【摘 要】
：
目的:成骨发育不全(OI)是一种最常见的遗传性骨骼脆性增加的疾病,以骨脆性增加或畸形,蓝色巩膜,身材矮小,牙本质发育不全及听力损害为主要特征.大多数病例是编码Ⅰ型胶原α链的COL1A1或COL1A2基因突变所致,一般呈常染色体显性遗传方式.本研究针对一中国成骨不全家系进行基因突变分析,以明确其致病突变.方法:在签署知情同意后,采集该家系中患者和表型正常个体外周静脉血3ml,使用Universal
【机 构】
：
中国医科大学医学基因组学教研室教育部医学细胞生物学重点实验室 沈阳市骨科医院骨科研究所病房
【出 处】
：
中国遗传学会第九次全国会员代表大会暨学术研讨会
论文部分内容阅读
目的:成骨发育不全(OI)是一种最常见的遗传性骨骼脆性增加的疾病,以骨脆性增加或畸形,蓝色巩膜,身材矮小,牙本质发育不全及听力损害为主要特征.大多数病例是编码Ⅰ型胶原α链的COL1A1或COL1A2基因突变所致,一般呈常染色体显性遗传方式.本研究针对一中国成骨不全家系进行基因突变分析,以明确其致病突变.方法:在签署知情同意后,采集该家系中患者和表型正常个体外周静脉血3ml,使用Universal Genomic DNA Extraction Kit Ver.3.0(TaKaRa)试剂盒提取基因组DNA,对COL1A1和COL1A2基因外显子进行PCR扩增,对扩增产物进行测序分析;同时采用PCR-RFLP方法通过聚丙烯酰胺凝胶电泳验证所发现的变异在人群中存在的频率;在COL1A1基因及其上下游区域选取两个微卫星标记WI-6632,D7S657和一个短串联重复AC*21,在COL1A2基因及其上下游区域选取5个微卫星标记D17S1293,D17S1868,D17S1180,D17S1319和D17S788,PCR扩增以上遗传标记并进行单体型分析;针对可能的致病突变,进行RNA水平的分析,Trizol法提取新鲜外周静脉血中总RNA,使用PrimeScript@RT reagent Kit (TaKaRa)反转录并用基因特异性引物扩增突变位点所在区域,琼脂糖凝胶电泳检测扩增产物大小,同时进行克隆测序分析.结果:在先证者COL1A2基因发现杂合突变c.3313G＞A(p.GIy 1105Ser),但在家系中该突变不与患者共分离;在先证者COL1A1基因发现杂合突变c.1299+5G＞C,该突变在家系中与患者共分离.在146个无关正常个体中均未发现存在以上两个突变.单体型分析结果支持该家系中COL1A1基因致病,不支持COL1A2基因致病.针对c.1299+5G＞C突变,进行RNA剪切分析,琼脂糖凝胶电泳显示患者COL1A1基因的扩增子与家系中正常个体大小一致.对扩增产物进行克隆测序分析显示存在剪接改变,在原始剪接位点前缺失8个碱基,即c.1292_ 1299del(p.Asn432*).结论:以上结果在DNA水平和RNA水平初步证实该成骨不全家系为COL1A1基因致病,其致病突变c.1292_1299del为国际首报;COL1A2基因突变c.3313G＞A在国际上已经报道过2次,其在成骨不全致病机制中的作用有待进一步研究,推测其也可能为罕见的SNP位点。
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