Interindividual variability in stable warfarin doses is largely attributed to VKORC1 and CYP2C9 variants. On the basis of a recent finding of the role of GATA4 in control of CYP2C9 expression, we tested a possible effect of GATA4 genotypes on variability in warfarin response using 201 Korean patients with prosthetic cardiac valves. Two single-nucleotide polymorphisms (SNPs), rs2645400 (G>T) and rs4841588 (G>T), were significantly associated with stable warfarin doses in patients carrying CYP2C9 wild-type homozygotes; homozygote carriers of these two SNPs required higher doses than those with other genotypes (5.94±1.73 versus 5.34±1.88 mg, P=0.026; 5.94±1.66 versus 5.37±1.92, P=0.036, respectively). Multivariate analysis showed that two GATA4 combinations, rs867858 (G>T)/rs10090884 (A>C) and rs2645400 (G>T)/rs4841588 (G>T), increased contribution to the overall warfarin dose variability from 36.4 to 40.9%. This study revealed that GATA4 can be predictive of stable warfarin dose and extended warfarin pharmacogenetics further to the regulation of CYP2C9 expression.
Warfarin, a vitamin K antagonist, has been indicated for the prophylaxis and a major therapy to prevent thrombosis and thromboembolism, and to reduce the recurrence of myocardial infarction. As the drug shows a narrow therapeutic window and a wide range of inter- and intraindividual variability in dose requirements, careful dosage adjustment should be taken into consideration, based on the international normalized ratio (INR) of prothrombin time. It has been known that environmental and clinical factors, such as age, height, body weight, co-medications and dietary vitamin K intake, influence warfarin dose requirements.1, 2, 3, 4 In addition, the contribution of genetic factors to interindividual variability has become increasingly appreciated in recent years. Over 40% of the variance in warfarin dose could be attributed to genetic variants in both cytochrome P450 (CYP) 2C9 and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes. The FDA updated the labelling information for warfarin to include dosing recommendations based on genotypes of CYP2C9 and VKORC1. CYP4F2 variants contribute to another 1–2% of the variability.5 Accounting for these genetic variations as well as environmental and clinical factors could explain about 55% of the variability in warfarin dose and the rest of the dose variability remains to be explained.
GATA4, a liver-specific transcription factor, recently showed to have an important role on the regulation of CYP2C9 gene expression.6 GATA4 belongs to the GATA family, a group of zinc-finger proteins that are conserved in metazoans. GATA family transcriptional regulators and their faithful cofactors, Friend-of-GATA proteins, collaborate to control cell fate and differentiation of diverse tissue types in metazoans including human.7 There are six GATA factors and two Friend-of-GATA proteins.8, 9, 10, 11 All six GATA factors recognize the consensus sequence (A/T)GATA(A/G) as the name implies; however, each shows a specific expression pattern during development. GATA1/2/3 are dominantly expressed in hematopoietic cells,12 whereas GATA4/5/6 are not expressed in hematopoietic cells. Instead, they are key factors in development of diverse tissues including heart, lung and muscle.13 GATA4 is also specifically expressed in the liver and herein takes part in the regulation of many detoxifying enzymes (drug-metabolizing enzymes) and transporters.14, 15, 16 The proximal promoter region of CYP2C9 has some important regulatory elements including several GATA4-binding motifs,6 which enable the interaction between the two proteins.
Encouraged by a report showing GATA4-mediated regulation of CYP2C9 expression,6 we entertained a possibility that the variation of the GATA4 genotype could explain some of the unexplained rest of the variabilities in the warfarin dose. In this study, to test this possibility we investigated the effects of GATA4 gene variants on the stable warfarin dose in Korean patients with mechanical heart valves.
Subjects and methods
Study patients were enrolled from the EAST (EwhA-Severance Treatment) Group of Warfarin. It comprises 229 patients who have undergone prosthetic mechanical heart valve replacements between January 1982 and December 2009 at Severance Cardiovascular Hospital of Yonsei University College of Medicine. Among them, 201 patients who maintained a stable warfarin dose (INR of 2–3 for at least three consecutive times) were selected.
The study protocol was approved by the Ethics Committee of the Severance Hospital Institutional Review Board (IRB No. 2009-4-0283), and informed consent was obtained from all patients before participation in this study.
