多重引物HRMA对成骨不全患者COL1A1/2基因突变的筛查与分析

Identification of mutations in COL1A1/2 in patients with osteogenesis imperfect using multiple primer high resolution melting analysis

目的建立多重引物高分辨熔解曲线分析(high resolution melting analysis,HRMA)方法并运用该方法筛查两例成骨不全(osteogenesis imperfecta,OI)COL1A1/2的突变基因。方法收集两例OI患者临床资料,采集患者及50例正常对照的血液标本。设计COL1A1、COL1A2基因103个外显子所对应的引物,将满足一定条件的两对引物相互配对,与单份样本DNA混合作为扩增体系。运用HRMA筛查产物突变情况,基因测序确定突变位点。结果本研究中共涉及到COL1A1和COL1A2的103个外显子,其中成功配对39对,未配对成功的有3个COL1A1外显子和22个COL1A2外显子,成功率为75.73%。先证者1筛查结果HRMA熔解曲线在COL1A141号外显子上存在异常,测序结果为c.2877delT杂合突变,即cDNA2877位碱基T缺失,编码的缬氨酸变成色氨酸。先证者2筛查结果HRMA熔解曲线在COL1A116号外显子上存在异常,测序结果为c.1028delC杂合突变,即cDNA1028位碱基C缺失,编码的脯氨酸变成亮氨酸。两种突变类型在中国人群中均未见报道,为新发现的两种剪切突变,且与传统HRMA筛查结果一致。结论多重引物HRMA方法在传统HRMA基础上进行了创新,将两对引物与单份样本DNA混合作为扩增体系,HRMA筛查产物突变情况,且与传统HRMA筛查结果一致,具有一定创新性和可行性,为成骨不全患者基因突变及其他遗传病致病基因的筛查提供了新的思路。

Objective To establish a multiple primer high resolution melting analysis(Multi-HRMA)and identify gene mutation in patients with osteogenesis imperfecta(OI).Methods The clinical data of two patients was collected.Blood samples from two patients,as well as 50 normal controls,were also collected.Design primers of 103 exons in COL1A1 and COL1A2 individually and mix two of them up in some conditions with one piece of DNA sample.Then the mutation was screened using HRMA and validated by the gene sequence.Results We succeeded in matching 39 pairs of primers accounting for 75.73%in all 103 exons,including 48 exons in COL1A1 and 30 exons in COL1A2.And there were 3 COL1A1 exons and 22 COL1A2 exons unpaired.The detection of Multi-HRMA showed the abnormal result of COL1A141 exon in proband from family one.The sequencing result was c.2877delT,which meant that cDNA of 2877 base T disappeared.The mutation transformed the amino acid valine into tryptophan.The detection of Multi-HRMA showed the abnormal result of COL1A116 exon in proband from family two.The sequencing result was c.1028delC,which meant that cDNA of 1028 base C disappeared.The mutation transformed the amino acid proline into leucine.Both mutations were novel mutations,the same as the traditional HRMA.Conclusion Multiple primer high resolution melting analysis identifying gene mutation has higher innovation and feasibility than traditional PCR.Not only does it decrease the workload,but it is cheaper and improves efficiency.It is really a new thought for the gene mutation screening in patients with OI and other genetic disorders.