Alexander Weise

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. 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. 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. 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.

Abstract: The polymorphic cytochrome P450 2D6 (CYP2D6) enzyme, a member of the cytochrome P450 mixed-function oxidase system, is one of the clinically most important enzymes involved in the metabolism of drugs and other xenobiotics. This highly polymorphic enzyme exhibits vastly different phenotypes: poor metabolizers show no enzyme activity, whilst ultrarapid metabolizers exhibit a significantly higher activity. Between these two extremes, there are the extensive and intermediate metabolizers, two heterogeneous groups with overlapping boundaries concerning CYP2D6 activity. These different phenotype groups can be correlated partly to the genotype of an individual. More than 70 different CYP2D6 alleles have been described to date, which encode for null alleles, have a decreased activity or carry gene duplications. However, there is a need for guidelines to translate different CYP2D6 allele combinations into phenotypes. We hereby summarize the current state of the knowledge concerning the relationship between CYP2D6 genotype and phenotype. Clinical relevance of CYP2D6 variation is highlighted with respect to both drugs and putative endogenous ligands. Looking forward, we present a practical genotype interpretation tool, which may help to implement CYP2D6 pharmacogenetics in medical practice. Additionally, we discuss CYP2D6 activity measurement in the context of the recent efforts for multiplexed phenotyping of drug metabolism in vivo.

The cytochrome P450 1A2 (CYP1A2) gene encodes one of the most important enzymes of the Phase I drug metabolism, which is involved in the metabolism of many lipophilic xenobiotics, such as haloperidol, theophylline, phenacetine, and others. The recently discovered single nucleotide polymorphisms CYP1A2*1C (-3860G-->A) in the 5' flanking region of the gene and CYP1A2*1F (-163C-->A) in intron 1 seem to interfere with the expression rate or catalytic function of the enzyme. Polymorphism carriers may either have a risk of reduced drug degradation and side effects, or may present with an increased induction of enzymatic activity resulting in clinical non-response to the prescribed therapy. We investigated two populations, a mental disease group and a healthy control group, to identify whether these two genetic variants are correlated with the general development of a mental disorder and if they could potentially be used as predictive markers for manifestation of the same. Using specifically designed primers, we established a high-throughput multiplex screening realtime PCR method for the two polymorphisms on the CYP1A2 gene with the Roche LightCycler instrument. We analysed the two cohorts to identify whether one of the two described genetic variants may be associated with the manifestation of a mental disorder in general. For the CYP1A2*1C variant, we identified an allele frequency of 1.7% for both cohorts. For the CYP1A*1F polymorphism, we found an allele frequency of 74.5% for the mental disease group and 68.6% for the healthy control group. This new diagnostic method of multiplex detection may be helpful to routinely identify carriers of CYP1A2 variants and to improve the therapeutic effectiveness by selection of the most appropriate therapeutic regimen. As a result of this pilot study, there appeared to be no significant correlation between the existence of one of the investigated genetic variants and the development of a mental disorder.

Summary: Recently, Ghana has changed the first‐line treatment of uncomplicated malaria from chloroquine to amodiaquine (AQ) plus artesunate. AQ may cause adverse events such as agranulocytosis and hepatoxicity. The pro‐drug AQ is transformed by cytochrome P450 CYP2C8 to the active metabolite N‐desethylaminodiaquine. Several polymorphic variants of CYP2C8 are known, some with reduced activity. In 200 randomly selected children from Northern Ghana, we determined the allele frequencies of the CYP2C8 variants CYP2C8*1 (wild type), CYP2C8*2, CYP2C8*3, and CYP2C8*4. We did not detect CYP2C8*3 and CYP2C8*4, but CYP2C8*2 showed an allele frequency of 0.1675. AQ metabolism in patients with CYP2C8*2 may be impaired, and with an increase of AQ based treatment the risk of severe adverse events may mount.