心肌病相关基因剪接变异预测软件的评估与临床应用  

Evaluating the clinical application of computational methods for identifying splice variants in cardiomyopathy-associated genes

作　　者：王梓懿 刘浩[1] 吴东洋 赵春霞 WANG Ziyi;LIU Hao;WU Dongyang;ZHAO Chunxia(Division of Cardiology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,Hubei,China)

机构地区：[1]华中科技大学同济医学院附属同济医院心血管内科,湖北武汉430030

出　　处：《癌变．畸变．突变》2024年第2期85-93,共9页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金面上项目(81873506)。

摘　　要：目的:发生在非经典剪接区域的基因突变是遗传性心肌病的重要致病因素。目前研究者已经开发了多种软件来预测变异对可变剪接的影响。然而,这些预测软件在遗传性心肌病基因诊断中的应用尚不明确。因此,本研究拟评估相关软件的预测性能,并探讨这些预测软件在肥厚型心肌病(HCM)患者中的临床应用。方法:对1 212名散发性肥厚型心肌病患者进行全外显子组测序(WES),并从公开发表研究中收集引起HCM相关基因可变剪接的突变位点。基于剪接区域特异性的策略来评估剪接变异预测软件的性能,找到针对各个区域的最优预测软件来筛选候选剪接变异,最后在体外HEK293细胞中通过Minigene实验来验证这些变异的剪接结果。结果:性能评估表明,各个剪接区域的最佳预测软件分别是Pangolin(深外显子、核心供体和扩展供体区)、MLCsplice(扩展供体、核心受体和扩展受体区)、MMSplice和SpliceAI(扩展供体区)。本研究在4.5%(54/1 212)的散发性肥厚型心肌病病例中预测得到43个“优先变异”,其中有23个变异经实验证实会导致异常剪接。结论:基于剪接区域特异性预测方法可以有效识别剪接变异,从而提高遗传性心肌病基因检测的诊断率。OBJECTIVE:Non-canonical splice variants are increasingly recognized as key contributors to inherited cardiomyopathies.Several computational methods have been developed to prioritize the effects of variants on splicing.However,their application in the genetic diagnosis of cardiomyopathies remains unclear.Thus,the performance of computational methods for cardiomyopathy-associated genes and of their clinical applications in hypertrophic cardiomyopathy(HCM)patients were evaluated.METHODS:Whole-exome sequencing was performed on 1212 unrelated patients with HCM,and splice-site variants in cardiomyopathyassociated genes were collected from scientific publications.A splicing-region-specific strategy was employed to evaluate performance of the computational methods and to used optimal methods for prioritizing candidate splice variants.Splicing effects of these variants were assessed using in vitro Minigene assays.RESULTS:The performance evaluation showed that the optimal methods were Pangolin(deep exonic,core donor,and extended donor regions),MLCsplice(extended donor,core acceptor,and extended acceptor regions),MMSplice,and SpliceAI(extended donor region).Using these optimal methods,53.5%(23/43)of the variants were prioritized and confirmed to cause aberrant splicing.A total of 23 splice variants were found in 4.5%(54/1212)of the unrelated HCM cases.CONCLUSION:Based on the splicing-region-specificity,the prediction strategy effectively identified splice variants,thereby improving the diagnostic value of genetic testing for inherited cardiomyopathies.

关 键 词：可变剪接 肥厚型心肌病 全外显子组测序 Minigene实验 致病性验证 

分 类 号：R542.2[医药卫生—心血管疾病]