寻常型银屑病易感基因GJB2功能性SNP的确定和生物效应研究

Psoriasis is a skin disease with complex genetic basis. Recently, the development of genome-wide association study (GWAS) leaded to accumulate a mass of valuable data in the field of psoriasis genetic research. In current study, we focused on the GJB2, a susceptibility gene of the psoriasis resulting from genome-wide association studies in Chinese Han population. Firstly, we repeatedly examined the association between the SNP rs3751385 of GJB2 gene and psoriasis in south Chinese Han population using case-control assay, and analysed the haplotypes of the GJB2 gene to observe that GJB2 haplotypes might be associated with in psoriatic properties/phenotype including the clinical severity of psoriasis. For seeking the potential functional SNPs of GJB2 gene, on the one hand, we will screen association between the functional variants of influenced Cx26 protein which is encoded by GJB2 gene and psoriasis. On the other hand, the location and frequency of GJB2 gene SNPs in coding and regulatory region will validate by direct full-length sequencing from the genome DNA of 100 normal subjects in south Chinese Han population, then, we will incorporate functional predictions of GJB2 gene SNPs in coding and regulatory region using the methods of haplotype construction and bioinformatics softwares. At same time, we will confirm the functional predictions of GJB2 gene SNPs association with in psoriasis by case-control assay. After that, we will select the functional SNPs to carry out a series of functional experiments including the construction of genotype carrier, immunofluorescence, dyes transportion and cell biology and so on. We will determinate the biologic effects of the functional GJB2 SNPs. Finally, the purpose of this study is to explain the biologic functions and the clinical phenotype effects of psoriasis susceptibility GJB2 functional SNPs in the pathogenesis of psoriasis.

银屑病是一类具有复杂遗传背景的皮肤疾病，随着全基因组关联分析(GWAS)的发展，银屑病遗传研究领域积累了许多有价值数据。本研究拟锁定中国人群银屑病GWAS研究已公布易感基因GJB2，首先在中国南部汉族人群重复验证其多态位点rs3751385与银屑病的关联，并开展易感基因单倍型与银屑病性状/表型如：疾病严重程度的关联分析。为寻找银屑病易感基因GJB2的功能性SNP，先筛选能影响GJB2基因编码蛋白Cx26功能的SNP，并对100例南部汉族正常人基因组DNA进行包括GJB2基因编码区和调控区的全长测序，明确其SNP分布情况后，利用单倍型构建、生物信息学和关联分析等方法发现GJB2潜在功能性SNP，再选取功能性SNP，运用基因型载体构建、免疫荧光、染料转运及细胞生物学等方法，证实GJB2功能性SNP的生物学效应。旨在初步解答银屑病易感基因GJB2功能性SNP发挥的具体作用和影响疾病表型的机制。

本项目立足于中国人群寻常型银屑病全基因组关联研究已公布的GWAS研究数据，锁定寻常型银屑病易感基因GJB2。1）首先通过病例-对照关联方法，成功验证寻常型银屑病易感基因GJB2的SNP位点rs3751385：C>T与疾病易感存在明显关联性；2）其次，通过在100例中国南部正常人群基因组中扩增GJB2基因编码区和部分调控区的片段，直接测序，获知GJB2基因存在的SNP位点与Hapmap数据库公布的一致，包括多态位点rs2274084：C>T，rs2274083：A>G，和rs3751385：C>T，除位点rs3751385在银屑病与正常对照的关联研究中呈现显著差异，其他多态位点并无显著差异；3）免疫组织化学研究亦显示寻常型银屑病患者皮损内GJB2基因编码的CX26蛋白水平较正常对照明显增高，同时以GJB2基因多态位点rs3751385：C>T作为分型依据，将银屑病患者分为：TT，CC和CT三种基因型，发现能增加银屑病患病风险的TT基因型患者皮损组织GJB2的mRNA和蛋白水平均较其他两种基因型明显增加，并且与银屑病严重程度有相关性，及TT基因型患者PASI评分较CC和CT基因型增加，疾病严重程度增加。4）比较寻常型银屑病患者与正常对照人群血浆GJB2的水平，结果显示银屑病患者GJB2浓度明显低于正常对照组，而血浆GJB2浓度与银屑病患者PASI积分呈负相关(r=-0.247, p=0.036)；5）功能研究显示，银屑病易感基因GJB2多态位点rs72474224：C>T，改变其编码蛋白CX26氨基酸序列，但并不影响细胞增殖及细胞周期功能。通过本研究项目，我们证实GJB2基因与中国南部人群寻常型银屑病易感性相关；明确了中国南部人群GJB2基因编码区及部分调控区内SNP分布情况；同时初步发现，易感基因GJB2多态位点rs72474224：C>T这一能改变编码蛋白CX26氨基酸序列（Val→Ile）的位点，可能不是通过影响角质形成细胞的增殖能力，发挥其易感基因的生物学效应，参与到银屑病发病环节。