中国汉族人特应性皮炎易感基因FLG的精细定位与功能研究

Atopic dermatitis (AD) is a common chronic relapsing, inflammatory skin disorder,often accompanies with severe pruritus, which may severely affect patients’physical and mental health. The proposer’s institution at the graduate level have found that FLG was associated with AD through a genome-wide association study.Based on which, the proposer performed an in-depth study,identified a FLG variant C.3321delA, and carried out genotype-phenotype associated analysis which revealed that the variant C.3321delA was associated with several complications of AD. However，the FLG region is complex.To date,over 60 variants of FLG have been reported around the world，but the true functional causal variants of FLG remain unclear.In order to identify the causal variants within FLG and reveal the potential roles in the mechanism of AD in Han Chinese population,we plan to perform high overlay sequencing within FLG using new sequencing technique and build the haploid reference template. We will perform genotype imputation analysis based on the previous data, Sequenom validation with another sample for FLG, and screen out the true causal variants by this fine mapping way. In addition, a series of functional studies on the identified causal variants at variant and gene levels will also be performed to make sure the true causal variants of FLG gene for AD, and it will provide a new theoretical basis for genetic pathogenesis of AD.

特应性皮炎（AD）是一种常见的慢性复发性炎症性皮肤病，常伴剧烈瘙痒，严重影响患者身心健康。申请人读研期间所在单位通过全基因组关联研究发现FLG基因与AD相关，申请人在此基础上进行深入研究，鉴定出FLG变异位点C.3321delA，基因型-表型的相关性分析结果表明该突变与AD的多种伴发症相关。然而该基因区域复杂，目前国内外已报道了60多个不同的FLG变异位点，但真正的功能性致病变异位点尚未明确。为了鉴定出FLG基因与汉族人AD易感性真正关联的致病变异位点，并揭示其在AD发病中的相关作用机制，本项目拟采用新一代测序技术对FLG区域进行高覆盖度测序并构建单倍型参考模板,在前期基因分型数据基础上进行基因型推算(imputation)，利用Sequenom基因分型在独立样本中验证，通过这种精细定位的方法筛选出真正的致病变异，并从变异位点水平、基因水平对其开展功能学研究，最终鉴定出FLG区域真正的功能

特应性皮炎是一种常见的炎症性皮肤疾病。大量证据表明，非编码RNA在特应性皮炎的发生发展中起重要作用。然而，血浆microRNA在特应性皮炎中的作用尚不清楚。本研究旨在鉴定关键的血浆microRNA，并探讨其在特应性皮炎中的潜在作用。通过miRNA-seq对5例儿童特应性皮炎和5例儿童对照的血浆microRNA进行测序，鉴定了40个差异表达的microRNA，其中上调26个，下调14个。5个差异表达的microRNA在30例儿童特应性皮炎和15例儿童对照中进行进一步实时定量PCR验证。其中hsa-miR-194-5p的倍数变化最为显著。ROC曲线分析表明hsa-miR-194-5p可作为特应性皮炎的诊断性生物标志物(特异性96.7%，敏感性53.3%)。因此，我们选择hsa-miR-194-5p进行进一步的研究。将hsa-miR-194-5p过表达人表皮角蛋白细胞，通过RNA-Seq鉴定出22个下调基因。基因功能与通路数据库分析表明，这些下调基因可以显著富集于角质细胞分化和皮肤屏障的建立功能通路，这与特应性皮炎的发病机制有关。hsa-miR-194-5p过表达人表皮角蛋白细胞后，HS3ST2基因的表达下调。通过免疫印迹分析表明HS3ST2是hsa-miR-194-5p的靶基因，荧光素酶报告分析显示hsa-miR-194-5p可以直接与HS3ST2的3’-UTR结合。本研究结果表明hsa-miR-194-5p可能通过抑制HS3ST2参与特应性皮炎的发病机制，为进一步分析AD的发病机制提供了重要依据。

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