Background: a higher odds of being grouped as

Background: Warfarin is the most
common oral anticoagulant prescribed to prevent thromboembolic events in
patients with mechanical heart valve replacements. However, warfarin therapy is
challenging due to the wide inter-individual variability in therapeutic
response. This study aimed to assess the effect of the VKORC1 (-1639G>A) rs9923231
polymorphism on the variations in mean daily dose and to evaluate its ability
to categorize warfarin-treated patients to high, intermediate or low dose

Materials & Methods: Two
hundred and twenty two warfarin-treated patients of South Indian origin were
genotyped for the VKORC1(-1639G>A) rs9923231 polymorphism. Variations in the
mean daily dose among different genotypes were compared using one-way ANOVA and
linear regression analysis. The discriminatory ability of the rs9923231
polymorphism to bin the patients into different dose categories was evaluated
by estimating the proportional odds ratios using the ordered logit regression

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Results:The frequency of the AA
genotype and A allele in the study sample was found to be 1.8% and 9.23%,
respectively. The mean daily dose required to achieve the optimum international
normalized ratio (INR) was significantly lower in AA homozygous genotype
carriers (3.99±1.67mg/day) and GA heterozygous (4.26±1.57mg/day) compared to
the GG genotype carriers (5.61±2.41mg/day), p-value=0.004. The A allele
carriers had a higher odds of being grouped as a low dose requiring category
(adjusted OR 3.31, 95% CI 1.52 – 7.21, p-value=0.003), compared to

Conclusion: These preliminary
results supports the use of the VKORC1 (-1639G>A) rs9923231 polymorphism for
genetically guided initial warfarin dosing in South Indian patients with heart
valve replacements.


Genotypic variations in the
VKORC1 gene contribute significantly to warfarin sensitivity among patients 1,
2. Inherent differences in allele frequencies have revealed varying dose
requirements for warfarin across populations 3.  Various studies have reported significant
geographic differentiation in the observed allele frequencies for the VKORC1
and CYP2C9 gene polymorphisms from Asian populations 4. Distinct Indian
subpopulations have also shown a great degree of pharmacogenetic variations in
the VKORC1, CYP2C9 and CYP4F2 genes 5. In this context,
subpopulation-specific studies designed to capture the genotypic distribution
of pharmacogenetic variants are essential to assist clinicians in optimizing
dose regimes. Moreover, incorporating the ethnic background of the patient as
an additional variable can significantly improve the predictability of
anticoagulant dosing algorithms 6.

The polymorphisms in the VKORC1
gene explains up to 25-50% of variance in dose 7-10.The strong association
between haplotypes of the VKORC1 gene polymorphisms and warfarin dose
phenotypes are distinguished by the tag-SNP rs9923231 present in the promoter
of the gene. At the transcriptional level, the A allele of the rs9923231 SNP
has been found to be associated with a 70% reduction of VKORC1 mRNA levels
compared to the G allele, by abolishing an E-box consensus sequence in the
promoter region 11, 12. A few studies from South India have published data
regarding the VKORC1 (-1639G>A) rs9923231 polymorphism and its effect on
daily warfarin dose requirement 13-18. Meanwhile, other studies on South Indian
populations restricted their findings to report allele and genotypic
frequencies without accessing association with dosage requirements 5,19, 20.
Comparative studies on North Indian populations have reported conflicting
allele and genotype frequencies possibly due to reduced sample sizes 19, 21,
22. At present, there are no reports on the VKORC1 (-1639G>A) rs9923231
polymorphism and its association with warfarin dose requirements exclusively
from Kerala, the southernmost state of India with a predominant Dravidian
population. Therefore, the aims of this study were to determine the frequency
of the VKORC1 (-1639G>A) rs9923231 polymorphism in the South Indian
population of Kerala, to assess variation of mean daily dose among carriers of
the three genotypes and to determine its ability to discriminate patients
belonging to high, intermediate and low dose categories.


Materials and Methods


Patient Sample

A total of 222 patients having a
stable therapeutic INR were recruited from the INR clinic of the Cardiology
Department, Sree Chitra Tirunal Institute for Medical Sciences and Technology
(SCTIMST), Trivandrum, Kerala. Stable therapeutic INR was defined as having at
least two consecutive INR measurements between 2.5 and 3.5 on the same
anti-coagulant dose measured at least one week apart. Patients over the age of
18 years who needed anticoagulation with warfarin or acenocoumarol after
prosthetic mitral valve replacement with tilting disc valve for rheumatic heart
disease and having normal prosthetic valve function were included in this
study. Patients with renal dysfunction, hepatic dysfunction and patients on
other anti-coagulants such as Dindevan (Phenindione) were excluded from the
study. All participants belonged to the Malayalam speaking South Indian population
of Kerala. The study was approved by the Institutional Ethics Committee of the
SCTIMST and conformed to the guidelines set by the Declaration of Helsinki.



DNA isolation and PCR-RFLP

Genomic DNA was isolated from
three millilitres of whole blood samples taken from patients after obtaining
written informed consent, using the modified Rapid-Method 23. A
PCR-restriction fragment length polymorphism method (RFLP) was designed to
identify the VKORC1 -1639G>A (rs9923231) SNP. A 15 ?l PCR reaction mixture was
prepared to amplify 100 ng gDNA mixed with 1 ?l 10× Taq buffer, 200 ?M dNTPs
(Invitrogen), 250 ?M MgCl2, 0.5U Taq Polymerase (NEB) and 10 ?M of each forward
and reverse primers. PCR cycling conditions consisted of an initial
denaturation for 2 min at 95 °C; followed by 35 cycles of denaturation at 95 °C
for 30 s, annealing at 60 °C for 45 s and extension at 72 °C for 45 s, with a
10 min final extension at 72 °C, done using Biorad MJ MiniTM thermal cycler.
Human-specific primers were VKORC1-F, 5?-TTGCTGCCCACGCCATAAACTA and VKORC1-R,
5?-ATCACAGACGCCAGAGGAAGAG, designed using the Primer Premier v.5 software.