Genome-wide association studies of autoimmune vitiligo identify 23 new risk loci and highlight key pathways and regulatory variants.

Vitiligo is an autoimmune disease in which depigmented skin results from the destruction of melanocytes, with epidemiological association with other autoimmune diseases. In previous linkage and genome-wide association studies (GWAS1 and GWAS2), we identified 27 vitiligo susceptibility loci in patients of European ancestry. We carried out a third GWAS (GWAS3) in European-ancestry subjects, with augmented GWAS1 and GWAS2 controls, genome-wide imputation, and meta-analysis of all three GWAS, followed by an independent replication. The combined analyses, with 4,680 cases and 39,586 controls, identified 23 new significantly associated loci and 7 suggestive loci. Most encode immune and apoptotic regulators, with some also associated with other autoimmune diseases, as well as several melanocyte regulators. Bioinformatic analyses indicate a predominance of causal regulatory variation, some of which corresponds to expression quantitative trait loci (eQTLs) at these loci. Together, the identified genes provide a framework for the genetic architecture and pathobiology of vitiligo, highlight relationships with other autoimmune diseases and melanoma, and offer potential targets for treatment.

Hypomethylation within gene promoter regions and type 1 diabetes in discordant monozygotic twins.

Genetic susceptibility to type 1 diabetes (T1D) is well supported by epidemiologic evidence; however, disease risk cannot be entirely explained by established genetic variants identified so far. This study addresses the question of whether epigenetic modification of the inherited DNA sequence may contribute to T1D susceptibility. Using the Infinium HumanMethylation450 BeadChip array (450k), a total of seven long-term disease-discordant monozygotic (MZ) twin pairs and five pairs of HLA-identical, disease-discordant non-twin siblings (NTS) were examined for associations between DNA methylation (DNAm) and T1D. Strong evidence for global hypomethylation of CpG sites within promoter regions in MZ twins with TID compared to twins without T1D was observed. DNA methylation data were then grouped into three categories of CpG sites for further analysis, including those within: 1) the major histocompatibility complex (MHC) region, 2) non-MHC genes with reported T1D association through genome wide association studies (GWAS), and 3) the epigenome, or remainder of sites that did not include MHC and T1D associated genes. Initial results showed modest methylation differences between discordant MZ twins for the MHC region and T1D-associated CpG sites, BACH2, INS-IGF2, and CLEC16A (DNAm difference range: 2.2%-5.0%). In the epigenome CpG set, the greatest methylation differences were observed in MAGI2, FANCC, and PCDHB16, (DNAm difference range: 6.9%-16.1%). These findings were not observed in the HLA-identical NTS pairs. Targeted pyrosequencing of five candidate CpG loci identified using the 450k array in the original discordant MZ twins produced similar results using control DNA samples, indicating strong agreement between the two DNA methylation profiling platforms. However, findings for the top five candidate CpG loci were not replicated in six additional T1D-discordant MZ twin pairs. Our results indicate global DNA hypomethylation within gene promoter regions may contribute to T1D; however, findings do not support the involvement of large DNAm differences at single CpG sites alone in T1D.

Changes in Zinc Transporter 8 Autoantibodies Following Type 1 Diabetes Onset: The Type 1 Diabetes Genetics Consortium Autoantibody Workshop.

Zinc transporter 8 autoantibodies (ZnT8A) were analyzed in sera from 1,504 subjects as part of the Type 1 Diabetes Genetics Consortium (T1DGC) Autoantibody Workshop. For these participants with type 1 diabetes (T1D), samples were collected within 3 years of T1D diagnosis. ZnT8A were detected in 862 subjects (57.3%), with the highest frequencies and median titers being associated with the shortest duration of disease. ZnT8A were present at similar frequencies in non-Hispanic whites, non-Hispanic blacks, and Hispanics, but significantly less prevalent in those of Asian ancestry. Sera containing ZnT8A selectively recognizing at least one of the SLC30A8 single nucleotide polymorphisms (encoding ZnT8A) were detected in all populations; however, Trp-specific sera were much less frequent in non-Hispanic blacks, consistent with the anticipated lower frequency of the SLC30A8 rs13266634 T allele in African American populations. ZnT8A positivity was associated with HLA-DQ8, but this was primarily due to the DRB1*0404-DQ8 haplotype. This was in contrast to autoantibodies to IA-2 that were strongly associated with DRB1*0401-DQ8. These effects appeared essentially independent of racial or ethnic background. The DRB1*0401-DQ8 and DRB1*0404-DQ8 haplotypes were associated with T1D subjects positive for GAD65, IA-2, and ZnT8A. In contrast to DRB1*0401-DQ8, there was no significant association of DRB1*0404-DQ8 with single or dual autoantibody positivity. The DRB1*0404-DQ8 haplotype was also associated with T1D subjects whose sera recognized both polymorphic variants of zinc transporter 8, an effect not seen for DRB1*0401-DQ8.

