CYP2C9 polymorphisms and CYP2C9*2 genotyping primers

The primers first suggested by Wang et al.[1] for the genotyping of the CYP2C9 Arg144Cys mutation were based on the gene structure of CYP2C9 prior to knowledge of the gene structure of the closely related CYP2C19. Early studies cannot be faulted for using these primers, since the gene structure of CYP2C19 was not known. The primers utilized by Wang et al.[1] differ from the gene structure of CYP2C19 by only a few bases (underlined) [5′-GAATATGAAGCAGTGAAGGAA3–3′ (forward primer) and 5′-GGCCTTGGTTTTTCTCAACTC-3′ (reverse primer)]. In particular, mismatches at the 5′-end are not generally considered to confer much specificity to primers, since many laboratories add restriction sites in this way. The primers later described by Sullivan-Klose et al.[2, 5′-TACAATACAATGAAAATACATG-3′ and 5′-CTAACAACCAGACTCATAATG-3′] included a number of mismatches with CYP2C19 (underlined) including two at the 3′-end of each primer. Thus, I have always considered them to be more specific for CYP2C9 and to be less likely to amplify the closely related CYP2C19. Since both CYP2C9 and CYP2C19 contain an arginine at 144, any spurious amplification of CYP2C19 would make it more difficult to recognize homozygous CYP2C9*2 individuals. However any comments of mine concerning the use of the primers described by Wang [1] by any subsequent particular group were unfortunate and not very tactful. Many groups have used these primers. Dr Daly's group has had extensive experience with genotyping tests, and she states that the annealing temperatures she used allowed her to avoid the coamplification problems. Her laboratory in fact identified three homozygous CYP2C9*2 individuals. Other laboratories including mine and those of Yasar et al.[3] have had problems with specificity using the primers described by Wang et al.[1]. The study of Yasar et al.[3] (Figure 1, panel 1) indicates heavy contamination by non-CYP2C9 genes, not slight contamination as suggested by Dr Daly. AvaII should not digest the PCR products of an individual homozygous for the CYP2C9*2 allele, but when Yasar et al.[3] used the primers described by Wang, approximately 50% of the PCR products appeared to be cleaved. Such results would have led to the incorrect identification of the individual as heterozygous for CYP2C9*2. Yasar et al.[3] were not able to achieve specificity using these primers by changing either the annealing temperature or Mg2+. I would simply like to point out that the choice of alternate primers containing more base differences with CYP2C19 would seem to be a prudent precaution in the genotyping of CYP2C9*2 in future studies. The use of more specific primers may help many laboratories to achieve the specificity necessary for the identification and study of homozygous CYP2C9*2 individuals. The identification and clinical testing of metabolism of drugs by individuals homozygous for the CYP2C9*2 alleles is an important issue. Compared with CYP2C9*3, there is a paucity of studies in the literature concerning rates of metabolism of drugs in CYP2C9*2 homozygous individuals. I hope my review indicated that I agree with Dr Daly that CYP2C9*2 is indeed a defective allele with lower clearances for many drugs than the wild-type CYP2C9. Although our early cDNA expression studies did not detect differences between the catalytic activity of the CYP2C9*2 allele and the wild-type allele in a yeast cDNA expression, this may have been due to the cDNA expression system used. Of the CYP2C9 alleles reported to date, CYP2C9*3, CYP2C9*4, and CYP2C9*5 have dramatically impaired catalytic activity [4]. The CYP2C9*4[4] (Ile359Thr) and CYP2C9*5 (Asp360Glu) [5] like CYP2C9*3 (Ile359Leu) alleles all contain changes at amino acids 359 and 360 which are predicted to lie in a putative substrate binding site. These alter either the Km or Vmax toward various substrates. A new null allele of CYP2C9 has been discovered with even more deleterious effects in vivo[6]. However, numerous studies have verified that the CYP2C9*2 protein has moderately impaired catalytic activity in vitro perhaps due to impaired binding of the reductase [7]. There are also studies of heterozygous individuals showing some impairment in metabolism of certain substrates in vivo. This is important because of (a) the relatively high frequency of the CYP2C9*2 allele (approximately 11%), the high frequency of heterozygotes, and (b) the fact that CYP2C9 mediates metabolism of several clinically important drugs with narrow therapeutic indices such as phenytoin and warfarin.