title: Development and validation of a rapid and reliable method for TPMT genotyping using real-time PCR.
abstract: BACKGROUND
  It is widely accepted that many medications exhibit inter-individual variability in their efficacy and toxicity due to polymorphisms in genes encoding drug-metabolising enzymes. One of the most often cited examples in this context is thiopurine S-methyltransferase (TPMT) polymorphism. TPMT is a phase 2 detoxification enzyme that catalyzes the S-methylation of thiopurine drugs such as thioguanine and 6-mercaptopurine. Approximately 11% of the Caucasian population carry a heterozygous deficiency of this enzyme causing intermediate enzyme activity, whereas 0.3% show a homozygous deficiency. In both cases, severe myelosuppression can develop upon treatment with thiopurines. These are commonly used in the treatment of leukemia. Therefore, genotyping of patients before treatment is absolutely necessary. Development of a fast and reliable real-time PCR application for TPMT genotyping would greatly improve thiopurine treatment regimens and allow the avoidance of adverse drug reactions.
  
  
  METHODS
  Blood was obtained from a Caucasian cohort of 143 individuals. After extraction of DNA, all samples were genotyped for TPMT polymorphisms *2, *3A, *3B, and *3C by real-time PCR as well as by PCR-RFLP as the reference method, in order to validate the new method.
  
  
  RESULTS
  Four different genotypes were found in the population studied. Of the 143 individuals investigated, 1 was heterozygous for TPMT*2 (0.70%), 2 were heterozygous for TPMT*3B (1.40%), and 8 heterozygous for TPMT-*3C (5.60%). No homozygous genotype could be identified. In total, 7.7% of the individuals carried mutations. Results from the newly developed real-time PCR were 100% concordant with those obtained using standard PCR-RFLP analysis, leading to 100% sensitivity and specificity. The hands-on time is approximately one third of the time needed for standard PCR-RFLP methods.
  
  
  CONCLUSIONS
  A new high-throughput genotyping method could be successfully established and optimised for the commonly found mutant alleles TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A), and TPMT*3C (A719G) via real-time PCR on the LightCycler (Roche) instrument and using the standard PCR-RFLP as reference method.
authors.affiliation: Institute for Clinical Research and Development (IKFE) GmbH, Mainz, Germany. marion.lorenz@pharmgenomics.com
authors.forename: Marion
authors.initials: M
authors.lastname: Lorenz
authors.forename: Alexander
authors.initials: A
authors.lastname: Weise
authors.forename: Stefan
authors.initials: S
authors.lastname: Prause
authors.forename: Marco
authors.initials: M
authors.lastname: Klemm
authors.forename: Moritz
authors.initials: M
authors.lastname: Eidens
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Luchi
authors.forename: Thomas
authors.initials: T
authors.lastname: Forst
authors.forename: Andreas
authors.initials: A
authors.lastname: Pfützner
authors.forename: Matthias M
authors.initials: MM
authors.lastname: Weber
mesh_terms: D015331:Cohort Studies; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008297:Male; D008780:Methyltransferases / Q000235:genetics*; D008875:Middle Aged; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D008780:Methyltransferases; C022745:thiopurine methyltransferase