Katherine Gowan

Variant of TYR and Autoimmunity Susceptibility Loci in Generalized Vitiligo

BACKGROUND Generalized vitiligo is an autoimmune disease characterized by melanocyte loss, which results in patchy depigmentation of skin and hair, and is associated with an elevated risk of other autoimmune diseases. METHODS To identify generalized vitiligo susceptibility loci, we conducted a genomewide association study. We genotyped 579,146 single-nucleotide polymorphisms (SNPs) in 1514 patients with generalized vitiligo who were of European-derived white (CEU) ancestry and compared the genotypes with publicly available control genotypes from 2813 CEU persons. We then tested 50 SNPs in two replication sets, one comprising 677 independent CEU patients and 1106 CEU controls and the other comprising 183 CEU simplex trios with generalized vitiligo and 332 CEU multiplex families. RESULTS We detected significant associations between generalized vitiligo and SNPs at several loci previously associated with other autoimmune diseases. These included genes encoding major-histocompatibility-complex class I molecules (P=9.05x10(-23)) and class II molecules (P=4.50x10(-34)), PTPN22 (P=1.31x10(-7)), LPP (P=1.01x10(-11)), IL2RA (P=2.78x10(-9)), UBASH3A (P=1.26x10(-9)), and C1QTNF6 (P=2.21x10(-16)). We also detected associations between generalized vitiligo and SNPs in two additional immune-related loci, RERE (P=7.07x10(-15)) and GZMB (P=3.44x10(-8)), and in a locus containing TYR (P=1.60x10(-18)), encoding tyrosinase. CONCLUSIONS We observed associations between generalized vitiligo and markers implicating multiple genes, some associated with other autoimmune diseases and one (TYR) that may mediate target-cell specificity and indicate a mutually exclusive relationship between susceptibility to vitiligo and susceptibility to melanoma.

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.

Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell–precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

Novel Vitiligo Susceptibility Loci on Chromosomes 7 (AIS2) and 8 (AIS3), Confirmation of SLEV1 on Chromosome 17, and Their Roles in an Autoimmune Diathesis

