CYP4F2 rs2108622: a minor significant genetic factor of warfarin dose in Han Chinese patients with mechanical heart valve replacement

Abstract

AIMS

The objective of this study was to assess the effect of the CYP4F2 on the daily stable warfarin dose requirement in Han Chinese patients with mechanical heart valve replacement (MHVR).

METHODS

From March 2007 to November 2008, 222 Han Chinese MHVR patients were recruited in our study. VKORC1 3673G>A, 5417G>T, CYP2C9*3 and CYP4F2 rs2108622 were genotyped by using the polymerase chain reaction restriction fragment length polymorphism method (PCR-RFLP). Polymorphisms of VKORC1 9041G>A were detected by direct sequencing. Multiple linear regression analysis was used to investigate the contribution of CYP4F2.

RESULTS

The CYP4F2 rs2108622 CT/TT group took a significantly higher stable warfarin dose (3.2 mg day⁻¹) than the CC group (2.9 mg day⁻¹, 95% CI 0.2, 1.0, P= 0.033). The multiple linear regression model included VKORC1 3673G>A, CYP2C9, CYP4F2 genotypes and clinical characteristics. The model could explain 56.1% of the variance in stable warfarin dose in Han Chinese patients with MHVR. CYP4F2 contributed about 4% to the variance in the warfarin dose. There was no variation in the SNPs of VKORC1 5417G>T.

CONCLUSION

CYP4F2 is a minor significant factor of individual variability in the stable warfarin dose in Han Chinese patients with MHVR. The effect of CYP2C9 and VKORC1 genotypes on variability in the stable warfarin dose had also been confirmed.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Genetic polymorphisms of VKORC1 and CYP2C9 are known to influence warfarin dosage.

• Recent studies among Caucasians showed that polymorphisms of CYP4F2 also play a role in warfarin pharmacogenetics.

• The contribution of CYP4F2 variants to the variability inwarfarin dose requirement in Chinese subjects remains to be investigated.

WHAT THIS STUDY ADDS

• This research was to study the effect of CYP4F2 variants on warfarin requirements in the Han Chinese population.

• This study developed a multiple regression model including CYP2C9, VKORC1 3673G>A, CYP4F2 genotypes and age, weight, combination use of amiodarone which could explain 56.1% of the individual variability in warfarin dose CYP4F2 could explain 4% of the variance in warfarin dose.

• We found that one novel genotypic polymorphism 5417G>T for Asp36Tyr, which was identified as an important marker of warfarin resistance, was absent in the Han Chinese population in our study.

Introduction

Warfarin, a coumarin derivative, exerts its anticoagulant effect by inhibiting vitamin K epoxide reductase (VKORC1), hence preventing vitamin K 2,3- epoxide reducing to vitamin K hydroquinone which is essential for the activation of vitamin K-dependent clotting factors [FII (or (pro)thrombin), FVII, FIX, and FX]. It is the most commonly used oral anticoagulant for patients with mechanical heart valve replacement (MHVR) to prevent thromboembolism. However, due to the narrow therapeutic index and the wide inter-individual variation, it usually takes a long time to reach the target international normalized ratio (INR). To minimize the adverse events and to shorten the time to reach the target INR, algorithms including patients' clinical characteristics and genetic polymorphism information have been explored.

It has been reported that genetic variants in the gene encoding VKORC1 result in altered sensitivity to warfarin [1]. A promoter polymorphism, 3673 G>A was detected in the upstream region of VKORC1 and VKORC1 3673 A has been found to be associated with warfarin-‘sensitive’ individuals who required lower warfarin doses [1]. Recently, a novel genotypic polymorphism 5417G>T for Asp36Tyr has been identified as an important marker of warfarin resistance [2]. This polymorphism has been detected in Ashkenazi and Sephardi Jewish populations (allele frequency 4.2% and 0.6%, respectively) [3] and also Ethiopian populations (allele frequency 15%) [4], but has not yet been identified in other populations.

Warfarin is mainly metabolized by CYP2C9. The common variants of CYP2C9 (CYP2C9*2 and CYP2C9*3) are known to decrease warfarin maintenance dose requirement in patients undergoing anticoagulation therapy, and detecting the polymorphism of CYP2C9 has been proved to be helpful in optimizing the administration of warfarin in several studies [5, 6].

In the past few years, research on the pharmacogenetics of warfarin has mostly focused on VKORC1 and CYP2C9. Together with weight, age and other clinical factors, about half of the interindividual variability of warfain response can be explained. However, there is still a large part of warfarin response variability that cannot be explained.

