title: Development and validation of a rapid and reliable real-time PCR method for CYP3A5 genotyping.
abstract: BACKGROUND
  A prominent example for inter-individual differences in drug-metabolizing enzymes is the cytochrome P450 family. These monooxygenases comprise enzymes responsible for metabolism of about 90% of common medications. CYP3A5 and CYP3A4 account for 50% of hepatic cytochrome P450 and conversion of about half of all their substrates. CYP3A5 is the predominant extra-hepatic CYP enzyme and shows varying inter-individual expression attributable to genetic variations in the corresponding gene. CYP3A5*2 and *3 are the most common among Caucasian populations. Among CYP3A5 substrates are cyclosporine and tacrolimus prescribed after organ transplantations. A high incidence of nephrotoxic events after administering these drugs is related to low expression of the CYP3A5 enzyme. A fast and reliable genotyping method for the CYP3A5 gene would help avoid unwanted adverse drug reactions.
  
  
  METHODS
  Blood samples from 143 Caucasian subjects were genotyped by means of a real-time PCR multiplex assay testing the two CYP3A5 variations, CYP3A5S*2 and *3. This assay was validated against RFLP-PCR.
  
  
  RESULTS
  Both mutations examined could be found in the study. No sample was homozygous for CYP3A5*2, but 2 out of 143 showed heterozygosity (allele frequency: 0.7%). For the CYP3A5*3 variant 17 samples were heterozygous and 115 were homozygous (allele frequency 86.4%). The multiplex real-time PCR yields shorter hands-on time and reduced cost compared to RFLP-PCR.
  
  
  CONCLUSIONS
  Establishment of a multiplex real-time PCR has been successful as could be proven by correctly identifying the desired mutations CYP3A5*2 and CYP3A5*3 against a standard reference method.
authors.forename: Moritz
authors.initials: M
authors.lastname: Eidens
authors.forename: Alexander
authors.initials: A
authors.lastname: Weise
authors.forename: Marco
authors.initials: M
authors.lastname: Klemm
authors.forename: Merle
authors.initials: M
authors.lastname: Fleischer
authors.forename: Stefan
authors.initials: S
authors.lastname: Prause
mesh_terms: D000368:Aged; D000483:Alleles; D016572:Cyclosporine / Q000737:chemistry; D051544:Cytochrome P-450 CYP3A / Q000097:blood* / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005260:Female; D005787:Gene Frequency; D014644:Genetic Variation; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D012150:Polymorphism, Restriction Fragment Length; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D016559:Tacrolimus / Q000737:chemistry; D044465:White People
chemical_list: D007166:Immunosuppressive Agents; D016572:Cyclosporine; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; D016559:Tacrolimus