外显子捕获-高通量测序技术用于法洛四联症胎儿遗传学检测

Application of exon-capture high-throughput sequencing in genetic test of fetal tetralogy of Fallot

目的用外显子捕获-高通量测序(exon-capture high-throughput sequencing,EC-HTS)技术寻找法洛四联症(tetralogy of fallot,TOF)胎儿可能存在的遗传学致病性证据,并探讨其在产前分子遗传学诊断中的价值。方法选择经超声心动图发现且G显带和微阵列比较基因组杂交(array-based comparative genomic hybridization,a CGH)分析结果均正常的9例TOF胎儿作为研究对象,以63个人类已知的先天性心脏病致病基因作为检测靶点制作捕获芯片,用EC-HTS技术进行检测,数据经过Calling、数据库比对、软件分析得到最终病理性突变位点;并用Sanger测序对其进行验证。结果在9例胎儿中共检测到单核苷酸多态性(SNP)位点732个,结果经过生物信息学分析得到致病性突变位点3个:CITED2 c.574_579 del AGCGGC(p.S192_G193del纯合突变,CHD7c.2550_2554 del GAGAA(p.K850Nfs*6)杂合突变,NOTCH2 c.4918T>C(p.F1640L)杂合突变。Sanger测序结果验证了这3个突变位点的真实存在,父母溯源检测发现这3个点突变均为新发突变。结论用靶向性EC-HTS技术发现了3例TOF胎儿存在病理性基因突变,可能为其致病的遗传学病因。

Objective To explore the possible pathogenic genetic evidence of tetralogy of Fallot（ TOF） and evaluate the effects of exoncapture high-througput sequencing technology in molecular genetic examination of prenatal diagnosis. Methods Nine fetus with tetralogy of Fallot,whose results of G-banding and comparative genomic hybridization（ array-based Comparative Genomic Hybridization,a CGH） analysis were normal,were included in this study. A customized capture microarray targeting sixty-three genes which were well-known congenital heart disease（ CHD） associated genes were made and captured DNA fragments were sequenced by high-throughput sequencing.Through base- calling,database comparison and software analysis,we obtained the final pathologic mutations which were validated by Sanger sequencing. Results We found 732 single nucleotide polymorphism sites in 9 fetus,in which three mutations were considered as pathogenic sites,including CITED2 c. 574_579del AGCGGC（ p. S192_G193del）,CHD7 c. 2550_2554del GAGAA（ p. K850 Nfs ＊ 6）,NOTCH2 c. 4918 T C（ p. F1640L）. Sanger sequencing confirmed the three sites were de nove mutation. Conclusion This study revealed three pathological mutations in the fetal with TOF and the mutations might be the genetic causes of TOF.