A 20 year history of clinical and genetic study of thyroid autoimmunity in a Tunisian multigenerational family: Evidence for gene interaction.

Autoimmune thyroid diseases (AITD), which include Hashimoto thyroiditis (HT), Graves' disease (GD) and primary idiopathic myxoedema (PIM), are recognized by their clinical and genetic heterogeneity. In this study, we have carried on a global approach gathering 20 year genetic and clinical data on a Tunisian multigenerational family (Akr). Our purpose was search for a combined genotype involved in AITD susceptibility using 33 gene polymorphisms. The Akr pedigree is composed of more than 400 members distributed on 10 generations. Clinical follow-up was performed by appreciation of the thyroid gland and measurement of both thyroid hormone and auto antibody levels. We used FBAT software to test for association between gene polymorphisms and AITDs. Clinical follow-up has showed that the number of AITD patients has increased from 25 to 78 subjects subdivided on 51 cases of GD, 22 PIM and 5 HT. Concerning genetic analysis, our study has revealed new gene association when compared with our previous analysis (considering single genes). Thus, PTPN22, TG and VDR gene polymorphisms have became associated with p-values ranging from 4.6  10(- 2) to 4  10(- 3) when considered with other genes on the same chromosome; giving evidence for gene interaction. The most significant association was found with the MHC region (p = 7.15 10(- 4)). Moreover, and among gene polymorphisms explored, our analysis has identified some of them as AITD biomarkers. Indeed, PDS gene polymorphisms were associated with either exophthalmia or goiter (p-values from 10(- 2) to 10(- 3)). In conclusion, our study gives evidence for gene interaction in AITD development confirming genetic complexity of these diseases.

IL-1ß and TSH disturb thyroid epithelium integrity in autoimmune thyroid diseases.

Pro-inflammatory cytokines such as IL-1ß and TNFα are known to affect thyroid function. They stimulate IL-6 secretion and modify epithelium integrity by altering junction proteins. To study the role of cytokines on thyroid epithelia tightness in autoimmune thyroid diseases (AITD), we analyzed the expression profiles of junction proteins (ZO-1, Claudin, JAM-A) and cytokines in human thyroid slices and also investigated the effect of IL-1ß on the epithelium integrity in primary cultures of human thyrocytes. Junction proteins expression (ZO-1, Claudin, JAM-A) has been analyzed by immunohistochemistry on thyroid slices and by Western blot on membrane proteins extracted from thyrocytes of patients suffering from Graves and Hashimoto diseases. The high expression of junction proteins we found on Graves' disease thyroid slices as well as in cell membrane extracts acknowledges the tightness of thyroid follicular cells in this AITD. In contrast, the reduced expression of JAM and ZO-1 in thyroid cells from patients suffering from Hashimoto thyroiditis is in agreement with the loss of thyroid follicular cell integrity that occurs in this pathology. Concerning the effects on epithelium integrity of TSH and of the pro-inflammatory cytokine IL-1ß in primary cultures of human thyroid cells, TSH appeared able to modify JAM-A localization but without any change in the expression levels of JAM-A, Claudin and ZO-1. Inversely, IL-1ß provoked a decrease in the expression of- and a redistribution of both, Claudin and ZO-1 without modifying the expression and sub-cellular distribution patterns of JAM-A in thyroid cells. These results demonstrate (i) that Hashimoto's- and Graves' diseases display different junction proteins expression patterns with a loss of epithelium integrity in the former and (ii) that IL-1ß modifies thyroid epithelial tightness of human thyrocytes by altering the expression and localization of junction proteins. Therefore, IL-1ß could play a role in the pathogenesis of thyroid autoimmunity.

IL-1ß a potential factor for discriminating between thyroid carcinoma and atrophic thyroiditis.

Interactions between cytokines and others soluble factors (hormones, antibodies...) can play an important role in the development of thyroid pathogenesis. The purpose of the present study was to examine the possible correlation between serum cytokine concentrations, thyroid hormones (FT4 and TSH) and auto-antibodies (Tg and TPO), and their usefulness in discriminating between different thyroid conditions. In this study, we investigated serum from 115 patients affected with a variety of thyroid conditions (44 Graves' disease, 17 Hashimoto's thyroiditis, 11 atrophic thyroiditis, 28 thyroid nodular goitre and 15 papillary thyroid cancer), and 30 controls. Levels of 17 cytokines in serum samples were measured simultaneously using a multiplexed human cytokine assay. Thyroid hormones and auto-antibodies were measured using ELISA. Our study showed that IL-1ß serum concentrations allow the discrimination between atrophic thyroiditis and papillary thyroid cancer groups (p = 0.027).

