DETECTION OF THE POOR METABOLIZER-ASSOCIATED CYP2D6(D) GENE DELETION ALLELE BY LONG-PCR TECHNOLOGY

The cytochrome P450 enzyme debrisoquine 4-hydroxylase metabolizes many different classes of commonly used drugs, such as antidepressants and neuroleptics. Deficient hydroxylation of debrisoquine, known as the p oor metabolizer (PM) phenotype, affects 5-10% of Caucasians and may le ad to adverse reactions upon administration of drugs in standard doses . This autosomal recessive metabolic deficiency is caused by the posse ssion of two PM-associated mutations in the human CYP2D6 gene locus co ding for the enzyme. These mutations include at least four different s ingle base mutations and two different large gene deletion alleles, Th e single base mutations can be rapidly detected by PCR methods. In con trast, the large gene deletions have so far only been directly identif ied by RFLP analysis, By the use of sequence data previously published by others, we report here an alignment of different CYP2D alleles to focus on the presence of almost completely identical sequences immedia tely downstream of both CYP2D7 and CYP2D6 which may seriously complica te and interfere with PCR-based detection of the gene deletion. Based on this analysis, we have developed a rapid assay which, for the first time, detects the 13kb (also called 11.5 kb) Xba I gene deletion alle le by the use of long-PCR technology. The primers were designed to amp lify a 3.5 kb PCR product in the presence of this D6(D) allele. We hav e evaluated the method on 23 different DNA samples heterozygous (n = 2 2) or homozygous (n = 1) for the 13 kb gene deletion allele (previousl y typed by RFLP analyses). All samples were correctly identified by th e assay, The PCR method did not detect the rare 11 kb Xba I gene delet ion allele (n = 5), and there was no false positive amplification from deletion negative DNA samples (n = 47). This sensitive and specific P CR-based assay for detection of the D6(D) allele will improve the scie ntific and clinical use of CYP2D6 genotyping.