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To determine if copy number variants contribute to warfarin dose requirements, we investigated CYP2C9, VKORC1, CYP4F2, GGCX and CALU for deletions and duplications in a multiethnic patient population treated with therapeutic doses of warfarin.
DNA samples from 178 patients were subjected to copy number analyses by multiplex ligation-dependent probe amplification or quantitative PCR assays. Additionally, the CYP2C9 exon 8 insertion/deletion polymorphism (rs71668942) was examined among the patient cohort and 1750 additional multiethnic healthy individuals.
All patients carried two copies of CYP2C9 by multiplex ligation-dependent probe amplification and no exon 8 deletion carriers were detected. Similarly, quantitative PCR assays for VKORC1, CYP4F2, GGCX and CALU identified two copies in all populations.
These data indicate that copy number variants in the principal genes involved in warfarin dose variability (CYP2C9, VKORC1), including genes with lesser effect (CYP4F2, GGCX), and those that may be more relevant among certain racial groups (CALU), are rare in multiethnic populations, including African–Americans.
Warfarin is a widely used oral anticoagulant commonly prescribed for prevention of thrombotic disorders among patients with deep vein thrombosis, pulmonary embolism, atrial fibrillation and other indications . The drug has a narrow therapeutic index and wide interindividual variability in therapeutic dose requirements, which is influenced by a number of clinical factors (e.g., age, race, bodyweight and gender amongst others) [2,3]. Several candidate gene studies have identified common sequence variants in CYP2C9 and VKORC1 that also significantly influence warfarin dose requirements [4–6]. These genes are involved in warfarin pharmacokinetics and pharmacodynamics, respectively, and since their identification, pharmacogenetic regression algorithms have been developed that include variant CYP2C9 and VKORC1 alleles and other clinical variables to predict therapeutic warfarin doses [7–15]. Importantly, a recent study has retro spectively shown that pharmacogenetic-guided dose prediction is more accurate than both empiric dosing (i.e., 5 mg/day) and the US FDA-approved dosing guidelines listed on the warfarin product insert .
Given that CYP2C9, VKORC1 and other clinical (e.g., age and weight) and environmental (e.g., concomitant medications) factors together only account for approximately 35–50% of interindividual warfarin dose variation [5,6,13,17], many studies have attempted to identify additional sources of genetic variation contributing to warfarin dose variability. For example, four genome-wide association studies (GWAS) using Caucasian and Japanese patient cohorts identified a single nucleotide variant in a third gene implicated in warfarin dosing, CYP4F2 [18–21]. Interestingly, no other statistically significant signals were reported among these GWAS; each of which involved approximately 500–2000 patients. However, other candidate gene studies have reported on the role of rare CYP2C9 variants in warfarin dosing, particularly in non-Caucasian racial and ethnic groups [22–25]. Moreover, nucleotide variants in GGCX and CALU, additional genes involved in the warfarin anticoagulation pathway, also may be involved in interindividual warfarin dose variability [26–30].
In addition to SNPs, recent attention has focused on the role of heritable copy number variants (CNVs) in disease susceptibility and complex traits [31–41]. CNVs are heritable duplication and deletion alleles, which can have functional consequences on protein abundance and/or enzyme activity. It is estimated that up to approximately 12% of the human genome harbors CNVs , which include several known pharmacogenetic genes: CYP2D6 [42,43], GSTT1 and GSTM1 [44,45], SULT1A1 [46,47] and UGT2B17 . Although one GWAS on warfarin dosing incorporated CNV detection in a subset of their cases , the probe density of their genome-wide platform had low resolution for copy number analyses, limiting the ability to detect smaller CNVs that potentially could influence therapeutic warfarin dose requirements. To date, no study has directly interrogated the copy number of important genes involved in warfarin dose variability. Therefore, we performed a pilot study to determine the role of CNVs in CYP2C9, VKORC1, CYP4F2,GGCX and CALU in a multiethnic patient population treated with therapeutic doses of warfarin, as well as in healthy individuals from different racial and ethnic groups.
