多重连接探针扩增和二代测序技术检测Duchenne型肌营养不良基因分析

Genetics analysis of patients with Duchenne muscular dystrophy using multiplex ligation-dependent probe amplification and next-generation sequencing

目的探讨联合应用MLPA和二代基因测序技术对Duchenne型肌营养不良症分子遗传学诊断的作用。方法收集2012-01-2019-12在中山大学孙逸仙纪念医院儿童神经内分泌专科就诊经临床和肌肉活检明确诊断Duchenne型肌营养不良症患儿,联合应用MLPA和二代测序技术,分析其DMD基因单个外显子的缺失情况。结果纳入Duchenne型肌营养不良症患儿59例,均为男性,就诊年龄(4.72±2.42)岁。58例患儿确定了致病基因突变位点,基因诊断阳性率为98.30%。其中缺失突变28例(47.46%),以第45-55号外显子缺失频率最高;重复突变7例(11.86%),23例(38.98%)微小突变,其中包括无义突变8例(13.55%),移码突变6例(10.17%),错义突变6例(10.17%),微缺失3例(5.08%)。结论联合应用MLPA和二代测序技术是明确Duchenne型肌营养不良症致病基因突变位点最佳策略,能为Duchenne型肌营养不良症的精准诊治和家系成员的遗传咨询提供准确的依据。

Objective To investigate the role of multiplex ligation-dependent probe amplification and next-generation sequencing in molecular genetics diagnosis of Duchenne muscular dystrophy.Methods All patients with DMD were examined and diagnosed based on clinical features and muscular biopsy at Children’s Neurology Department of Sun Yat-sen Memorial Hospital,Sun Yat-sen University from January.Multiplex ligation-dependent probe amplification(MLPA) was first performed to screen large deletions/duplications of DMD exons in the patients,and then,next-generation sequencing(NGS) was carried out to detect small mutations in the MLPA-negative patients.Results A total of 59 unrelated hospitalized children(all boys mean age(4.72±2.42) years) with a clinically suspected diagnosis of DMD were enrolled in this study in Sun Yat-sen Memorial Hospital,Sun Yat-sen University from January 2012 to December 2019.Out of the mutations,35 large mutations encompassing 28(47.46%) deletions and 7 duplications(11.86%) were identified by MLPA;23 small mutations including 8(13.55%) nonsense mutation,6(10.17%)frameshift mutation,6(10.17%)missense mutation and 3(5.08%) small deletions were found by NGS.Large mutations were found most frequently in the hotspot region between exons 45 and 55.Conclusion Our data indicated that the MLPA plus NGS can be a comprehensive and effective tool for precision diagnosis,potential treatment and genetic counseling of DMD.