High-throughput analysis of informative CYP2D6 compound haplotypes

Abstract

We describe a high-throughput protocol for detecting key polymorphisms in the drug-metabolizing enzyme gene CYP2D6 and a number of linked microsatellites that is both fast and relatively inexpensive to perform. This approach employs GeneScan technology to enable a researcher to determine rapidly the status of seven simple nucleotide polymorphisms in CYP2D6 and also to assay repeat number variation at five closely linked dinucleotide microsatellite loci. The method requires only three PCRs and two GeneScan runs per sample. We anticipate that this will be of value to researchers in three different ways: (1) rapid discrimination of common CYP2D6 alleles, (2) high-resolution haplotyping for association studies involving chromosome 22q13.1 using microsatellite variation, and (3) generation of compound haplotypes for investigating the evolution of CYP2D6 variation. We also report compound haplotype frequencies for an Ashkenazi Jewish and a British sample. © 2003 Elsevier Science (USA). All rights reserved.Keywords: CYP2D6; Pharmacogenetics; Genotyping; Haplotype; Microsatellites; SNPs; Jews; British; Population; Variation It is now clear that much of the observed variation in drug efficacy and safety has a hereditary basis, arising from polymorphisms in genes coding for drug-metabolizing enzymes (DMEs). The frequency of DME alleles can vary greatly between different human populations, and this has implications for both medical and evolutionary studies. Consequently, there has been growing academic and commercial interest in the extent, nature, and causes of DME variation. However, it is currently unknown to what extent interpopulation variability is due to selection, drift, or other processes. Studies seeking to elucidate this have been held back by the absence of efficient low-cost high-throughput procedures for characterizing variability at the molecular level in and around DME loci. Ultimately, the identification of the molecular basis of pharmacogenetically relevant variation combined with the technology to screen individuals for the presence of specific alleles allows for the prediction of drug response and individualized treatment.Polymorphism in debrisoquine/sparteine oxidation is arguably the most highly studied pharmacogenetic trait. The DNA sequence encoding the enzyme has been localized to the 4.3-kb, nine-exon cytochrome P450 2D6 (CYP2D6) gene found at chromosome 22q13.1. To date more than 48 mutations and 53 alleles of CYP2D6 have been characterized in European populations [1]. The poor metabolizer (PM) phenotype follows an autosomal recessive pattern of inheritance [2]. An allele duplication consisting of multiple functional copies of CYP2D6 confers the ultrarapid metabolizer (URM) phenotype [3]. Individuals who demonstrate normal levels of CYP2D6 activity are referred to as extensive metabolizers. Intermediate metabolizers (IMs) typically produce lower than normal levels of functional enzyme.Medical interest in CYP2D6 polymorphism arises because the CYP2D6 enzyme has been found to process more t...

Αναλύσεις Υβριδισμού Νουκλεϊκών Οξέων Με Νανοσωματίδια

“…Η παρουσία των ενζύµων αυτών είναι ουσιαστική για τη σωστή λειτουργία και οµοιόσταση του οργανισµού. 15 κυτοχρώµατα P450 (οµάδες CYP1, Κεφάλαιο Το γονίδιο, µεγέθους 4,3 kbp, το οποίο κωδικοποιεί το CYP2D6 βρίσκεται στο χρωµόσωµα 22q13.1 και παρουσιάζει περίπου 80 πολυµορφισµούς76,77 . Η ύπαρξη των πολυµορφισµών έχει ως αποτέλεσµα την έκφραση ενζύµων CYP2D6 που παρουσιάζουν αυξηµένη, µειωµένη ή µηδενική ενεργότητα.…”