Yoshiyuki Ban

Common and Unique Susceptibility Loci in Graves and Hashimoto Diseases: Results of Whole-Genome Screening in a Data Set of 102 Multiplex Families

The autoimmune thyroid diseases (AITDs), comprising Graves disease (GD) and Hashimoto thyroiditis (HT), develop as a result of a complex interaction between predisposing genes and environmental triggers. Previously, we identified six loci that showed evidence for linkage with AITD in a data set of 56 multiplex families. The goals of the present study were to replicate/reject the previously identified loci before fine mapping and sequencing the candidate genes in these regions. We performed a whole-genome linkage study in an expanded data set of 102 multiplex families with AITD (540 individuals), through use of 400 microsatellite markers. Seven loci showed evidence for linkage to AITD. Three loci, on chromosomes 6p, 8q, and 10q, showed evidence for linkage with both GD and HT (maximum multipoint heterogeneity LOD scores [HLOD] 2.0, 3.5, and 4.1, respectively). Three loci showed evidence for linkage with GD: on 7q (HLOD 2.3), 14q (HLOD 2.1), and 20q (LOD 3.3, in a subset of the families). One locus on 12q showed evidence of linkage with HT, giving an HLOD of 3.4. Comparison with the results obtained in the original data set showed that the 20q (GD-2) and 12q (HT-2) loci continued to show evidence for linkage in the expanded data set; the 6p and 14q loci were located within the same region as the previously identified 6p and 14q loci (AITD-1 and GD-1, respectively), but the Xq (GD-3) and 13q (HT-1) loci were not replicated in the expanded data set. These results demonstrated that multiple genes may predispose to GD and HT and that some may be common to both diseases and some are unique. The loci that continue to show evidence for linkage in the expanded data set represent serious candidate regions for gene identification.

Amino acid substitutions in the thyroglobulin gene are associated with susceptibility to human and murine autoimmune thyroid disease

The 8q24 locus, which contains the thyroglobulin (Tg) gene, was previously shown to be strongly linked with autoimmune thyroid disease (AITD). We sequenced all 48 exons of the Tg gene and identified 14 single-nucleotide polymorphisms (SNPs). Case control association studies demonstrated that an exon 10-12 SNP cluster and an exon 33 SNP were significantly associated with AITD (P < 0.01). Haplotype analysis demonstrated that the combination of these two SNP groups was more significantly associated with AITD (P < 0.001). Gene-gene interaction studies provided evidence for an interaction between HLA-DR3 and the exon 33 SNP, giving an odds ratio of 6.1 for Graves disease. We then sequenced exons 10,12, and 33 of the mouse Tg gene in 19 strains of mice. Fifty percent of the strains susceptible to thyroiditis had a unique SNP haplotype at exons 10 and 12, whereas none of the mouse strains that were resistant to thyroiditis had this SNP haplotype (P = 0.01). We concluded that Tg is a susceptibility gene for AITD, both in humans in and in mice. A combination of at least two Tg SNPs conferred susceptibility to human AITD. Moreover, the exon 33 SNP showed evidence for interaction with HLA-DR3 in conferring susceptibility to Graves disease.

Arginine at position 74 of the HLA-DR β 1 chain is associated with Graves' disease

Graves disease (GD) is associated with HLA-DR3 (DRB1*03) in Caucasians, but the exact amino-acid sequence in the DR β1 chain conferring susceptibility to GD is unknown. Therefore, the aim of our study was to identify the critical sequence among the HLA-DRB1 amino-acid residues occupying the peptide-binding pocket, which conferred susceptibility to GD. We sequenced the HLA-DRB1 locus in 208 Caucasian GD patients and 149 Caucasian controls. Sequence analysis showed an increased frequency of DR β-Arg-74 in GD patients compared to controls (41.8 and 13.4%, respectively; P=2.3 × 10−8, OR=4.6). Moreover, subset analyses showed that DR β-Arg-74 was also significantly more frequent in the HLA-DR3 negative GD patients than in controls (7.6 vs 0.8%, P=0.02, OR=10.5), suggesting that the association with DR β-Arg-74 is independent of the association with HLA-DR3. Structural modeling studies demonstrated that the change at position 74 from the neutral amino acids Ala or Gln to the positively charged amino-acid Arg significantly modifies the three-dimensional structure of the DR peptide-binding pocket. Our results suggested that structural heterogeneity of the DR β-chain peptide-binding pocket P4 at residue 74 predispose some at risk individuals to GD.