CYP4F2 is a member of cytochrome P450. It is involved in the ω-hydroxylation of arachidonic acid and vitamin E [7, 8]. Recently, McDonald et al[9] reported that CYP4F2 is involved in VK1 metabolism, and the polymorphism of CYP4F2 rs2108622 could affect VK1 oxidase activity. In a comparison of VK1 oxidase activity in genotyped human liver microsomes (HLM), the HLM-CC pool (CYP4F2 CC) exhibited the highest VK1 oxidase activity, while the HLM-TT pool (CYP4F2 TT) exhibited a 75% reduction and the HLM-CT pool displayed an intermediate activity [9]. Therefore, the CYP4F2 rs2108622 T carriers are likely to have higher hepatic levels of VK1, and thus require a higher warfarin dose [9]. The effect of CYP4F2 in clinic has been studied in several studies. It was reported that a polymorphism of CYP4F2 rs2108622 could affect warfarin dose requirement and explain approximately 2%∼7% of the variability [10, 11]. A genome-wide association study (GWAS) with 1 053 Swedish subjects also considered CYP4F2 as a minor predictor of warfarin dose [12]. These studies proved that CYP4F2 rs2108622 T carriers had a higher warfarin dose. CYP4F2 may be the third genetic predictor for warfarin dose.

The consideration of pharmacogenetics may help optimize the warfarin dose and minimize the risk of adverse reactions. However, because of the different genetic backgrounds and environment, the same genetic factor may exert different effects in different populations. The Chinese population is known to be warfarin sensitive, and thus needs lower warfarin doses than other populations. It is important to know the effect of these genetic factors on the warfarin response in Chinese. Also there are many patients, such as rheumatic heart disease patients, undergoing mechanical heart valve replacement in China. These patients can also suffer from thromboembolism and haemorrhage due to inappropriate warfarin dose. Therefore, it is necessary to know whether this genetic factor plays a key role in Chinese patients and how much it can explain the interindividual variability in warfarin dose.

Taking the above-mentioned into consideration, the aim of our study was to investigate the effect of CYP4F2 polymorphisms on the stable warfarin dose and to confirm the contribution of VKORC1, CYP2C9 and CYP4F2 genetic polymorphisms by multiple regression model analysis in Han Chinese patients with MHVR.

Methods

Study population and data collection

From March 2007 to November 2008, 222 Han Chinese MHVR patients were recruited from the Cardiovascular Clinic of the First Affiliated Hospital, Sun Yat-sen University Guangzhou, PR China. Patient demographics, medical history, medication used and daily warfarin dose were recorded. The routine INR values were also recorded to determine the stable dose, defined as a constant dose for at least 3 months with the INR measurements within the range of 1.5–3.0 for multiple time periods. Patients who were younger than 18 years, or who also received a platelet aggregation inhibitor, such as aspirin, clopidogrel or dipyridamole or other drugs known to interact with warfarin (according to the report of Holbrook et al, such as celecoxib [13]) with the exception of amiodarone, were excluded from this study.

Blood samples (2 ml) were collected in evacuated tubes containing ethylenediaminetetraacetic acid (EDTA) for DNA extraction. The study was approved by the Ethics Committee of Sun Yat-sen University, Guangzhou, PR China. Written informed consent was obtained from all subjects.

Genotyping

Total DNA was extracted from peripheral leukocytes by the phenol-chloroform extraction method as described [14].

All PCRs were carried out in a 25 µl volume containing 50 ng genomic DNA, 2.5 mm dNTPs, 10 mm of primer F and primer R, 2.5 ml 10×Ex Taq buffer and 0.75 U Ex Taq DNA polymerase (Takara, Japan). Details on primer sequences, amplicon sizes and restriction enzymes for VKORC1, CYP2C9 and CYP4F2 are shown in Table 1.

Table 1.

