家族性高胆固醇血症纯合表型患儿及家系致病基因突变分析

Pathogenic Gene Mutation Analysis of A Homozygous Phenotype Child with Familial Hypercholesterolemia and His Pedigree

目的对1例临床确诊为纯合型家族性高胆固醇血症(FH)患儿及其家系进行基因检测和系谱分析,探讨该家系可能存在的致病基因突变。方法于先证者家中收集该家系3代共47例血标本及临床资料。对先证者及其家系成员进行血脂测定和心电图检查;酚氯仿法提取患者基因组DNA并鉴定,PCR扩增基因外显子和内含子,采用变性高效液相色谱(HPLC)法及扩增产物直接序列分析检测其低密度脂蛋白受体(LDL-R)基因的全部18个外显子和启动、载脂蛋白B(ApoB100)R3500Q位点、枯草溶菌素9(PCSK9)基因全部12个外显子有无点突变;用多重连接探针扩增法(MLPA)检测先证者有无LDL-R基因的长片段的缺失或插入突变;核苷酸序列分析结果与GenBank比对寻找突变。结果先证者临床确诊为纯合子FH。根据血脂和临床表现,先证者家系47例中16例诊断为高胆固醇血症,其中临床确诊1例纯合子FH,6例杂合子FH;系谱分析该家系遗传方式符合常染色体显性遗传规律。先证者核苷酸序列分析排除LDL-R、ApoB100、PCSK9基因点突变;MLPA法排除了先证者LDL-R基因的长片段缺失。结论该家系为遗传性高胆固醇血症家系,通过对该家系进行基因突变检测和家系分析排除了已知的致FH基因突变,认为先证者的FH样表型可能是一种未知的基因突变,尚需采用进一步的全基因扫描方法寻找其致病基因。

Objective To detect gene mutation and pedigree analysis of one patient with homozygous familial hypercholesterolemia （FH） and his family, and analyze the possible mutation genes by gene testing and genetic analysis. Methods Peripheral blood and clinical data of the pedigree in proband＇s family, including 47 cases in 3 generations were collected. All family members took conventional clinical examination including blood - fat trait and electrocardiogram examination. Genomic DNA was extracted with phenol - trichlormethane method and all exons and introns were amplifyed by PCR. Eighteen exons and 1 promoter of low density lipoprotein - receptor （ LDL - R） gene ,R3500Q of apolipoprotein （ApoB100） gene, 12 exons of bacillus bacteriolytic- 9 （PCSK9） gene were amplified by PCR respectively. PCR products were analyzed by denaturing high - performance liquid chromatography （ HPLC ） and direct sequence analysis. Larger gDNA deletions and duplications in LDL - R gene by multiplex ligation - dependent probe amplification（MLPA） were identified and compared with Gen Bank to detect mutations. Results According to clinical data and lipid,16 cases were diagnosed as hypercholesterolemia,the proband diagnosed as homozygous FH clinically,6 cases were diagnosed as heterozygous FH and others as hypercholesterolemia. The genetic pedigree manner was consistent with autosomal dominant inheritance by pedigree analysis. According to gene sequencing ,the proband excluded LDL- R, ApoB100 and PCSK9 gene mutations. Larger gDNA deletions and duplications were excluded by MLPA. Conclusions This genetic pedigree manner is consistent with autosomal dominant inheritance. According to genetic analysis and pedigree analysis,the FH phenotype of proband may be an unknown gene mutation. Subsequently,the gene mutation will be found by genome wide Scan.