| Background Generalized vitiligo is an acquired, non-contagious disorder in which progressive, patchy loss of pigmentation of skin and often overlying hair, and mucous membranes, results from loss of melanocytes from the involved areas. It is one of the most common pigmentary disorders affecting about 0.09%-2.7% of Chinese Han population. Although many different etiologic hypotheses have been suggest for vitiligo, the most compelling of which involves a combination of environment and genetic factors interacting to contribute to autoimmune melanocyte destruction. Vitiligo is strongly influenced by genetic factors. In the past years, researches mainly used linkage studies, candidate genes studies or other research strategies to search vitiligo susceptibility genes and some progress was made. However, there are limitations in these studies. For example, there are few microsatellite markers in linkage studies and the targeted region is large, which is why it is often difficult to identify the real susceptibility genes. Meanwhile, the genome linkage method is a family-based approach, which identified the diseases susceptibility locus. Whether it can represent the sporadic cases is unknown. Similar to candidate genes studies, researchers often selected candidate genes based on available data which suggest an implication of this gene in the disease. So, there are some limitations in candidate genes study, which could not uncover susceptibility genes of vitiligo with a genome wide coverage. In recent years, completion of the Human Genome Project (HGP) and the Human Genome Haplotype Mapping Project (HapMap) facilitated the rapid development of high-throughput gene genotyping technologies and the reduction of genotyping costs, which made genome-wide association study become possible in large-scale approaches. The method of genome wide SNP analysis is highly effective to uncover disease susceptibility genes. GWAS is one of the most effective ways currently described to identify susceptibility genes of complex diseases. The success of a large number of GWAS shows a strong efficiency to uncover susceptibility genes of complex diseases, and GWAS of vitiligo in Chinese Han and Caucasian have identified a spectrum of new associated genes/loci: TYR, C1QTNF6, RERE, LPP, UBASH3A, GZMB, PTPN22 and 6q27, 10p15.1. Most of these loci were immune related loci, seven of them have been reported associate with other autoimmune diseases.Object To explore the susceptibility variants for vitiligo, we carried out a candidate gene study in Chinese populations.Methods (1) We conducted a genome-wide association study of generalized vitiligo in Chinese Han population by genotyping 1,149 cases and 1,701 controls using Illumina Human 610-Quad BeadChips. We took the most promising SNPs for replication in Chinese Han (5,740 cases and 10,324 controls) and Chinese Uygur (713 cases and 758 controls) using Sequenom MassArray.(2) Replication stage:①Replication in the first stage: After quality control and statistical analysis, we selected 67 SNPs for replication in two independent samples of Chinese Han (2,827 cases and 3,876 controls) by using the Sequenom(?) MassARRAY research platform.②Replication in the second stage: we further genotyped the most promising 12 (of the 45 selected) SNPs that showed supportive association evidence in the initial validation study in additional 2,913cases and 6,448 controls using Sequenom(?) MassARRAY research platform. Then, for statistical analysis we combined GWAS, replication of the first stage and replication of the second stage together and found 3 SNPs reached genome-wide significance. In addition, one promising SNP showed a genome-wide association barely below significance.③Replication in the third stage: 12 most significant SNPs of the 45 SNPs were further genotyped in Chinese Uygur (713 cases and 758 controls) by using TaqMan assays. (3) RNA from 17 pairs of full-thickness vitiligo skin biopsies were used in DNA microarray analysis.(4) We also analysis the relationship between SNPs and genes in the loci.Results (1) After stringent quality control, 493,909 SNPs were analyzed in 1,117 cases and 1,701 controls by using Cochran-Armitage trend test to assess the genotype-phenotype association. (2) We identified one novel locus at 12q13 associated with vitiligo both in Chinese Han (rs10876864, P=8.07×10-12) and Chinese Uygur (rs10876864, P= P=1.01×10-2), which been reported to confer risk to several autoimmune disease, such as type 1 diabetes (T1D) and alopecia areata (AA). (3) We also identified one association evidence at 10q23 in Chinese Han (rs638893, P=2.47×10-9), this region have been reported to be associate with SLE. There are three genes in this region: PHLDB1, TREH,DDX6, PHLDB1 showed the differential expression between vitiligo and normal skin. (4) there are two genes in the region of 11q23: SLC29A3,CDH23 the most significant SNP was rs1417210 (P=1.83×10-8),we can not find the association between SNP rs1417210 and any gene of this region. (5) We also performed a repetation of previous reported vitiligo associated genes. Only one locus at 3q28 provided suggestive evidence (rs9851967, P=8.57×10-8)Conclusions We have performed a large scale GWAS for vitiligo in Chinese population, with replication in three independent Chinese population groups. Our study identifies three novel susceptibility loci at 12q13, 10q23, 11q23. In particular, it highlighted potential pathogenic overlap between vitiligo and other autoimmune diseases.
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