Polymerase chain reaction-restriction fragment length polymorphism genotyping

	Forward primer	Reverse primer	Amplicon size (bp)	Restriction enzyme	Reference
CYP4F2 rs2108622	5′-CGGAACTTGGACCATCTACA-3′	5′-CCTACTCTCCCACAGGCATTA-3′	439	PvuII
VKORC1 3673G>A	5′-ATCCCTCTGGGAAGTCAAGC-3′	5′-CACCTTCAACCTCTCCATCC-3′	636	BcnI	[15]
VKORC1 9041G>A	5′-TTTGCTTTGGCATGTGAGCCTTGC-3′	5′-ACAGTCCATGGCAGACACATGGTT-3′	282		[3]
VKORC1 5417G>T	5′-AACCTGGAGATAATGGGCAGCA-3′	5′-ACACCGATCCCAGACTCCAGAATA-3′	351	AfaI
CYP2C9*3	F1 5′-AATAATAATATGCACGAGGTCCAGA GATGC-3′	5′-GATACTATGAATTTGGGACTTC-3′	141	AvaIII	[16, 29]
	F2 5′-AATAATAATATGCACGAGGTCCAGA GGTAC-3′		141	KpnI

VKORC1 3673G>A, 9041G>A (rs7294) and the newly discovered SNP 5417G>T for Asp36Tyr were detected.

A PCR restriction fragment length polymorphism (RFLP) genotyping test to detect the 3673G>A base pair change was performed as described by Obayashi et al. [15]. The cycling prole consisted of the first step being held at 95°C for 5 min, followed by 35 cycles at 95°C for 60 s, 60°C for 30 s, and 72°C for 2 min and a 10 min nal extension at 72°C. The 636–base pair (bp) product was digested with BcnI at 37°C for 5 h and analyzed by 2.5% agarose gel electrophoresis.

For 5417G>T, the primers were designed by Primer premier 5.0. The cycling prole consisted of the first step being held at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 61°C for 30 s, and 72°C for 1 min and a 7 min final extension at 72°C. The product was digested with AfaI. 9041G>A genotype was sequenced by direct sequencing, using primers as described by Scott et al. [3].

Primers for CYP2C9*3 (rs1057910) were described by Sullivan et al. [16]. The cycling prole consisted of the rst step being held at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min and a 7 min nal extension at 72°C. PCR products were digested with AvaIII and KpnI and separated on 3% agarose gel.

CYP4F2 rs2108622 genotyping was also performed by the PCR-RFLP method. The primers were designed by Primer premier 5.0. The cycling prole consisted of the rst step being held at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 1 min and a 7 min final extension at 72°C. The product was digested with PvuII at 37°C over night.

Statistical analysis

Deviations from the Hardy–Weinberg equilibrium were assessed by the goodness-of-fit χ² test. Allele and genotype frequencies of CYP4F2 rs2108622 were compared with Hapmap reports using the χ² test. LD analyses of VKORC1 polymorphisms were performed by using Haploview.

Because the distribution of stable warfarin dose was skewed, logarithmic transformation was performed to better meet normality. Each genotype was assigned into two groups (VKORC1 AA group and AG/GG group; CYP2C9*1*3/*3*3 group and *1*1 group; CYP4F2 CC group and CT/TT group). Differences in the daily stable dose of warfarin of different genotype groups were evaluated by a two-sample t-test. Multiple linear regression analysis was performed to analyze the contribution of demographic characteristics (age, weight, height, gender and combination use of amiodarone) and genetic polymorphisms to the variation of daily stable warfarin dose. Variables were selected in a stepwise manner. All analyses were performed with the Statistical Package for Social Science (ver. 17.0; SPSS, Chicago, IL). A P value <0.05 was considered to be statistically significant.

Results

Patient characteristics

Table 2 summarizes the demographics and clinical characteristics of the 222 MHVR patients (104 males and 118 females, with a mean age of 45 ± 12 years). The mean daily warfarin dose requirement was 3.0 ± 1.1 mg day⁻¹ and ranged from 1.1 to 8.8 mg day⁻¹.

Table 2.

Demographic characteristics of MHVR patients

Demographic characteristic	Patients (222)
Age ± SD (range) (years)	45 ± 12 (17–77)
Gender [n (%)]
Male	104 (47)
Female	118 (53)
Weight ± SD (kg)	57 ± 10
Height ± SD (cm)	162 ± 7.50
Combination use of amiodarone [n (%)]	12 (5)
Stable warfarin dose ± SD (mg day−1)	2.9 ± 1.4
Target INR ± SD	2.1 ± 0.3

Genotypic analysis

The genotyping results showed that the polymorphism of VKORC1 5417G>T was absent in this study. The allelic frequencies of other SNPs (VKORC1 3673G>A, 9041G>A, CYP2C9*3 and CYP4F2 rs2108622) were accorded with Hardy–Weinberg equilibrium. Table 3 shows the allelic frequencies and genotype distributions in Han Chinese patients with MHVR. VKORC1 3673 G>A and 9041G>A SNPs were in strong linkage disequilibrium (LD) in this study (D' = 0.963, r²= 0.864).

