Endocrinol 1991; 97: 170–172.

8 Tomer Y, Davies T: Searching for the autoim-mune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev 2003; 24: 694–717.

9 Tomer Y, Greenberg D, Barbesino G, Con-ception E, Davies T: CTLA-4 and not CD28 is a susceptibility gene for thyroid autoanti-body production. J Clin Endocrinol Metab 2001; 86: 1687–1693.

10 Tomer Y, Ban Y, Concepcion E, Barbesino G, Villanueva R, Greenberg D, Davies T: Com-mon and unique susceptibility loci in Graves’ and Hashimoto diseases, results of whole-genome screening in a dataset of 102 multi-plex families. Am J Hum Genet 2003; 73: 736–747.

11 Ban Y, Tozaki T, Taniyama M, Tomita M, Ban Y: Association of a CTLA-4 3 � untrans-lated region (CT60) single nucleotide poly-morphism with autoimmune thyroid disease in the Japanese population. Autoimmunity 2005; 38: 151–153.

12 Zaletel K, Krhin B, Gaberscek S, Pirnat E, Hojker S: The influence of the exon 1 poly-morphism of the cytotoxic T lymphocyte an-tigen-4 gene on thyroid antibody production in patients with newly diagnosed Graves’ disease. Thyroid 2002; 12: 373–376.

13 Zaletel K, Krhin B, Gaberscek S, Hojker S: Thyroid autoantibody production is influ-enced by exon 1 and promoter CTLA-4 poly-morphisms in patients with Hashimoto’s thyroiditis. Int J Immunogenet 2006; 33: 87–91.

14 Sahin M, Gursoy A, Erdogan M: Cytotoxic T lymphocyte-associated molecule-4 poly-morphism in Turkish patients with Hashi-moto thyroiditis. Int J Immunogenet 2009; 36: 103–106.

15 Harper K, Balzano C, Rouvier E, Mattei M, Luciani M, Goldstein P: CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to se-quence, message expression, gene structure and chromosomal location. J Immunol 1991; 147: 1037–1044.

16 Kucharska A, Wisniewska A, Rymkiewicz-Kluczynska B: The frequency of CTLA-4 gene polymorphism at position 49 exon 1 in children with Hashimoto’s thyroiditis (in Polish). Endokrynol Diabetol Chor Przemi-any Materii Wieku Rozw 2006; 12: 163–166.

17 Kolar P, Knieke K, Hegel K, Quandt D, Bur-mester G, Hoff H, Brunner-Weinzierl M: CTLA-4 (CD152) controls homeostasis and suppressive capacity of regulatory T cells in mice. Arthritis Rheum 2009; 60: 123–132.

18 De Somer L, Fevery S, Bullens D, Rutgeerts O, Lenaerts C, Mathieu CH, Boon L, Kasran A, Waer M, Billiau A: Murine bone marrow chimeras developing autoimmunity after CTLA-4 blockade show an expansion of T-regulatory cells with an activated cytokine profile. Immunol Lett 2010; 133: 49–53.

19 Waterhouse P, Penninger J, Timms E, Wake-ham A, Shahinian A, et al: Lymphoprolifera-tive disorders with early lethality in mice de-ficient in CTLA-4. Science 1995; 270: 985–988.

20 Tivol E, Borriello F, Schweitzer A, Lynch W, Bluestone J, Sharpe A: Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 1995; 3: 541–547.

21 Dariavach P, Mattei M, Golstein P, Lefranc M: Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and hu-man CTLA-4 cytoplasmic domains. Eur J Immunol 1988; 18: 1901–1905.

22 Ueda H, Howson J, Esposito L, Heward J, et al: Association of the T-cell regulatory gene CTLA-4 with susceptibility to autoimmune disease. Nature 2003; 423: 506–511.

23 Kavvoura F, Akamizu T, Awata T, Ban Y, et al: Cytotoxic T-lymphocyte-associated anti-gen 4 gene polymorphisms and autoimmune thyroid disease: a meta-analysis. J Clin En-docrinol Metab 2007; 92: 3162–3170.

24 Kouki T, Sawai Y, Gardine C, Fisfalen M, Alegre M, DeGroot L: CTLA-4 gene poly-morphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and con-tributes to the pathogenesis of Graves’ dis-ease. J Immunol 2001; 165: 6606–6611.

25 Stęchły T, Kula D, Steinhof K, Jurecka-Lu-bieniecka B: TNF and CTLA-4 genes in Hashimoto disease (in Polish). Endokrynol Pol 2005; 1: 96–98.

Kucharska   /Wisniewska   /Popko   /Demkow  

Horm Res Paediatr 2012;78:67–7272

26 Kula D, Bednarczuk T, Jurecka-Lubieniecka B, Polańska J, et al: Interaction of HLA-DRB1 alleles with CTLA-4 in the predisposi-tion to Graves’ disease: the impact of DRB1 * 07. Thyroid 2006; 16: 447–453.

27 Stafford EA, Rose NR: Newer insights into the pathogenesis of experimental autoim-mune thyroiditis. Int Rev Immunol 2000; 19: 501–533.

28 Jacobson E, Tomer Y: The CD40, CTLA-4, thyroglobulin, TSH receptor, and PTPN22 gene quintet and its contribution to thyroid autoimmunity: back to the future. J Autoim-mun 2007; 28: 85–98.

29 Weetman AP, McGregor AM: Autoimmune thyroid disease: further developments in our understanding. Endocr Rev 1994; 15: 788–830.

30 McLachlan S, McGregor A, Smith B, Hall R: Thyroid-autoantibody synthesis by Hashi-moto thyroid lymphocytes. Lancet 1979; 1: 162–163.

31 McLachlan S, Pegg C, Atherton M, Middle-ton S, et al: Subpopulations of thyroid auto-antibody secreting lymphocytes in Graves’ and Hashimoto thyroid glands. Clin Exp Immunol 1986; 65: 319–328.

32 Radetti G, Gottardi E, Bona G, Corrias A, Salardi S, Loche S, and Study Group for Thy-roid Diseases of the Italian Society for Pedi-atric Endocrinology and Diabetes (SIED/ISPED): The natural history of euthyroid Hashimoto’s thyroiditis in children. J Pedi-atr 2006; 149: 827–832.

33 Rodien P, Madec A, Ruf J, Rajas F, Bornet H, Carayon P, Orgiazzi J: Antibody-dependent cell-mediated cytotoxicity in autoimmune thyroid disease: relationship to antithyro-peroxidase antibodies. J Clin Endocrinol Metab 1996; 81: 2595–2600.

34 Yoshida H, Amino N, Yagawa K, Uemura K, Satoh M, Miyai K, Kumahara Y: Association of serum antithyroid antibodies with lym-phocytic infiltration of the thyroid gland: studies of seventy autopsied cases. J Clin En-docrinol Metab 1978; 46: 859–862.

35 Mazziotti G, Premawardhana L, Parkes A, Adams H, et al: Evolution of thyroid autoim-munity during iodine prophylaxis – the Sri Lankan experience. Eur J Endocrinol 2003; 149: 103–110.