Between July 2007 and July 2011, adult patients taking warfarin and managed at institutional anticoagulation clinics at the Mount Sinai Hospital (NY, USA) or Elmhurst Hospital Center in New York City (NY, USA), or referred to the study investigators, were assessed for enrollment. Subjects were included in the analysis if their target international normalized ratios (INR) were between 2.0 and 3.0 or between 2.5 and 3.5, and at least two therapeutic measurements were separated by at least 1 week. Thus, the study sample was comprised of patients with established warfarin dose requirements. Prior to participation, all subjects signed a written informed consent approved by the institutional review board (IRB) governing research involving human subjects. Subsets of these specimens were used in previously described studies [13,22].
In addition, peripheral blood samples from healthy donors who indicated their racial and ethnic background (African–American, Hispanic, Asian or Caucasian) and gave informed consent for the use of their DNA for research were obtained from the New York Blood Center (NY, USA) with IRB approval [22,49]. Blood samples were also obtained with informed consent from unrelated healthy 100% Ashkenazi Jewish individuals from the greater New York metropolitan area [50,51]. All personal identifiers were removed from these samples and isolated DNA was tested anonymously. Genomic DNA was isolated using the Puregene® DNA Purification kit (Qiagen, CA, USA) according to the manufacturer's instructions.
Multiplex ligation-dependent probe amplification (MLPA) of CYP2C9 was performed using the P128 CYP450 MLPA kit (Version A1; MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer's instructions. In brief, all DNA samples were diluted to 200 ng with Tris-EDTA buffer and denatured in a thermal cycler for 5 min at 98°C. After cooling to 25°C, probemix (including CYP2C9 probes for exons 1, 7 and 9; Figure 1) and MLPA buffer were added to each sample, mixed and incubated for 1 min at 95°C followed by a 16 h hybridization at 60°C. The ligation reaction was performed at 54°C by adding 32 μl of Ligase-65 mix to each tube followed by heating for 5 min at 98°C. PCR buffer, water and MLPA ligation reactions were mixed in new tubes and maintained in a thermalcycler at 60°C while polymerase mix was added to each tube. Exon-specific probes with universal-tagged primers underwent PCR consisting of 35 amplification cycles (95°C for 30 s, 60°C for 30 s and 72°C for 60 s), followed by a 20 min incubation at 72°C. Amplified products were separated by capillary gel electrophoresis and data were analyzed using GeneMarker v1.90 software (SoftGenetics, PA, USA).
The 2226-bp CYP2C9 exon 8 insertion/deletion polymorphism (rs71668942) was genotyped using a three-primer PCR and standard agarose gel electrophoresis. The use of three primers allowed for a deletion-specific amplicon as illustrated in Figure 1. PCR reactions were performed in 25 μl containing approximately 100 ng of DNA, 1X PCR buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.4 μM of the forward primer (FWD: 5′-GCACATAGTTTCAGTATCCTGTTACTTTCC-3′) and 0.2 μM of each reverse primer (REV-1: 5 ′-C A G G T C A A G A T C A T C A GGTCAGATG-3′; REV-2: 5′-GATTGAATCATGGGGGATAATTTCC-3′), and 1.0 unit of Platinum® Taq DNA Polymerase (Invitrogen, CA, USA). The PCR amplification conditions consisted of a denaturation step at 94°C for 5 min followed by 35 amplification cycles (94°C for 30 s, 60°C for 30 s and 72°C for 30 s) and a final incubation at 72°C for 5 min. Aliquots of all PCR products were loaded onto 1.5% agarose gels, electrophoresed and visualized under UV light. Representative amplification products were confirmed by bi directional sequencing using individual amplification primers.