Blood samples of study patients used for genotyping were collected during their regular outpatient clinic visits. To determine stable warfarin doses, defined as the mean doses for three or more consecutive visits with the INR of 2.0–3.0, routine INR values were recorded. Patient charts and electronic medical records from June 1983 to May 2012 were reviewed. Patients’ data collected included age, gender, age at the time of operation, body weight, body mass index, duration of warfarin therapy, position of valve prosthesis, valve types, co-medications, comorbidity and stable warfarin doses during warfarin therapy.
For the selection of the GATA4 single-nucleotide polymorphisms (SNPs), genetic information of the GATA4 gene was incorporated into the Haploview program.17 There were 12 SNPs in the GATA4 gene with a minor allele frequency (MAF)⩾20% in the Japanese and Han Chinese population. Linkage disequilibrium blocks were constructed following the D’-method by Gabriel et al. in Haploview.18 The Tagger function within Haploview was used to assign Tag SNPs. Seven Tag SNPs in the GATA4 gene (rs13273672, rs2645400, rs4841588, rs867858, rs17153755, rs10090884 and rs2898292) captured the common variations within the gene and the surrounding area with a minimum r2 of 0.80. Four SNPs (rs10086064, rs3735814, rs2740434 and rs904018) that presented a MAF⩾10% in the Asian population were further selected from previously published study.19 Of the 11 GATA4 SNPs selected for study, nine SNPs were in the intron region of the GATA4 gene, and two SNPs were in the 3’ untranslated region (UTR; Table 1).
In addition to the selected GATA4 SNPs, VKORC1 rs9934438 (C>T), CYP2C9 rs1957910 (A>C) and CYP4F2 rs2108622 (G>A) polymorphisms were further included in this study. A total of 15 SNPs were evaluated for the study.
Genomic DNA of study patients was prepared from ethylenediaminetetraacetic acid-blood samples using QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Hilden, Germany) according to standard procedures recommended by the manufacturer. Genotyping of VKORC1, CYP2C9 and CYP4F2 polymorphisms was conducted using single-base primer extension assay using ABI PRISM SNaPShot Multiplex kits (ABI, Foster City, CA, USA) according to the manufacturer’s recommendation. Genotyping of 11 GATA4 SNPs was performed with the TaqMan genotyping assay, using the RT-PCR system (ABI 7300, ABI).
Univariate and multivariate analyses were performed to compare the association of genotypes with stable warfarin doses. One-way analysis of variance (ANOVA) followed by post hoc Bonferroni test was used to see associations between the mean stable doses among three different genotype groups. Independent-sample t-test was used to compare stable warfarin doses between two groups. Multiple linear regression analysis was used to investigate the factors that independently affected the interindividual variability of warfarin dose requirements. Stepwise selection of variables was entered into the regression method that meet the default criteria of P<0.05 for entry and P>0.1 for removal.20 All analyses were performed with the IBM SPSS Statistics version 20 Software (International Business Machines Corp, New York City, NY, USA). A P-value of <0.05 was considered statistically significant.
The patients were followed up until May 2012 after their heart valve replacements. The follow-up period ranged from 1.0 to 29.7 years. The mean stable warfarin dose was 5.4±1.9 mg per day with a large interindividual variation among the study patients, ranging from 2.2 to 14.1 mg per day. The demographic characteristics and warfarin doses are shown in Table 2.
Effects of each of 11 genetic variants of GATA4 on the interindividual variability of warfarin dose requirements were evaluated. The observed genotype frequencies were consistent with the Hardy–Weinberg equilibrium for all SNPs. The associations between the mean stable doses of warfarin and grouped genotypes are shown in Table 3.
Stratification for CYP2C9 showed that genetic variations in two GATA4 SNPs, rs2645400 (G>T; MAF=36%) and rs4841588 (G>T; MAF=38%), significantly correlated with stable warfarin doses in the CYP2C9*1/*1 group (P=0.026 and P=0.036, respectively) but not in the CYP2C9 variant group (Figures 1 and 2). In wild-type homozygote (*1/*1) carriers of CYP2C9, patients who carried the variant-type homozygote (TT) of either SNP (rs2645400 and rs4841588) in GATA4 required a higher dose of warfarin than those with the other GATA4 genotypes. Of note, all the SNPs that showed associations with warfarin dose requirements in our cohort had over 30% of MAF.