TNF gene polymorphisms in Graves' disease: TNF-308 A/G meta-analysis.

Autoimmune thyroid diseases (AITDs), including Graves' disease (GD) and Hashimoto thyroiditis, are associated with human MHC polymorphisms. The present study analysed two polymorphisms within tumour necrosis factor (TNF) genes (TNF-308 A/G SNP and TNFb (CT)n microsatellite) in a sample of 106 GD patients and 199 controls from the Tunisian population. The present study was designed to investigate genetic association of these polymorphisms (taken separately or considered as a haplotype) with GD development. Statistical analysis confirmed the association between the TNF-308 A allele and GD (p = 0.002), previously reported in a Tunisian familial study. The data from the present study suggest that the TNF-308 A allele plays a role in GD pathogenesis in the Tunisian population. This association was further confirmed by a meta-analysis on eight published studies (p < 0.0001). Haplotype analysis with GD revealed an associated haplotype (TNFb3-TNF-308 G haplotype: chi2 = 13.16; p = 0.0003).

A potential role of TNFR gene polymorphisms in autoimmune thyroid diseases in the Tunisian population.

Autoimmune thyroid diseases (AITDs) including Graves disease (GD) and autoimmune hypothyroidism (AH) are associated with TNF genes polymorphisms. TNF molecules bind to TNFRI and TNFRII. No genetic association was reported between TNFR and AITDs. In this study, we have analysed two polymorphisms in TNFRI gene (TNFRI+36A/G SNP and a microsatellite (GT)(17) (GA)(n)) and one polymorphism in TNFRII gene (TNFRII +676 T/G). All these polymorphisms were studied in a large Tunisian family with high prevalence of AITDs, and on a case-control sample of 91 GD patients and 165 controls. The present study was undertaken to investigate the genetic association of these polymorphisms with AITDs development. We reported the implication of TNFRIA3 allele in AITDs pathogenesis in familial and case control studies, respectively (chi(2)=4.13, p=0.042; chi(2)=9.26, p(c)=0.005). In addition, Case-control study has revealed for the first time that TNFRII+676G allele was associated with GD (chi(2)=11.53; p=0.0007). Two TNFRI haplotypes were found to be associated with GD: TNFRI+36G-A8, TNFRI+36A-A3 (chi(2)=88.07; p=6.32x10(-21), chi(2)=16.78; p=4.2x10(-5), respectively). Our data showed that TNFRI polymorphisms have an important role in AITDs pathogenesis in both familial and case-control samples and that TNFRII was rather implicated in GD development in the Tunisian population.

Association analysis of interleukin gene polymorphisms in autoimmune thyroid diseases in the Tunisian population.

Autoimmune thyroid diseases (AITDs), including Graves' disease (GD) and autoimmune hypothyroidism (AH), are inherited as complex traits. Among the genes contributing to AITDs susceptibility are genes of the IL-1 family. IL-1 regulates T and B lymphocyte maturation, including the induction of several cytokines and cytokine receptors. Therefore, disturbances of this balance may not only play a role in inflammation but also in the pathogenesis of autoimmunity. In order to investigate genetic association of IL-1 gene polymorphisms with AITDs, we performed both a familial study in a large Tunisian pedigree with high prevalence of AITDs (64 patients and 176 controls), and a case-control study (131 GD unrelated patients and 225 healthy controls). PCR and PCR-RFLP methods were used to analyse respectively a VNTR in the IL-1RN gene and three SNPs in both IL-1B genes (-511 C/T and +3954 C/T) and IL-1A (-889 C/T). The family-based association study showed an association of the IL-1B+3954 C/T polymorphism (p=0.02) and two haplotypes IL-1RN*3/C/T/T and IL-1RN*1/C/T/T (p=0.009 and p=0.047 respectively) with AITDs. The case-control study is the first study revealing a significant association of the IL-1A-889 C/T polymorphism (chi2=10.23; p=0.0014) with susceptibility to GD. Our data suggest that the IL-1 gene cluster may harbour susceptibility genes for AITDs and GD pathogenesis in the Tunisian population.