The copy number of selected genes known to influence warfarin dose requirements (VKORC1, CYP4F2, GGCX and CALU) was interrogated using commercially available TaqMan® real-time quantitative PCR (qPCR) copy number assays (Applied Biosystems, CA, USA) as per the manufacturer's instructions. In brief, FAM-labeled VKORC1, CYP4F2, GGCX and CALU TaqMan minor-groove binding probes and unlabeled PCR primers (Table 1) were individually run in a duplex qPCR with a VIC-labeled RNase P TaqMan Copy Number Reference Assay (catalog number 4403326, Applied Biosystems). Quadruplicate experiments were each performed in 10 μl containing 10 ng of DNA, 1X TaqMan Genotyping Master mix, 0.5 μl each of TaqMan Copy Number and Reference Assays in 384 well plates. Covered plates were run in a 7900HT Fast Real-Time PCR System (Applied Biosystems) and the amplification consisted of a denaturation step at 95°C for 10 min followed by 40 amplification cycles (95°C for 15 s and 60°C for 60 s). Data were captured using absolute quantitation with a manual CT threshold and autobaseline, followed by analysis using CopyCallerTM v1.0 Software (Applied Biosystems) where the number of copies of target sequence was determined by relative quantitation with the comparative CT (ΔΔCT) method. This method measures the CT difference (ΔCT) between target and reference sequences, and then compares the ΔCT values of test samples to a calibrator sample with two copies of the target sequence. The copy number of the target was calculated as twice the relative quantity.
Patient characteristics are listed in Table 2. The mean age was 65 ± 15 years; 106 participants (61%) were male. A total of 88 subjects (49%) were Caucasian, 37 (21%) African–American, 21 (12%) Hispanic and 30 (17%) Asian. The most frequent indications for anticoagulation were atrial fibrillation in 117 patients (66%), and venous thromboembolism in 23 patients (13%). The median therapeutic warfarin dose among all patients was 35 mg/week and the distribution of therapeutic warfarin doses among the INR 2.0–3.0 and 2.5–3.5 cohorts are illustrated in Figure 2. Among the warfarin-treated patient population, therapeutic dose requirements ranged from 7–280 mg/week; 21% had therapeutic warfarin dose requirements of less than 21 mg/week, 47% were between 21 and 49 mg/week and 31% were >49 mg/week. Previous analyses of the majority of patients in this cohort with target INR values of 2.0–3.0 identified significant associations between common CYP2C9 and VKORC1 variant alleles and warfarin dosing, consistent with other studies [13,22].
To interrogate CYP2C9 copy number, MLPA was performed on all 178 patient DNA samples. The P128 Cytochrome P-450 MLPA kit (Version A1, MRC-Holland, The Netherlands) contained probes for CYP2C9 exons 1, 7, and 9 (Figure 1). All 178 patient patient samples had two copies of CYP2C9 based on the interrogated exons compared with control reference probes. A representative MLPA analysis of 50 patient samples is shown in Figure 3.
Given that the CYP2C9 MLPA probes did not interrogate exon 8 of the gene and a large (~2.2 kb) insertion/deletion polymorphism (rs71668942) encompassing this exon is cataloged in the NCBI SNP database (dbSNP) , additional genotyping was performed to interrogate CYP2C9 exon 8. Using the three-primer PCR strategy detailed in the ‘Patients & methods’ section, in the presence of a wild-type CYP2C9 sequence, the three CYP2C9 primers generate two amplicons of 457 and 2528 bp; however, the PCR conditions did not allow for amplification of the larger 2528-bp product (Figure 1). A deletion corresponding to rs71668942 would result in a single 302-bp amplification product. Among all 178 warfarin-treated patient samples tested, no rs71668942 deletion carriers were identified. A representative gel image for 18 patient samples is shown in Figure 3. In addition, 350 DNA samples, each from healthy anonymous African–American, Asian, Hispanic, Caucasian and Ashkenazi Jewish individuals were interrogated and no CYP2C9 exon 8 deletion polymorphism carriers were identified.
