第二代测序技术在一中国先天性白内障家系致病基因检测中的应用

Application of next-generation sequencing in detection of mutation gene in a Chinese pedigree with congenital cataract

背景 某些遗传性疾病具有高度遗传异质性,因此传统的Sanger测序技术已经不能满足医学研究及临床工作的需求.第二代测序（NGS）技术由于具有费用低及检测速度快的优点而得到广泛应用,但在先天性眼病突变基因的检测中的应用效果有待验证.目的 探讨NGS技术对先天性白内障患者进行致病基因诊断和产前诊断的可行性.方法 于2013年1月收集来自河南省洛阳市的一汉族先天性白内障家系,抽取家系中3例患者（Ⅱ2、Ⅲ3、Ⅲ4）和3名表型正常者（Ⅱ3、Ⅲ1、Ⅲ2）的外周血各2 ml,EDTA抗凝,在河南省医学遗传研究所应用NGS技术对先证者进行基因突变位点检测,并采用Sanger测序技术对NGS结果进行验证,然后用Sanger测序技术对该家系其他成员的外周血样本进行突变位点的序列分析,根据确定的致病突变位点对先证者的胎儿进行产前诊断.本研究遵循赫尔辛基宣言,检测方案经河南省人民医院医学伦理委员会批准,所有研究对象均签署知情同意书.结果 该家系4代14位成员中共5例患者,其中男2例,女3例,分布于Ⅰ、Ⅱ、Ⅲ代中,其他家系成员表型正常,符合常染色体显性遗传方式.NGS检测发现先证者Ⅲ3CRYBB1基因第6外显子上存在c.682T＞C（p.S228P）杂合突变,Sanger法验证了该点突变.Sanger法检测发现家系中患者均存在CRYBB1基因c.682 T＞C突变,而家系中表型正常者CRYBB1基因的c.682位点基因型为T/T野生型.产前诊断结果显示胎儿（Ⅳ1）CRYBB1基因c.682位点基因型为T/T野生型.结论 NGS可用于先天性白内障基因突变的快速检测,该家系致病性基因为CRYBB1基因c.682T＞C突变,应用NGS技术结合一代测序技术成功对先证者进行了产前诊断.

Background Due to the genetic heterogeneity of many diseases,the Sanger sequencing technology is far from satisfying the needs of scientific research and clinical applications.The next-generation sequencing （NGS） technology is being widely used in relevant studies because of its lower cost and much higher throughput.Objective This study was to explore the feasibility of NGS technology for the detection of genetic cause of congenital cataract.Methods A Chinese congenital cataract pedigree was collected from Luoyang city in Medical Genetic Institute of Henan Province in January,2013.The peripheral blood of 2 ml was obtained from each 3 patients with congenital cataract （Ⅱ 2,Ⅲ 3,Ⅲ 4） and 3 subjects with normal phenotype （Ⅱ 3,Ⅲ 1,Ⅲ 2） in this pedigree respectively using EDTA anticoagulant tube.The mutant gene of proband was detected by NGS,and the result was verified by Sanger sequencing.Sanger sequencing was employed to determine the mutation sites of other subjects in this pedigree and further to perform the prenatal diagnosis.The research followed the Declaration of Helsinki,and the protocol was approved by the Medical Ethics Committee of Henan Provincial People＆#39;s Hospital.Written informed consent was obtained from each subject prior to any medical examination.Results Total 5 patients were found in 14 family members of 4 generations in this pedigree,including 2 males and 3 females in generation of Ⅰ,Ⅱ,Ⅲ,and the other family members showed normal phenotype,which followed autosomal dominant inheritance pattern.NGS revealed that the proband occurred a heterozygous mutation of c.682 （p.S228P） in the exon 6 of CRYYB1 gene,and the outcome was further confirmed by Sanger sequencing.In addition,this heterozygous mutant gene was also found in other patients of this pedigree.However,the genotype of c.682 in the exon 6 of CRYYB1 gene was T/T wild type in the subjects with normal phenotype in this pedigree.The genotype of CRYYB1 gene was T/T wild type in the fetus （Ⅳ 1） by prenatal diagnosis.Conclusions NGS is a useful tool for the detection of mutant gene in congenital cataractous pedigree.The heterozygous mutation of c.682T〉C （p.S228P） in the exon 6 of CRYYB1 gene is the causative mutation in this pedigree.NGS combined with Sanger sequencing enables prenatal diagnosis of the disease.