Flexible peptide recognition by HLA-DR triggers specific autoimmune T-cell responses in autoimmune thyroiditis and diabetes.

Autoimmune polyglandular syndrome 3 variant (APS3v) refers to the co-occurrence of autoimmune thyroiditis (AITD) and type 1 diabetes (T1D) within the same individual. HLA class II confers the strongest susceptibility to APS3v. We previously identified a unique amino acid signature of the HLA-DR pocket (designated APS3v HLA-DR pocket) that predisposes to APS3v. We hypothesized that both thyroid and islet peptides can be presented by the unique APS3v HLA-DR pocket, triggering AITD + T1D together. To test this hypothesis we screened islet and thyroid peptides for their ability to bind to the APS3v HLA-DR pocket. Virtual screen of all possible thyroglobulin (Tg), thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), insulin (Ins), and glutamic acid decarboxylase 65 (GAD65) peptides identified 36 peptides that bound to this unique pocket. In vitro binding assays using baculovirus-produced recombinant APS3v HLA-DR identified 11 thyroid/islet peptides (of the 36 predicted binders) that bound with high affinity. By immunizing humanized HLA-DR3 mice carrying the APS3v HLA-DR pocket we identified 4 peptides (Tg.1571, GAD.492, TPO.758, TPO.338) that were presented by antigen presenting cells and elicited T-cell response. We conclude that both thyroid and islet peptides can bind to this flexible APS3v HLA-DR pocket and induce thyroid and islet specific T-cell responses. These findings set the stage to developing specific inhibitors of the APS3v HLA-DR pocket as a precision medicine approach to treating or preventing APS3v in patients that carry this genetic HLA-DR pocket variant.

Genetic-epigenetic dysregulation of thymic TSH receptor gene expression triggers thyroid autoimmunity.

Graves disease (GD) is an autoimmune condition caused by interacting genetic and environmental factors. Genetic studies have mapped several single-nucleotide polymorphisms (SNPs) that are strongly associated with GD, but the mechanisms by which they trigger disease are unknown. We hypothesized that epigenetic modifications induced by microenvironmental influences of cytokines can reveal the functionality of GD-associated SNPs. We analyzed genome-wide histone H3 lysine 4 methylation and gene expression in thyroid cells induced by IFNα, a key cytokine secreted during viral infections, and overlapped them with known GD-associated SNPs. We mapped an open chromatin region overlapping two adjacent GD-associated SNPs (rs12101255 and rs12101261) in intron 1 of the thyroid stimulating hormone receptor (TSHR) gene. We then demonstrated that this region functions as a regulatory element through binding of the transcriptional repressor promyelocytic leukemia zinc finger protein (PLZF) at the rs12101261 site. Repression by PLZF depended on the rs12101261 disease susceptibility allele and was increased by IFNα. Intrathymic TSHR expression was decreased in individuals homozygous for the rs12101261 disease-associated genotype compared with carriers of the disease-protective allele. Our studies discovered a genetic-epigenetic interaction involving a noncoding SNP in the TSHR gene that regulates thymic TSHR gene expression and facilitates escape of TSHR-reactive T cells from central tolerance, triggering GD.

Analysis of immune regulatory genes' copy number variants in Graves' disease.

BACKGROUND: Copy number variants (CNVs) have recently been reported to be associated with several autoimmune conditions. Moreover, loci involved in immunity are enriched in CNVs. Therefore, we hypothesized that CNVs in immune genes associated with Graves' disease (GD) may contribute to the etiology of disease. METHODS: One hundred ninety-one North American Caucasian GD patients and 192 Caucasian controls were analyzed for CNVs in three major immune regulatory genes: CD40, PTPN22, and CTLA-4. Copy number was determined using quantitative-PCR (Q-PCR) assays specifically designed for determining copy numbers in genomic DNA. Additionally, a well-characterized CNV in the amylase gene was typed in a separate dataset of DNA samples that were derived from cell lines or blood. RESULTS: No CNVs could be confirmed in the CD40 and CTLA-4 genes, even though a CD40 CNV is cataloged in the Database of Genomic Variants. Only the PTPN22 CNV was confirmed in our cohort, but it was rare and appeared in only two individuals. A key finding was that the source of DNA has a significant effect on CNV typing. There was a statistically significant increase in amylase locus deletions in cell line-derived DNA compared to blood-derived DNA samples. CONCLUSIONS: We conclude that CNV analysis should be performed only using blood-derived DNA Samples. Additionally, the CTLA-4, CD40, and PTPN22 loci do not harbor CNVs that play a role in the etiology of GD.

