CYP2D6 Polymorphism in Patients of General Practice in Austria
Condition: Disorder Due Cytochrome P450 CYP2D6 Variant
Study type: Observational

Summary: 
      The CYP 2D6 enzyme metabolizes a significant number of drugs frequently prescribed in general
      practice/ family medicine. Various genetically different variants define if the patient is an
      ultra-rapid (UM), an normal (NM) (the normal case), an intermediate (IM) or a poor
      metabolizer (PM). It is estimated that approximately 20- 25 % of frequently described drugs
      are activated to more active or metabolized to ineffective or less effective drugs by CYP
      2D6. Substrates of CYP 2D6 are mainly antidepressants, neuroleptics, opioids (e.g. codeine),
      beta-blockers, anti-arrhythmic drugs and various other single drugs. In case of an UM a drug
      can be metabolized too rapidly losing its therapeutic effect, requiring a higher dosage, or
      it can have a toxic effect, if it is converted too rapidly in the effective form (e.g.
      codeine). If metabolized too slowly (PM) it can accumulate and reach toxic levels.

      In this observational study (1) data relating to the number of patients of a single Austrian
      general practice receiving one or more drugs metabolized by CYP 2D6 are collected by
      extracting their electronic records of the last 3 years. In addition (2) consecutive patients
      with unknown genetic status of their CYP 2D6 enzyme visiting the surgery for a routine blood
      test due to various reasons, are additionally tested for their CYP 2D6 metabolizing status,
      if they actually take a drug metabolized by CYP 2D6.

      The aim of the study is to generate CYP 2D6 polymorphism data from Caucasian patients of an
      average Austrian general practice for the first time, which allows to group patients
      according to their NM, UM, IM and PM status. This can be of considerable clinical relevance
      when prescribing specific drugs. This study tries to investigate in how many patients the
      knowledge of the CYP 2D6 metabolizing status could have an influence on choosing the actually
      prescribed drug. In addition we plan to describe the distribution of frequent and relevant
      CYP 2D6 alleles including their combinations in patients of an average Austrian general
      practice for comparison reasons with other Caucasian populations.
    

Detailed description: 
      Study population:

      Unselected consecutive patients of the practice office visiting the surgery for a routine
      blood sampling due to various medical conditions and who are prescribed or have been
      prescribed drugs metabolized by the CYP2D6 enzyme (or drugs being a strong inhibitor of CYP
      2D6) during the last 3 years. Only in these patients CYP2D6 polymorphism is determined using
      a fraction of the EDTA-blood sample. No further genetic investigations or additional blood
      collections are performed.

      Patients who are considered to be eligible to participate in the study have to sign an
      informed consent after being informed about the aims of the study.

      Blood sampling and processing of the specimens:

      A standardized blood sampling using a Vacutainer system with filling of an EDTA tube (4 ml)
      for the red and white blood count is performed. 300 microliters of the collected blood are
      used for further determination of the CYP2D6 polymorphism.

      Isolation of DNA:

      DNA is extracted from EDTA blood in the practice laboratory by using the Spin Micro
      Extraction Kit® (ViennaLab,Vienna). The concentration and quality of DNA is measured using a
      Bio Photometer plus (Eppendorf). The extracted DNA is stored at -81°C in an ultra-low
      temperature deep freezer (U101-86, New Brunswick Scientific Co., Inc) without any further
      additives.

      Real-time PCR for determination of copy number of the CYP2D6 gene:

      For the determination of the copy number of the CYP2D6 gene a real-time PCR in triplicates on
      an ABI StepOnePlus by using the CYP2D6 RealFast™ CNV Assay (ViennaLab) is performed in the
      practice laboratory.

