圆锥角膜双胞胎家系新致病基因的鉴定及功能研究

Keratoconus (KC) is a common blinding corneal disease, and heredity plays a key role in its development. Detection and identification of the pathogenic gene has being a research hotspot and difficulty in this field. However, the reported pathogenic genes explain only a small percentage of KC. The etiology of most patients is still unknown. Our previous study found that the reported genetic Loci in abroad cannot be repeated in Chinese keratoconus population, which suggested that there may be novel genetic loci in our population. . Our group collected two KC twins pedigrees, and excluded the reported candidate gene mutation by sequencing. Therefore, we did exome sequence on eight core members of these two families (twins and their parents) to find a new candidate gene. Through bioinformatics analysis, including data integration, filtering, function prediction and annotation, we detected 332 functional variations preliminarily. Next, we plan to further analyze the data (including genetic models, novel mutations and twin difference analyses), and find out the possible pathogenic gene mutations. Moreover, the gene will be validated in additional independent 500 sporadic KC cases and 500 normal controls in order to evaluate the contribution of the gene to KC. Finally, study the gene function in vivo and in vitro models and reveal the underlying pathogenic mechanism. This research will further clarify the mechanism of keratoconus and provide new clues for diagnosis and treatment of keratoconus.

圆锥角膜是一种常见的与遗传相关的致盲性角膜疾病。其致病基因的发掘与鉴定一直是圆锥角膜研究领域的热点和难点。已报道的致病基因仅能解释很少一部分人的发病，大部分病人的病因仍是不明。我们前期研究发现，国外已报道的相关位点在中国圆锥角膜人群中不能重复，提示中国群体中极可能有新的相关基因区域。临床上我们诊断了两个圆锥角膜双胞胎家系，前期基因筛查排除了已报道致病基因突变致病的可能。因此课题组利用全外显子组测序技术对2个家系中的8名核心成员进行了测序，并通过位点的整合、过滤、功能预测及注释等生物信息学手段初步筛选出了332个功能性变异，下一步拟对数据进一步分析（遗传模式、新生突变及双生子差异分析），以找出新的致病基因突变；并在500散发病例和500正常对照中进行验证，确定致病基因突变谱；最后通过体内外模型研究其作用机制。本研究将进一步阐明圆锥角膜的发病机理，为圆锥角膜早期诊断及治疗提供新靶点和思路。

圆锥角膜是一种常见的与遗传相关的致盲性角膜疾病。其致病基因的发掘与鉴定一直是圆锥角膜研究领域的热点和难点。临床上我们诊断了两个圆锥角膜双胞胎家系，课题组利用全外显子组测序技术对2个家系中的8名核心成员进行了测序，并通过位点的整合、过滤、功能预测、注释、遗传模式及新生突变等生物信息学手段进行了筛选分析，在两个家系中分别找到了新的致病基因突变TUBA3D c.C31T（p.Q11X）和FGA c.T709C（p.F237L）；随后在200散发病例和200 正常对照中对上述两个基因进行验证，结果分别在2 例散发病人中检测到了位于TUBA3D及FGA的其他杂合突变，在正常人中都未检测到变异，验证了其致病性。.进一步功能机制研究发现：1）TUBA3D突变双胞胎病人与其正常父母相比TUBA3D 基因表达水平降低，尿激酶型纤溶酶原激活物（UPA）及基质金属蛋白酶1（MMP1）表达升高；2）TUBA3D基因突变后，其蛋白变得不稳定，并且可导致人角膜成纤维细胞基质金属蛋白酶的高表达和氧化压力升高，从而减少了角膜中的细胞外基质，导致了圆锥角膜的发生；3) FGA突变双胞胎病人与其正常父母相比FGA基因表达水平升高, 与正常人原代角膜成纤维细胞（HCF）相比，先证者HCF 胶原蛋白 IV型表达降低； 4) FGA突变后可导致人角膜成纤维细胞基质金属蛋白酶的高表达，从而减少了角膜中的细胞外基质，导致了圆锥角膜的发生。总之，本研究通过外显子组测序发现了圆锥角膜的2个新的致病基因，并通过功能研究探讨其致病机制，进一步阐明了圆锥角膜的发病机理，为圆锥角膜早期诊断及治疗提供新靶点和思路。

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