Dr. István Balogh, Dr. János Kappelmayer, Dr. József Tőzsér (2011)
University of Debrecen
One of the most quickly developing field of molecular testing is pharmacogenetics. In the process of metabolism, drugs will be more water soluble, therefore more accessible for renal excretion. During drug metabolism, sometimes toxic compounds are formed. In many cases, metabolism is responsible for the activation of the prodrug. This process can be divided into two different types of reaction. Type I reactions are oxydation, reduction and hydrolysis. Type II (conjugation) reactions include sulfation, methylation, glucuronidation, acetylation. Both reactions – whose names do not indicate the succession of the reactions – normally make the originally lipophilic compound more hydrophilic (Figure 11.1.) The genes coding the proteins responsible for these processes are usually highly polymorphic. This means that in some cases, the individual response after the administration of a specific drug can be attributed to the genetic background of the patient. It also means that in some cases, the knowledge of the patient’s genetic status makes individualized therapy possible. Individualized therapy has two major goals: it might help not only in quickly establishing the correct dose of certain drug, but also in avoiding the dangerous, sometimes life-threatening side effects. Some pharmacogenetic examples are shown below.
CYP2D6 is involved in the metabolism of many drugs, therefore it is one of the main pharmacogenetic targets (Figure 11.2.). Its gene is located in the chromosome 22. Null alleles (mutations that lead to non-functioning protein product or no protein product al all), labelled with white boxes will lead to the poor metabilzer (PM) phenotype. 5-10% of the Caucasian population belongs to this group. When a normal dose of the drug is administered to these patients, some severe side-effect might be experienced as a consequence. There are mutations which decrease enzyme activity, even though they do not completely abolish it (dotted boxes). Patients (5-10% of the Caucasian population) with this genotype show intermediate (IM) phenotype. Side-effects are also expected, though to a lesser extent compared to the poor metabolizers. In the case of wild type alleles (black boxes) the resulting enzyme activity is normal. Those individuals (65-80% of the Caucasian population) are the extensive metabolizers (EM). The duplication and multiplication of the CYP2D6 gene might be present in 5-10% of the Caucasian population, resulting in ultrarapid metabolizer (UM) phenotype. The administration of the normal dose of the drug is completely ineffective in those patients. The right-hand side of the picture shows the plasma concentration of the drug and its therapeutic range.
Thiopurine methyltransferase (TPMT) polymorphisms.
Thiopurine methyltransferase (TPMT) together with xanthine oxidase are responsible for the degradation of the purine analogue drugs, including 6-thioguanine, 6-mercaptopurine and azathioprin. There are significant interindividual and ethnic differences between the enzyme activities. It is the polymorphism of the TPMT gene that is responsible for these differences (Figure 11.3.).The TPMT gene is located to chromosome 6 (6p22.3). It consists of 10 exons and 9 introns. Numerous different alleles are known, which differ from each other only in a few nucleotides. The most common (wild type) allele is TPMT*1. The presence of any other allele results in decreased enzyme activity, both in heterozygous, and in homozygous form. The prevalence of heterozygosity is 11% among Caucasians. 1 in 300 is homozygous (phenotypically). The most common known alleles are TPMT*3A, 3B, 3C, 3D, 2, 4, 5, 6, 7, of which TPMT*3A-D are the most prevalent.
Individuals with either low or intermediate enzyme activity (homozygous or heterozygous genotypes, respectively) require much less than the standard dose of the above-mentioned drugs. The administration of the normal dose of the drug might result, especially in the homozygotes, inlife-threatening side effects, such as myelosuppression and pancytopenia. The different common TPMT alleles are the followings: TPMT*1 is the wild type. TPMT*2 allele is a G238C replacement in the exon 5, resulting in an alanine-proline amino acid substitution. In TPMT*3A two point mutations are present in one allele: G460A in exon 7 (effect: alanine-threonine replacement) and A719G in exon 10 (tyrosine-cysteine substitution). G460A alone is TPMT*3B allele, while A719G is TPMT*3C. TPMT*3D allele has G292T mutation (exon 5, with a glutamine-stop consequence) in addition to the two mutations present in TPMT*3A. In TPMT*4 allele, the boundary of intron 9 - exon 10 is affected, with a G>A nucleotide substitution. TPMT*7 allele is T681G mutation in exon 10, resulting in a histidine-glutamine amino acid replacement.
The pharmacogenetic aspects of the anticoagulant terapy.
The most commonly used oral anticoagulant worldwide is warfarin. Its administration is lifelong in some patients groups (e.g., for those with artificial heart valves). One of the sister drugs of warfarin is acenocoumarol (syncumar), which is very commonly used in Hungary (the use of warfarin is app. 10-15%). Warfarin interferes with the vitamin-K cycle (Figure 11.4.). The Vitamin-K cycle is a critical process for the posttranslational modifications of some proteins (the majority of them are involved in blood coagulation). The vitamin-K cycle makes possible the generation of gamma-carboxy-glutamate residues in these proteins, which is necessary for the membrane binding. Such proteins are factor II (prothrombin), factor VII, factor IX, factor X, protein C and protein S. In the absence of correct gamma-carboxylation the membrane binding, and as a consequence the blood coagulation is severely impaired. One of the key enzymes of the vitamin-K cycle is VKOR (vitamin-K epoxide reductase). The main function of VKOR is the reduction of the epoxide form of vitamin-K. This is a two-step process, where first the kinon, then the hydrokinon is formed. Warfarin interferes with both steps, as it binds to the same site where vitamin-K does and this site cannot accommodate both at the same time. INR is used for monitoring the effect of warfarin.
Pharmacogenetics of the warfarin/syncumar type of anticoagulant drugs is important from the point of view of both individual and public health. Both drugs exist in a mixture of optical isomers, but there are substantial differences. In the case of warfarin, the S isomer is responsible for most part of the anticoagulant effect, while in syncumar, it is the R. The key enzyme of the metabolism of the S isomers is CYP2C9. It has two common variants with decreased enzyme activity (CYP2C9*2 and *3 alleles). Although the VKORC1 gene is highly polymorphic, testing one functional variant, namely -1639G>A is sufficient. A wide international collaboration has resulted in a warfarin dosing algorithm, which was based on - in addition to other factors - the pharmacogenetic data. This dosing formula is available as an Excel worksheet.
In conclusion, the genetic status of CYP2C9 will affect the availability of the active drug (warfarin) and the genotype of VKORC1 will affect the efficiency of the vitamin-K cycle.