      The test is based on the fluorogenic 5' nuclease assay, also known as TaqMan® assay. Each
      reaction contains gene-specific primer pairs for amplification of CYP2D6 and endogenous
      control (EC) gene fragments with 141 bp each. Further components are two dual-labeled,
      gene-specific hydrolysis probes, the FAM-labeled CYP2D6 probe and the HEX-labeled EC probe,
      which hybridize to an internal sequence of the amplified fragments. The proximity of the
      5'-fluorescent reporter and 3'-quencher dye on intact probes prevents the reporter from
      fluorescing. During the extension phase of PCR the 5' - 3' exonuclease activity of Taq DNA
      polymerase cleaves the 5'-fluorescent reporter from the hybridized probe. The physical
      separation of the fluorophore from the quencher dye generates a fluorescent signal in
      real-time, which is proportional to the accumulated PCR product. The CYP2D6 RealFast™ CNV
      Assay is a relative quantitation assay and compares the amount of both nucleic acid targets
      (CYP2D6 and EC) in relation to the CYP2D6 CNV Calibrator. The EC gene is used to normalize
      fluorescence signals between different samples and serves as a PCR positive control.

      For additional normalization of data ROX dye to a final concentration of 1 microliter to the
      2 x Probe Mix is added.

      RT- PCR cycling conditions for the ABI StepOneplus cycler: Initial denaturation: 95°C 10 min
      1 cycle; denaturation 95°C 15 sec 40 cycles; annealing /extension 60°C 1 min.

      PCR and hybridisation for CYP2D6 allele determination by the PGX-CYP2D StripAssay™:

      This assay is used for a subset of samples in this study and covers only 3 polymorphic loci:
      1795delT (2D6*6), 1934G>A (2D6*4) and 2637delA (2D6*3). The test principle and procedure are
      similar to the PGX-CYP2D6 XL StripAssay® as described below but using different cycling
      conditions for the Palm-Cycler (Corbett Life Science): pre-PCR: 94°C/2 min; thermocycling:
      94°C/15 sec.- 58°C/ 30 sec.-72°C/30 sec (35 cycles); final extension: 72°C/3 min.

      PCR and hybridisation for CYP2D6 allele determination by the PGX-CYP2D6 XL StripAssay®:

      For the determination of 19 clinically relevant CYP2D6 alleles (*1; *2 A, *2 B-M; *3, *4A-H,
      K, L or P, *4J or N, *4M; *5; *6 A, B or D, *6C; *7, *8; *9,; *10A or B, *10 C or D; *11;
      *12; *14; *15; *17; *29; *35; *39; *40 or *58; *41) a PCR amplification on a Palm-Cycler
      (Corbett Life Science, Eight Mile Plains, QLD 4113, Australia) using biotinylated primers is
      first performed. The amplification products are further hybridized to a test strip containing
      allele-specific oligonucleotide probes immobilized as an array of parallel lines. Bound
      biotinylated sequences are detected using streptavidin-alkaline phosphatase and color
      substrates. The evaluation of this reaction is done manually by using the
      StripAssay®-Evaluator (ViennaLab,Vienna), a proprietary PC program to determine the
      homozygous or heterozygous genotype.

      Cycling conditions for the Palm-Cycler (Corbett Life Science): pre-PCR: 95°C/4 min;
      thermocycling: 95°C/25 sec.- 60°C/ 45sec.-72°C/1min (36 cycles); final extension: 72°C/3 min.

      Statistics:

      The data were recorded and analyzed using Microsoft Excel Version 10 and the R Language and
      Environment for Statistical Computing and Graphics, Version 2.9. Standard methods are used
      for the description of data (frequencies and percentages for categorical data, mean and
      standard deviation for continuous data). To compare frequencies of CYP2D6-specific drugs
      prescribed in the single practice with the total number of the respective prescriptions in
      Lower Austria, Spearman's rank correlation-coefficient is calculated. Differences between
      groups were calculated by paired t-test. A p-value < 0.05 was considered to indicate
      statistical significance.

      The Hardy-Weinberg- equilibrium was calculated using Fisher´s Exact Chi-Square test due to
      the small numbers of genotypes.
    

Observational model: Other
Time perspective: Other
Sampling method: Non-Probability Sample
Study population: 
        Unselected consecutive patients of an Austrian general practice suffering from chronic
        diseases visting the practice office for a routine blood test
      
Biospecimen description: 
      DNA extraction from peripheral white blood cells
    
Keywords: Cytochrome P 450 CYP2D6 Variants; Genetic polymorphism; Practice, Family; Drug metabolism