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Blood, 1 April 2003, Vol. 101, No. 7, pp. 2896-2896
CORRESPONDENCE
To the editor:
CYP2C9 exon 4 mutations and warfarin dose
phenotype in Asians
It is well established that functionally defective variant
alleles of CYP2C9 have a major impact on
anticoagulation-related outcomes during warfarin therapy.1
A recent paper by Leung et al2 reported the discovery of 4 new single nucleotide polymorphisms (SNPs) in the coding region of the
CYP2C9 gene: L208V, Q192P, H184P, and I181L, with allele
frequencies of 0.09-0.75 that may be associated with a low warfarin
maintenance dose in Hong Kong Chinese patients. This report is
significant because Asian patients have long been recognized to require
only about 50% of the average warfarin dose used in whites to
elicit the same degree of anticoagulation.3 Asian patients
in the Leung study who were heterozygous or homozygous for V208
exhibited 26%-34% reductions in their mean warfarin dose relative to
"wild-type" subjects, although the differences were not
statistically significant.2 Nonetheless, because the L208V mutation is located in a putative substrate-binding site of the enzyme,
alterations in the catalytic efficiency of this variant could be
anticipated, thereby providing a plausible metabolic explanation for
the low warfarin dose phenotype in Asians. Given our interests in genetic variability at this locus, we
incorporated these SNPs into our own panel of assays. We have now
screened for these variants in a total of 62 individuals of Asian
descent: 52 Chinese and 10 Koreans. Subjects were healthy volunteers,
none of whom were on anticoagulant therapy. Our screens were based on
direct sequence analysis or a single-base extension assay. Two separate
sets of primers located within intron 3 and intron 4 were used to
amplify exon 4 of CYP2C9: forward 1: 5'-AAT ACA GTG TTT TAT
ATC TAA AGT TTA ATA-3'; reverse 1: 5'-ATG CAA TTC AGA GCT TGA TC-3';
forward 2: 5'-TGT TAA GGG AAT TTG TAG G-3'; reverse 2: 5'-AAT TTT GGA
TTT GTC AGA A-3'. All primers were checked against the entire
CYP2C locus to ensure specificity and yielded single
amplification products of the expected size. We detected no
CYP2C9 exon 4 SNPs in these 2 Asian populations or in banked white DNA samples (n = 50). Moreover, Zarza et al4
failed to identify these SNPs in 106 Spanish subjects, and
Goldstein5 has not reported CYP2C9 exon 4 mutations from preliminary resequencing in 90 subjects from 3 racial groups. In assessing variations in the experimental protocols that might
underlie the interlaboratory differences, we re-examined the primers
chosen for amplification of exon 4 by Leung et al.2 The
first 10 bases of the forward primer and 8 bases of the reverse primer
(5' to 3') appeared to have been included to incorporate a unique
restriction endonuclease site into the resulting amplicon. Analysis of
the remaining primer sequence revealed that the forward primer exhibits
a 100% match to exon 4 sequences within CYP2C8, CYP2C9, CYP2C18, and CYP2C19. The
reverse primer exhibits a 100% match to exon4/intron4 sequences within
CYP2C9 and CYP2C19, although the 3' end of the
primer shows considerable identity to sequences with CYP2C18
and CYP2C19 and might well have resulted in some amplification at those loci as well. In any event, it is clear that the
primers used by Leung et al2 are not adequate for SNP
discovery, as they will result in a mixed template from multiple loci.
These observations call into question the presence of the L208V, Q192P,
H184P, and I181L variants of CYP2C9 and their association with a low
warfarin dose phenotype in Asian patients.
Allan E. Rettie, Guoying Tai, David L. Veenstra, Fred M. Farin, Sengkeo Srinouanprachan, Yvonne
S. Lin, Kenneth E. Thummel, and Ronald N. Hines
Correspondence: Allan E. Rettie, Department of
Medicinal Chemistry, University of Washington, Seattle, WA 98195;
e-mail: rettie{at}u.washingon.edu.
References
1.
Higashi MK, Veenstra DL, Kondo LM, et al.
Association between CYP2C9 genetic variants and anticoagulation-related outcomes during warfarin therapy.
JAMA.
2002;287:1690-1698[Abstract/Free Full Text].
2.
Leung AY, Chow HC, Kwong YL, et al.
Genetic polymorphism in exon 4 of cytochrome P450 CYP2C9 may be associated with warfarin sensitivity in Chinese patients.
Blood.
2001;98:2584-2587[Abstract/Free Full Text].
3.
Chenhsu RY, Chiang SC, Chou MH, Lin MF.
Long-term treatment with warfarin in Chinese population.
Ann Pharmacother.
2000;34:1395-1401[Abstract].
4.
Zarza J, Hermida J, Montes R, Alberca I, Lopez ML, Rocha E.
Leu208Val and Ile181Leu variants of cytochrome P450 CYP2C9 are not related to the acenocoumarol dose requirement in a Spanish population.
Blood.
2002;100:734[Free Full Text].
5.
Goldstein J.
Polymorphisms in the human CYP2C subfamily [abstract].
Drug Metab Rev.
2002;34(suppl 1):5Abstract 9.
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