The Effect Of VNTR-ZNF And C-14T Variant On Transcription Activity In The Promoter Region Of ATP-binding Cassette Transporter 1

ObjectiveTo investigate the frequency distribution of VNTR-ZNF ACCCC deleted/inserted polymorphism and C-14T polymorphism in the promoter region of ATP-binding cassette Transporter l(ABCAl) in Tianjin district and the association between polymorphisms with coronary heart disease (CHD) and with the plasma level of high-density lipoprotein (HDL). To study the effect of VNTR-ZNF and C-14T variant for transcription activity of ABCAlgene in vitro.Methods1. A total of 265 cases were recruited from Tianjin Chest hospital during the period of 2007 and 2009, including166 patients with CHD and 99 healthy donors. The polymorphism of VNTR-ZNF ACCCC inserted/deleted was detected by polymerase chain reaction (PCR) and polyarcrylamide gel electrophoresis. The polymorphism of C-14T was detected by polymerase chain reaction-restricted fragments length polymorphism.2. To construct recombinants by ligating DNA fragment containing VNTR-ZNF ACCCC inserted/deleted allele with or without-14C/T substitution gene fragments with PGL2-basic vector containing luciferase report gene. Recombinants were transfected to HepG2 cells by using the cationic lipid method.48h after transfection cells were collected and luciferase activity expressed was detected.Results1. The frequency was 0.313 for inserted allele and 0.687 for deleted allele of VNTR-ZNF. The genotypic frequencies of VNTR-ZNF were 6.7%,45.5%,51.8% for inserted homozygote, deleted homozygote and the inserted/deleted heterozygote respectively. No significant difference of allele and genotype frequency of VNTR-ZNF between CHD and control groups. HDL plasma level did not show significant difference in three genotypes.2. The frequency of C allele was 0.691 and T allele was 0.309; the genotypic frequencies were 44.2%for CC gene type,6.0%for TT gene type and 49.8%for CT gene type. The frequency distribution of allele and genotype also didn't show significantly differences between CHD and control groups and there were no significant differences of HDL level in three different genotypes.3. Six of recombinants were constructed, they were named as PGL2-ZNF-ACCCCDel, PGL2-ZNF-ACCCCIns (containing only VNTR-ZNF allele) PGL2-ZNFDel-14C, PGL2-ZNFDel-14T, PGL2-ZNFIns-14C, PGL2-ZNFIns-14T (containing both VNTR-ZNF and-14bp allele).48h after transfection reported gene activity of recombinant expressed in HepG2 cells was measured Luciferase activity of PGL2-ZNF-ACCCCDel was higher than that of PGL2-ZNF-ACCCCIns. Luciferase activity of PGL2-ZNFDel-14C was higher than that of PGL2-ZNFDel-14T, PGL2-ZNFIns-14C, PGL2-ZNFIns-14T.Conclusion1. The variation of VNTR-ZNF and C-14T in ABCA1 gene promoter region were not associated with plasma level of HDL and coronary heart disease. The frequency of allele and genotype of VNTR-ZNF was markedly different between the population of Tianjin in China and Europe.2. Transcription activity of the ACCCC deleted type of VNTR-ZNF was higher than that of inserted type, when-14bp allele was combined simultaneously with ACCCC deleted type the gene transcription activity was markedly higher than ACCCC deleted alone. The C allele maybe enhanced the transcription activity of the ACCCC deleted type of VNTR-ZNF.