To the Editor: Generalized vitiligo (MIM 193200) is a common acquired disorder in which patches of white skin and overlying hair result from loss of pigment-forming melanocytes (reviewed in Bolognia et al. [1998], Kovacs [1998], and Hann and Nordlund [2000]), apparently because of a noninflammatory, T cell autoimmune response (reviewed in Ongenae et al. [2003]). Vitiligo occurs in 0.38% of whites (Howitz et al. 1977) and, in 23% of cases, is associated with other autoimmune disorders, particularly including autoimmune thyroid disease, pernicious anemia, systemic lupus erythematosus, Addison disease (Alkhateeb et al. 2003), and adult-onset insulin-dependent diabetes mellitus (our unpublished data). This complex of associated multiple autoimmune diseases most likely results from combinations of genes, some predisposing to an inherited autoimmune diathesis and others to specific forms of autoimmune disease. In the past, the association of various multiple autoimmune diseases has been termed “autoimmune polyendocrine syndrome, type 2” (APS2 [MIM 269200]), with a number of descriptive subcategories, the biological bases for which are uncertain (reviewed in Betterle et al. [2002]). Vitiligo is a polygenic, multifactorial disorder (Majumder et al. 1988; Nath et al. 1994; Arcos-Burgos et al. 2002; Alkhateeb et al. 2003), with frequent family clustering (Mehta et al. 1973; Carnevale et al. 1980; Goudie et al. 1983; Hafez et al. 1983; Das et al. 1985; Majumder et al. 1993; Alkhateeb et al. 2003), and ∼20% of probands having at least one affected first-degree relative (Alkhateeb et al. 2003). We previously described a genome scan of 71 white multiplex families with vitiligo, and we mapped AIS1 (autoimmune susceptibility 1), a locus in chromosome segment 1p31.3-p32.2 that apparently confers susceptibility to generalized vitiligo and associated autoimmune disorders, as well as seven additional suggestive linkage signals on chromosomes 1, 7, 8, 11, 19, and 22 (Alkhateeb et al. 2002; Fain et al. 2003). We have now extended this study to a total of 102 white multiplex families with vitiligo, providing substantially increased power for genomewide linkage analysis. Families were ascertained principally from the Vitiligo Society (U.K.) and the National Vitiligo Society (U.S.A.), as described elsewhere (Fain et al. 2003). Phenotypes were checked carefully by history, lesion maps, and, in most cases, physical examination and/or photographs; individuals for whom the phenotype was at all questionable were excluded. DNA was prepared from peripheral blood by standard methods, and genotyping was performed on a total of 660 individuals (300 affected with vitiligo; 192 females, 108 males) for 382 autosomal microsatellite markers from the ABI Prism 10-cM Linkage Mapping Set LMSv2-MD10, with manual checking of all genotypes by at least two people to minimize data errors, as described elsewhere (Fain et al. 2003). Multipoint nonparametric linkage analyses were performed using Allegro (Gudbjartsson et al. 2000). Heterogeneity testing between autoimmune and nonautoimmune families was performed using a predivided sample test (Morton 1956; Ott 1999). As shown in table 1, analysis of the extended 102-family cohort provides continued strong support for AIS1 at 73.7 cM on chromosome 1p (LOD=5.59; P=.000000279) (all positions have been updated in accordance with the deCODE genome map [Kong et al. 2002]). In addition, two other signals that previously were only suggestive now achieve threshold criteria for significant linkage (Lander and Kruglyak 1995). These loci, now designated “AIS2” at 89.4 cM on chromosome 7 (LOD=3.73; P=.0000208) and AIS3 at 54.2 cM on chromosome 8 (LOD=3.36; P=.0000418), thus represent candidates for confirmation by analysis of a replicate family cohort. Our data also provide support for a locus at 4.3 cM on chromosome 17 (LOD=3.07; P=.0000852), most likely corresponding to SLEV1, a locus detected in multiplex families with lupus that also segregate cases of vitiligo (Nath et al. 2001). Three linkage signals that were suggestive in our previous analysis of 71 families (Fain et al. 2003)—at 12.2 cM on chromosome 1p, 4.1 cM on 11p, and 107.8 cM on 19q—fell below the threshold for suggestive linkage at the 102-family level. However, we detected two new linkage signals that met criteria for suggestive linkage (Lander and Kruglyak 1995)—at 88.1 cM on chromosome 9q (LOD=2.34; P=.000238) and at 109.4 cM on 13q (LOD=2.30; P=.000563)—that represent candidates for follow-up extension and replication linkage studies. Table 1 Suggestive and Significant LODs (and P Values) for 71 and 102 Multiplex White Families with Vitiligo[Note] The 102 study families were selected solely on the basis of having multiplex cases of vitiligo. Nevertheless, half of the families segregated only vitiligo, whereas the other half also segregated various others of the vitiligo-associated autoimmune diseases (autoimmune thyroid disease, pernicious anemia, lupus, Addison disease, or adult-onset autoimmune diabetes mellitus) (Alkhateeb et al. 2002) (table 2). This provided an obvious basis for phenotypic stratification of the 102 families into autoimmunity-associated versus nonautoimmunity-associated family subgroups and analysis of the four a priori significant linkage signals in each subgroup. As shown in table 3, the AIS1, AIS2, and SLEV1 linkage signals derive principally from the autoimmunity-associated family subgroup and thus may predispose to a vitiligo-associated autoimmunity diathesis. Indeed, the AIS1 and SLEV1 LODs increased substantially on family stratification, even though the number of families was reduced by half. AIS1 was originally mapped in a large family with vitiligo, Hashimoto disease, and 21-hydroxylase autoantibody positivity (a preclinical marker for Addison disease) (Alkhateeb et al. 2002), and SLEV1 was originally mapped in families segregating lupus and vitiligo (Nath et al. 2001), so the derivation of these linkage signals from the autoimmunity-associated family subgroup is not unexpected. In contrast, the AIS3 linkage signal appears to derive principally from the nonautoimmunity-associated family subgroup. AIS3 may thus predispose to vitiligo per se, rather than to an autoimmune diathesis, although, of course, the basis of generalized vitiligo even in these families might still be autoimmune in nature. Table 2 Vitiligo-Associated Autoimmune Diseases in the 51 Autoimmunity-Associated Families[Note] Table 3 LODs (and P Values) on Phenotypic Stratification of 102 Multiplex Families with Vitiligo into Autoimmunity-Associated and Nonautoimmunity-Associated Family Subgroups Heterogeneity testing did not quite exclude the possibility that AIS1 might also contribute to vitiligo susceptibility in the nonautoimmune families, since the total LOD score did not decrease significantly (P=.084) when the autoimmune and nonautoimmune family subgroups are combined, compared with the LOD in the autoimmune families alone. This was also the case for AIS2 and AIS3 (P=.362 and .192, respectively). These results suggest the possibility of allelic heterogeneity at these loci between these two groups of families. However, for SLEV1, there was a significant (P=.018) decrease in the LOD score when the autoimmune and nonautoimmune family subgroups were combined, a result that suggests linkage to SLEV1 in the autoimmune families and nonlinkage in the nonautoimmune families. Furthermore, although SLEV1 was originally detected in multiplex families with lupus with at least one case of vitiligo (Nath et al. 2001), there was only a single case of possible lupus among all of our 51 autoimmune families. Thus, linkage to SLEV1 in these families indicates that SLEV1 confers susceptibility to a broader range of autoimmune diseases than just lupus and vitiligo. Our data thus indicate that generalized vitiligo can be divided into at least two distinct phenotypic subcategories that apparently involve different loci or alleles. Vitiligo associated with a specific constellation of autoimmune diseases is linked with AIS1, AIS2, and SLEV1, whereas vitiligo unassociated with other autoimmune diseases is linked with AIS3. These findings begin to elucidate the genetic underpinnings of vitiligo and to dissect the contributions of individual loci to the vitiligo-associated autoimmune disease diathesis.

