成骨不全家系患者相关基因突变筛查及分析

Screening and analysis gene mutations in families patients with osteogenesis imperfecta

目的筛查4例成骨不全（0I）家系先证者I型前胶原α链基因（COL1A1／COLIA2）基因突变，分析基因型与表型的关系。方法收集先证者及家系成员临床资料，采集先证者、家系成员及50例正常对照的血液标本。采用聚合酶链反应（PCR）．高分辨率熔解曲线分析（HRMA）筛查先证者COL1A1／COL1A2基因突变，基因测序确定突变位点。结果HRMA显示先证者1分别在COL1A2基因12外显子、19外显子区域结果异常，标准熔解曲线与正常对照存在明显差异。测序结果，先证者1分别在COL1A2基因12外显子、19外显子及18内含子发生突变：c．578G〉T、c．948C〉T、c．937-3T〉C；其中c．948C〉T、c．937-3T〉C为单核苷酸多态性（SNP）位点，c．578G〉T为新发现的突变位点。先证者2、3、4，分别在COL1A2基因的38外显子、COL1A1基因的6外显子、23外显子区域的标准熔解曲线与正常对照存在明显差异。测序验证先证者2COL1A2基因突变：c．2305G〉T。先证者3、4均在COLlAl基因发生突变：C．517G〉T、c．1588G〉A。其中先证者3COL1A1基因c．517G〉T为新发现的突变位点。先证者1、2．4基因突变，导致I型胶原蛋白3股螺旋区域甘氨酸（Gly）被替换，可引起较为严重的表型，先证者3的无义突变，可造成I型胶原合成量的改变，临床表型较轻。结论患者COL1A2基因c．578G〉T、COL1A1基因c．517G〉T均为新发现的突变位点，患者的基因型与临床表型存在一定的联系。

Objective To investigate type I collagen alpha chain gene （COL1A1 and COL1A2） gene mutation and analyze the correlation between genotype and clinical phenotype in four families with os- teogenesis imperfecta （OI）. Methods A survey was taken out of families history of OI probands along with the clinical data. We collected blood samples from the proband and their families, as well as 50 normal controls peripheral blood samples. Polymerase chain reaction analysis and high resolution melting （PCR- HRMA） method was used to screen blood samples COLIAI and COL1A2 genes and validated by gene sequencing. Results PCR - HRMA method showed abnormal results from proband 1 in the COLIA2 gene exon 12 and exon 19. Standard melting curves of proband 1 were differences from normal controls. Se- quencing analysis showed that proband 1 with COL1A2 gene mutation, c. 578G 〉 T, c. 948 C 〉 T and c. 937 -3T 〉 C. That COLIA2 gene mutation c. 948 C 〉T and c. 937 -3T 〉 C were the SNP. The study found a new mutation of COL1A2 gene, c. 578 G 〉 T. PCR - HRM method showed abnormal results from prohand 2, 3 and 4 in the COL1A2 gene exon 38, COLIA1 gene exon 6 and exon 23, respectively. Stand- ard melting curves of prohands were differences from normal controls. Sequencing analysis showed that COLIA2 gene mutation c. 2305G 〉 T, COL1 A1 gene mutation c. 517 G 〉 T, c. 1588 G 〉 A, respectively. The study found a new mutation of COLIA1 gene, c. 517 G 〉 T. Genetic mutations from Proband 1,2 and 4 with OI in collagen type I helix structure domain glyeine （Gly） alternative, which cause a more severe clinical phenotype. Missense mutations in Prohand 3 may result in type I OI, which clinical phenotype is lighter. Conclusion COLIA2 gene mutation c. 578G 〉 T, and COLIA1 gene mutation c. 517 G 〉 T in patients with OI, are newly discovered mutation. Genotypes and clinical phenotypes in patients with OI may have some connection.