Genetics of Autoimmune Thyroiditis in Type 1 Diabetes Reveals a Novel Association With DPB1*0201: Data From the Type 1 Diabetes Genetics Consortium.

BACKGROUND: Autoimmune thyroiditis occurs in 10-25% of patients with type 1 diabetes (T1D). Most of these patients are also positive for thyroid peroxidase (TPO) antibodies. Thyroid dysfunction complicates T1D metabolic control and is a component of the autoimmune polyglandular syndrome (APS, type 2 or 3). Previous studies of isolated T1D and of T1D combined with other autoimmune disorders showed genetic susceptibility for alleles in HLA-DQB1 and -DRB1 and also CTLA4 and PTPN22. RESEARCH DESIGN AND METHODS: We analyzed the Type 1 Diabetes Genetics Consortium Autoantibody Workshop data by differentiating those T1D probands with and without TPO antibodies or thyroid disease with respect to polymorphisms in HLA, CTLA4, INS, PTPN22, and VDR, taking into account the ethnic origin. Genotype and clinical/immunogenic phenotype data were analyzed by gene counting methods and logistic regression analysis. RESULTS: The presence of TPO antibodies (25.2%) and thyroid disease (8.4%) was associated with older age, female sex, and presence of other autoantibodies (GAD65, ATPase, 21-OH) (all P<0.001). The highest prevalence was in patients of Hispanic ancestry (31%) and the lowest in those of African ancestry (8%). In T1D non-Hispanic whites, HLA-DRB1*0101 is significantly (P<0.0001) less frequent in TPO-positive than in TPO-negative individuals, whereas HLA-DRB1*0404, -DQB1*0301, and -DPB1*0201 are significantly (P<0.0001) more frequent. Subjects with a high titer of TPO autoantibodies and with thyroid disease were associated with female sex and older age and negatively associated with DRB1*0401-DQB1*0302 (P<0.0001). No significant differences were observed for an association of TPO positivity or thyroid disease with single nucleotide polymorphisms in the INS, CTLA4, or VDR loci, with nominal significance (P=0.01) for PTPN22 R620W variant. CONCLUSIONS: Thyroid autoimmunity is highly prevalent in T1D patients of non-Hispanic white, Asian, or Hispanic origin. The strongest disease risk is conferred by female sex and older age. This risk is modulated by HLA-DRB1 and HLA-DPB1 loci. The immunogenetic profile for T1D with thyroid autoimmunity may identify distinct pathways regulating polyglandular autoimmunity and disease.

ATPase4A Autoreactivity and Its Association With Autoimmune Phenotypes in the Type 1 Diabetes Genetics Consortium Study.

Autoantibodies targeting the H+/K+-ATPase proton pump of the gastric parietal cell (parietal cell antibodies [PCA]) are diagnostic of atrophic body gastritis (ABG) leading to pernicious anemia (PA). PCA, ABG, and PA occur in increased frequency in patients with type 1 diabetes and their relatives and are considered "minor" components of forms of autoimmune polyglandular syndrome (APS). A customized radioimmunoprecipitation assay was applied to 6,749 samples from the Type 1 Diabetes Genetics Consortium to measure ATP4A autoreactivity. Autoantibody prevalence was correlated with variants in HLA class II, PTPN22, and CTLA4 genes. With an ATP4A radioimmunoprecipitation assay, PCA were detected in sera from 20.9% of affected individuals. PCA prevalence increased with age and was greater in females (25.3%) than males (16.5%) and among Hispanics (36.3%) and blacks (26.2%) compared with non-Hispanic whites (20.8%) and Asians (16.7%). PCA and other organ-specific autoantibodies GAD65, IA-2, thyroid peroxidase (TPO), 21-hydroxylase (21-OH), and transglutaminase (TG) clustered within families with heritability estimates from 71 to 95%. PCA clustered with TPO, 21-OH, and persistent GAD65 autoantibodies but not with celiac (TG) or IA-2 autoantibodies. PCA-positive subjects showed an increased frequency of DRB1*0404, DPB1*0201, and PTPN22 R620W (rs2476601-T) and a decreased frequency of DRB1*0101, DPB1*0301, and CTLA4 CT60 (rs3087243-T). Genetic variants accounted for 4-5% of the heritable risk for PCA. The same alleles were associated with other autoantibody phenotypes in a consistent pattern. Whereas most of the heritable risk for PCA and other antibodies reflects genetic effects that are tissue specific, parietal cell autoimmunity is a major pathogenetic contributor in APS2.

