The Study On Association And Function Between Genetic Variants In The Promoter Region Of FABP1 Gene And Serum Lipid

Background:In recent years, with the rapid development of national economy, there are many great changes in people's lifestyles. Recent studies showed that the prevalence of dyslipidemia in Chinese population was increasing and became a major public health problem. Furthermore, dyslipidemia was the main risk factors of some diseases such as ischemic cardiovascular disease(ICVD), obesity and insulin resistance. Therefore, exploring the possible risk factors of dyslipidemia was important in preventing and controlling of dyslipidemia and the related diseases. Objective: 1. To explore the distribution characteristics of rs2919872G>A and rs2970903A>G genotypes and allele frequencies at the promoter region of FABP1 gene in Fuzhou Han population.2. To analyze the association between the FABP1 rs2919872G>A and rs2970903A>G genotypes and serum lipid levels.3. To explore the potential mechanism of promoter SNPs(rs2919872 and rs2970903) of FABP1 gene on serum lipid. Method: 1. The association between the promoter polymorphism and the risk for dyslipidemia in the Han population was assessed using a cross-sectional survey. A total of 1,182 subjects(male/female: 817/365, aged 18~72 years) were recruited from among individuals visiting the Union Hospital of Fujian Medical University for regular medical check-ups between August, 2012 to January, 2013. Differences in mean values between FABP1 rs2919872G>A or rs2970903A>G genotypes were assessed using unpaired t-tests, one-way ANOVA or the Mann-Whitney U-test, as appropriate. In addition, multiple linear regression analysis using TG levels as the dependent variable were done in order to find out the variables that were independently associated with serum TG.2. The p GL3-rs2919872 G and p GL3-rs2970903 A plasmid harboring the human FABP1 promoter with the rs2919872 G and rs2970903 A alleles were constructed by ligation of the PCR-generated full length human FABP1 promoter(nucleotides-2125 to +50, relative to the transcription start site) into the p GL3-Basic luciferase reporter plasmid. Fusion PCR was used to create p GL3-rs2919872 A, p GL3-rs2970903 G and p GL3-rs2919872A/rs2970903 G alleles of the FABP1 promoter using p GL3-rs2919872 G and p GL3-rs2970903 A as a template, respectively. The human hepatoblastoma cell line Hep G2 and the hepatoma cell line Huh7 were seeded(2×105 cells/well) in 12-well plates and transfected with the empty p GL3-Basic vector(a promoterless control) or with different haplotypes constructs using Lipofectamine 2000. Luminescence measurement was carried out on a luminometer to analyze the effects of transcriptionactivity of FABP1 in different haplotypes.3. Preparation of nuclear extracts from sub-confluent Hep G2 cell cultures and Electrophoretic mobility shift assay(EMSA) was utilized to find out whether the G to A change of rs2919872 alters the ability of the polymorphism region to bind transcription factors. Moreover, widely used software for TF binding site prediction were used to predict possible transcription changes due to the presence of A variant of rs2919872. Results: 1. The results of allele-specific phenotype analysis showed that the rs2919872G>A variant was significantly associated with serum TG concentration(P =0.032).Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration. In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum TG. However, we found no significant association between rs2970903A>G and serum lipids.2. The three haplotypes of FABP1 gene promoter were successfully cloned into the p GL3-basic luciferase reporter plasmid respectively. Furthermore, the results of dual-luciferase reporter assay showed that cells transfected with p GL3-rs2919872 A or p GL3-rs2919872A/rs2970903 G displayed much lower luciferase activity than that of cells transfected with p GL3-2125 haplotype. However, there was no significant difference in the luciferase expression between the p GL3-2125 haplotype and p GL3-rs2970903 G haplotype. These results suggested that the rs2919872 A allele dramatically reduced FABP1 promoter activity.3. The results of EMSA showed noticeable differences in affinity for nuclear proteins between the two alleles of rs2919872. Widely used software for TF binding site prediction were used to predict possible transcription changes due to the presence of a variant and the results showed that there was a GR-alpha binding sites only for rs2919872 G, and a TFIID binding sites only for rs2919872 A.Conclusion: The results of this study demonstrate that rs2919872G>A, a common polymorphism in the promoter region of FABP1, may be of functional importance in regulating the expression of the FABP1 gene and influences the serum TG concentrations in a Chinese Han population. Considering the association of increased serum TG levels with increased risk of metabolic syndrome and CVD, this variant still represents an interesting potential target for future lipid-lowering therapies to reduce the risk of CVD.