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目的 检测中国汉族家族性高胆固醇血症 (FH)家系低密度脂蛋白受体 (LDLR)基因突变类型 ,研究基因型与表型间的关系 ,探讨FH发病的分子病理机制。方法 先证者及家系成员进行血脂测定、心电图、心脏及大血管彩色多普勒超声检查后采用聚合酶链反应 ( polymerasechainreaction ,PCR) 变性高效液相色谱 (DHPLC)法结合扩增产物直接序列分析检测LDLR基因启动子和全部 18个外显子片段 ,结果与GenBank公布的该基因正常序列比对找出突变并检索FH突变数据库(www .ucl.ac .uk/fh)。此外 ,采用PCR 限制性内切酶技术 ,检测载脂蛋白B10 0 (ApoB10 0 )基因Q35 0 0R突变 ,以排除家族性ApoB10 0 缺陷症 (FDB)。结果 DHPLC分析发现该患儿及其父母LDLR基因第 3外显子存在一异常波峰 ,DNA测序证实该患儿第 3内含子 5′剪接位点存在G→A纯合剪接突变 ,其父母相同位点表现为野生型和突变型杂合现象 ;同时未检测出患儿及其父母ApoB10 0Q35 0 0R突变。结论 国内首次发现LDLR基因第 3内含子G→A纯合剪接突变 ;该突变可能是FH发病的分子基础并导致其严重的临床表型 ;PCR DHPLC法可用于FH可疑人群的确诊
Objective To detect the type of low density lipoprotein receptor (LDLR) gene mutation in familial hypercholesterolemia (FH) family members in Han Chinese and investigate the relationship between genotypes and phenotypes and the molecular pathogenesis of FH. Methods Proximals and their family members were measured for blood lipid, ECG, heart and blood vessels by color Doppler ultrasound examination by polymerase chain reaction (PCR) denaturing high performance liquid chromatography (DHPLC) combined with amplification products direct sequence analysis The promoter and all 18 exons of LDLR gene were detected. The results were compared with the normal sequence of the gene published in GenBank to find out the mutation and to search the FH mutation database (www. Ucl.ac.uk / fh). In addition, Q3500R mutation of apolipoprotein B10 0 (ApoB10 0) gene was detected by PCR restriction endonuclease assay to exclude familial ApoB10 0 deficiency (FDB). Results DHPLC analysis showed that there was an abnormal peak in exon 3 of LDLR gene in this patient and its parents. DNA sequencing confirmed that there was a G → A homozygous splicing mutation in the 5 ’splice site of the third intron in this patient, with the same parents Site showed wild-type and mutant heterozygous phenomenon; at the same time did not detect children and their parents ApoB10 0Q35 0 0R mutation. Conclusion The first intron G → A homozygous splicing mutation of LDLR gene was found in China. This mutation may be the molecular basis of FH and lead to its severe clinical phenotype. PCR DHPLC can be used to diagnose suspicious FH population