Association of generalized vitiligo with MHC class II loci in patients from the Indian subcontinent.

TO THE EDITOR Generalized vitiligo is a disease in which patches of depigmented skin and overlying hair result from autoimmune destruction of melanocytes in involved regions (Spritz, 2012). Clinic-based studies cite high prevalence of vitiligo in India, up to 8.8% (e.g., Handa and Kaur, 1999), although population-based surveys report much lower prevalence, 0.46% in Calcutta (Das et al., 1985) and 1.79% in South Gujarat (Mehta et al., 1973). Vitiligo is a distressing cosmetic problem in individuals of dark skin phototypes, owing to striking contrast between lesions and unaffected skin. This may explain the reported high prevalence of vitiligo in India and the negative impact on perceived quality of life in this population (Parsad et al., 2003). Indeed, vitiligo has long been recognized in India (Singh et al., 1974), the specific use of UV light treatment was pioneered in India (Menon, 1945), and some of the earliest genetic studies of vitiligo were carried out there: of ABO blood groups, α1-antitrypsin, and haptoglobin, and subsequent candidate gene studies, including GCH1, ACE, CAT, CTLA4, GPX1, IL4, MBL2, and PTPN22, most yielding negative or conflicting results. Recently, Singh et al. (2012) tested the genetic association of vitiligo in Indian patients with HLA-A, -B, -C in the major histocompatibility complex (MHC) class I region and HLA-DRB1 in the class II region, identifying primary genetic association with HLA-DRB1*07:01. Here, we describe a more comprehensive genetic association study of generalized vitiligo on the Indian subcontinent, using the Immunochip (Cortes and Brown, 2011) to screen 196,524 single-nucleotide polymorphisms (SNPs) in 128 loci previously implicated in autoimmune and inflammatory diseases, including 9,441 SNPs spanning the extended MHC on chromosome 6p. Our results suggest that there are at least two independent association signals in the MHC class II region, one located upstream of HLA-DRA and the other located between HLA-DRB1 and HLA-DQA1, generally similar to what we previously found in a genome-wide association study of vitiligo in European-derived whites (EUR) (Jin et al., 2010). Our initial study group consisted of 255 patients with generalized vitiligo and 377 unrelated non-vitiligo controls of Indian subcontinent (Pakistan, India, Sri Lanka, and Bangladesh) derivation. After quality control procedures, data for 120,724 remaining SNPs from 251 remaining cases were compared with those from 349 remaining controls. Suggestive association signals were considered as clusters of nearby SNPs with trend P-values <10−5. The International Immunochip Consortium has agreed on a genome-wide significance criterion of P<5 × 10−8 for studies utilizing the Immunochip (Cortes and Brown, 2011). As shown in Figure 1a and Supplementary Table S1 online, the only highly suggestive association signals were in the MHC class II gene region (Figure 1b), from rs3134942 (chr6:32168770) to rs2856674 (chr6:32659644), spanning the upstream part of NOTCH4 through HLA-DQB1. The principal region of association encompassed c6orf10--BTNL2--HLA-DRA--HLA-DRB5--HLA-DRB1--HLA-DQA1 (Figure 1b), with extensive linkage disequilibrium (LD) through this region in this population (Figure 1c). One SNP, rs482044, located toward the centromeric end of the region, between HLA-DRB1 and HLA-DQA1, achieved genome-wide significance (G allele; P=1.94 × 10−8, odds ratio (OR)=1.93; Table 1), remaining significant (P=4.86 × 10−8) even after correction for the observed genomic inflation factor λ=1.06. To determine which SNPs in the MHC class II region represent primary association with vitiligo versus are signals secondary to LD, we applied a backward regression procedure, comparing a model including the seven most significant MHC class II SNPs to alternative models in which each SNP was removed one by one. This analysis suggested that this region contains two independent associated loci, one represented by rs482044-G (located between HLA-DRB1 and HLA-DQA1) and the other represented by rs3129859-C (located 6680 nt upstream of HLA-DRA). Forward regression analysis of these two SNPs showed that the model composed of rs3129859 was significantly (P=4.4 × 10−5) improved by adding rs482044, and that the model composed of rs482044 was significantly improved (P=6.0 × 10−4) by adding rs3129859. In contrast to our previous findings in EUR (Jin et al., 2010), we observed no apparent association of vitiligo with SNPs in the MHC class I region in this Indian–Pakistani population (Figure 1a and b). Furthermore, considering loci represented on