遗传性乳光牙本质家系致病基因突变的鉴定  

Identification of a novel mutation of DSPP gene in a Chinese family affected with dentinogenesis imperfecta shields type Ⅱ

作　　者：刘彦山[1] 黄颖之[1] 高劲松[2] 李闪[1] 赵秀丽[1] 张学[1] 

机构地区：[1]中国医学科学院基础医学研究所一北京协和医学院基础学院医学遗传学系,医学分子生物学国家重点实验室,北京100005 [2]北京协和医院妇产科

出　　处：《中华医学遗传学杂志》2016年第1期34-37,共4页Chinese Journal of Medical Genetics

基　　金：国家科技支撑计划（2006BA105A03）

摘　　要：目的对一个遗传性乳光牙本质（dentinogenesis imperfecta shieldstype Ⅱ ，DGI-Ⅱ）家系进行DSPP基因的突变分析。方法采集家系成员外周血或胎儿绒毛组织，用酚氯仿法提取基因组DNA。应用聚合酶链反应（polymerasechain reaction，PCR）-Sanger测序方法鉴定先证者DSPP基因第2～5外显子及外显子／内含子衔接区序列，并进行突变分析；针对突变位点设计错配引物引入AluI酶切位点，通过限制性酶切和琼脂糖凝胶电泳方法在该家系正常人及60名无关正常个体中进行致病突变验证；构建含微型DSPP基因的pcDNA3．1基因表达载体，在体外培养细胞中验证突变致病性。结果该家系3例患者和一名胎儿均携带DSPP基因内C．52—1G〉A的杂合突变，突变造成该基因第3外显子5’端剪接点变异；60名对照者和家系正常个体均未携带该突变；微小基因（Minigene）体外表达显示e．52—1G〉A导致DSPP基因转录产物第3外显子的跳跃剪接。结论本研究在一个DGI-Ⅱ家系中发现了DSPP基因内一个新的致病剪接突变（c．52—1G〉A），并在此基础上为先证者提供了产前基因诊断。Objective To identify the causative mutation in a Chinese family affected with dentinogenesis imperfecta shields type Ⅱ （DGI-Ⅱ）. Methods With informed consent obtained from all participants, peripheral blood or chorionic villi samples were collected from the family members. Genomic DNA was extracted using a standard SDS-proteinase K-phenol/chloroform method. The whole coding region and exon/intron boundaries of the DSPP gene were amplified with polymerase chain reaction （PCR） and subjected to Sanger sequencing. To confirm the pathogenicity of the identified mutation, an Alu I recognition sequence was introduced into the mutant allele using mismatch primers by semi-nested PCR. Restriction fragment length polymorphism （RFLP） analysis was then carried out for all family members and 60 unrelated healthy controls. Meanwhile, mini-DSPP constructs were conducted to confirm the effect of the mutation in vitro. Results A splicing site mutation, c. 52-1G〉A, which was located upstream of exon 3, was found in all three patients and the fetus of the proband. Restriction analysis confirmed that all unaffected individuals and the 60 healthy controls did not carry the same mutation. The expression of minigene showed that the exon 3 of the DSPP gene was skipped during the transcription. Conclusion A novel pathogenic splicing-mutation c. 52-1G〉A has been detected in a Chinese family affected with DGI-Ⅱ, which enabled prenatal diagnosis for the fetus of the proband.

关 键 词：乳光牙 DSPP基因 剪接点突变 微小基因 

分 类 号：R781.2[医药卫生—口腔医学]