Table 3.

Allelic frequency and genotype frequency distribution of VKORC1, CYP2C9 and CYP4F2 in Chinese MHVR patients

Gene	SNP	Genotype	Frequency number (%)	Allele	Frequency number (%)
VKORC1	3673 G>A	GG	4 (2)	G	61 (14)
		AG	53 (24)	A	383 (86)
		AA	165 (74)
	9041G>A	GG	162 (73)	G	380 (86)
		AG	56 (25)	A	64 (14)
		AA	4 (2)
	5417G>T	GG	160	G	320
		GT	0	T	0
		TT	0
CYP2C9	42613A>C	*1*1	203 (91)	*1	424 (96)
		*1*3	18 (8)	*3	20 (4)
		*3*3	1 (1)
CYP4F2	rs2108622	CC	115 (52)	C	322 (73)
		CT	92 (41)	T	122 (27)
		TT	15 (7)

The CYP4F2 rs2108622 genotype frequency was not significantly different from Han Chinese (Beijing) HapMap populations [17]. There were only 15 patients carrying the TT genotype.

Impact of VKORC1, CYP2C9 and CYP4F2 polymorphisms on stable warfarin dose

The associations of stable warfarin dose and VKORC1, CYP2C9 and CYP4F2 genotypes are shown in Table 4.

Table 4.

Comparison of mean stable warfarin doses for the four studied SNPs

Gene	SNP	Genotype	Number of patients (%)	Mean stable warfarin dose (mg day−1± SD)	95% CI*	95% CI on the difference**	P value
VKORC1	3673 G>A	GG/AG	57 (26)	4.0 ± 1.3	3.7, 4.4	1.3, 1.6	<0.001
		AA	165 (74)	2.7 ± 0.8	2.6, 2.8
	9041G>A	GG	162 (73)	2.7 ± 0.8	2.6, 2.8	1.3, 1.6	<0.001
		AG/AA	60 (27)	4.0 ± 1.3	3.7, 4.3
CYP2C9	42613A>C	*1*1	203 (91)	3.1 ± 1.1	3.0, 3.3	1.3, 1.7	<0.001
		*1*3/*3*3	19 (9)	2.1 ± 0.6	1.8, 2.4
CYP4F2	rs2108622	CC	115 (52)	2.9 ± 1.1	2.7, 3.1	0.2, 1.0	0.033
		CT/TT	107 (48)	3.2 ± 1.1	3.0, 3.4

^*95% confidence interval;

^**95% CI on the difference: on the difference between different genotype groups.

For VKORC1 3673 G>A genotype, patients with the AA genotype required a lower dose of warfarin (2.6 mg day⁻¹), AG/GG carriers required a higher dose (4.0 mg day⁻¹) (95% CI 1.3, 1.6, P < 0.001). Conversely, a variant genotype of VKORC1 9041G>A (AG/AA groups) had a higher stable warfarin dose than that of the GG groups (95% CI 1.3, 1.6, P < 0.001).

Patients with theCYP2C9 variant genotype *3*3/*1*3 required much less warfarin than wild type patients (2.1 mg day⁻¹vs. 2.9 mg day⁻¹; 95% CI 1.3, 1.7, P < 0.001). The dose of the one patient carrying *3*3 was 1.3 mg day⁻¹.

CYP4F2 rs2108622 genotype also had a significant correlation with stable warfarin dose. The CT/TT group (CYP4F2 T carriers) took a significantly higher stable warfarin dose (3.2 mg day⁻¹) than the CC group (2.9 mg day⁻¹; 95% CI 0.2, 1.0, P= 0.033).

Multiple regression analysis was used to depict the impact of clinical characteristics and genotypes on stable warfarin dose. The result shows that the model including VKORC1 3673G>A, CYP2C9*3, CYP4F2 rs2108622, age, weight and combination use of amiodarone together could explain 56.1% of individual differences in stable warfarin dose (Table 5). In this analysis, VKORC1 and CYP2C9 contributed most to the interindividual variability in warfarin dose, accounting for 40.1% and 26.3%, respectively. CYP4F2 was a minor but significant factor, which could explain approximately 4% of individual differences in stable warfarin dose.

Table 5.

