Development and evaluation of a rapid and reliable method for cytochrome P450 2C8 genotyping.
The analysis of gene polymorphisms has started to become an interesting field of new services to be provided by clinical laboratories for both, clinical research and routine determinations. To expand the use of these methods and to increase the benefit for patients and the society, cheap and reliable methods for genotyping also allowing for high-turnover analysis need to be established in clinical laboratories. The presented investigation was performed to develop and evaluate a fast real-time PCR method for the detection of three different allele mutations of Cytochrome P450 isoenzyme 2C8 (CYP2C8*2, *3, and *4), which have been demonstrated to influence drug metabolism (and thus the efficacy) of a variety of drugs. The DNA of 122 Caucasian subjects (56 male, 66 female, age (mean +/- STD): 50 +/- 16 years) was analyzed for these mutations by means of classical RFLP-PCR and a new protocol developed for the LightCycler real-time PCR method. The polymorphisms within the CYP2C8 gene were detected by use of two primer pairs and three different pairs of hybridization probes. The results of both methods were perfectly concordant and comparable to results published in the literature (allele frequencies: CYP2C8*2: 0.016, *3: 0.140, *4: 0.074). A subsequent analysis of the related costs revealed comparable resource requirements but substantially longer time needs for RFLP-PCR, resulting in higher overall analysis costs for the older method. In conclusion, the elaborated protocol for real-time PCR analysis of gene mutations CYP2C8*2, *3, and *4 is reliable and cost effective, and thus, suitable for routine laboratory use.
Weise
A
A
Institute for Clinical Research and Development, IKFE GmhH, Mainz, Germany.
Grundler
S
S
Zaumsegel
D
D
Klotzek
M
M
Gröndahl
B
B
Forst
T
T
Pfützner
A
A
D001189:Aryl Hydrocarbon Hydroxylases / Q000145:classification / Q000235:genetics*; D001483:Base Sequence; D065727:Cytochrome P-450 CYP2C8; D004252:DNA Mutational Analysis; D005260:Female; D005453:Fluorescence; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006801:Humans; D007527:Isoenzymes / Q000145:classification / Q000235:genetics; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010597:Pharmacogenetics / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results
D007527:Isoenzymes; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8