第二代测序技术在先天性巨结肠症家系致病基因筛查中的应用

Next generation sequencing technology for detecting a new RET gene mutation in two Hirschsprung＇s disease families

目的筛查2个先天性巨结肠症（Hirschsprung’s disease，HSCR）家系的致病基因突变。方法对2个来自辽宁的HSCR家系（家系1和家系2）进行基因分析。在获得家系成员的同意后，采集外周静脉血，提取全基因组DNA。应用第二代测序技术（next-generation sequencing technology，NGS）对各个家系成员的全外显子组DNA进行基因突变筛选和拷贝数变异（copy number variation，CNV）分析，结合突变的危害性和致病性分析家系成员的临床表型，依据测序结果筛选可疑致病基因，运用Sanger测序法进行验证。结果家系1中4人，子代2人均发病，母亲有类似临床症状，但未做检查诊断，父亲正常。在过滤掉常见变异及同义突变后，全外显子测序检测出633个SNP和35个InDel突变。在候选基因RET中，发现14号外显子一个新的无义突变c.2599G〉T，经突变的蛋白危害性和致病性分析并结合遗传方式及临床特征等多因素分析后高度怀疑该突变是该家系的致病突变，经Sanger法验证后认为该突变为该家系的致病突变。家系2中4人，子代2人发病，但其中1人新生儿期死亡未能得到外周血样标本，父母正常，测序结果提示609个SNP和30个InDel突变，结合突变影响、遗传方式及临床特征等多因素综合分析未能发现意义明确的致病突变。结论第二代测序技术可以筛选出新的与先天性巨结肠相关的基因突变信息，为HSCR的病因研究提供一种新的途径和方法。

ObjectiveTo identify the disease-causing gene mutation in two families of Hirschsprung’s disease （HSCR）.MethodsTwo HSCR families from Liaoning Province were collected to analyze genomic DNA. Whole-exome genome mutation screening and copy number variation （CNV） analysis were performed on whole-genome DNA extracted from blood using next-generation sequencing （NGS） technology. Combining the hazard and pathogenicity analysis of mutation and clinical phenotype of family members screening susceptible pathogenic gene for sequencing results. Then classical Sanger’s method was applied for verifying the obtained results.ResultsAmong 4 persons in family 1, both children were affected, the mother had similar clinical manifestations but the diagnosis was absent, the father had no phenotype. After filtering out common mutations and synonymous mutations, 633 SNPs and 35 InDel mutations were detected by whole-exome sequencing. The sequencing results revealed heterozygous mutation c. 2599G〉T in exon 14 encoding region of RET gene and it was confirmed as a pathogenicity mutation through the hazard and pathogenicity analysis of mutated protein. Among 4 persons in family 2, both offsprings were identified as HSCR, but one of them dying in neonatal period failed to obtain a peripheral blood sample. Sequencing results revealed 609 SNPs and 30 InDel mutations. Comprehensive analysis of mutations, genetic patterns, clinical features and other factors failed to detect a distinct pathogenic mutation in this family.ConclusionsNGS can screen out new HSCR-related gene mutations and provide etiological insights of HSCR.