Multiple linear regression model for stable warfarin dose based on age, weight, CYP2C9, VKORC1 3673G>A, CYP4F2 and combination use of amiodarone

Variable	Beta-standardized coefficients	P value	Partial r2
VKORC1 3673 G>A	−0.551	<0.001	0.401
CYP2C9*3	−0.401	<0.001	0.263
CYP4F2 rs2108622	0.135	0.003	0.040
Age	−0.096	0.036	0.020
Weight	0.102	0.026	0.023
Amiodarone	−0.343	<0.001	0.211

General r² model: 56.1% = percentage of stable interindividual variability explained by this model. Partial-univariate r² values (%) are also shown.

Discussion

In our study, the association between CYP4F2 and stable warfarin dose was investigated, and the allele frequency of CYP4F2 rs2108622 between Chinese and other ethnic groups was also compared. To our knowledge, this is the first research to study the effect of CYP4F2 variants on warfarin requirements in Han Chinese population. Until now no relevant research has been done in other ethnic groups besides Caucasians [12, 18–20]. The Chinese population is known to be warfarin sensitive, and thus needs a lower warfarin dose than Caucasians. These two populations have quite different genetic backgrounds [21, 22]and therefore it is very important to investigate role of CYP4F2 in the Chinese population. In this study, only Han Chinese patients with mechanical heart valve replacement were included. The study confirmed the association of CYP4F2 rs2108622 with warfarin dose in a Chinese population and this association could be explored to use in the clinic.

As we know, distribution of the VKORC1 3673G>A genotype is significantly different between Chinese and Caucasians, which could explain the different dosage between these two ethnic groups [1, 21]. However, in our study, there was no significant difference in the allele frequency of the CYP4F2 rs2108622 between Han Chinese (28%) and Caucasians (23%). Thus, CYP4F2 is definitely not the genetic factor for ethnic differences in warfarin dose.

However, CYP4F2 still could be the genetic factor for interindividual variability in warfarin dose. We found in this study that CYP4F2 rs2108622 T carriers required about 0.3 mg day⁻¹ more warfarin dose than patients with C homozygous. This variability is minor but significant. It is smaller than that reported by Caldwell et al. [11] and Brgiani et al. [10], who demonstrated that patients who were T homozygous required more than 1 mg day⁻¹ more warfarin than patients who were C homozygous. However, in our study CYP4F2 rs2108622 explained about 4% of the intervariability in stable warfarin dose, which is more than that reported by Caldwell et al. [11] and Takeuchi et al. [12], who demonstrated that CYP4F2 rs2108622 could explain 2% and 1.5% of intervariability in warfarin response, respectively. This may due to the ethnic differences. It is well known that the Chinese population is warfarin sensitive and most patients thus need a lower warfarin dose. It makes the overall interindividual variability in stable warfain dose in the Chinese smaller than that in Caucasians. Even though the variability between different CYP4F2 rs2108622 genotype groups is smaller than other reports, this genetic factor contributes more to interindividual variability of warfarin response in Han Chinese population.

Most of the previous reports included various kinds of indications for warfarin use, while our study only included patients with mechanical heart valve replacement. Since INR is known to be a kind of sensitive index, which is easily affected by patients' conditions, such as combination diseases and medication [23–26], our study may be less affected by such confounding factors and can reflect the effect of CYP4F2 more accurately.

In this study, we also confirmed the impact of VKORC1 and CYP2C9 on stable warfarin dose. We investigated the effects of VKORC1 3673G>A, 9041G>A, and a new SNP VKORC1 5417G>T. However, VKORC1 5417G>T did not exist in our studied population. Previous studies have shown that this mutation was common in Ethiopian (15%) and existed in Jewish populations [3, 4]. This polymorphism is relevant to warfarin resistance, but Han Chinese people are known to be warfarin sensitive. Thus, it is not surprising that we did not observe this polymorphism. Together with VKORC1 3673G>A, CYP2C9, CYP4F2 genotypes and age, weight, combination use of amiodarone, the multiple linear regression model could explain about 56.1% of the variation in the stable warfarin doses.

Most Chinese patients are warfarin sensitive, and are at risk of suffering haemorrhages. Many attempts have been made to use the pharmacogenentic approach to predict warfarin dose [27, 28]. VKORC1 and CYP2C9 are definitely the most important genetic factors, which can explain about 40% of intervariability in stable warfarin dose. Our research has confirmed a new meaningful genetic predictor (CYP4F2 rs2108622) of the warfarin dose required for Chinese patients, which could explain about 4% of the intervariability in the stable warfarin dose. In the future, CYP4F2 rs2108622 together with other genetic factors could be applied to prospective studies to evaluate further its power and to guide personalized pharmacotherapy in Han Chinese patients with MHVR.

Competing interests

There are no competing interests to declare.