摘要
目的应用聚合酶链反应-高分辨率熔解曲线分析（polymease chain reaction-high-resolutionmelting analysis, PCR-HRMA）技术筛查成骨不全症（osteogenesis imperfecta, OI）患者COL1A1/COL1A2基因的突变，并探讨突变位点与疾病的关系。方法采集家系成员（患儿、患儿父母）以及50名正常对照的血液样本，应用PCR-HRMA技术筛查患儿家系及正常对照者的COL1A1／COLIA2基因突变，并用基因测序验证。应用蛋白预测软件PolyPhen、SIFT及AlignGVGD对两个杂合突变进行预测。结果该患儿及其父母COL1A1基因第45外显子区域的PCR-HRMA结果显示异常，标准熔解曲线和差异熔解曲线与正常对照比较均存在明显差异。测序结果显示，患儿COL1A1基因第45外显子区域发生两个杂合突变（c．3235G〉A、c．3247G〉A），临床诊断为Ⅳ型OI。c．3235G〉A来源于患有OI的父亲，使α螺旋结构域1079位氨基酸由甘氨酸（Gly）突变为丝氨酸（Ser）。c．3247G〉A来源于表型正常的母亲，使1083位氨基酸由丙氨酸（Ala）突变为苏氨酸（Thr）。50名正常对照均未发现这两种突变。3种蛋白预测软件PolyPhen、SIFT及AlignGVGD均预测c．3235G〉A突变可能影响蛋白的功能。而c．3247G〉A突变，PolyPhen软件预测其可能为良性。结论COL1A1基因第45外显子上同时存在c．3235G〉A、c．3247G〉A突变的病例在人类胶原突变数据库中未见报道。COL1A1基因c．3235G〉A突变可能是导致该患儿成骨不全的主要原因。
Objective To detect potential mutations of COL1A1 and COL1A2 genes with polymerase chain reaction - high-resolution melting analysis （PCR-HRMA） in a proband diagnosed with osteogenesis imperfecta （OI）. Methods Peripheral blood samples were collected from the proband and members of his family as well as healthy controls. The mutations were detected by PCR-HRMA and confirmed by direct sequencing. Potential effects of the mutations were predicted using softwares including PolyPhen, SIFT and Align GVGD. Results The PCR-HRMA has indicated mutations in exon 45 of the COL1A1 gene in the prohand as well as his parents, which were presented as the difference in the melting curves between the patients and the control samples. Sequencing analysis confirmed that the proband has carried two heterozygous mutations （c. 3235G〉A, p. Gly1079Ser and c. 3247G〉A, p. Ala1083Thr） in exon 45 of the COL1A1 gene. Among them, c. 3235G〉A was predicted to have impeded alpha helix structure domain, which was inherited from the father who also had OI. c. 3247G〉A was inherited from mother who had a normal phenotype. All three softwares predicted that the c. 3235G〉 A mutation can interfere with thefunction of the protein, while the c. 3247G〉A may have a benign effect by PolyPhen analysis. Conclusion The study identified two mutations（c. 3235G〉 A and c. 3247G〉 A） occurred simultaneously in COLIA1 gene in a case. The case is the first reported in human collagen mutation database. As identified,mutation of c. 3235G〉A may be the major cause of the disease in the proband.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2016年第2期140-144,共5页
Chinese Journal of Medical Genetics
基金
国家高技术研究发展计划（863计划）（2012AA021003）
国家自然科学基金（21177091）
天津市卫生局科技基金（2013KZ072）