Analysis of the CTLA-4, CD28, and inducible costimulator (ICOS) genes in autoimmune thyroid disease.

The cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) gene on 2q33 is associated with autoimmune thyroid diseases (AITDs). Our earlier study in 56 families showed linkage of 2q33 to the presence of thyroid antibodies (TAbs). The goals of this study were to confirm the linkage of the 2q33 region to TAbs, to fine map this region, and study the ICOS gene. We performed a linkage study in an expanded data set of 99 multiplex AITD-TAb families (529 individuals). The highest two-point LOD score of 2.9 was obtained for marker D2S325 on 2q33. To fine map this locus, we genotyped 238 Caucasian AITD patients and 137 controls for five additional markers in the linked locus, which contained the CTLA-4, CD28, and ICOS genes. The A/G single-nucleotide polymorphism at position 49 of CTLA-4 was associated with AITD (P=0.01, OR=1.5), while markers inside CD28 and ICOS were not. Functional studies have shown that the G allele was associated with reduced inhibition of T-cell proliferation by CTLA-4. We concluded that: (1) the AITD gene in the 2q33 locus is the CTLA-4 gene and not the CD28 or ICOS genes; and (2) the G allele is associated with decreased function of CTLA-4.

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.

Association of a thyroglobulin gene polymorphism with hashimoto's thyroiditis in the Japanese population

objectiveu2002 The aetiology of the autoimmune thyroid diseases (AITDs), Graves’ disease (GD) and Hashimotos thyroiditis is largely unknown. However, genetic susceptibility is believed to play a major role. Two whole genome scans from Japan and from the USA identified a locus on chromosome 8q24 which showed evidence for linkage with AITD and HT. Recent studies have demonstrated an association between a Tg polymorphisms and AITD, suggesting that Tg is the susceptibility gene on 8q24.

Susceptibility Genes in Thyroid Autoimmunity

The autoimmune thyroid diseases (AITD) are complex diseases which are caused by an interaction between susceptibility genes and environmental triggers. Genetic susceptibility in combination with external factors (e.g. dietary iodine) is believed to initiate the autoimmune response to thyroid antigens. Abundant epidemiological data, including family and twin studies, point to a strong genetic influence on the development of AITD. Various techniques have been employed to identify the genes contributing to the etiology of AITD, including candidate gene analysis and whole genome screening. These studies have enabled the identification of several loci (genetic regions) that are linked with AITD, and in some of these loci, putative AITD susceptibility genes have been identified. Some of these genes/loci are unique to Graves disease (GD) and Hashimotos thyroiditis (HT) and some are common to both the diseases, indicating that there is a shared genetic susceptibility to GD and HT. The putative GD and HT susceptibility genes include both immune modifying genes (e.g. HLA, CTLA-4) and thyroid specific genes (e.g. TSHR, Tg). Most likely, these loci interact and their interactions may influence disease phenotype and severity.

Association of the Protein Tyrosine Phosphatase Nonreceptor 22 Haplotypes with Autoimmune Thyroid Disease in the Japanese Population