In addition to CYP2C9 copy number analyses using MLPA and insertion/deletion genotyping, VKORC1 copy number was interrogated using TaqMan copy number assays with specific probes and primer pairs for all three VKORC1 exons. All 178 patients analyzed had two copies of VKORC1 and the calculated copy number data for 50 representative patient samples are shown in Figure 4. In addition, three other genes recently shown to influence warfarin dosing, CYP4F2, GGCX and CALU, were analyzed with TaqMan copy number assays and all patients again harbored two copies of each gene (Figure 5).
Given that variant alleles known to influence warfarin dosing (e.g., CYP2C9*2, *3 and VKORC1 c.-1639G>A) are less frequent among African–Americans compared with Caucasians  and account for less of the dosing variability in the African–American population , an additional 50 DNA samples from healthy African–American individuals were tested for CYP2C9, VKORC1, CYP4F2, GGCX and CALU copy number [22,49]. Consistent with the multi ethnic warfarin-treated patient cohort and the 37 warfarin-treated African–American patients, two copies of each tested gene were detected in the additional healthy African–American samples tested using MLPA and TaqMan copy number assays.
Previous studies investigating the pharmacogenetics of interindividual warfarin dose variability have focused on the role of common SNPs within candidate genes involved in warfarin pharmacokinetics and pharmacodynamics. The paucity of data on copy number in the warfarin pharmacogenetics literature prompted our study to interrogate CNVs in major genes associated with warfarin dose requirements. Using quantitative molecular techniques, we found that heritable variation in the copy number of CYP2C9, VKORC1, CYP4F2, GGCX and CALU was not common in a multiethnic patient population treated with therapeutic doses of warfarin and additional healthy control individuals. Based on our study sample sizes, CNVs in the tested genes have allele frequencies in the African–American, Asian, Caucasian and Hispanic populations of less than 0.5, 2.5, 0.5 and 2.0%, respectively.
Common CYP2C9 variant alleles with reduced activity result in delayed warfarin elimination, decreased therapeutic warfarin doses and increased bleeding risks [53,54]. The VKORC1 enzyme is the direct target of warfarin, and common VKORC1 haplotypes that result in reduced gene expression have been reproducibly implicated as the major genetic determinant of warfarin dose variability [7,8,11,26,52,55]. Together, CYP2C9 and VKORC1 variant alleles account for approximately 35% of the interindividual dose variability [8,11], prompting several GWAS and candidate gene studies dedicated to identifying additional genes and/or variants involved in warfarin dose variability. These studies have confirmed the principal roles of CYP2C9 and VKORC1 and also have identified a single variant allele in the CYP4F2 gene (p.V433M; rs2108622) that accounts for between 2 and 5% of dosing variability [18–21]. Notably, with the exception of one of these GWAS , copy number analyses have not previously been incorporated into warfarin pharmacogenetic studies.
Because CYP2C9 and VKORC1 are the primary genes known to influence warfarin dosing, our CNV study attempted to cover as much of the coding region of these genes as possible. For CYP2C9, exons 1, 7 and 9 (of nine exons) were assessed by MLPA, and for VKORC1, all three exons were interrogated by qPCR. Although MLPA would easily detect any full-gene CYP2C9 deletion or duplication, it would not detect smaller exon-specific copy number variants not interrogated by the probe mix, including the approximately 2.2-kb exon 8 insertion/deletion (rs71668942) polymorphism. This variant allele was previously identified by computational analyses of DNA resequencing data originally generated for SNP discovery projects [56,57]. Although this particular insertion/deletion variant was not directly validated when first identified, the authors reported a 97% validation rate when confirming other novel insertion/deletion variants . No allele frequencies currently exist for rs71668942 on dbSNP; however, our study indicates that the CYP2C9 exon 8 deletion allele is rare (<0.1%) in the African–American, Asian, Hispanic, Caucasian and Ashkenazi Jewish populations. Of note, the newer version of the P128 Cytochrome P-450 MLPA kit (Version B1; MRC-Holland, The Netherlands) now contains two additional CYP2C9 probes that target exon 8 based on these data.