The effects of combined genotypes of the GATA4 gene on the warfarin dose requirements were further analyzed (Table 4). Patients who carried wild-type genotypes in both GATA4 rs867858 (GG) and rs10090884 (AA) sites required significantly lower stable warfarin dose (4.32±1.43 mg; P=0.025) than those with the other combinations of genotypes (5.52±1.88 mg). The mean doses of other combined genotypes ranged from 5.41±1.97 to 5.56±1.87 mg. Patients with the wild-type genotypes in both rs10090884 (AA) and rs2645400 (GG) sites received significantly lower warfarin dose (4.27±1.56 mg; P=0.041) than those with the other combined genotypes (5.51±1.87 mg). The mean doses of other combined genotypes ranged from 5.27±1.74 to 5.75±2.32 mg. Patients with either GT or TT genotype in the rs2645400 (G>T) along with the GG genotype in the rs4841588 (G>T) showed significantly lower warfarin dose (3.29±0.13 mg; P=0.045) requirements than those with the other combined genotypes (5.48±1.87 mg). The mean doses of other combined genotypes ranged from 4.96±1.76 to 5.50±1.83 mg. Taken together, these results postulate that the combined effects of these two GATA4 SNPs are much stronger than the effect of each SNP alone and suggest that the two SNPs make effect on warfarin metabolism.
Multivariate regression analysis showed that GATA4 SNP combinations of rs867858 (G>T)/rs10090884 (A>C) and rs2645400 (G>T)/rs4841588 (G>T) were significantly associated with stable warfarin doses after adjustment for potential confounding factors (Table 5). These GATA4 genotype combinations accounted for 3.3% and 1.2%, respectively, of the overall interindividual variability in warfarin dose requirements. In total, the final model explained 40.9% of the overall interindividual variability in warfarin dose requirements among study patients; VKORC1 genotype accounted for 25.4% of the variability, GATA4 genotype for 4.5%, CYP2C9 genotype for 4.3%, age for 4.1% and CYP4F2 genotype for 2.6%. Therefore, the contribution of the GATA4 genotype to interindividual variability in warfarin dose requirements is comparable to that of CYP2C9 genotype.
Lines of evidence from various functional studies suggest that GATA4 is a transcription factor that participates in the regulation of CYP2C9 gene expression. Using luciferase gene reporter assays, Mwinyi el al.6 showed that GATA4 cotransfection in Huh-7 cells strongly upregulates wild-type CYP2C9 promoter constructs. Mutations on GATA4-binding sites significantly reduced this induction. Our study was based on the hypothesis that allelic variants of GATA4 would elicit differences in interaction with the CYP2C9 gene and consequently result in altered warfarin sensitivity. This study revealed that variance in transcription factors of CYP2C9 might also take a part in the interindividual variability of stable warfarin dose requirements.
The stable warfarin dose was defined as the mean dose for three or more consecutive visits with the INR of 2.0–3.0. Although the American College of Chest Physicians’ guidelines 2012 suggest INR of 2.5∼3.5 in patients with mitral valve replacements,21 many studies of Asian populations suggested considerably lower intensities of warfarin therapy after mechanical valve prostheses including mitral valves.22, 23, 24
To our knowledge, this is the first report to investigate the effect of genetic variation of GATA4 in Asian populations. In our study, a total of nine intron SNPs and two 3’UTR SNPs were selected. Notably, this study showed that some of GATA4 gene variants could, although at the modest level, contribute to variation in warfarin dose and provided an evidence that another genetic factor, GATA4 variants, could make an impact on warfarin dose, presumably through altered expression levels of the CYP2C9 gene.
Some combinations of certain GATA4 allelic variants manifested a significant alteration of warfarin sensitivity. Genotypes, when grouped based on stable warfarin doses, revealed the clinical significance of differences in warfarin dose requirements. The stable warfarin dose in patients with wild-type homozygotes in both rs867858 (GG) and rs10090884 (AA) was significantly lower than that in the other combinations. On the contrary, in the combination of rs2645400 and rs4841588, patients carrying a variant allele (T allele) in rs2645400 and wild-type homozygote in rs4841588 (GG) showed significantly reduced stable warfarin dose compared with the other combinations. This is in accordance with the results of former multivariate models and the stratification analysis.
Associations shown in the 3’UTR variant rs867858 (G>T) might be explained by the role of its location in post-transcriptional regulation. As stability and transport of mRNA transcripts are dependent on a properly configured 3’UTR,21 sequence variants in the 3’UTR region of the GATA4 gene might lead to altered function of the protein products by affecting the secondary structure of the mRNA and alternating its gene expression. It has been reported that polymorphisms in the intron region can also affect altered mRNA stability and degradation, gene expression and alternative splicing resulting in different protein isoforms.22 Therefore, it could be speculated that combined polymorphisms such as rs867858 (G>T)/rs10090884 (A>C) and rs2645400 (G>T)/rs4841588 (G>T) might cause an allelic imbalance that could alter gene regulation and expression. Up to now, however, no study has been conducted on the effects of GATA4 SNPs not only on GATA4 expression levels or functions but on CYP2C9 blood concentrations. Further functional analyses are required to establish the mechanism underlying these associations.
