摘要目的 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.

abstractsObjective 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.