外显子捕获测序技术用于室间隔缺损胎儿遗传学病因检测

Genetic analysis of fetus with ventricular septal defect using targeted Exon-capture and Sequencing technique

目的:探讨外显子捕获-高通量测序技术检测室间隔缺损(VSD)胎儿遗传学病因的可行性。方法:选择超声心动图诊断为VSD且微阵列比较基因组杂交(a CGH)和G显带检测结果均正常的19例胎儿作为研究对象;将已知的63个人类先心病致病基因作为检测靶点制作成捕获芯片,对各个样本DNA进行外显子捕获后高通量测序,数据经过滤、数据库比对、生物学软件分析得到最终病理性突变位点;病理性突变位点用Sanger测序验证。结果:19例VSD胎儿中共检测到1540个基因突变位点,数据库比对、生物信息学分析后得到8个病理性突变位点:JAG1 c.1078T>G(p.C360G),CHD7 c.6718G>T(p.D2240Y),NOTCH2c.6131G>A(p.R2044H),MYH7 c.77C>T(p.A26V),ZFPM2 c.2107A>C(p.M703L),CHD7 c.7198C>T(p.R2400W),HAND2 c.341G>A(p.S114N),MLL2 c.12140_12168del GGCCGTTAGCAATAGGAACTACCCCTGAG(p.G4047Vfs*5),其中MYH7、ZFPM2两个突变点为已报道突变,其余6例未见报道。8个突变位点的Sanger测序结果与高通量测序结果一致。父母溯源分析均为新发突变。结论:外显子捕获芯片用于VSD胎儿遗传学检测可提高阳性检出率,具有较好的临床应用价值。

Objective：To explore possible pathogenic genetic mutations of fetus with ventricular septal defect using exon-capture high-throughput sequencing technology. Methods： Nineteen fetuses with ultrasound detected VSD and normal amniotic fluid cell G-banding and aCGH results were included in this study. A customized exon-eapture gene-chip targeting 63 human congenital heart disease associated genes were made. Captured DNA fragments using our gene-ehip were sequenced by high-throughput sequencing technology. After a series of analysis including filtering data, database comparison, bio-software, we obtained the final pathological mutation sites,which were all verified by Sanger sequencing. Results： We found totally 1540 gene mutations in the 19 fetus with VSD. Eight mutations were considered as pathogenic mutations including lAG1 c. 1078T〉G （ p. C360G）, CHD7 c. 6718G〉T （ p. D2240Y）, NOTCH2c. 6131G〉A（p. R2044H）, MYH7 c. 77C〉T（p. A26V）, ZFPM2 c. 2107A〉C （ p. M703L）, CHD7 e. 7198C 〉 T （p. R2400W）, HAND2 c. 341G 〉 A （p. Sl14N）, MLL2 e. 12140_ 12168delGGCCGTTAGCAATAGGAACTACCCCTGAG（ p. G4047Vfs ＊ 5 ）. Two of the eight mu- tation including MYH7 c. 77C〉T and ZFPM2 e. 2107A〉C have been reported previously and the remaining six mutation have not been reported as far as we know. Sanger sequencing con- firmed the eight mutations detected by exon-eapture and sequencing, and revealed all of the eight mutations are de novo. Conclusion：Customized exon-eapture and sequencing technology may be promising in the future clinical genetic diagnosis.