全基因组外显子测序搜寻新疆一维吾尔族特大EPPK家系致病基因及功能初探

Epidermolytic Palmoplantar Keratoderma(EPPK,OMIM 144200) is a heterogeneous disease characterized by thickening, and marked hyperkeratosis, of the epidermis of the palms and soles. In 1992, Reis first mapped a gene for EPPK to chromosome 17q11-q23, with linkage analysis using microsatellite DNA-polymorphisms in a single large family of 7 generations. Keratins are the major structural proteins of the epidermis and associated appendages. They are normally expressed in pairs consisting of one of each type, in a tissue and differentiation-specific manner. KRT9 specifically expressed in palmoplantar skin, so it naturally is considered a candidate gene in EPPK. So far, researchers in various racial or ethnic EPPK family have been found more than 20 different KRT9 mutations. We investigated in a large Uygur EPPK family from Xinjiang region with 257 pedigrees of 6 generations, including 195 immediate family. Blood samples were collected and genomic DNA was extracted from 48 members of the family. Six microsatellite repeat sequences on chromosome region 17q12-q21 and 12q13 was selected. Two-point linkage analysis and haplotype analysis were performed. The disease gene of the EPPK is located 17q 21.2（between marker 17/TG/36620115 and D17S846）. Chromosome 12q13 region was excluded with the negative Lod score obtained in marker D12S96 (Lod=- - ∞). There are not pathogenic mutation found in the gene keratin 9 as well as keratin 14, keratin 16, keratin 17. Based on previous study, we will apply whole-exome sequencing to identify causative pathogenic gene. Furthermore, the effect of causal gene silence,using silencing of specific gene by small interfering RNAs (siRNAs), in HaCaT cell were confirmed by real-time quantitative PCR and western blot respectively. In addition, Immunofluorescence analysis will indicate the keratin intermediate filament structure changed by the causal gene alternation.We can explore the role of the causal gene in the development of the EPPK. This research will help us to gain a better understanding on Uygur population EPPK both on the clinical and genetic aspects.

表皮松解性掌跖角化病（Epidermolytic Palmoplantar Keratoderma，EPPK）（OMIM 144200）是一种以掌跖表皮角化过度为主要特征的常染色体显性遗传病，也是一种具有遗传异质性的单基因遗传病，其发病与角蛋白基因9（KRT9）及其与之配对表达的角蛋白基因1（KRT1）的突变密切相关。我们前期收集到一个完整罕见的维吾尔族6代257人的EPPK大家系，通过微卫星标记连锁分析，将此家系致病基因定位在17q21.2上（chr17:36620083-37146934）约1Mb区域内，并对此区域内KRT9基因进行测序筛查未发现突变，除外候选致病基因KRT1、KRT9。本研究将在之前工作的基础上采用全基因组外显子测序技术搜寻新的致病基因，并采用质粒介导的RNA干扰等技术使EPPK致病基因沉默，从分子水平阐明新疆EPPK发病机制，为该病的产前诊断、基因治疗奠定实验基础。

表皮松解性掌跖角化症(epidermolytic palmoplantar keratoderma，EPPK，OMIM : 144200)是一种常染色体显性遗传性皮肤病，具有遗传异质性，未见维吾尔族人群EPPK致病基因及发病机制的相关报道。本研究收集一罕见维吾尔族6代257人EPPK大家系，绘制家系图，分析维吾尔族EPPK遗传特征；通过全基因组外显子测序结合前期连锁分析明确此维吾尔族EPPK大家系的致病基因为KRT9 c.C487T（p.R163W）。建立新疆维吾尔族EPPK致病基因cDNA文库。利用KRT9 R163W重组质粒与siRNA共转染至角质形成细胞，观察转染前后细胞骨架结构变化，KRT9 R163W高表达时影响中间丝组装，正常角质形成细胞骨架结构呈杂乱无序状态，部分细胞骨架结构消失。免疫酶免疫组化法检测KRT9R16W突变的EPPK石蜡组织中KRT1、KRT5、KRT6、KRT16、KRT10、KRT14及KRT12等角蛋白的表达量及表达位置的变化，结果显示KRT6表达位置由表皮的基底表达迁移至棘层及颗粒层，且在颗粒变性细胞周围表达量增多，而KRT1、KRT5、KRT16、KRT10、KRT14及KRT12表达位置及表达量未见明显变化。本研究丰富了不同民族EPPK的遗传学研究，为EPPK的产前诊断、靶向治疗提供一定的理论基础和实验依据。

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