目标外显子捕获技术在初级纤毛相关疾病基因检测中的应用  

Application of targeted exome capture in identifying genetic mutations in ciliopathies

作　　者：张蔓丽[1] 周红辉[1] 卢彦平[1] 李芮冰 游艳琴[1] 叶明侠[1] 汪淑娟[1] 黄柯[1] 谢潇潇[1] 李亚里[1] 

机构地区：[1]解放军总医院妇产科,北京100853 [2]解放军总医院临床检验科,北京100853

出　　处：《中华围产医学杂志》2016年第6期422-426,共5页Chinese Journal of Perinatal Medicine

基　　金：志谢减挚感谢参与本研究的各位患者及家属.感谢国家人口和计划生育委员会科学技术研究所罗敏娜博士提供病例5,同时感谢其对病例3及病例5致病基因位点的确定提供了宝贵帮助

摘　　要：目的探讨利用目标外显子捕获结合高通量测序技术检测初级纤毛相关疾病的致病基因。方法采取2009年7月至2014年12月在解放军总医院收集的6例初级纤毛相关疾病疑似病例（包括婴儿型多囊肾、麦克尔综合征、Joubert综合征等）先证者组织或脐带血及其父母外周肘静脉血样。设计了包含113个基因、针对初级纤毛相关疾病的目标外显子基因捕获试剂盒，组织或血提取DNA后，利用该试剂盒结合高通量测序技术，进行初级纤毛相关疾病致病基因的筛查，对可疑似点进行一代测序验证。结果5例先证并俭测出致病基因，其中PKHD1基因突变2例（例1突变碱基位点为c.609ldelG、c．865delC，例2突变碱基位点为c．11042T〉G、c．5137G〉T）：TCTN2基因突变1例（例3），突变位点为c．343G〉T、c．1540C〉T；CEP290基因突变1例（例5），突变位点为c.7328—7332del、c．4897C〉T。上述基因突变均为复合杂合子。TMEM67基因1例（例4），突变位点为c．1645C〉T，为纯合子、一代测序证实这些突变分别来自于其父母。1例先证者（例6）未发现明确致病基因。6例染色体核型分析均未见异常。结论利用目标外显子捕获结合高通量测序技术可为5例初级纤毛相关疾病家庭明确致病基因，从而有助于再次妊娠的遗传咨询及产前诊断。Objective To develop an approach based on next generation sequencing to determine the genetic defects in ciliopathies precisely and effectively. Methods Six suspected cases ofciliopathies were enrolled from July 2009 to December 2014 in the PLA General Hospital. Cord blood or tissue of the probands and peripheral blood samples of all the parents were collected for karyotyping analysis. Tissue or blood DNA from the six cases was scanned on 113 kinds of genes related to ciliopathies using targeted exome capture with high throughput sequencing. Mutations detected from the related genes were confirmed by direct Sanger sequencing reactions. Results Karyotype was normal in all the six cases. Pathogenic genes were found in five cases, including PKHDI mutation in two cases （case one with c.6091delG and c.865delC, and case two with c.11042T〉G and c.5137G〉T）; TCTN2 mutation in one case （c.343G〉T and c.1540C〉T） and CEP290 mutation in one case （c.7328 7332del and c.4897C〉T）. All were compound heterozygous mutations. TMEM67 mutation was found in one case （c.1645C〉T）, which was homozygous mutation. First-generation sequencing confirmed that these mutations originated from their parents. No definite pathogenic courses were found in one case （case six）. Conclusions Targeted exome capture with high throughput sequencing is a new approach to find pathogenic gene mutations in ciliopathies. The methodology provides a reliable strategy for routine gene diagnosis and genetic counseling on ciliopathies.

关 键 词：多囊肾 常染色体隐性 纤毛运动障碍 脑膨出 畸形 多发性 外显子 高通 量核苷酸序列分析 

分 类 号：R440[医药卫生—诊断学] R714.5[医药卫生—临床医学]