一代二代联合测序快速鉴定肝豆状核变性及家系溯源研究

Combining next-generation sequencing and Sanger sequencing for rapid identification of the genetic mutation diagnosis with family tracing of hepatolenticular degeneration

摘要:

目的：探索二代测序筛选和一代测序验证的快速准确诊断罕见遗传病肝豆状核变性的检测方法和流程。方法：提取先证者DNA并构建高通量测序文库后通过二代测序鉴定其可能致病位点；再提取包括先证者在内的该家系13位成员DNA，设计引物并对目标片段PCR扩增后进行双相一代测序验证。结果：先证者的ATP7B基因外显子8中检测出1个杂合错义突变（rs121907990；chr13：51937570）；家系成员共查出3位携带同一杂合错义突变者，先证者ATP7B突变来源于母系。结论：本研究设计的联合二代测序检测与一代测序验证的肝豆状核变性基因突变位点的快速鉴定技术和流程图，可能为罕见遗传病基因突变致病位点提供可借鉴的检测模式，为早期高效精准确诊罕见遗传病提供帮助。

Abstract:

Objective：To investigate an early，accurate and rapid gene detection method and procedure using next-generation sequencing（NGS） full screening and Sanger sequencing based verification for diagnosing a rare genetic disease，hepatolenticular degeneration. Methods：The proband Ⅲ-3 DNA was extracted and the bioinformatic data of high throughout genomic sequences（HTGS） was established to confirm the possible pathogenic sites by using the NGS；then the DNA of all 13 members of the family was extracted，including the proband，in order to design the primer and amplify the target fragment by PCR，then the possible pathogenic sites were verified by using the biopolar generation sequencing. Results：A heterozygous missense mutation（rs121907990；chr13：51937570） was detected on the proband Ⅲ-3’s ATP7B gene exon 8. Three family members，Ⅱ-4，Ⅱ-5 and Ⅲ-4，carried the same heterozygous sense mutation，and their genetic pattern showed mutation located on the same chromosome. The proband’s ATP7B mutation came from the matriarchal origin. Conclusion：The rapid identification method and procedural flow chart designed by our research，which combines the NGS technique and Sanger sequencing based verification technique for the pathogenic site in genealo－gy，has the potential to provide reference for gene diagnosis procedure，and contribute to the timeliness and accuracy of early diagnosis of rare genetic disease.