一个成骨不全家系COL1A1基因的突变筛查

Screening and analysis of a new mutation of COL1A1 gene in a family with osteogenesis imperfecta

目的筛查1个成骨不全（osteogenesisimperfecta，OI）家系中COL1A1基因的突变，并分析基因型与临床表型的关系。方法收集先证者及家系成员临床资料，采集先证者、随诊家属及50名正常对照的外周血标本，应用PCR-高分辨率熔解曲线（highresolutionmelting，HRM）分析筛查COL1A1基因突变，基因测序确定突变位点。结果PCR-HRM分析显示，先证者COL1A1基因第33～34外显子结果异常，Tm值为87．7℃，比正常对照的Tm值（87．9℃±0.06℃）低0．2℃，先证者与正常对照标准熔解曲线及差异熔解曲线均有明显差异。测序结果提示，先证者COL1A1基因第33～34外显子C．232ldelC，移码突变，导致此后第334位氨基酸提前出现终止密码子（UAA），即p．Pr0774 LeufsX334杂合突变。先证者之父、祖父基因测序均具有相同的突变位点。50名正常对照无此突变。该突变位点在人类胶原突变数据库中未见报道。家系图谱显示为常染色体显性遗传，先证者及家系患者临床诊断均为I型OI。结论成骨不全COL1A1基因c．2321delC为新的突变位点。移码突变导致提前形成终止密码子，使I型胶原合成量减少，临床表型较轻。

Objective To investigate mutation of COL1A1 gene and analyze the relationship between genotype and clinical phenotype in a family with osteogenesis irnperfecta （OI）. Methods The family history of an OI pedigree, along with clinical data, was collected. Blood samples from the proband and his families, as well as 50 normal controls, were collected. Mutation of COL1A1 gene was screened using PCR-high resolution melting （PCR-HRM） and validated by sequencing. Results PCR-HRM method showed an abnormal result in proband COL1A1 33_34 exons, which Tm was 87.7℃, in contrast to the normal control （wt） Tm of 87.9 ℃±0.06℃. There was a significant difference between the proband and the normal control with the standardization curve and the difference curves. DNA sequencing showed that COL1A1 gene exons 33_34 has lost a C base （c. 2321delC）, which resulted in a frameshift mutation and caused an premature termination codon （UAA） at amino acid 334, i. e., p. Pro774LeufsX334. The father and grandfather of the proband, both suffered from OI, were verified to be heterozygous for the same mutation. The same mutation was not found in 50 normal controls. Database search confirmed this to be a novel mutation. Pedigree analysis suggested that it has an autosomal dominant inheritance. The proband and patients from the family were clinically diagnosed as OI type I. Conclusion The study has identified a novel mutation of COLIA1 gene, c. 2321de1C. This frameshift mutation has caused a premature stop codon and reduced collagen type I synthesis, characterized by a lighter OI clinical phenotype.