利用外显子捕获测序技术鉴定肥厚型心肌病新致病突变的研究

Hypertrophic cardiomyopathy is a common inherited heart disease and recognized to be an important cause of arrhythmias, sudden death and heart failure. Due to the limitations of traditional detection methods, so far pathogenic mutations remain unknown in 35% of patients with familial hypertrophic cardiomyopathy and 60% of sporadic patients. We performed targeted capture and high throughput sequencing for a case in a family with hypertrophic cardiomyopathy. No reported mutations were detected, and four novel single point variants were screened. These candidate pathogenic mutations will be validated in all members of the pedigree. If no variant will segerate with disease, further studies are needed to screen pathogenic mutation in this pedigree by performing whole-exome sequencing. After the novel mutation is identified, it will be validated in a large number of cases with family history and sporadic cases as well as the normal population, and genotype-phenotype correlation will be studied. The function of the novel mutation in the pathogenesis of hypertrophic cardiomyopathy will be explored by infecting recombinant adenovirus expression vector into cardiomyocytes and specifically silencing the mutation expression in cardiomyocytes. The purpose of this study is to look for and validate novel pathogenic mutation associated with hypertrophic cardiomyopathy and provide new targets for the prevention and treatment of hypertrophic cardiomyopathy.

肥厚型心肌病是一种常见的遗传性心脏疾病，是心律失常、猝死和心脏衰竭的重要原因。受传统检测方法的限制，目前有35%的肥厚型心肌病家系和60%的散发病例的致病突变尚未发现。我们前期工作对一个肥厚型心肌病大家系中的一例患者进行了目标基因的捕获测序，经分析后排除了已知致病突变的作用，同时筛选出4个可能导致该家系疾病的新突变位点。本课题将在该家系内全体成员中对这4个位点进行验证，如验证后未发现与疾病共分离的新致病突变，则对该家系成员进行全外显子测序以找到致病突变。确定致病突变后，选取大量汉族肥厚型心肌病家系患者、散发病例以及正常人群，验证致病突变在患者和正常人群中的分布，并且探讨该致病突变与患者临床表型的关系。利用携带该突变位点的基因的腺病毒载体转染心肌细胞及RNA干扰技术特异性沉默该基因突变位点的表达，探索该致病突变在肥厚型心肌病发病机制中的作用，为肥厚型心肌病的防治提供新思路。

肥厚型心肌病是一种常见的遗传性心脏疾病，是心律失常、猝死和心脏衰竭的重要原因。受传统检测方法的限制，目前有 35%的肥厚型心肌病家系和 60%的散发病例的致病突变尚未发现。我们前期工作对一个肥厚型心肌病大家系中的一例患者进行了目标基因的捕获测序，经分析后发现MYBPC3 Y842X位点是导致该家系的致病位点。MYBPC3 基因是HCM一个重要的致病基因，能解释35%的HCM患者。为了验证MYBPC3在中国肥厚型心肌病患者的致病情况，我们选取了114个肥厚型心肌病患者进行了验证，进行了包括MYBPC3在内的24个基因进行了目标基因的捕获测序，最后在25个患者中发现8个新突变和12个已知突变。同时也进行了基因型与表型的分析，发现带有两个突变和终止突变的患者与带有错义突变的患者相比显示严重临床表型，也验证了MYBPC3 p.Y842X是中国肥厚型心肌病患者的一个热点突变，呈现严重临床表型。成果于2015年以SCI文章的形式发表于Scientif ic Reports（Sci Rep. 2015 Jun 19;5:11411）上。同时，我们对患者也进行了24个目标基因的捕获测序，开展了中国肥厚型心肌病的致病突变谱研究,对基因型-临床表型的关系进行深入研究。经过上述研究未发现致病突变的家系进行全外显子捕获测序，并进行候选基因与心肌病相关性排序和候选基因功能富集，鉴定肥厚型心肌病候选新致病基因。后续研究利用Cas9技术制备热点突变MYBPC3 Y842X以及新致病基因的Knockin小鼠进行功能学的验证。

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