【作者】 王绿娅； 蔺洁； 陈立伟； 潘晓冬； 杜兰平； 秦彦文； 刘舒； 陈保生；

【机构】 北京市心肺血管疾病研究所动脉粥样硬化研究室； 中国医学科学院基础医学研究所国家医学分子生物学重点实验室；

【摘要】 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)1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1临床诊断为家族性高胆固醇血症的汉族家系共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,PCR方法扩增LDLR基因包含启动子和全部基因编码区(1-18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,采用变性高效液相色谱(DHPLC)技术检测了中国LDLR基因,对洗脱曲线异常者进行核苷酸序列测定分析。结果经变性高效液相色谱该家系中发现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4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。更多还原

【Abstract】 Objective To establish and evaluate temperature - modulated high performance liquid chromatography ( DHPLC) as a rapid and efficient technique of detecting low density lipoprotein receptor (LDLR) gene mutations on familial hypercholesterolemia (FH) . Method Collect a FH pedigree including 37 family members, among them 30 family members are first or second degree relatives; the other 7 in law relatives are regard as control. The genome DNA was extracted. The LDL - R 18 exons and promoter were amplified by PCR. The total 21 fragments were detected by using DHPLC and sequence analysis. The result werecompared against the normal sequences in GenBank to find the mutation and the heterozygotes. Result The twenty - one aimed fragments of LDLR gene can be detected simultaneously by DHPLC and 4 heterozygotes were found in the FH family. The mutation was proved by sequence analysis. Conclusion The DHPLC technique provides an efficient, sensitive and rapid method for detect LDLR gene mutation.更多还原