the Immunochip that have been reported to be associated with vitiligo in previous candidate gene studies from India, no SNPs in the ACE (3 SNPs), CTLA4 (505 SNPs), or IL4 (103 SNPs) gene regions showed even nominal association in the present study. To confirm the association of generalized vitiligo with MHC class II region SNPs in the Indian subcontinent, we carried out a replication study of rs3129859 and rs482044, as well as the third most significant Immunochip SNP, rs3096691 (located just upstream of NOTCH4) (Figure 1b). These three SNPs were genotyped in 685 unrelated generalized vitiligo cases and 774 unrelated controls from Gujarat state, India. All three were in Hardy–Weinberg equilibrium in the controls, and all three achieved at least nominal significance in the replication study (Table 1). The most significant association in the replication study was observed for rs3129859-C (P=9.48 × 10−9), with no significant heterogeneity of OR between the two studies (PBreslow–Day=1.15 × 10−1). Cochran–Mantel–Haenszel meta-analysis of the rs3129859 data from the Immunochip screen and replication study similarly yielded the strongest overall association (P=4.30 × 10−14, OR=1.67; 95% confidence interval 1.46–1.91). Association was also confirmed in the replication study for rs482044 (P=1.11 × 10−4), with only nominal association for rs3096691 (P=2.32 × 10−2), although both of these SNPs exhibited heterogeneity of OR. Both rs482044 (P=1.58 × 10−2) and rs3129859 (P=1.20 × 10−6) remained significant when each was conditioned on the other. Overall, our findings thus generally confirm the association of vitiligo with at least two independent loci in the MHC class II region. In a previous genome-wide association study of generalized vitiligo in EUR subjects, we found that both vitiligo susceptibility (Jin et al., 2010) and age of onset (Jin et al., 2011) are similarly associated with at least two independent loci in the MHC class II region. To assess whether the MHC class II loci observed in the Indian subcontinent and EUR populations might correspond ancestrally, we carried out trans-ethnic meta-analysis using MANTRA (Morris, 2011), which indicated that the MHC association signal represented by rs482044 in the Indian subcontinent population apparently corresponds to the MHC signal represented by rs532098 in EUR (Jin et al., 2010) (Supplementary Table S2 online). In contrast, rs3129859 is not significantly associated with vitiligo in EUR (Jin et al., 2010), and the correspondence between the association signals upstream of HLA-DRA observed in both populations remains uncertain. Our findings thus highlight both similarity and differences of vitiligo MHC genetic associations in subjects from different major world populations. In the Indian subcontinent, this study and that of Singh et al. (2012) support the association of vitiligo with loci in the MHC class II region, but show no primary association in the MHC class I region. Similarly, in the EUR population, vitiligo is also associated with multiple signals in the MHC class II region, at least one of which, between HLA-DRB1 and HLA-DQA1, appears to correspond to one in the Indian subcontinent population. However, in the EUR population vitiligo shows primary association with HLA-A in the distal class I region (Jin et al., 2010), specifically HLA-A*02:01 (Jin et al., 2012). In addition, studies in the Chinese population show principal MHC association in the class III region (Quan et al., 2010) and in the proximal class I region, between HLA-B and HLA-C (Liu et al., 2012). Together, these similarities and differences of principal MHC genetic associations with generalized vitiligo among different populations may partly underlie differing prevalence of this autoimmune disease in different groups around the world. This study was approved by the Combined Institutional Review Board (COMIRB) of the University of Colorado–Denver, Aurora, CO, USA; by the Ethics Committee of King Edward Medical University, Lahore, Pakistan; and by the Institutional Ethical Committee for Human Research (IECHR) of The Maharaja Sayajirao University of Baroda, Gujarat, India, and was conducted in accord with the Declaration of Helsinki Principles. Written informed consent was obtained from all study subjects. The authors state no conflict of interest. This work was supported by grants AR045584 and AR056292 from the National Institutes of Health and by a generous gift from the Doshi Family Foundation. We thank Drs Matt Brown and Adrian Cortes for advice regarding the subset of Immunochip SNPs appropriate to use for population stratification analysis. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper