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PCR-HRM分析筛查成骨不全一家系患儿基因突变

白雪1,李克秋2,任秀智1,何晓波3,王毅4,官士珍1,景亚青3,李光5   

  1. 1. 天津市天津医院
    2. 天津医科大学基础医学院生物学教研室
    3. 天津医科大学
    4. 天津医院
    5. 天津医科大学基础医学院
  • 收稿日期:2014-01-07 修回日期:2014-02-16 出版日期:2014-07-15 发布日期:2014-07-15
  • 通讯作者: 白雪

PCR-HRM Analysis for Gene Mutation Screening in a Child with Osteogenesis Imperfecta

  • Received:2014-01-07 Revised:2014-02-16 Published:2014-07-15 Online:2014-07-15

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摘要：

【摘要】目的 采用PCR-高分辨率熔解曲线（HRM）分析筛查成骨不全（OI）一家系患儿（先证者）COL1A1基因突变位点，探讨其基因型与临床表型的联系。方法对先证者进行家族史及临床资料的调查，采集先证者、家属及 50名正常对照者血液标本，应用PCR-HRM分析筛查先证者及正常对照者COL1A1基因突变，基因测序确证突变位点。结果先证者COL1A1基因17外显子筛查结果异常，其熔解温度（Tm）值比正常对照者Tm值低约0.4℃。先证者与正常对照者的标准熔解曲线及差异熔解曲线均有明显差异。测序结果为c.1138G>A，突变导致380位氨基酸由甘氨酸（Gly）变成丝氨酸（Ser）：p.（Gly380Ser），为错义突变。先证者父亲、祖母均具有相同突变位点。先证者母亲及正常对照者基因测序结果无此突变。该突变在中国人群中未见报道。该家系遗传特征为常染色体显性遗传，先证者临床诊断为Ⅳ型OI，临床表型较严重。结论PCR-HRM分析是有效的OI基因筛查新方法。COL1A1基因 c.1138G>A突变在中国人群中为新发现的突变位点。α螺旋结构域Gly被替换可能导致较严重的临床表型。 COL1A1

关键词: 成骨不全, COL1A1基因, 基因突变, 基因诊断, 高分辨率熔解曲线分析

Abstract:

[Abstract] Objective To investigate COL1A1gene mutation by PCR-high resolution melting (PCR-HRM) and an? alyze the correlation between genotype and clinical phenotype in a child (proband) with osteogenesis imperfecta (OI).Meth? ods The family history of OI pedigree along with the clinical data was collected. Blood samples from the proband and his family members, as well as 50normal controls, were collected. The mutation of COL1A1gene was screened using PCR HRM and validated by the gene sequence.Results The detection of PCR-HRM showed the abnormal result of COL1A1 17exon in proband with a lower melting temperature (Tm) value than that of normal controls by0.4℃. There were signifi? cant differences in the standardization melting curve and the different melting curve between the proband and the normal controls. The sequencing result was c.1138G>A, which meant that cDNA of1138base G mutation into A. The mutations transformed the amino acid glycine into a serine at amino acid380（P. Gly380Ser), which resulted in missense mutations. The proband’s father and grandmother had the same mutation of COL1A1gene. The mutation was not found in the proband’s mother and normal controls. There was no report for such mutation in Chinese population. Pedigree analysis showed the fami? ly genetic characteristics of autosomal dominant inheritance. The proband was clinically diagnosed as OI type Ⅳwith more severe clinical phenotype.Conclusion PCR-HRManalysis is a new effective method for genetic screening of OI. COL1A1 mutation of c.1138G>A is a newly discovered mutation in Chinese population. Gly replaced inαhelical domain may lead to a more severe clinical phenotype.

Key words: Osteogenesis imperfecta, COL1A1 gene, Gene mutation, Genetic diagnosis, High-resolution melting