The prediction of warfarin dose requirement in children is difficult and over-anticoagulation during initiation of warfarin is not uncommon. The role of genetic polymorphism in determining a response to warfarin during initiation therapy is well established in adult patients; individuals who possess the CYP2C9 and VKORC1 polymorphisms have a greater sensitivity to warfarin and are more likely to have a supra-therapeutic International Normalized Ratio (INR) and serious bleeding during warfarin initiation than those who do not [1,2]. Recent reports have indicated that the VKORC1 and CYP2C9 genotypes influence the warfarin maintenance dose in children [3,4] but their influence on outcomes during warfarin initiation has not been investigated. This cross-sectional study evaluated the influence of the VKORC1 and CYP2C9 polymorphisms on an anticoagulation response to warfarin in children during initiation of therapy.
Subjects were recruited from three UK centres (Birmingham Children’s Hospital; The Newcastle upon Tyne Hospitals NHS Foundation Trust; and Royal Manchester Children’s Hospital) and The Hospital for Sick Children, Toronto, Canada. The study recruited children, aged 18 years and under, who were anticoagulated with warfarin for at least 3 months to a target INR range of 2.0–3.0 or 2.5–3.5 and in whom retrospective data were available for the first 3 months of warfarin therapy. The study was approved by the Regional Ethics Committee and the Institutional Review Boards for each of the participating sites. Written informed consent was obtained from patients aged 16 years or over and from a parent/carer of children < 16 years old. The study complied with the Declaration of Helsinki. Details of indication for anticoagulation with warfarin, target INR range, INR values and corresponding warfarin doses were obtained by questionnaire and review of patients’ medical records. A venous blood sample (4–8 mL) was collected from each patient and stored in EDTA tubes at −80 °C for later genotyping. Genomic DNA was extracted from whole blood samples according to an established method . Genotyping for VKORC1 (−1639G > A; rs9923231) and CYP2C9 (*2 and *3 alleles; rs1799853 and rs1057910) was performed . Statistical analyses were carried out using MiniTab v15.0 (Minitab, Inc., Coventry, UK). Associations between the VKORC1 and CYP2C9 genotypes and outcome variables during the initiation phase of warfarin therapy were evaluated. Analysis was performed on the square root of peak INR during the first week of therapy which resulted in an approximate Normal distribution. The association between the CYP2C9 genotype and peak INR was examined using the t-test and between the VKORC1 genotype and peak INR using linear regression analysis. The association between the incidence of supra-therapeutic INR during the first month of warfarin therapy and CYP2C9 genotype was evaluated using a Mann–Whitney test and the VKORC1 genotype using regression analysis. Results are presented as mean ± standard deviation (SD) unless stated otherwise. A P-value of < 0.05 was taken as statistically significant.
Fifty-one children were studied with a median age at the time of first warfarin dose of 4 years (range: 1–17). There were 39 males (76.5%) and 33 children (64.7%) of white Caucasian origin. The most frequent indications for anticoagulation were the Fontan procedure (28 patients: 54%), a prosthetic heart valve (seven patients: 13.7%) and cardiomyopathy (five patients: 9.8%). The target INR range was 2.0–3.0 in 46 (90.2%) and 2.5–3.5 in the remainder. The mean warfarin dose given on days 1 and 2 of initiation therapy was 0.14 mg kg−1 (± 0.06), based on the recommended dose of 0.2 mg kg−1 (to a maximum of 5 mg) . Genotype frequencies for VKORC1 (−1639G > A; rs9923231) were: GG, 19 patients (37.3%); GA, 26 (51%); AA, 6 (11.8%), and for CYP2C9 were: *1/*1, 35 patients (68.6%); *1/*2, 6 (11.8%); *1/*3, 9 (17.6%); *2/*3, 1 (2%). All genotypes were in Hardy–Weinberg equilibrium. A mean of 3.9 (± 1.9) and 16.1 (± 8.1) INR measurements were taken within the first week and month of therapy, respectively.
Children with a variant CYP2C9 allele (*2 or *3) had a higher mean peak INR during week 1 than those with wild-type CYP2C9, 4.1 ± 1.7 vs. 3.2 ± 1.4, respectively (P = 0.04; Student’s t-test). The child with the CYP2C9 *2/*3 genotype had a peak INR in week 1 of 5.5 (Fig. 1A). Children with the VKORC1 AA genotype had a higher mean peak INR (5.1 ± 2.1) than those with the GA (3.5 ± 1.4) or GG (3.0 ± 1.3) genotype (P = 0.01, regression analysis) (Fig 1B). Children with a variant CYP2C9 allele had a greater proportion of INR values above the target therapeutic range during month 1 of warfarin therapy than those with wild-type CYP2C9; 19.3% vs. 15.9%, respectively (P = 0.08; Mann–Whitney U-test). The single child with two variant CYP2C9 alleles (*2 and *3) had 63.1% of the INR values above the target range during the same period. Children with the VKORC1 AA genotype had a greater proportion of INR values above the target range during the first month of warfarin therapy (21.5%) than those with the GA (17.6%) or GG (17.2%) genotype although this was not statistically significant. There was no association between genotype and time to first therapeutic INR, time to first supra-therapeutic INR, number of INR values > 4.0 (during the first month or the first 3 months of warfarin therapy), time to stable warfarin dose or number of warfarin dose changes. No hemorrhagic or thrombotic events occurred during the initiation of warfarin therapy in this cohort in spite of a peak INR of > 5.0 in 10 patients.
Currently accepted dosing regimes for warfarin initiation in children are based only on the patient’s weight . Although concurrent medication and liver function are also often considered, adjustments in warfarin dose according to other factors which may influence the warfarin dose requirement are not uniformly applied. The role of genetic polymorphisms in determining a response to warfarin during the initiation phase of therapy is well established in adult patient populations with studies showing that individuals with CYP2C9 variant alleles have a higher incidence of supra-therapeutic INR (> 4.0) [1,2,7,8] and a higher rate of serious bleeding events [1,2] during the first 3 months of warfarin therapy in addition to taking longer to reach a stable warfarin dose [2,8].
Only one small study in 29 children has previously examined the influence of the CYP2C9 genotype on anticoagulation outcomes in children; it showed that those who were heterozygous for a variant CYP2C9 gene (n = 7) achieved a target INR sooner, taking on average 3.7 days to do so compared with 7 days for children who were wild-type for CYP2C9 . In addition, heterozygosity for the CYP2C9 genotype was associated with a greater proportion of INR measurements above the target range . The effect of the VKORC1 genotype on an anticoagulation response was not reported.
The present study identified that the CYP2C9 and VKORC1 genotypes both have a significant effect on the anticoagulation response at an early stage of warfarin initiation in children as detailed above. However, we found no relationship between the CYP2C9 and VKORC1 genotypes and the proportion of INR values above the target range beyond the first month of therapy suggesting that adjustment of warfarin doses prior to months two and three of initiation therapy had counteracted the influence of the CYP2C9 and VKORC1 genotypes.
This study was limited by the relatively small patient cohort (n = 51) and the retrospective nature of data collection. The results of this study are mostly in keeping with the published reports of the impact of the CYP2C9 and VKORC1 genotypes on the anticoagulation response during warfarin initiation therapy in adult patient populations and provide further support to the development of personalized pharmacogenetics-based warfarin dosing algorithms in children.