Shared molecular amino acid signature in the HLA-DR peptide binding pocket predisposes to both autoimmune diabetes and thyroiditis.

There is strong genetic association between type 1A diabetes (T1D) and autoimmune thyroid disease (AITD). T1D and AITD frequently occur together in the same individual, a condition classified as a variant of the autoimmune polyglandular syndrome type 3 (APS3). Because T1D and AITD are individually strongly associated with different HLA class II sequences, we asked which HLA class II pocket sequence and structure confer joint susceptibility to both T1D and AITD in the same individual (APS3v). We sequenced the HLA-DR gene in 105 APS3v patients and 153 controls, and identified a pocket amino acid signature, DRß-Tyr-26, DRß-Leu-67, DRß-Lys-71, and DRß-Arg-74, that was strongly associated with APS3v (P = 5.4 × 10(-14), odds ratio = 8.38). Logistic regression analysis demonstrated that DRß-Leu-67 (P = 9.4 × 10(-13)) and DRß-Arg-74 (P = 1.21 × 10(-13)) gave strong independent effects on disease susceptibility. Structural modeling studies demonstrated that pocket 4 was critical for the development of T1D+AITD; all disease-associated amino acids were linked to areas of the pocket that interact directly with the peptide and, therefore, influence peptide binding. The disease-susceptible HLA-DR pocket was more positively charged (Lys-71, Arg-74) compared with the protective pocket (Ala-71, Gln-74). We conclude that a specific pocket amino acid signature confers joint susceptibility to T1D+AITD in the same individual by causing significant structural changes in the MHC II peptide binding pocket and influencing peptide binding and presentation. Moreover, Arg-74 is a major amino acid position for the development of several autoimmune diseases. These findings suggest that blocking the critical Arg-74 pocket might offer a method for treating certain autoimmune conditions.

Molecular amino acid signatures in the MHC class II peptide-binding pocket predispose to autoimmune thyroiditis in humans and in mice.

Hashimoto's thyroiditis (HT) is associated with HLA, but the associated allele is still controversial. We hypothesized that specific HLA-DR pocket-sequence variants are associated with HT and that similar variants in the murine I-E locus (homologous to HLA-DR) predispose to experimental autoimmune thyroiditis (EAT), a classical mouse model of HT. Therefore, we sequenced the polymorphic exon 2 of the HLA-DR gene in 94 HT patients and 149 controls. In addition, we sequenced exon 2 of the I-E gene in 22 strains of mice, 12 susceptible to EAT and 10 resistant. Using logistic regression analysis, we identified a pocket amino acid signature, Tyr-26, Tyr-30, Gln-70, Lys-71, strongly associated with HT (P = 6.18 x 10(-5), OR = 3.73). Lys-71 showed the strongest association (P = 1.7 x 10(-8), OR = 2.98). This association was seen across HLA-DR types. The 5-aa haplotype Tyr-26, Tyr-30, Gln-70, Lys-71, Arg-74 also was associated with HT (P = 3.66 x 10(-4)). In mice, the I-E pocket amino acids Val-28, Phe-86, and Asn-88 were strongly associated with EAT. Structural modeling studies demonstrated that pocket P4 was critical for the development of HT, and pockets P1 and P4 influenced susceptibility to EAT. Surprisingly, the structures of the HT- and EAT-susceptible pockets were different. We conclude that specific MHC II pocket amino acid signatures determine susceptibility to HT and EAT by causing structural changes in peptide-binding pockets that may influence peptide binding, selectivity, and presentation. Because the HT- and EAT-associated pockets are structurally different, it is likely that distinct antigenic peptides are associated with HT and EAT.