In the coagulation pathway, GGCX is directly involved in the post-translational carboxylation and activation of hypofunctional clotting factors with reduced vitamin K serving as an essential cofactor. Variant GGCX alleles (e.g., rs11676382) have been associated with warfarin dosing in some studies with modest effect sizes, but have not been consistently replicated [26–28,58]. Although a GGCX CNV is cataloged in the Database of Genomic Variants (Variation_35863) [31,59], no previous studies on GGCX and warfarin dosing have directly interrogated its copy number. Despite the single entry for GGCX in the Database of Genomic Variants, we did not identify any patients or additional population samples that harbored deletion or duplication alleles of this gene, suggesting that GGCX CNVs are uncommon in the general population.
GGCX is inhibited by the chaperone protein CALU, found in the endoplasmic reticulum. Interestingly, a conserved intronic CALU SNP (c.582+133A>G; rs339097) was reported to predict higher warfarin doses in African–Americans , which was recently confirmed in a study of Egyptian patients . We did not detect CNVs in the CALU gene among the multiethnic patient population or the additional African–American subjects. Since rs339097 is more frequent in African– Americans (~15%) than in Caucasians (<1%), and it is unclear if the allele is a functional or haplotype-tagging SNP, future studies on CALU might benefit from extensive sequencing and/or fine mapping in different ethnic and racial patient populations. Microsomal EPHX1 might also be involved in the warfarin pathway; however, it was not included in our CNV study given the inconsistent reports on its putative association with warfarin dose requirements [60,61].
CNVs in genes involved in drug metabolism and response have received increasing attention since the identification of heritable deletion and duplication alleles [62–65], most notably for CYP2D6 [42,43]. This is the first study to directly and systematically interrogate the role of CNVs in warfarin pharmacogenetics. However, our data indicate that CNVs in genes involved in warfarin dose variability (CYP2C9 and VKORC1), including genes with lesser effect (CYP4F2 and GGCX), and those that may be more relevant among certain racial groups (CALU), are rare in multiethnic patient populations. It is still possible that CNVs in these genes may be present in certain ethnic populations that were not studied here. Additionally, we cannot rule out the possibility of other small exon-specific insertion/deletion polymorphisms that were not interrogated by the primers and probes used in the current study. Moreover, CNVs in as yet unidentified genes in the warfarin pharmaco kinetic and pharmacodynamic pathways may still play an important role in interindividual warfarin dose variability.
As the major genetic (e.g., CYP2C9 and VKORC1) and nongenetic (e.g., age, weight, concomitant medication) components of warfarin dose variability only account for approximately 50% of interindividual variation, it is likely that additional genes and variants will soon be discovered that contribute to dose variability. Although CNVs of known genes involved in warfarin pharmacokinetics and pharmaco dynamics do not appear to play a major role in interindividual dose variability, it is still possible that CNVs of currently unidentified genes will significantly influence warfarin dosing. Ongoing GWAS will likely identify additional genes and common variants with moderate effects on warfarin dosing in different racial and ethnic groups. Moreover, forthcoming whole-exome and/or whole-genome sequencing studies have the potential to identify novel genes and rare variants with significant effect sizes , as well as influential small and large CNVs. As these discoveries are made, pharmacogenetic-guided warfarindosing algorithms will undoubtedly incorporate the new genetic and nongenetic variables for more accurate, refined and personalized warfarin dose prediction [8,11,12,102].
CYP2C9, VKORC1, CYP4F2, GGCX & CALU copy number
Financial & competing interests disclosure
This research was supported in part by grant KL2 RR029885 (SAS) from the NIH.
Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
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