To assess whether CYP2C9 alleles other than CYP2C9*2 and CYP2C9*3 are associated with a low warfarin dose requirement, CYP2C9 exons, intron-exon boundaries and 3 kb of upstream sequence in 20 patients requiring  1.5 mg warfarin day^-1 and with apparently homozygous wild-type or heterozygous CYP2C9*2 genotypes were screened for novel polymorphisms. Evidence for the existence of polymorphism at eight different upstream sites was obtained. Five of these polymorphisms had been described previously but three others were novel. We found that the majority of the upstream polymorphisms were in complete linkage disequilibrium with previously described coding region polymorphisms. However, two polymorphisms, T-1188C and the novel ΔG-2664 ΔT-2665, occurred both in individuals who were otherwise wild-type and in individuals positive for coding region polymorphisms. Genotyping assays for novel upstream and other known polymorphisms were developed and used to screen a larger patient population (n = 153) of known CYP2C9*2 and *3 genotype requiring a range of warfarin doses.

Evidence for the existence of 11 haplotypes, including eight with frequencies  0.01 was obtained. In individuals negative for coding region polymorphisms, neither individual genotypes for T-1188C nor ΔG-2664 ΔT-2665 nor particular combinations of haplotype pairs were predictive of dose requirement or S-warfarin total clearance, suggesting neither upstream polymorphism was functionally significant. Dose requirements in individuals heterozygous for CYP2C9*11 haplotypes were not statistically significantly different from homozygous wild-type individuals.

We conclude that the coding region nonsynonymous polymorphisms associated with the CYP2C9*2 and CYP2C9*3 alleles are the major CYP2C9-related factor affecting warfarin dose requirement in UK Caucasians. Upstream CYP2C9 polymorphisms do not appear to be important independent determinants of dose requirement. There is extensive linkage disequilibrium within the CYP2C9 gene but genotyping for three polymorphisms should enable the majority of common haplotypes to be assigned.