Interestingly, a study on the Netherlands population showed that GATA4 rs3735814 has a correspondence with daily acenocoumarol dose, which is a derivative of warfarin.19 The mean dosages decreased from 2.92 mg per day for the patients having the wild homozygote alleles to 2.65 mg per day for the patients carrying one variant allele and to 2.37 mg per day for patients carrying two GATA4 variant alleles (P=0.004). This study reported that genetic variations in this SNP explain an additional 1.1% of the acenocoumarol dose requirements in patients with the CYP2C9 common genotype alongside VKORC1 genotype, age, height, bodyweight, sex and amiodarone use. On the contrary, the rs3735814 variation did not show any correlation in our study. This is possibly because of the lack of the wild-type homozygous genotype (CC) in the Korean population.
Previous studies from the United Kingdom and the United States reported that the CYP2C9 gene polymorphisms (*2 and *3) explain about 15–27% of the interindividual warfarin dose variation.25, 26 In an Asian study, the predictive contribution of CYP2C9 was 3.94%, which was significantly lower compared with that of the Caucasian population.27 In our cohort, CYP2C9 variability accounted for about 4.3% of the overall warfarin dose variability. It is possible that the relatively low contributions of the CYP2C9 gene on the Asian population might have obscured the effects of the GATA4 variations. Stratification analysis was performed to see the impact of GATA4 variation in different CYP2C9 genotype groups, and rs2645400 (G>T) and rs4841588 (G>T) showed significant associations with stable warfarin doses in CYP2C9 wild-type homozygote carriers. However, the lack of the CYP2C9 variant alleles gave limitations for full comparisons. Furthermore, although the SNPs in our study were carefully selected, it is possible that other SNPs in GATA4 might also contribute to warfarin dose variation.
It should be noted that all the SNPs that showed associations with warfarin dose requirements in this study had over 30% of MAF. Accordingly, our findings will give a high real-world clinical implications in Korea and possibly other East Asian populations. In this study, 40.9% of the variance in warfarin dose in Korean patients who underwent mechanical valve surgery was explained. The genetic variations in transcription factors of the associated genes have rarely been studied in warfarin pharmacogenetics yet. Findings of this study present the possible influence of transcription factor gene variants on warfarin dose requirements. This study is expected to encourage other studies aimed at investigating other genetic variants of transcription factors associated with warfarin sensitivity.
Penning-van Beest FJA, Geleijnse JM, van Meegen E, Vermeer C, Rosendaal FR, Stricker BHC . Lifestyle and diet as risk factors for overanticoagulation. J Clin Epidemiol 2002; 55: 411–417.
Carlquist JF, Horne BD, Muhlestein JB, Lappe DL, Whiting BM, Kolek MJ et al. Genotypes of the cytochrome p450 isoform, CYP2C9, and the vitamin K epoxide reductase complex subunit 1 conjointly determine stable warfarin dose: a prospective study. J Thromb Thrombolysis 2006; 22: 191–197.
Schalekamp T, van Geest-Daalderop JHH, Kramer MHH, van Holten-Verzantvoort ATM, de Boer A . Coumarin anticoagulants and co-trimoxazole: avoid the combination rather than manage the interaction. Eur J Clin Pharmacol 2007; 63: 335–343.
Gage BF, Eby C, Milligan PE, Banet GA, Duncan JR, McLeod HL . Use of pharmacogenetics and clinical factors to predict the maintenance dose of warfarin. Thromb Haemost 2004; 91: 87–94.
Takeuchi F, McGinnis R, Bourgeois S, Barnes C, Eriksson N, Soranzo N et al. A genome-wide association study confirms VKORC1, CYP2C9, and CYP4F2 as principal genetic determinants of warfarin dose. PLoS Genet 2009; 5: e1000433.
Mwinyi J, Nekvindova J, Cavaco I, Hofmann Y, Pedersen RS, Landman E et al. New insights into the regulation of CYP2C9 gene expression: the role of the transcription factor GATA-4. Drug Metab Dispos 2010; 38: 415–421.
Chlon TM, Crispino JD . Combinatorial regulation of tissue specification by GATA and FOG factors. Development 2012; 139: 3905–3916.
