扩张型心肌病候选致病基因NAG的遗传及功能研究

Dilated cardiomyopathy (DCM) represents the third most common cause of heart failure after coronary artery disease and hypertension. The pathogenic mechanisms of DCM remain unclear, the progress of treatment is quite slow, and the patients have poor prognosis. Genetic study could offer good opportunity in discovering new mechanism of diseases, which could be completely novel. The underlying genetic causes of roughly 60% of familial DCM (FDCM) cases remain unknown. Previously in a FDCM pedigree in which disease-causing variations in known cardiomyopathy genes have been ruled out, we identified a mutation in neuroblastoma amplified gene (NAG, c.A5227G) as the candidate disease-causing mutation of this family. This mutation is absent from 550 ancestry-matched normal Han Chinese individuals, and not found in the public databases including dbSNP, the 1000 Genome, ESP6500, and the ExAC databases. Also in 22 DCM patients not harboring disease-causing mutations in known cardiomyopathy genes, we sequenced the whole exomes and exome-intron boundaries of NAG gene, and found 1 patient present with this c.A5227G mutation. NAG is a critical regulator of the alternative splicing-nonsense mediated mRNA decay (AS-NMD) process, which is important and always involved in genetic diseases. Particularly, a number of AS-NMD factors, including RBM20, Smg1 and hnRNP U, have been reported to cause DCM phenotype in mice once they were mutated or knocked out. In our future work, we will confirm that NAG is a novel disease-causing gene of DCM from two aspects—the clinical genetic aspect and functional molecular aspect. We will focus on the splicing and transcriptional regulation of NAG on cardiomyocyte specific genes, and endoplasmic reticulum stress and cell apoptosis. We will also for the first time describe the atlas of clinical genetic of human dilated cardiomyopathy in Chinese population.

扩张型心肌病（DCM）发病率高达1/250，发病机制并不完全清楚，导致治疗进展缓慢。遗传研究在发现疾病新致病机制中有先驱作用。DCM大部分遗传学基础尚不清楚，已知的致病基因只能解释40%的家族性DCM。我们对一排除已知致病基因的DCM家系，采用全外显子测序的方法，鉴定出NAG基因c.A5227G突变为唯一候选致病突变；该突变不存在于550例同族对照人群中。在22例排除已知致病基因的DCM患者中，1例同样携带NAG c.A5227G突变。NAG是选择性剪切-无义介导的mRNA降解（AS-NMD）调节过程的关键分子，后者在遗传疾病中有重要作用：多个AS-NMD调节分子如RBM20、Smg1、hnRNP U等功能改变均可导致心肌病。申请人拟完善遗传和功能证据，证明NAG是DCM新的致病基因，主要在调节心脏特异性分子的剪切和转录及细胞凋亡的方面阐述其致病机制；并首次阐述中国DCM患者致病基因图谱。

扩张型心肌病（DCM）发病率高达1/250，发病机制并不完全清楚，导致治疗进展缓慢。遗传研究在发现疾病新致病机制中有先驱作用。DCM大部分遗传学基础尚不清楚，已知的致病基因只能解释40%的家族性DCM。我们对一排除已知致病基因的DCM家系，采用全外显子测序的方法，鉴定出NAG基因c.A5227G, p.Met1743Val突变为唯一候选致病突变。对90例特发性并且已排除已知致病基因的DCM患者，利用Sanger测序法对NAG基因测序，发现一例患者携带上述NAG c.A5227G突变，另一患者携带NAG c.C2300T, p.Thr767Ile突变。两突变均位于物种的高度保守区域，不存在于公共数据库和550例同族对照人群中。NAG是选择性剪切-无义介导的mRNA降解（AS-NMD）调节过程的关键分子，后者在遗传疾病中有重要作用。通过构建突变型病毒，我们发现NAG可调节心脏特异性分子的剪切，如CAMK2D和RyR2。因此，NAG可能通过影响心肌病相关基因剪切导致DCM发生。.此外，我们对118例DCM患者进行心肌病相关基因的靶向二代测序，发现41例患者携带有致病基因突变，占比34.7%，其中包括22个INDEL突变和18个错义突变。32个突变为新发突变。除LMNA: c.568C>T p.R190W和 RBM20: c.2017C>T p.R673W突变均被两例患者携带外，其他突变均只有一例患者携带。没有患者同时携带2种致病突变。突变的基因分布不均，其中TTN基因占比最大，达31%；之后为LMNA占比14.3%；RBM20和NEXN，各占比4.8%；其余突变基因均占比2.4%。我们比较携带突变和不携带突变患者的临床特点及预后，发现患者性别、年龄、临床药物的使用、左室射血分数和心脏结构等在两者无显著差异。我们以心源性死亡或需要接受心脏移植为终点，发现携带突变和不携带突变的患者发生上述终点的风险无差异。同样，以TTN突变、LMNA突变单独分析，亦未发现与其对照有显著差异。因此，我们首次阐述中国DCM患者致病基因图谱以及突变与预后的关系。