Two Single-Tube Tetra-Primer Assays to Detect the CYP2C19*2 and *3 Alleles of S-Mephenytoin Hydroxylase

Abstract

Cytochrome P450 2C19 (CYP2C19) metabolizes several therapeutic agents, such as S -mephenytoin, omeprazole, propranolol, and imipramine. Interindividual differences in CYP2C19 activity divide the population into extensive metabolizers and poor metabolizers (PMs). PMs may suffer adverse effects when treated with a routine clinical dose of a drug inactivated by CYP2C19 or may not gain therapeutic benefit from prodrugs activated by CYP2C19. For example, the antimalarial drug proguanil is administered as the prodrug and requires activation by CYP2C19. PMs were found to totally lack the active metabolite cycloguanil in their plasma and are at risk for failed protection from plasmodium infection by proguanil (1)(2). The PM phenotype occurs in 2–5% of Caucasians and Africans and 10–23% of Orientals (3). Seven alleles have been described in the CYP2C19 gene that produce an inactive CYP2C19 enzyme; however, two alleles account for the majority of the PM phenotypes. The CYP2C192 and the CYP2C193 alleles are found in ∼87% of PMs in Caucasians and ∼98% of PMs in Orientals (4). CYP2C192 is the most prevalent PM allele, with a G-to-A nucleotide substitution in exon 5, which produces an aberrant splice site (5). CYP2C193 is found mainly in Orientals, with a G-to-A nucleotide substitution at position 636 in exon 4, which produces a premature stop codon (6). Several methods based on PCR amplification of the CYP2C19 locus are used to genotype the PM alleles. The most widely used methods are restriction fragment length polymorphism (RFLP) analysis (7)(8) and allele-specific amplification (ASA) (9). However, ASA and RFLP include transfer steps that increase hands-on time and the chance of contamination or confusion of samples. To decrease hands-on time and to facilitate genotyping of the CYP2C19 alleles that predict the majority of …