Heritability of thyroid peroxidase autoantibody levels in type 1 diabetes: evidence from discordant twin pairs.

AIMS/HYPOTHESIS: The discordance status of (autoimmune) type 1 diabetes within monozygotic twin pairs points to the importance of environmental factors. The aim of this study was to investigate whether the environmental events causing type 1 diabetes influence thyroid autoimmunity. METHODS: Monozygotic and dizygotic twins discordant for type 1 diabetes from the UK and USA were tested for thyroid peroxidase autoantibodies (TPOA) by radioimmunoassay. Using quantitative genetic model fitting of a liability-threshold model we estimated the contribution of genetic (heritability) and environmental factors to TPOA. RESULTS: TPOA positivity was higher in females than in males in both cohorts and was associated with later age at diagnosis in the UK and combined cohorts (p < 0.01). TPOA did not specifically segregate with type 1 diabetes in the twin pairs (p > 0.2 in all groups). The best-fitting models showed heritability (95% CI) estimates for TPOA of 63% (37%, 80%) for the UK and 80% (51%, 92%) for US twins, while the best-fitting meta-analysis model of the two twin cohorts combined included additive genetic and unique environmental factors with a heritability estimate of 69% (50%, 82%). CONCLUSIONS/INTERPRETATION: Risk of thyroid autoimmunity, defined by TPOA, in the context of autoimmune diabetes is, substantially, genetically determined in discordant twin pairs. Environmental factors leading to type 1 diabetes were not the same as those involved with thyroid autoimmunity. It follows that it is as important to investigate for thyroid autoimmunity in relatives of type 1 diabetes patients as it is in the patients themselves.

Whole exome sequencing identifies a troponin T mutation hot spot in familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) commonly causes heart failure and shows extensive genetic heterogeneity that may be amenable to newly developed next-generation DNA sequencing of the exome. In this study we report the successful use of exome sequencing to identify a pathogenic variant in the TNNT2 gene using segregation analysis in a large DCM family. Exome sequencing was performed on three distant relatives from a large family with a clear DCM phenotype. Missense, nonsense, and splice variants were analyzed for segregation among the three affected family members and confirmed in other relatives by direct sequencing. A c.517T C>T, Arg173Trp TNNT2 variant segregated with all affected family members and was also detected in one additional DCM family in our registry. The inclusion of segregation analysis using distant family members markedly improved the bioinformatics filtering process by removing from consideration variants that were not shared by all affected subjects. Haplotype analysis confirmed that the variant found in both DCM families was located on two distinct haplotypes, supporting the notion of independent mutational events in each family. In conclusion, an exome sequencing strategy that includes segregation analysis using distant affected relatives within a family represents a viable diagnostic strategy in a genetically heterogeneous disease like DCM.

NLRP1 haplotypes associated with vitiligo and autoimmunity increase interleukin-1ß processing via the NLRP1 inflammasome.

Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a key regulator of the innate immune system, particularly in the skin where, in response to molecular triggers such as pathogen-associated or damage-associated molecular patterns, the NLRP1 inflammasome promotes caspase-1-dependent processing of bioactive interleukin-1ß (IL-1ß), resulting in IL-1ß secretion and downstream inflammatory responses. NLRP1 is genetically associated with risk of several autoimmune diseases including generalized vitiligo, Addison disease, type 1 diabetes, rheumatoid arthritis, and others. Here we identify a repertoire of variation in NLRP1 by deep DNA resequencing. Predicted functional variations in NLRP1 reside in several common high-risk haplotypes that differ from the reference by multiple nonsynonymous substitutions. The haplotypes that are high risk for disease share two substitutions, L155H and M1184V, and are inherited largely intact due to extensive linkage disequilibrium across the region. Functionally, we found that peripheral blood monocytes from healthy subjects homozygous for the predominant high-risk haplotype 2A processed significantly greater (P < 0.0001) amounts of the IL-1ß precursor to mature bioactive IL-1ß under basal (resting) conditions and in response to Toll-like receptor (TLR) agonists (TLR2 and TLR4) compared with monocytes from subjects homozygous for the reference haplotype 1. The increase in basal release was 1.8-fold greater in haplotype 2A monocytes, and these differences between the two haplotypes were consistently observed three times over a 3-mo period; no differences were observed for IL-1α or TNFα. NLRP1 RNA and protein levels were not altered by the predominant high-risk haplotype, indicating that altered function of the corresponding multivariant NLRP1 polypeptide predisposes to autoimmune diseases by activation of the NLRP1 inflammasome.

