Rapid detection of CYP2D6 null alleles by long distance- and multiplex-polymerase chain reaction.
The CYP2D6 gene on human chromosome 22 encodes a cytochrome P450 responsible for oxidative metabolism of over 30 clinically used drugs. The CYP2D6 gene is highly polymorphic with more than 20 alleles described to date. Some of these harbour loss-of-function mutations which lead to the poor metabolizer phenotype in 5-10% of Caucasians. These individuals are at increased risk of suffering from adverse side effects or to experience therapeutic failure following drug treatment. Phenotype determination requires ingestion of a probe drug and has other inherent problems. Due to the increasing number of alleles known, comprehensive CYP2D6 genotyping using the conventional assays has become cumbersome and time consuming. We have therefore developed a streamlined and more rapid CYP2D6 genotyping procedure. Use of long distance PCR allowed the amplification of a 4666 bp fragment which contains the entire CYP2D6 gene. The 4.7 kb fragment serves as a template for a multiplex allele-specific PCR assay to simultaneously identify the five PM-associated alleles, CYP2D6*3 (A), *4 (B), *6 (T), *7 (E), and *8 (G). Together with the CYP2D6 deletion allele CYP2D6*5 (D), which can be detected in a separate PCR assay, these alleles are responsible for the PM phenotype in approximately 99% of Caucasian individuals. We tested the reliability of the procedure by analysing DNA from more than 80 individuals with known CYP2D6 genotypes. Twelve different genotypes were present among these samples and all of them were correctly identified.
Stüven
T
T
Dr Margarete Fischer-Bosch-Institut für Klinische Pharmakologie, Stuttgart, Germany.
Griese
E U
EU
Kroemer
H K
HK
Eichelbaum
M
M
Zanger
U M
UM
D000483:Alleles; D002892:Chromosomes, Human, Pair 22; D019389:Cytochrome P-450 CYP2D6 / Q000145:classification / Q000235:genetics*; D017931:DNA Primers; D005838:Genotype; D005858:Germany; D006801:Humans; D009154:Mutation; D004364:Pharmaceutical Preparations / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D044465:White People
D017931:DNA Primers; D004364:Pharmaceutical Preparations; D019389:Cytochrome P-450 CYP2D6