Arceci RJ, King AAJ, Simon MC, Orkin SH, Wilson DB . Mouse GATA-4 - a retinoic acid-inducible gata-binding transcription factor expressed in endodermally derived tissues and heart. Mol Cell Biol 1993; 13: 2235–2246.
Evans T, Reitman M, Felsenfeld G . An erythrocyte-specific dna-binding factor recognizes a regulatory sequence common to all chicken globin genes. Proc Natl Acad Sci USA 1988; 85: 5976–5980.
Yamamoto M, Ko LJ, Leonard MW, Beug H, Orkin SH, Engel JD . Activity and tissue-specific expression of the transcription factor nf-e1 multigene family. Genes Dev 1990; 4: 1650–1662.
Tsang AP, Visvader JE, Turner CA, Fujiwara Y, Yu CN, Weiss MJ et al. FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation. Cell 1997; 90: 109–119.
Weiss MJ, Orkin SH . Gata transcription factors - key regulators of hematopoiesis. Exp Hematol 1995; 23: 99–107.
Molkentin JD . The zinc finger-containing transcription factors GATA-4,-5, and-6 - ubiquitously expressed regulators of tissue-specific gene expression. J Biol Chem 2000; 275: 38949–38952.
Zhu QS, Qian B, Levy D . Regulation of human microsomal epoxide hydrolase gene (EPHX1) expression by the transcription factor GATA-4. Biochim Biophys Acta 2004; 1676: 251–260.
Kwintkiewicz J, Cai ZL, Stocco C . Follicle-stimulating hormone-induced activation of Gata4 contributes in the up-regulation of Cyp19 expression in rat granulosa cells. Mol Endocrinol 2007; 21: 933–947.
Sumi K, Tanaka T, Uchida A, Magoori K, Urashima Y, Ohashi R et al. Cooperative interaction between hepatocyte nuclear factor 4 alpha and GATA transcription factors regulates ATP-binding cassette sterol transporters ABCG5 and ABCG8. Mol Cell Biol 2007; 27: 4248–4260.
Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B et al. The structure of haplotype blocks in the human genome. Science 2002; 296: 2225–2229.
van Schie RMF, Wessels JAM, Verhoef TI, Schalekamp T, le Cessie S, van der Meer FJM et al. Evaluation of the effect of genetic variations in GATA-4 on the phenprocoumon and acenocoumarol maintenance dose. Pharmacogenomics 2012; 13: 1917–1923.
Jacobson PA, Oetting WS, Brearley AM, Leduc R, Guan WH, Schladt D et al. Novel polymorphisms associated with tacrolimus trough concentrations: results from a multicenter kidney transplant consortium. Transplantation 2011; 91: 300–308.
Whitlock RP, Sun JC, Fremes SE, Rubens FD, Teoh KH . Antithrombotic and thrombolytic therapy for valvular disease antithrombotic therapy and prevention of thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012; 141: E576S–E600S.
Sun XA, Hu SS, Qi GQ, Zhou YY . Low standard oral anticoagulation therapy for Chinese patients with St. Jude mechanical heart valves. Chin Med J 2003; 116: 1175–1178.
Matsuyama K, Matsumoto M, Sugita T, Nishizawa J, Yoshida K, Tokuda Y et al. Anticoagulant therapy in Japanese patients with mechanical mitral valves. Circ J 2002; 66: 668–670.
Yoon IK, Lee KE, Lee JK, Chang BC, Gwak HS . Adequate intensity of warfarin therapy for Korean patients with mechanical cardiac valves. J Heart Valve Dis 2013; 22: 102–109.
Caldwell MD, Awad T, Johnson JA, Gage BF, Falkowski M, Gardina P et al. CYP4F2 genetic variant alters required warfarin dose. Blood 2008; 111: 4106–4112.
Sconce EA, Khan TI, Wynne HA, Avery P, Monkhouse L, King BP et al. The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen. Blood 2005; 106: 2329–2333.
Zhong SL, Yu XY, Liu Y, Xu D, Mai LP, Tan HH et al. Integrating interacting drugs and genetic variations to improve the predictability of warfarin maintenance dose in Chinese patients. Pharmacogenet Genomics 2012; 22: 176–182.
The authors declare no conflict of interest.
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Cite this article
Jeong, E., Lee, K., Jeong, H. et al. Impact of GATA4 variants on stable warfarin doses in patients with prosthetic heart valves. Pharmacogenomics J 15, 33–37 (2015). https://doi.org/10.1038/tpj.2014.36
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