BACKGROUNDnA missence single-nucleotide polymorphism (SNP) in the protein tyrosine phosphatase nonreceptor 22 (PTPN22) gene known as R620W (rs2476601) was recently reported to be associated with several autoimmune diseases including Graves disease (GD). The association was repeatedly confirmed in the populations of North European ancestry. However, this amino acid was reported to be nonpolymorphic in the Asian populations. Since the gene confers an impact on autoimmune diseases, we attempt to explore an association between the PTPN22 gene and autoimmune thyroid disease (AITD) in a Japanese population without restricting to rs2476601. Previous investigations have also demonstrated that two intronic SNPs (rs706778 and rs3118470) in the interleukin-2 receptor-alpha (IL2RA) gene were associated with type 1 diabetes in the Japanese population.nnnPATIENTS AND METHODSnWe genotyped the five SNPs (rs12760457, rs2797415, rs1310182, rs2476599, and rs3789604) of the PTPN22 and the two SNPs (rs706778 and rs3118470 in the IL2RA gene) in 456 Japanese patients with AITD (286 with GD, 170 with Hashimotos thyroiditis) and 221 matched Japanese control subjects. Seven SNPs were analyzed by either the SNAPshot method or the high-resolution melting and unlabeled probe methods. Case-control association studies were performed using the chi(2) and Fishers exact tests with Yates correction. Haplotype was conducted using the expectation-maximization algorithm.nnnRESULTSnNo association was found between any of the individual SNPs of the PTPN22 gene and AITD. Permutation analysis revealed that the distribution of one haplotype is significantly different between patients with AITD and controls (p = 0.0036). A novel protective effect of a haplotype containing five SNPs was observed (p < 0.0001 for AITD, p < 0.0001 for GD, and p < 0.0001 for Hashimotos thyroiditis, respectively). The GG allele of rs3118470 in the IL2RA gene was significantly associated with GD (p = 0.03), although the association was weak.nnnCONCLUSIONSnSignificant difference in the distribution of the haplotype suggests that the PTPN22 gene rather than rs2476601 is involved in the development of AITD in the Japanese population.

The Effects of Alpha Interferon on the Development of Autoimmune Thyroiditis in the NOD H2h4 Mouse

Alpha interferon (αIFN) therapy is known to induce thyroid autoimmunity in up to 40% of patients. The mechanism is unknown, but Th1 switching has been hypothesized. The aim of our study was to examine whether αIFN accelerated the development of thyroiditis in genetically susceptible mice. We took advantage of NOD-H2h4, a genetically susceptible animal model, which develops thyroiditis when fed a high iodine diet. Six to eight week old male NOD H2h4 mice were injected with mouse αIFN (200 units) or with saline three times a week for 8 weeks. All mice drank iodinated water (0.15%). Mice were sacrificed after 8 weeks of injection. Their thyroids were examined for histology and blood was tested for antithyroglobulin antibody levels. T4 and glucose levels were also assessed. In the IFN-injected group, 6/13 (46.2%) developed thyroiditis and/or thyroid antibodies while in the saline-injected group, only 4/13 (30.8%) developed thyroiditis and/or thyroid antibodies (p=0.4). The grade of thyroiditis was not different amongst the two groups. None of the mice developed clinical thyroiditis or diabetes mellitus. Our results showed that αIFN treatment did not accelerate thyroiditis in this mouse model. This may imply that αIFN induces thyroiditis in a non-genetically dependent manner, and this would not be detected in a genetically susceptible mouse model if the effect were small. Alternatively, it is possible that αIFN did not induce thyroiditis in mice because, unlike in humans, in mice αIFN does not induce Th1 switching.

SEL1L Microsatellite Polymorphism in Japanese Patients with Autoimmune Thyroid Diseases

The autoimmune thyroid diseases (AITDs), comprising Graves disease (GD) and Hashimotos thyroiditis (HT), appear to develop as a result of complex interactions between predisposing genes and environmental triggers. A recently performed genome-wide linkage study identified six loci that showed evidence for linkage to AITD. One locus, GD-1, on chromosome 14q31 was mapped to within 2 centimorgans (cM) of the recently reported multinodular goiter (MNG)-1 locus. Furthermore, microsatellite markers for the thyroid stimulating hormone receptor gene on chromosome 14q31 were associated with AITDs in the Japanese population. A newly isolated growth factor, SEL1L, was recently mapped to 14q31, and we considered it an interesting candidate gene to examine with respect to both GD and MNG. We therefore have analyzed a dinucleotide (CA)n repeat polymorphism in the intron 20 of the SEL1L gene in patients with AITDs and in normal subjects. The polymorphic marker was analyzed by polymerase chain reaction (PCR) followed by electrophoresis on denaturing polyacrylamide gels. There was no significant difference in the distributions of SEL1L alleles between patients and controls. The present results do not support an association between a dinucleotide repeat polymorphism in intron 20 of the SEL1L gene and AITD in Japanese women.