Luo Xiaomei, Wang Ruifang, Fan Yanjie, Gu Xuefan, Yu Yongguo
Department of Pediatric Endocrinology/Genetics, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Institute for Pediatric Research, Shanghai, P.R. China.
Department of Pediatric Endocrinology/Genetics, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Institute for Pediatric Research, 200092 Shanghai, P.R. China.
J Pediatr Endocrinol Metab. 2018 Aug 28;31(8):927-931. doi: 10.1515/jpem-2018-0088.
Background Tandem mass spectrometry (MS/MS) has been used for newborn screening (NBS) of inherited metabolic diseases (IMDs) for decades. However, the traditional approach can yield false-positive or false-negative results and is affected by biochemical substrate-level fluctuations. To overcome the current limitations, we explored the possibility of using next-generation sequencing (NGS) as a second-tier diagnostic test to detect gene mutations in samples with abnormal MS/MS results. Methods Genomic DNA was extracted from dried blood spots and we designed a multigene panel, comprising 77 genes related to over 40 IMDs, for NBS. The prepared libraries were sequenced on the Ion Personal Genome Machine (PGM) platform. Thirty-eight samples identified as abnormal by MS/MS were tested for the diagnostic accuracy of NGS compared with Sanger sequencing. Results The concentration of DNA extracted from the 38 dried blood spots was sufficient for library preparation. The coverage and depth of the sequencing data were sufficient for the analysis. For all samples, the NGS results were consistent with the Sanger sequencing results. Conclusions The genomic DNA extracted from dried blood spots could be used for NGS, generating reliable sequencing results, and NGS may function as a second-tier diagnostic test for NBS. Ion PGM could facilitate the molecular diagnosis of IMDs with appropriate primers designed for candidate genes.
背景 几十年来，串联质谱（MS/MS）一直用于遗传性代谢疾病（IMD）的新生儿筛查（NBS）。然而，传统方法可能会产生假阳性或假阴性结果，并且会受到生化底物水平波动的影响。为了克服当前的局限性，我们探索了使用下一代测序（NGS）作为二级诊断测试来检测MS/MS结果异常的样本中基因突变的可能性。方法 从干血斑中提取基因组DNA，并设计了一个包含与40多种IMD相关的77个基因的多基因检测板用于NBS。制备的文库在Ion Personal Genome Machine（PGM）平台上进行测序。将38个被MS/MS鉴定为异常的样本与桑格测序法进行比较，测试NGS的诊断准确性。结果 从38个干血斑中提取的DNA浓度足以用于文库制备。测序数据的覆盖度和深度足以进行分析。对于所有样本，NGS结果与桑格测序结果一致。结论 从干血斑中提取的基因组DNA可用于NGS，产生可靠的测序结果，并且NGS可作为NBS的二级诊断测试。Ion PGM可以通过为候选基因设计合适的引物来促进IMD的分子诊断。