Interleukin (IL)-23 receptor is a major susceptibility gene for Graves' ophthalmopathy: the IL-23/T-helper 17 axis extends to thyroid autoimmunity.

CONTEXT: IL-23 and its receptor (IL-23R) guide T cells toward the T-helper 17 phenotype. IL-23R single nucleotide polymorphisms (SNPs) have been associated with several autoimmune diseases, including Crohn's disease and rheumatoid arthritis. OBJECTIVE: Our objective was to determine whether variants in the IL-23R gene are associated with Graves' disease (GD) and Graves' ophthalmopathy (GO). DESIGN AND PARTICIPANTS: A total of 216 North American Caucasian GD patients and 368 healthy controls were genotyped for four SNPs spanning the IL-23R gene. SNPs rs11209026 and rs7530511 were genotyped using the TaqMan allelic discrimination assays (Applied Biosystems, Foster City, CA), and SNPs rs2201841 and rs10889677 were genotyped using a fluorescent-based restriction fragment length polymorphism method. RESULTS: The A allele of rs2201841 was present in 78.8% of GD patients with GO and 64.7% of controls [P=1.1x10(-4); odds ratio (OR)=2.04]; the AA genotype was also significantly increased in GO patients compared with controls (62.5 and 41%, respectively; P=1.0x10(-4); OR=2.4). The C allele of rs10889677 was present in 78.6% of GO patients and 64.5% of controls (P=1.3x10(-4); OR=2.03), and the CC genotype was also significantly increased in GO patients vs. controls (62.1 and 41.0%, respectively; P=1.4x10(-4); OR=2.36). The TT genotype of rs7530511 was significantly associated with GD, but not specifically with GO; it was present in 2.5% of GD patients and 0.3% of controls (P=0.02; OR=9.4). The rs11209026 SNP, which is the most strongly associated with Crohn's disease, was not associated with GD or GO in our data set. CONCLUSIONS: Variants in the IL-23R gene are strongly associated with GO. These variants may predispose to GO by changing the expression and/or function of IL-23R, thereby promoting a proinflammatory signaling cascade.

The regulatory T cell gene FOXP3 and genetic susceptibility to thyroid autoimmunity: an association analysis in Caucasian and Japanese cohorts.

FOXP3 is a key gene in the development of regulatory T cells (Treg). FOXP3 expression commits naïve T cells to become Treg cells. Indeed, mutations in the FOXP3 gene cause severe systemic autoimmune diseases in humans and in mice. Therefore, we hypothesized that the FOXP3 gene may be associated with thyroid autoimmunity which is among the typical autoimmune diseases that develop in individuals with FOXP3 mutations. Moreover, the FOXP3 gene is located within an X-chromosome locus (Xp11.23) previously shown to be linked with autoimmune thyroid diseases (AITD). We tested the FOXP3 gene locus for association with AITD in two large cohorts of US Caucasians and Japanese AITD patients. We analyzed 269 Caucasian AITD patients (52 males and 217 females) and 357 Caucasian controls (159 males and 198 females), as well as 377 female Japanese AITD patients and 179 female Japanese controls. The FOXP3 gene locus was analyzed using four microsatellite polymorphisms [(GT)n; (TC)n; DXS573; DXS1208] flanking the FOXP3 gene locus. Interestingly, while no association was found between FOXP3 polymorphisms and AITD in the Japanese cohort there was a significant association in the Caucasian cohort. There was a significant association of the (TC)n polymorphism with AITD in the Caucasian male AITD patients (p=0.011; 5 degrees of freedom [df]). Similarly, there was an association between the DXS573 microsatellite and AITD in the Caucasian female AITD patients (p=0.00023; 4 df). These results suggest that polymorphisms of the FOXP3 gene may play a role in the genetic susceptibility to AITD in Caucasians, perhaps by altering FOXP3 function and/or expression.

