变性高效液相色谱技术在家族性高胆固醇血症低密度脂蛋白受体基因突变检测中的应用

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(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。

Analysis of temperature-modulated high performance liquid chromatography for detecting mutations of LDLR gene in a familial hypercholesterolemia pedigree

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).Methods 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 LDLR 18 exons and promoter were amplified by PCR. The total 21 fragments were detected by using DHPLC and sequence analysis. The result (were compared against the normal sequences in GenBank to find the mutation and the heterozygotes.ResultsKG*2])The twenty-one aimed fragments of LDLR gene can be detected simultaneously by DHPLC and 1 monozygote 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.