Identification of a synonymous polymorphism within the cytochrome P4502C9 gene that interferes with identification of the CYP2C9*2 allele

Cytochrome P450 2C9 (CYP4502C9) genotyping is useful in dosage adjustments for several critical drug therapies, including warfarin. Potential interference compromising these genotyping results could lead to inappropriate dose adjustments that may result in adverse drug reactions. During routine clinical CYP4502C9 genotyping using multiplex allele-specific primer extension, an ambiguous result was obtained for determination of the CYP2C9 430C>T substitution, which defines the CYP2C9*2 allele. In this one patient sample submitted for CYP2C9 genotyping, the ratio for the variant 430T allele signal to the total signal (C+T alleles) was 0.29. This is above the expected ratio to be classified as wild-type (<0.15) and below the minimum expected ratio (>0.3) when the genotype is heterozygous at the 430 position. The mean fluorescence intensity for the 430C allele was consistent with that observed in subjects who are heterozygous at this nucleotide position. However, the corresponding signal for the 430T allele indicated the absence of the CYP2C9*2 allele. This suggests the assay was not able to determine the correct nucleotide at position 430 for one of the two alleles in this patient. Subsequent sequencing to investigate the allele-specific primer extension failure revealed the presence of a rare C>T nucleotide substitution at position 429. We tested this subject's CYP2C9 genotype using AvaII restriction endonuclease digestion and found that this rare substitution causes false-positive identification of the CYP2C9*2 allele when using this method. We developed a DpnII endonuclease digestion assay to specifically detect the CYP2C9 429C>T substitution and tested 100 randomly selected samples obtained from unrelated individuals. The 429C>T polymorphism was not identified in this sample set, which indicates an allele frequency of less than 2.0% (95% confidence interval, 0.0-1.8%) in the general population. Despite the rarity of this polymorphism, it has important implications for the accuracy of assays using allele-specific primers and the Ava II restriction endonuclease, when it occurs, which are two common methods currently applied for detecting the presence of the CYP2C9*2 allele.