Possible interaction between HLA-DRbeta1 and thyroglobulin variants in Graves' disease.

Graves' disease (GD) is influenced by two major susceptibility loci, HLA-DR3 and thyroglobulin (Tg). Recently we have shown that specific HLA-DR and Tg gene sequences predispose to Graves' disease. Individuals carrying at least one arginine at position 74 of the DRbeta1 chain (denoted the R- genotype) have a significantly increased risk of GD, as do individuals homozygous for the single nucleotide protein (SNP) in exon 33 of the Tg gene (denoted the CC genotype). Therefore, for the current study we hypothesized that these two genes may interact to influence the etiology of GD. To test this hypothesis, we analyzed the genotypes of 185 Caucasian patients with GD and 143 Caucasian controls for both genes. We tested for an interaction effect, that is, is one gene's effect on GD greater when the other gene is also present than when the other gene is absent? A logistic regression analysis yielded an estimate of 4.31 for the interaction term (p = 0.053). Our results may suggest an interaction between the R- and CC variants in conferring susceptibility to GD. These results, if confirmed, may imply that these two variants interact biologically to increase the odds of GD.

Analysis of immune regulatory genes in familial and sporadic Graves' disease.

Graves' disease (GD) is seen in apparently sporadic and familial forms. At least two immune regulatory genes are associated with GD, human leukocyte antigen (HLA) and cytotoxic T lymphocyte antigen-4 (CTLA-4). The aim of our study was to examine the contributions of HLA and CTLA-4 to the familial clustering of GD by analyzing them for association with familial and sporadic GD. We analyzed 160 Caucasian GD patients (69 familial and 91 sporadic), and 150 matched controls. Analysis of all GD patients demonstrated significant associations between GD and HLA-DR3 [P = 9.0 x 10(-7); relative risk (RR) = 3.8] and two CTLA-4 single nucleotide polymorphisms (SNPs), A/G(49) SNP (P = 0.03; RR = 1.5), and CT60 SNP (P = 0.03; RR = 1.4). Moreover, there was evidence for joint susceptibility to risk between HLA-DR3 and CTLA-4, giving a combined RR of 5.9. Subset analysis demonstrated no significant difference between the frequencies of HLA-DR3 and the susceptibility alleles of CTLA-4 A/G(49) and CT60 SNPs in the familial and sporadic GD subsets (P > 0.05). These results suggested that HLA-DR3 and CTLA-4 conferred a general increased risk for GD in both the sporadic and familial forms, and that the risk conferred by them was additive. However, HLA-DR3 and CTLA-4 did not have a stronger effect in the familial GD patients, suggesting that additional genes must contribute to the aggregation of GD within families.

A C/T single nucleotide polymorphism at the tyrosine kinase domain of the insulin receptor gene is associated with polycystic ovary syndrome.

OBJECTIVE: To examine whether the insulin receptor (INSR) gene contributes to genetic susceptibility to the polycystic ovary syndrome (PCOS). DESIGN: Case-control study. SETTING: Academic endocrinology clinic. PATIENT(S): Ninety-nine women with PCOS as defined by the National Institutes of Health consensus and polycystic ovaries on ultrasonography, and 136 healthy controls. MAIN OUTCOME MEASURE: Frequency of genotypes of a single nucleotide polymorphism of the INSR gene in patients and controls. RESULT(S): After stratification of participants by body mass index, the frequency of the uncommon T allele of the INSR single nucleotide polymorphism was significantly increased in lean patients with PCOS (body mass index < or =27 kg/m2) compared with lean controls (relative risk, 2.1). CONCLUSION(S): The INSR gene is a susceptibility gene for PCOS among lean patients with PCOS. It remains to be determined whether the exon 17 C/T single nucleotide polymorphism is the susceptibility single nucleotide polymorphism for PCOS or whether it is in linkage disequilibrium with another INSR gene polymorphism.

