A novel mutation in proprotein convertase subtilisin/kexin type 9 gene leads to familial hypercholesterolemia in a Chinese family  

作　　者：LIN Jie WANG Lu-ya LIU Shu WANG Xu-min YONG Qiang YANG Ya DU Lan-ping PAN Xiao-dong WANG Xu JIANG Zhi-sheng 

机构地区：[1]Institute of Cardiovascular Disease,Key Laboratory for Arteriosclerology of Hunan Province,University of South China,Hengyang,Hunan 421001,China Beijing Anzhen Hospital,Beijing Institute of Heart,Lung and Blood Vessel Diseases,Capital Medical University,Beijing 100029,China [2]Beijing Anzhen Hospital,Beijing Institute of Heart,Lung and Blood Vessel Diseases,Capital Medical University,Beijing 100029,China [3]Beijing Institute of Genomics,Chinese Academy of Sciences,Beijing 100029,China [4]Institute of Cardiovascular Disease,Key Laboratory for Arteriosclerology of Hunan Province,University of South China,Hengyang,Hunan 421001,China

出　　处：《Chinese Medical Journal》2010年第9期1133-1138,共6页中华医学杂志（英文版）

基　　金：This study was supported by grants from the National Natural Science Foundation of China （No. 30470722, 30771986, 30772356）, Beijing Natural Science Foundation （No. 7092016, 7062010, 7052021）, and Key Project for Applicable Basic Research of Hunan Province （No. 2008FJ2006）.

摘　　要：Background Familial hypercholesterolemia （FH） is an autosomal disorder associated with elevated plasma low density lipoprotein （LDL） levels leading to premature coronary heart disease （CHD）. As a result of long-term hyperlipemia, FH patients will present endarterium thickening and atherosclerosis. In the present study we scanned the related gene of a clinically diagnosed autosomal genetic hypercholesterolemia family for the possible mutations and established eukaryotic expression vector of mutation of proprotein convertase subtilisin/kexin type 9 （PCSK9） gene with gene recombination technique to investigate the contributions of the variation on low density lipoprotein receptor （LDL-R） metabolism and function alternation.Methods Mutation detection was conducted for LDL-R, apolipoprotein B100 （apoB100） and PCSK9 gene with nucleotide sequencing in a Chinese FH family. The full-length cDNA of wild type PCSK9 gene （WT-PCSK9） was obtained from Bel-7402. Site mutagenesis was used to establish the recombinant eukaryotic expression vector carrying pathogenic type of PCSK9 gene and the inserted fragment was sequenced. With the blank vector as control, liposome transfection method was used to transfect the Bel-7402 cells with recombinant plasmid. The expression of LDL-R mRNA was examined by RT-PCR. PCSK9 and the expression of LDL-R protein were determined by Western blotting. Results The G→T mutation at the 918 nucleotide of PCSK9 gene resulted in the substitution of the arginine by a serine at the codon 306 of exon 6. After sequencing, it was confirmed that the inserted fragment of established expression vector had correct size and sequence and the mutant was highly expressed in Bel-7402 cells. There was no significant variation in the levels of LDL-R mRNA. LDL-R mature protein was decreased by 57% after the cells were transfected by WT-PCSK9 plasmid. Mature LDL-R was significantly decreased by 12% after the cells were transfected by R306S mutant as evidenced by gray scale scanning, suggestingBackground Familial hypercholesterolemia （FH） is an autosomal disorder associated with elevated plasma low density lipoprotein （LDL） levels leading to premature coronary heart disease （CHD）. As a result of long-term hyperlipemia, FH patients will present endarterium thickening and atherosclerosis. In the present study we scanned the related gene of a clinically diagnosed autosomal genetic hypercholesterolemia family for the possible mutations and established eukaryotic expression vector of mutation of proprotein convertase subtilisin/kexin type 9 （PCSK9） gene with gene recombination technique to investigate the contributions of the variation on low density lipoprotein receptor （LDL-R） metabolism and function alternation.Methods Mutation detection was conducted for LDL-R, apolipoprotein B100 （apoB100） and PCSK9 gene with nucleotide sequencing in a Chinese FH family. The full-length cDNA of wild type PCSK9 gene （WT-PCSK9） was obtained from Bel-7402. Site mutagenesis was used to establish the recombinant eukaryotic expression vector carrying pathogenic type of PCSK9 gene and the inserted fragment was sequenced. With the blank vector as control, liposome transfection method was used to transfect the Bel-7402 cells with recombinant plasmid. The expression of LDL-R mRNA was examined by RT-PCR. PCSK9 and the expression of LDL-R protein were determined by Western blotting. Results The G→T mutation at the 918 nucleotide of PCSK9 gene resulted in the substitution of the arginine by a serine at the codon 306 of exon 6. After sequencing, it was confirmed that the inserted fragment of established expression vector had correct size and sequence and the mutant was highly expressed in Bel-7402 cells. There was no significant variation in the levels of LDL-R mRNA. LDL-R mature protein was decreased by 57% after the cells were transfected by WT-PCSK9 plasmid. Mature LDL-R was significantly decreased by 12% after the cells were transfected by R306S mutant as evidenced by gray scale scanning, suggesting

关 键 词：proprotein convertase subtilisin/kexin type 9 gene familial hypercholesterolemia coronary heart disease low density lipoprotein receptor gain of function 

分 类 号：R[医药卫生]