基于目的基因捕获的二代测序技术对质谱检测阳性患儿的分子诊断

目的运用基于目的基因捕获的二代测序技术,对质谱检测阳性的遗传代谢病疑似患儿进行基因检测,探讨二代测序技术在遗传代谢性疾病诊断的应用价值。方法回顾性收集复旦大学附属儿科医院遗传代谢专科门诊收治的、质谱检测阳性遗传代谢病疑似患儿的剩余干血滴滤纸片,行目的基因捕获的二代测序,行Sanger进行验证。结果 2013年6月至2014年5月共有48例质谱检测阳性的遗传代谢病疑似患儿纳入本文分析,40例完成二代测序,男性22例。40例测序标本中,29例(72.5%)检测到与临床症状相符的基因变异,包括甲基丙二酸血症6例,citrin缺陷病6例,高苯丙氨酸血症5例,戊二酸血症3例,其他遗传代谢病9例。2例疑似citrin缺陷病的标本在SLC25A13基因上仅检测到单个致病性突变,进一步行长片段PCR,检测到2例标本均存在3 kb的杂合插入突变。结论基于目的基因捕获的二代测序技术在对临床疑似遗传代谢病的检测中,有着较高的阳性诊断率,不仅能为临床医生提供可靠的分子诊断依据,而且可以根据致病基因对疾病分型,有效地为后续的临床治疗和遗传咨询提供依据。

Application of targeted next generation sequencing in the molecular diagnosis of abnormal mass spectrometry analysis findings

ObjectiveTo evaluate the application value of targeted next generation sequencing for the molecular diagnosis in children with high risk of inherited metabolic disorders. MethodsFTA cards with dried blood spots were collected from the patients coming to genetic and metabolic specialist outpatient clinic, whose mass spectrometry analysis(MAS) was abnormal. Next generation sequencing (NGS) of a panel of 89 genes known to cause genetic disorders in children was uesed to find the genetic cause of the diseases. All mutations detected by NGS were subsequently confirmed by Sanger sequencing. ResultsA total of 48 patients with abnormal MAS findings were recruited retrospectively from June 2013 to May 2014. Forty patients completed the study finally, including 22 males. Among those samples, 29 samples accounting for 72.5 percent of the total samples, were detected mutations on the candidate genes that were consistent with the clinical symptoms, including 6 methylmalonic acidemia, 6 citrin deficiency, 5 phenylketonuria, 3 glutaric acidemia and 9 miscellaneous genetic disorders. Single pathogenic mutation was found in gene SLC25A13 by NGS in 2 suspected citrin deficiency cases. Long range PCR was done after that and another 3 kb insertion mutation was found in both of the cases. ConclusionApplication of targeted next generation sequencing in children with high risk of inherited metabolic disorders can provide reliable molecular diagnosis in a cost- and time-efficient manner. Identification of disease-causing mutations may have benefit in clinical practice and is essential for genetic counseling.