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Rapid detection of CYP2D6 null alleles by long distance- and multiplex-polymerase chain reaction
T Stüven1, E U Griese, H K Kroemer
1Dr Margarete Fischer-Bosch-Institut für Klinische Pharmakologie, Stuttgart, Germany.
Abstract:
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.
Insights
A new CYP2D6 genotyping method offers a faster, streamlined approach to identify poor metabolizer phenotypes. This improves drug safety and efficacy for individuals with specific genetic variations in drug metabolism.
Area of Science:
- Pharmacogenomics
- Molecular Biology
- Clinical Chemistry
Background:
- The CYP2D6 gene is crucial for metabolizing over 30 drugs, exhibiting high polymorphism with over 20 alleles.
- Loss-of-function CYP2D6 mutations cause a poor metabolizer phenotype in 5-10% of Caucasians, increasing risks of adverse drug reactions or treatment failure.
- Current phenotype determination methods are problematic, and comprehensive genotyping is time-consuming due to numerous known alleles.
Purpose of the Study:
- To develop a streamlined and rapid CYP2D6 genotyping procedure.
- To efficiently identify key CYP2D6 alleles associated with the poor metabolizer phenotype in Caucasian populations.
Main Methods:
- Developed a long-distance PCR to amplify the entire 4666 bp CYP2D6 gene.
- Utilized a multiplex allele-specific PCR assay on the amplified fragment to simultaneously detect five poor metabolizer-associated alleles (*3, *4, *6, *7, *8).
- A separate PCR assay was used to detect the CYP2D6 deletion allele (*5).
Main Results:
- The method successfully identified five key poor metabolizer-associated alleles (*3, *4, *6, *7, *8) and the deletion allele (*5).
- These targeted alleles account for approximately 99% of poor metabolizer phenotypes in Caucasians.
- The procedure demonstrated high reliability, correctly genotyping over 80 individuals with 12 different known CYP2D6 genotypes.
Conclusions:
- The developed CYP2D6 genotyping procedure is rapid, streamlined, and accurate.
- This method facilitates efficient identification of individuals at risk due to poor drug metabolism.
- It offers a significant improvement over conventional genotyping assays for clinical and research applications.