The influence of human leucocyte antigen (HLA) genes on autoimmune thyroid disease (AITD): results of studies in HLA-DR3 positive AITD families.

OBJECTIVE: Population-based, case-control studies have consistently shown association of Graves' disease (GD) with human leucocyte antigen (HLA)-DR3 in Caucasian populations. HLA association studies in Hashimoto's thyroiditis (HT) have also suggested an association with DR3, as well as with other HLA alleles. In contrast, HLA linkage studies in autoimmune thyroid disease (AITD) have been largely negative. The aim of the present study was to investigate the role of HLA in AITD and to explain the observed associations, but lack of linkage, by examining only AITD families with the associated allele, DR3. PATIENTS: We studied 99 probands (60 with GD and 39 with HT) from 99 multiplex, multigenerational Caucasian AITD families, and 135 age- and sex-matched Caucasian controls in association studies. In addition, a dataset of 34 Caucasian AITD families (out of the 99 families) with HLA-DR3 positive probands were analysed in linkage studies. DESIGN: HLA typing was performed using the technique of group-specific polymerase chain reaction-amplification with restriction enzyme digestion. Whole genome screening was performed using the ABI microsatellite panels. For fine mapping of the HLA region, we used the following markers: D6S276, D6S464, D6S439, D6S273, tumour necrosis factor alpha and D6S1610. LOD scores were calculated using the LIPED and GeneHunter programs. RESULTS: Case-control association analyses using the probands from our 99 Caucasian families showed an association of GD with DRB1*03 [P = 0.00032, relative risk (RR) = 3.4]. Linkage analysis for the HLA region in the 34 DR3 positive AITD families showed negative LOD scores throughout the region. The two-point LOD score at marker D6S273 (the closest to HLA-DRB1) was -3.0, and the multipoint LOD score was -7.6, demonstrating strong evidence against linkage to the HLA region in the subset of DR3 positive families. Whole genome screening in the subset of 34 DR3 positive families revealed one locus showing evidence for linkage to AITD: D3S1580 on chromosome 3q27 with a maximum two-point LOD score of 2.1. CONCLUSIONS: The HLA locus did not cosegregate with disease in DR3 positive families, suggesting that HLA genes are not major genes for AITD expression even within DR3 positive families; Hence, although HLA-DR3 was associated with GD in the probands, it was most likely a modulating gene and not causative; and, as the DR3 positive families showed evidence for linkage with D3S1580, it may imply that the DR3 gene modulated the effect of a susceptibility gene within the D3S1580 locus.

A germline single nucleotide polymorphism at the intracellular domain of the human thyrotropin receptor does not have a major effect on the development of Graves' disease.

Graves' disease (GD) is caused by an interplay of genetic factors and environmental triggers. The major antigen in GD is the thyrotropin receptor (TSHR) on the surface of the thyroid epithelial cell. Population-based case-control studies have largely shown no association of GD with the D36H (Asp to His) and P52T (Pro to Thr) single nucleotide polymorphisms (SNPs) in the N-terminal region of the extracellular domain of the TSHR gene in Caucasian populations. Recently, a D727E (Asp to Glu) SNP in the intracellular C-terminal domain of the TSHR was reported to be associated with GD in a Russian population. In the present study we assessed whether the codon 727 SNP is associated with GD in a Caucasian population. We found no significant differences in codon 727 SNP frequencies between GD patients and controls. In addition, our results did not show an effect of the SNP on the GD phenotype and on disease severity. Further analysis showed no evidence that the TSHR 727 SNP modulated the risk for GD conferred by HLA (DR3) and/or CTLA-4 (SNP 49 G allele) genes. A meta-analysis combining our data and those of 2 previous studies showed a very weak association between the D727E SNP and GD (p = 0.03, relative risk = 1.6). Therefore, we concluded that the TSHR gene is not a major gene for GD in our population.