Association And Functional Characterization Of Single Nucleotide Polymorphisms In CYP4F2 Gene Regulatory Region With Essential Hypertension

ObjectiveEssential hypertension (EH) is a common complex disease and its patho-genesis remains largely unknown. As a multifactorial disease, EH is the combination effect of individual genetic predisposition and environmental factors. Therefore, association study and functional identification of EH candidate gene relevant to environmental metabolism, would throw light on the pathogenesis of this common complex disease.Cytochrome P450 4F2 ( CYP4F2) is an environmental metabolic enzyme and the major renal ω - hydroxylase in human which convert arachidonic acid ( AA) to 20 - hydroxyeicosatetraenoic acid (20 - HETE). 20 - HETE is a va-soactive metabolite and involved in the maintenance of renal water - salt homeo-stasis. A study using chromosomal substitution and cDNA microarray revealed differential expression of CYP4 gene in renal of salt - sensitive hypertensive mice. Recently, one functional SNP (T8590C)of aother 20 - HETE synthase gene expressed in human kidney, CYP4A11 gene, is reported to be associated with EH. However, association of CYP4F2 gene with EH as well as functional investigation were not observed to date.In this work, we focused on single nucleotide polymorphism ( SNP) in CYP4F2 gene regulatory region, genotyped a relatively isolated population from hypertension prevalent region in Northeastern China, and undertook case - control and family - based association study of CYP4F2 gene with EH. Furthermore, we analyzed the CYP4F2 gene regulatory region by software prediction, reporter vector construction and CAT detection to elucidate the role of P450pathway in EH pathogenesis.Materials and Methods1. All subjects were Han Chinese in origin and from the prevalent region of hypertension in Liaoning province Northeastern China. Five hundred and fouty -eight subjects were recruited for case - control study, including 271 unrelated hypertensives (128 males and 143 feamles) and 277 unrelated normotensives (128 males and 149 females). Sixty - six EH families including 275 subjects were also selected for the family - based association study.2. Genomic DNA was isolated from whole - blood samples. CYP4F2 gene intron 1 transcriptional region ( + 260 ~ + 887) was amplified by polymerase chain reaction (PCR). One hundred and thirty PCR products from case -control population were randomly selected to perform DNA sequencing. The rest case - control population and EH family were genotyped by restriction fragment length polymorphism using restriction enzyme Apa I.3. A series of chloramphenicol acetyltransferase (CAT) reporter constructs were generated by PCR amplification and cloning into pCAT - Basic. Transient transfection was performed in HEK293 cells using liposome and CAT assay was undertook by enzyme linked immuno sobent assay. Lipopolysaccharide treatment was performed to stimulate NF - kB pathway.4. Electrophoretic mobility shift assay (EMSA) was conducted to identify transcription factor binding element in CYPAF1 gene. Nuclear extracts from HEK293 cells were prepared by a modified rapid micropreparation technique. Preparation for recombinant Myb was done by transforming c - Myb expression plasmid into BL21 ( DE3) host strain and induced by IPTG. The putative and mutant oligonucleotides according to CYP4F2 gene sequence with NF - kB and Myb responsive elements were radiolabeled with [7 -32P] ATP. For competition assay, 100 - fold and 500 - fold molar excess of the unlabeled double stranded oligonucleotides were added into reaction mixture. Recombinant Myb was used as positive protein control to identify Myb - DNA complex. NF - kB consensus oligonucleotide was used as a positive control and p50 antibody was usedin supershift assay.5. Hardy - Weinberg equilibrium was tested using x2 test- Adjusted odds ratios (ORs) with 95% confidence intervals from logistic regression analysis were used to estimate the relative risk of hypertension associated with genotype. Statistical analyses were done with SPSS 10.0 for windows. Clinical biochemical data were expressed as mean ± SD. P <0.05 was considered significant. Haplo-types were inferred using PHASE (version 2.0) program. Linkage disequilibrium was analyzed by HaploXT 1.1 and haplotype block was tested by HAPLOB-LOCK software. TDT - STDT Program 1. 