Genome-wide association analyses identify 13 new susceptibility loci for generalized vitiligo.

We previously reported a genome-wide association study (GWAS) identifying 14 susceptibility loci for generalized vitiligo. We report here a second GWAS (450 individuals with vitiligo (cases) and 3,182 controls), an independent replication study (1,440 cases and 1,316 controls) and a meta-analysis (3,187 cases and 6,723 controls) identifying 13 additional vitiligo-associated loci. These include OCA2-HERC2 (combined P = 3.80 × 10(-8)), MC1R (P = 1.82 × 10(-13)), a region near TYR (P = 1.57 × 10(-13)), IFIH1 (P = 4.91 × 10(-15)), CD80 (P = 3.78 × 10(-10)), CLNK (P = 1.56 × 10(-8)), BACH2 (P = 2.53 × 10(-8)), SLA (P = 1.58 × 10(-8)), CASP7 (P = 3.56 × 10(-8)), CD44 (P = 1.78 × 10(-9)), IKZF4 (P = 2.75 × 10(-14)), SH2B3 (P = 3.54 × 10(-18)) and TOB2 (P = 6.81 × 10(-10)). Most vitiligo susceptibility loci encode immunoregulatory proteins or melanocyte components that likely mediate immune targeting and the relationships among vitiligo, melanoma, and eye, skin and hair coloration.

Congruence as a measurement of extended haplotype structure across the genome.

BACKGROUND: Historically, extended haplotypes have been defined using only a few data points, such as alleles for several HLA genes in the MHC. High-density SNP data, and the increasing affordability of whole genome SNP typing, creates the opportunity to define higher resolution extended haplotypes. This drives the need for new tools that support quantification and visualization of extended haplotypes as defined by as many as 2000 SNPs. Confronted with high-density SNP data across the major histocompatibility complex (MHC) for 2,300 complete families, compiled by the Type 1 Diabetes Genetics Consortium (T1DGC), we developed software for studying extended haplotypes. METHODS: The software, called ExHap (Extended Haplotype), uses a similarity measurement we term congruence to identify and quantify long-range allele identity. Using ExHap, we analyzed congruence in both the T1DGC data and family-phased data from the International HapMap Project. RESULTS: Congruent chromosomes from the T1DGC data have between 96.5% and 99.9% allele identity over 1,818 SNPs spanning 2.64 megabases of the MHC (HLA-DRB1 to HLA-A). Thirty-three of 132 DQ-DR-B-A defined haplotype groups have > 50% congruent chromosomes in this region. For example, 92% of chromosomes within the DR3-B8-A1 haplotype are congruent from HLA-DRB1 to HLA-A (99.8% allele identity). We also applied ExHap to all 22 autosomes for both CEU and YRI cohorts from the International HapMap Project, identifying multiple candidate extended haplotypes. CONCLUSIONS: Long-range congruence is not unique to the MHC region. Patterns of allele identity on phased chromosomes provide a simple, straightforward approach to visually and quantitatively inspect complex long-range structural patterns in the genome. Such patterns aid the biologist in appreciating genetic similarities and differences across cohorts, and can lead to hypothesis generation for subsequent studies.

Replication and further characterization of a Type 1 diabetes-associated locus at the telomeric end of the major histocompatibility complex.

BACKGROUND: We recently reported an association between Type 1 diabetes and the telomeric major histocompatibility complex (MHC) single nucleotide polymorphism (SNP) rs1233478. As further families have been analyzed in the Type 1 Diabetes Genetics Consortium (T1DGC), we tested replication of the association and, with more data, analyzed haplotypic associations. METHODS: An additional 2717 case and 1315 control chromosomes have been analyzed from the T1DGC, with human leukocyte antigen (HLA) typing and data for 2837 SNPs across the MHC region. RESULTS: We confirmed the association of rs1233478 (new data only: P=2.2E-5, OR=1.4). We also found two additional SNPs nearby that were significantly associated with Type 1 diabetes (new data only rs3131020: P=8.3E-9, OR=0.65; rs1592410: P=2.2E-8, OR=1.5). For studies of Type 1 diabetes in the MHC region, it is critical to account for linkage disequilibrium with the HLA genes. Logistic regression analysis of these new data indicated that the effects of rs3131020 and rs1592410 on Type 1 diabetes risk are independent of HLA alleles (rs3131020: P=2.3E-3, OR=0.73; rs1592410: P=2.1E-3, OR=1.4). Haplotypes of 12 SNPs (including the three highly significant SNPs) stratify diabetes risk (high risk, protective, and neutral), with high-risk haplotypes limited to approximately 20,000 bp in length. The 20,000-bp region is telomeric of the UBD gene and contains LOC729653, a hypothetical gene. CONCLUSIONS: We believe that polymorphisms of the telomeric MHC locus LOC729653 may confer risk for Type 1 diabetes.

