成骨不全症患者COL1A1/2致病突变谱和基因诊断研究

Spectrum of COL1A1/2 mutations and gene diagnosis in Chinese patients with osteogenesis imperfecta

目的 在成骨不全症（OI）患者中鉴定Ⅰ型胶原基因（COL1A1和COL1A2）致病突变,构建中国人OI致病基因突变谱,并在此基础上完成OI高危胎儿的产前基因诊断.方法 应用酚/氯仿法提取先证者及家系成员外周血基因组DNA,联合应用聚合酶链反应（PCR）-Sanger DNA测序和靶向高通量测序技术对200例OI先证者进行COL1A1/2编码区及外显子/内含子衔接区的DNA序列分析;采用PCR-高分辨熔解曲线（HRM）技术对先证者及其家系成员进行突变验证;通过PCR-测序对散发病例的父母样本进行突变检测,进而确定突变来源;联合应用PCR-测序和微卫星标记等位基因分析的方法进行胎儿产前基因诊断.结果 在158例先证者中,发现COL1A1/2基因致病突变125种,包含COL1A1突变74种（91例）,COL1A2突变51种（67例）,阳性检出率79％ （158/200）;发现新突变63种,包括33种COL1A1突变和30种COL1A2突变.在上述125种COL1A1/2基因突变中,13种突变分别在2例以上先证者中重复出现（其中6种突变重复出现4次以上）,合计检出46次,检出率为29.11％ （46/158）;完成产前基因诊断74例,其中包括患儿40例,正常胎儿34例.结论 建立基于Sanger DNA测序、靶向高通量测序和PCR-高分辨熔解曲线分析技术的OI基因诊断平台.在中国人群中,构建了OI相关COL1A1/2基因致病突变谱,并在此基础上完成大样本OI患者和高危胎儿的基因诊断.

Objective To identify mutations of the type Ⅰ collagen genes （COL1A1 and COL1A2） in the affected with osteogenesis imperfecta （OI） , to establish the spectrum of COL1A1/2 mutations in Chinese OI patients, and to provide prenatal gene diagnosis to the fetuses at high risk.Methods Genomic DNA was extracted from peripheral blood by the standard SDS-proteinase K-phenol/chloroform method.All the coding regions and exon/intron boundaries of COL1A1/2 were screened in 200 OI cases by conventional Sanger sequencing and targeted next-generation sequencing （NGS） on Ion Torrent-personalized genome sequencing operation（Ion PGMTM）.For familial cases, candidate mutations were validated in all available family members using high resolution mehing analysis （HRM）.In sporadic cases, only parents were examined to determine the origin of the identified mutation.Prenatal gene diagnosis was carried out by PCR direct sequencing and linkage analysis using microsatellite markers.Results In total, the authors identified 125 differently pathogenic mutations, including 74 in COL1A1 and 51 in COL1A2, in 158 probands, with a mutation detection rate of 79% （158/200）.Among the 125 identified mutations, there were 63 novel mutations （33 in COL1A1 and 30 in COL1A2） and 13 recurrent mutations found in 46 probands （seven mutations recurring for two times, and the other six mutations recurring for more than 4 times）.They performed prenatal genetic testing in 74 fetuses and found that 40 ones carried COL1A1/2 mutations identified in the corresponding probands.Conclusions The authors have developed a combined approach for genetic testing of OI, extended the COL1A1/2 mutation spectrum in Chinese OI patients, and confirmed gene diagnosis in a relatively large cohort of OI probands and fetuses.