高分辨率熔解曲线分析在成骨不全相关基因突变筛查中的应用

The application of high-resolution melting analysis in gene mutation screening of osteogenesis imperfecta

目的探讨高分辨率熔解曲线分析（HRMA）在成骨不全（OI）相关基因突变筛查中的应用价值。方法收集2012年3月至12月于天津医院住院的5例0I患儿临床资料。采集患儿、有家族史患儿父母及查体健康者（正常对照）血液标本，PCR—HRMA筛查患儿COL1A1／COL1A2基因所有外显子及其侧翼序列，基因测序确定突变位点。有家族史患儿父母基因测序进行验证。结果PCR-HRMA显示，5例患儿分别在COL1A1基因11、39、8外显子及COL1A2基因19外显子区域结果异常，患儿熔解曲线与正常对照比较存在差异，提示患儿基因变异，标准熔解曲线显示患儿基因型均为杂合子。5例患儿基因测序结果分别为COL1A1基因c．768dupC、C．2644c〉T、C．635G〉A以及COL1A2基因c．982G〉A、C．948C〉T。患儿1、2基因突变导致提前形成终止密码子，临床诊断均为Ⅰ型OI。患儿3、4基因突变均使α螺旋结构域甘氨酸（Gly）被替代，临床诊断为Ⅳ型OI。患儿5基因变异未造成氨基酸的改变，该变异不是导致0I的原因，可能的致病原因有待研究。结论PCR—HRMA因具有成本低、操作简便快速、高通量、无污染等优势，成为0I基因筛查有效的新方法。COL1A1基因突变C．768dupC为新发现的突变位点。

Objective To explore the application of high-resolution melting analysis （HRMA） in gene mutations screening of osteogenesis imperfecta（OI）. Methods Clinical data of five children with OI was collected from March to December 2012 in Tianjin Hospital. Blood samples from five children with OI and their parents with a family history of OI were collected as well as normal controls. All exons and their flanking sequences of COL1A1 and COLIA2 gene were screened using PCR-HRMA and validated by the gene sequencing. Results PCR-HRMA showed abnormal results from five children in the COL1A1 gene exon 11 , exon 39 , exon 8 and the COL1A2 gene exon 19 screening area, respectively. Melting curves of children with OI were differences from normal controls, which showed the mutations of genes. Standard melting curve showed five children with mutations of heterozygous mutation. Sequencing analysis showed that children with COL1A1 gene mutation, c. 768dupC, c. 2644C 〉 T, c. 635G 〉 A and COLIA2 gene mutation c. 982G 〉 A, c. 948C 〉 T, respectively. COL1A1 gene mutation caused a premature stop codon in children 1, 2 and clinical diagnosis with type I OI. Genetic mutations in children 3, 4 with OI in alpha helix structure domain Gly ahernative, and clinical diagnosis with type IV OI. Children 5 gene mutation was nonsense mutations. This variation is not the cause of OI, Which possible causes need to be researched. Conclusions PCR-HRMA has a low cost, easy operation, fast, high flux, pollution-free advantages. PCRHRMA is a new effective method for OI mutation screening. The study found a new mutation of COLIA1 gene, c. 768dupC.