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目的以变性高效液相色谱(DHPLC),分析检测家族性高胆固醇血症(FH)一汉族家系成员的低密度脂蛋白受体(LDLR)基因突变,以明确诊断。方法收集临床诊断为家族性高胆固醇血症的汉族一个家系共37名成员,其中30人为一级和二级亲属,7名为亲属配偶作为对照,提取基因组DNA,聚合酶链反应(PCR)方法扩增LDLR基因包含启动子和全部基因编码区(118外显子)及临近的内含子序列共21个片段,琼脂糖凝胶电泳鉴定产物。采用DHPLC技术检测了LDLR基因,对洗脱曲线异常者进行核苷酸序列分析。结果该家系中发现4处变异,其中1处经核苷酸序列测定明确了突变的性质为第3内含子的剪接突变,并在此家系5名成员中得到证实,而对照组中未检出。结论成功地建立了以DHPLC筛查LDLR基因点突变的方法及技术参数,该方法简便,结果稳定,可作为大样本筛查突变位点的一种便捷可靠手段。
Objective To detect the mutations of low density lipoprotein receptor (LDLR) gene in familial hypercholesterolemia (FH) -Human family members by denaturing high performance liquid chromatography (DHPLC) to confirm the diagnosis. Methods A total of 37 Han family members with familial hypercholesterolemia were enrolled in this study. Among them, 30 were first and second-degree relatives and 7 were relatives of spouses as controls. Genomic DNA was extracted and polymerase chain reaction (PCR) The amplified LDLR gene contains 21 promoters and all the coding region (118 exons) and adjacent intron sequences, and the products were identified by agarose gel electrophoresis. The LDLR gene was detected by DHPLC and nucleotide sequence analysis was performed on those with abnormal elution curve. Results Four mutations were found in the pedigree. One of them was confirmed by nucleotide sequence determination that the mutation was the splicing mutation of the third intron, which was confirmed in five members of the pedigree, but not in the control group Out Conclusion The method and technical parameters for screening point mutations of LDLR gene by DHPLC were successfully established. The method is simple, stable and can be used as a convenient and reliable method to screen mutation sites in large samples.