CAJ | 학술논문

目的初步建立基于基因芯片捕获和高通量测序技术的迪谢内型肌营养不良(DMD)微小突变的临床检测新平台,验证该方法的敏感度和特异度,并分析中国人群微小突变特点.方法对41例符合临床诊断标准的连续门诊就诊的DMD和贝克型肌营养不良(BMD)患者(非大片段缺失或重复者),提取基因组DNA后,依次进行DNA建库、芯片捕获、高通量测序和测序数据分析,检测DMD基因微小突变.发现致病突变后,进行Sanger测序验证.未发现致病突变的患者,行肌肉活体组织检查免疫组织化学染色,明确是否为抗肌萎缩蛋白病.结果共38例患者检测出致病突变,经Sanger法验证全部正确.新报道突变21种,微小突变类型分布与国外人群相似.3例未发现微小突变的患者经肌肉病理检查,1例确诊为DMD,2例排除DMD.本方法检测敏感度97.4％,特异度100％.结论基因芯片捕获及高通量测序技术用于检测DMD微小突变,结果准确,方法便捷,具有很高的临床应用价值.
목적 초보건립기우기인심편포획화고통량측서기술적적사내형기영양불량(DMD)미소돌변적림상검측신평태,험증해방법적민감도화특이도,병분석중국인군미소돌변특점.방법 대41례부합림상진단표준적련속문진취진적DMD화패극형기영양불량(BMD)환자(비대편단결실혹중복자),제취기인조DNA후,의차진행DNA건고、심편포획、고통량측서화측서수거분석,검측DMD기인미소돌변.발현치병돌변후,진행Sanger측서험증.미발현치병돌변적환자,행기육활체조직검사면역조직화학염색,명학시부위항기위축단백병.결과 공38례환자검측출치병돌변,경Sanger법험증전부정학.신보도돌변21충,미소돌변류형분포여국외인군상사.3례미발현미소돌변적환자경기육병리검사,1례학진위DMD,2례배제DMD.본방법검측민감도97.4％,특이도100％.결론 기인심편포획급고통량측서기술용우검측DMD미소돌변,결과준학,방법편첩,구유흔고적림상응용개치.
Objective To set up a new diagnostic platform based on microarray exon-capture and next-generation sequencing for detecting small mutations in dystrophin gene.The sensitivity and specificity of the method were assessed in clinical settings and the distribution of small mutations in Chinese Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD) patients were also analyzed.Methods Forty-one DMD/BMD patients diagnosed by the clinical criteria without large deletion or duplication (≥ 1exon) were recruited from Peking Union Medical College Hospital consecutively.Genomic DNA was extracted from blood samples.The libraries were prepared.Then exon and intron-exon flanking sequences of DMD gene were captured by custom microarray.Targeted next-generation sequencing and Sanger Sequencing were conducted.The patients who were not detected any disease-causing mutation were performed muscle biopsy.Results Thirty-eight subjects were detected small mutations in DMD gene.All single nucleotide variants (SNVs) and insertion & deletions (INDELs) were validated by Sanger sequencing.Twenty-one novel mutations were reported.The distribution of SNVs and INDELs was similar to other international DMD databases.Upon immunohistochemistry staining of dystrophin protein,1 of 3 mutation-undetected patients was diagnosed as DMD,2 of them were excluded.The specificity of the method was 100％,while the sensitivity was 97.4％.Conclusions Our microarray-captured next-generation sequencing assay could detect SNVs and INDELs with high sensitivity and specificity.Its advantages are economic,time-saving and stable.The platform is suitable for clinical gene diagnosis.