1 was used for EH family analysis. Transmission - disequilibrium test ( TDT) was calculated using TDT - STDT Program 1.1. The potential transcription factor binding sites were searched with Match? ( public version 1.0) in computer analysis.ResultsIn the case - control population, body mass index, serum triglyceride, total cholesterol and low - density lipoprotein cholesterol level were significantly higher in EH group than in the control, and the elevated total cholesterol and low -density lipoprotein cholesterol level were revalent to EH in hypertensive families.Seven DNA variants located in CYP4F2 regulatory region were found in 130 samples, six in intron 1 (T378C, T392C, G421C, C426T, G446A and T456C) and one in exon 2 T502G (W12G). Four variants (T378C, G421C, T456C and T502G) were SNPs with fenquency 2s 1% . Two common SNP haplo-types, major - Hap(TGTT) and minor - Hap( CCCG) , were inferred after haplotype reconstruction. Complete linkage disquilibrium (LD) was found between SNPs, and one of them, G421C, could be used as a tag SNP.Genotype distribution of G421C was in Hardy - Weinberg equilibrium in both study population. In the case - control population, 421G allele frequency was higher in EH group (89.9% ) than in the control (85.9% ) (p =0.046) , and the frequency of homozygous 421GG genotype was pronouncedly higher in EH group than in control group (80.4% versus 72. 9% , p = 0. 038;OR = 1.53;95% CI 1.02 ~2.28). Logistic regression analysis revealed that homozy-gous 421GG genotype was one of the independent risk factors for EH (OR = 1. 85, 95% CI 1. 19 ~ 2. 88, P < 0. 05 ). Sorting by genotype demonstrated that mean level of systolic blood pressure in 421GG individual was 2. 85 mm Hg higher than other genotype group. TOT in family - based association study indicated that the 421G was transmitted more frequently to affected offsprings from their parents than expected (x2 =5.23, P <0. 05) , and combined score (z'= 2.70, P <0. 05) of TDT with S - TOT also showed a significant preferential transmission of 421G allele to affected subjects.Putative Myb transcription factor binding element in CYP4F2 gene intron 1 around G421C was identified by EMS A, showing that 421G probe could form protein - DNA complex with HEK239 cell extracts or c - Myb recombinant protein, however, 421C abolished this binding affinity. Fourty - nine base pair (bp) sequence encomparsing Myb response element at G421C ( +414 ~ + 372) were synthesized to construct single or double - copy insert CAT reporter vectors (p49G - CAT, p49C - CAT, p49G x 2 - CAT and p49C x 2 - CAT). CAT assay demonstrated that all four vectors have transcriptional activities, the report activity with 421G allele was lower than that of 421C, and the report activity with double - copy decreased compared with the single one.Another putative transcription factor binding site in CYP4F2 gene intron 1 around T378C, NF - kB response element, was also testified by EMS A, showing that 378T probe bound with both p65/p50 heterodimer and p50/p50 ho-modimer of NF - kB , while 378C could only form p65/p50 - DNA complex.A series of 5'- end deleted CAT reporter vectors for CYP4F2 gene regulatory region were constructed ( p564major - CAT, p564minor - CAT, p219major -CAT, p219minor - CAT, p67major - CAT and p67minor - CAT). CAT assay indicated that transcriptional activity decreased with 5' - end deletion, among them 564 - bp had the highest activity and 67 - bp exhibited the lowest. The remarkable activity difference between major - Hap and minor - Hap was found in p219 - CAT, and the activity of p219major - CAT was 35. 93% higher than p219minor - CAT ( P < 0. 05). Similar to p49 - CAT, major - Hap of p67 -LPS treatment up - regulated transcriptional activity of reporter vetectors containing NF - KB binding element (p564 - CAT and p219 - CAT), and the elevation of major - Hap haplotype was more remarkable than minor - Hap.Conclusions1. Seven DNA variations exist in CYP4F2 gene regulatory region, which constitute two common SNP haplotypes, major - Hap and minor - Hap. The major - Hap of CYP4F2 gene regulatory region is associated with enhanced secepti-bility of EH.2. G421C polymorphism in CYP4F2 gene intron 1 alters the binding affinity of Myb responsive element, and leads to decreased transcriptional activity of 421G allele regulatory fragments compared with 421C.3. T378C in CYP4F2 gene intron 1 influence the binding pattern of NF - kB responsive element. Transcriptional activity of major - Hap regulatory region is higher than minor - Hap due to the up - regulation of NF - kB p50/p50 binding , which surpasses the repressive effect of Myb response element.4. CYP4F1 gene maybe a EH causative gene. Different susceptibility for EH may be determined by variant of CYP4F2 gene regulatory region through NF- kB pathway.