Genome-wide analysis identifies a quantitative trait locus in the MHC class II region associated with generalized vitiligo age of onset.

Generalized vitiligo is a common autoimmune disease in which acquired patchy depigmentation of skin, hair, and mucous membranes results from loss of melanocytes from involved areas. Previous genetic analyses have focused on vitiligo susceptibility, and have identified a number of genes involved in disease risk. Age of onset of generalized vitiligo also involves a substantial genetic component, but has not previously been studied systematically. In this study, we report a genome-wide association study of vitiligo age of onset in 1,339 generalized vitiligo patients, with replication in an independent cohort of 677 cases. We identified a quantitative trait locus for vitiligo age of onset in the major histocompatibility complex (MHC) class II region, located near c6orf10-BTNL2 (rs7758128; P=8.14 × 10(-11)), a region that is also associated with generalized vitiligo susceptibility. In contrast, there was no association of vitiligo age of onset with any other MHC or non-MHC loci that are associated with vitiligo susceptibility. These findings highlight the differing roles played by genes involved in vitiligo susceptibility versus vitiligo age of onset, and illustrate that genome-wide analyses can be used to identify genes involved in quantitative aspects of disease natural history, as well as disease susceptibility per se.

Comprehensive association analysis of candidate genes for generalized vitiligo supports XBP1, FOXP3, and TSLP.

We previously carried out a genome-wide association study of generalized vitiligo (GV) in non-Hispanic whites, identifying 13 confirmed susceptibility loci. In this study, we re-analyzed the genome-wide data set (comprising 1,392 cases and 2,629 controls) to specifically test association of all 33 GV candidate genes that have previously been suggested for GV, followed by meta-analysis incorporating both current and previously published data. We detected association of three of the candidate genes tested: TSLP (rs764916, P=3.0E-04, odds ratio (OR)=1.60; meta-P for rs3806933=3.1E-03), XBP1 (rs6005863, P=3.6E-04, OR=1.17; meta-P for rs2269577=9.5E-09), and FOXP3 (rs11798415, P=5.8E-04, OR=1.19). Association of GV with CTLA4 (rs12992492, P=5.9E-05, OR=1.20; meta-P for rs231775=1.0E-04) seems to be secondary to epidemiological association with other concomitant autoimmune diseases. Within the major histocompatibility complex (MHC), at 6p21.33, association with TAP1-PSMB8 (rs3819721, P=5.2E-06) seems to derive from linkage disequilibrium with major primary signals in the MHC class I and class II regions.

Genome-wide analysis of copy number variation in type 1 diabetes.

Type 1 diabetes (T1D) tends to cluster in families, suggesting there may be a genetic component predisposing to disease. However, a recent large-scale genome-wide association study concluded that identified genetic factors, single nucleotide polymorphisms, do not account for overall familiality. Another class of genetic variation is the amplification or deletion of >1 kilobase segments of the genome, also termed copy number variations (CNVs). We performed genome-wide CNV analysis on a cohort of 20 unrelated adults with T1D and a control (Ctrl) cohort of 20 subjects using the Affymetrix SNP Array 6.0 in combination with the Birdsuite copy number calling software. We identified 39 CNVs as enriched or depleted in T1D versus Ctrl. Additionally, we performed CNV analysis in a group of 10 monozygotic twin pairs discordant for T1D. Eleven of these 39 CNVs were also respectively enriched or depleted in the Twin cohort, suggesting that these variants may be involved in the development of islet autoimmunity, as the presently unaffected twin is at high risk for developing islet autoimmunity and T1D in his or her lifetime. These CNVs include a deletion on chromosome 6p21, near an HLA-DQ allele. CNVs were found that were both enriched or depleted in patients with or at high risk for developing T1D. These regions may represent genetic variants contributing to development of islet autoimmunity in T1D.

Common variants in FOXP1 are associated with generalized vitiligo.

In a recent genome-wide association study of generalized vitiligo, we identified ten confirmed susceptibility loci. By testing additional loci that showed suggestive association in the genome-wide study, using two replication cohorts of European descent, we observed replicated association of generalized vitiligo with variants at 3p13 encompassing FOXP1 (rs17008723, combined P=1.04x10(-8)) and with variants at 6q27 encompassing CCR6 (rs6902119, combined P=3.94x10(-7)).