NLRP1 haplotypes associated with vitiligo and autoimmunity increase interleukin-1β 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-1β (IL-1β), resulting in IL-1β 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-1β precursor to mature bioactive IL-1β 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.

Multi-SNP Analysis of MHC Region: Remarkable Conservation of HLA-A1-B8-DR3 Haplotype

Technology has become available to cost-effectively analyze thousands of single nucleotide polymorphisms (SNPs). We recently confirmed by genotyping a small series of class I alleles and microsatellite markers that the extended haplotype HLA-A1-B8-DR3 (8.1 AH) at the major histocompatibility complex (MHC) is a common and conserved haplotype. To further evaluate the region of conservation of the DR3 haplotypes, we genotyped 31 8.1 AHs and 29 other DR3 haplotypes with a panel of 656 SNPs spanning 4.8 Mb in the MHC region. This multi-SNP evaluation revealed a 2.9-Mb region that was essentially invariable for all 31 8.1 AHs. The 31 8.1 AHs were >99.9% identical for 384 consecutive SNPs of the 656 SNPs analyzed. Future association studies of MHC-linked susceptibility to type 1 diabetes will need to account for the extensive conservation of the 8.1 AH, since individuals who carry this haplotype provide no information about the differential effects of the alleles that are present on this haplotype.

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 = 1.04 × 10−8) and with variants at 6q27 encompassing CCR6 (rs6902119, combined P = 3.94 × 10−7).

Genome-wide association study of generalized vitiligo in an isolated European founder population identifies SMOC2, in close proximity to IDDM8.

Generalized vitiligo is a common disorder in which patchy loss of skin and hair pigmentation principally appears to result from autoimmune loss of melanocytes from affected regions. We previously characterized a unique founder population in an isolated Romanian community with elevated prevalence of generalized vitiligo and other autoimmune diseases, including autoimmune thyroid disease, rheumatoid arthritis, and type I diabetes mellitus. Here, we describe a genome-wide association study (GWAS) of generalized vitiligo in 32 distantly related affected patients from this remote village and 50 healthy controls from surrounding villages. Vitiligo was significantly associated with single-nucleotide polymorphisms (SNPs) in a 30-kb LD block on chromosome 6q27, in close vicinity to IDDM8, a linkage and association signal for type I diabetes mellitus and rheumatoid arthritis. The region of association contains only one gene, SMOC2, within which SNP rs13208776 attained genome-wide significance for association with generalized vitiligo (P=8.51x10(-8)) at odds ratio 7.445 (95% confidence interval=3.56-15.53) for the high-risk allele and population attributable risk 28.00. SMOC2 encodes a modular extracellular calcium-binding glycoprotein of unknown function. Our findings indicate that SMOC2 is a risk locus for generalized vitiligo and perhaps other autoimmune diseases.

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.

Decreased Levels of BAG3 in a Family With a Rare Variant and in Idiopathic Dilated Cardiomyopathy

The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole‐exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10‐nucleotide deletion in the BCL2‐associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C‐terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end‐stage HF undergoing cardiac transplantation when compared with non‐failing controls. Diminished levels of BAG3 protein may be associated with both familial and non‐familial forms of dilated cardiomyopathy. J. Cell. Physiol. 229: 1697–1702, 2014.