| Background: Warfarin is widely used in the prevention and treatment of thromboembolic diseases. However, due to the risk of severe bleeding complications,its clinical application is severely limited. How to use warfarin for anticoagulant therapy in a safe and efficient way is still an urgent problem to be solved. In recent years, warfarin’s pharmacogenomics study found that besides clinical and environmental factors, genetic variants on the pathway of warfarin metabolism and vitamin K circle largely determined the efficacy of warfarin. It has been found that CYP4F2 has the function of oxidative hydrolysis of vitamin K. The vitamin K metabolic level in vitamin K cycle could be changed in carriers of CYP4F2 genetic variants, leading to the alteration of γ -carboxylation of coagulation factor. Therfore,CYP4F2 genetic variants may be associated with bleeding complications in warfarin treated patients. However, rare study has investigated the contribution and the functional mechanisms of CYP4F2 variants on warfarin related bleeding events.Part 1: The association between CYP4F2 candidate genetic variants and warfarin severe bleeding complications as well as bioinformatic analysis: cohort studyObjective: To screen and validate the association between CYP4F2 candidate gene variants and warfarin severe bleeding complications.Methods: Consecutive patients with AF on initial warfarin treatment were prospectivly recruited with 2 stages from Chinese PLA General Hospital, and follow-up for at least 3 months. The tag SNPs of CYP4F2 were screened by Hapmap and NCBI dsSNP database as well as the Haploview software. The peripheral blood DNA of all recruited patients was genotyped by Snapshot method. Logistic regression was used to screen and validate the association between CYP4F2 candidate gene variants and warfarin severe bleeding complications. Bioinformatics analysis was applied to find the potential biological function of warfarin major bleeding related genetic variants in CYP4F2.Results: A total of 312 warfarin treated patients were recruited from January 2008 to September 2011 as derivation cohort, while 241 patients from January 2014 to December 2015 as validation cohort. In all the patients, 410 with atrial fibrillation(74.14%), 106 (18.63%) with valve replacement, 12 (2.17%) with pulmonary embolism, 38 (3.25%) with venous thrombosis, and 9 (1.63%) with other indications were included. Eight tag SNPs of CYP4F2 were seleted and genotyped successfully.Logistic regression analysis in derivation cohort showed that the risk of major bleeding events was significantly higher in CYP4F2 rs3093168 CC carriers than in non-carriers (7.79% vs.2.13%, OR: 5.39, 95% CI: 1.28-23.123, p = 0.02). The similiar correlation was found in the validation group (6.45% vs. 1.68%, OR: 7.93, 95% CI:1.03-61.06, p = 0.04). The linkage disequilibrium analysis showed that there was a SNP rs2079288 linked to rs3093168, which was located in the 44,244 bps upstream of the CYP4F2 promoter. SNPalyze analysis found that rs2079288 was in the coding region of linc RNA (NONHSAGO25013.2), and its functional mechanism might be related to the regulation of CYP4F2 gene expression by cis regulation.Conclusion: The genotype of CYP4F2 rs3093168 CC is related to the complication of severe bleeding in warfarin. Bioinformatics analysis revealed that its functional mechanism may be related to the regulation of CYP4F2 gene expression.Part 2: Screening CYP4F2 genetic variants associated with warfarin major bleeding complications by target gene resequencing: a case-control studyObjective: To screen genetic variants of CYP4F2 associated with warfarin major bleeding complications by target gene resequencing.Methods: Patients with warfarin major bleeding events and matched patients without warfarin bleeding events were were recruited from PLA General Hospital for CYP4F2 gene was resequenced by Sanger method. The correlation between CYP4F2 genetic variants and warfarin major bleeding events was analyzed.Bioinformatic analysis was used to find the potential function of the major bleeding associated CYP4F2 variants.Results: A total of 64 CYP4F2 gene variants were found by resequencing analysis, of which 12 novel genetic variants were found, and all the genotypes were in accordance with the Hardy-Weinberg equilibrium. The newly found insertion/deletion mutation (CYP4F2-1705 ins AGAT-1733 del CAGA) located in the third intron of CYP4F2 was found only in cases with major bleedings. Mutation Taster software analysis found that insertion /deletion mutation is located downstream of the third intron and exon splicing site and may affect gene expression by affecting the splicing of mRNA.Conclusion: CYP4F2-1705 ins AGAT -1733 del CAGA is related to the major bleeding complication of warfarin. Bioinformatics analysis shows that its functional mechanism may be related to the transcription of CYP4F2 gene.Part 3: In vitro biological functional analysis of the association between CYP4F2 gene variants and warfarin major bleeding complicationsObjective: To analyze the biological function of CYP4F2 genetic variants associated with warfarin major bleeding complications in vitro.Methods: The healthy male volunteers were recruited and the peripheral blood were extracted. Warfarin major bleeding related genetic variants (rs3093168 and CYP4F2-1705 ins AGAT-1733 del CAGA) were genotyped, and the demographic and other genetic backgrounds factors were matched between the related genetic variants carriers and non-carriers. The effect of genetic variants on the transcription of CYP4F2 gene was analyzed by qRT-PCR, on the expression of CYP4F2 protein was analyzed by CYP4F2 Elisa kit, and on the activation of FIX was analyzed by FIXa Assay kit. The co-transfected plasmids of pIRES-FIX-CYP4F2 were constructed and identified by restriction enzyme digestion. The pIRES-FIX plasmid,pIRES-FIX-CYP4F2 plasmid and blank control were transfected into L02 cells respectively. The protein expression of CYP4F2 and FIX was detected by western-blot. The effect of CYP4F2 on FIX carboxylation was examined by FIXa assay kit.Results: The expression of CYP4F2 mRNA was not significantly different between rs3093168 CC genotype carriers and non-carriers. The expression level of CYP4F2 mRNA was significantly higher in CYP4F2-1705 ins AGAT-1733 del CAGA insertion/deletion carriers than in non-carrier. In comparison to the control group, the expression levels of CYP4F2 protein were significantly higher in rs3093168 CC genotype carriers and CYP4F2-1705 ins AGAT-1733 del CAGA carriers. In comparison to the control group, the activation of FIX was significantly lower in rs3093168 CC genotype and CYP4F2-1705 ins AGAT-1733 del CAGA insertion/deletion carriers. After the transfection of the co-expression plasmid, the protein of CYP4F2 and FIX was successfully overexpressed. FIXa activity in L02 cell culture supernatant and cell lysate overexpressing FIX was significantly higher than that in control group. At the same time, FIXa activity in supernatant and cell lysate was significantly higher in CYP4F2 overexpression group than that in control group.Conclusion: rs3093168 CC gene variants and CYP4F2 insert/deletion mutations may inhibit the activation of coagulation factors by increasing the expression of CYP4F2 protein, leading to an increased risk of major bleeding in warfarin threated patients. |
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