Genetic variation of promoter sequence modulates XBP1 expression and genetic risk for vitiligo

Abstract

Our previous genome-wide linkage analysis identified a susceptibility locus for generalized vitiligo on 22q12. To search for susceptibility genes within the locus, we investigated a biological candidate gene, X-box binding protein 1(XBP1). First, we sequenced all the exons, exon-intron boundaries as well as some 5' and 3' flanking sequences of XBP1 in 319 cases and 294 controls of Chinese Hans. Of the 8 common variants identified, the significant association was observed at rs2269577 (p_(trend) = 0.007, OR = 1.36, 95% CI = 1.09-1.71), a putative regulatory polymorphism within the promoter region of XBP1. We then sequenced the variant in an additional 365 cases and 404 controls and found supporting evidence for the association (p_(trend) = 0.008, OR = 1.31, 95% CI = 1.07-1.59). To further validate the association, we genotyped the variant in another independent sample of 1,402 cases and 1,288 controls, including 94 parent-child trios, and confirmed the association by both case-control analysis (p_(trend) = 0.003, OR = 1.18, 95% CI = 1.06-1.32) and the family-based transmission disequilibrium test (TDT, p = 0.005, OR = 1.93, 95% CI = 1.21-3.07). The analysis of the combined 2,086 cases and 1,986 controls provided highly significant evidence for the association (p_(trend) = 2.94x10(-6), OR = 1.23, 95% CI = 1.13-1.35). Furthermore, we also found suggestive epistatic effect between rs2269577 and HLA-DRB1*07 allele on the development of vitiligo (p = 0.033). Our subsequent functional study showed that the risk-associated C allele of rs2269577 had a stronger promoter activity than the non-risk G allele, and there was an elevated expression of XBP1 in the lesional skins of patients carrying the risk-associated C allele. Therefore, our study has demonstrated that the transcriptional modulation of XBP1 expression by a germ-line regulatory polymorphism has an impact on the development of vitiligo.

Figure 1. Difference of the promoter activity between the G and C allele of rs2296577 in Mel-RM cells and HaCat cells.

Each column represents mean±s.d. of at least three experiments. *Two-tailed P<0.05 by Student's t-test.

Figure 2. XBP1 mRNA expression in the paired lesional and non-lesional skins from 38 patients.

XBP1 mRNA was measured by using quantitative real-time PCR. Data represent the normalized XBP1 mRNA expression level by 18S rRNA. The normalized expression data was standardized so that the average of the normalized expression in the non-lesional skins is equal to 1. The comparison of XBP1 mRNA expression between the 38 paired lesional and non-lesional skins were performed by using nonparametric Wilcoxon Signed Ranks Test.

Figure 3. Relative expressions of XBP1 in the lesional and non-lesional skins (fold) of three patient groups with different genotypes of rs2269577: 12 patients with GG genotype, 20 patients with GC genotype, and 6 patients with CC genotype.

The Avg. indicates the mean of the difference between the paired lesional and non-lesional skins within each of three genotypes. The y axis is in log2 scale.

Figure 4. XBP1 protein level determined by immunohistochemical staining in the paired lesional and non-lesional skins from 37 patients with vitiligo.

Data represent the percentage of positive cells. The comparison of XBP1 protein level between the 37 paired lesional and non-lesional skins was done by using Paired Samples T Test.

Figure 5. The difference of XBP1 protein level (fold) between the paired lesional and non-lesional skins in three groups of patients with different genotypes of rs2269577: 7 patients with GG genotype, 24 patients with GC genotype, and 6 patients with CC genotype.

The Avg. indicates the mean of the difference between the paired lesional and non-lesional skins within each of three genotypes.

Figure 6. Immunohistochemical staining of XBP1 protein in the paired lesional (A,B) and non-lesional skin (C,D) of a patient with vitiligo (magnifications: (A,C)×200; (B,D)×400).