Pharmacogenetics is the study of variations in DNA sequence as related to drug response, i.e. pharmacokinetics, drug efficacy and adverse effects. The literature review of the thesis covers pharmacogenetics of analgesics. The most studied genetic variations affecting the analgesics response are the 118A>G variant of µ-opioid receptor gene (OPRM1) and several variations in the genes coding for cytochrome (CYP) P450 enzymes. Also variations in the COMT gene and the ABCB1 gene coding for P-glycoprotein have been shown to modify the response to analgesics. Genetic polymorphism of CYP2D6, CYP3A4 and CYP3A5 enzymes was studied in the experimental part of the thesis. The aim of the study was to determine if the allele and haplotype frequencies of the CYP2D6, CYP3A4 and CYP3A5 gene variations are different between Finnish breast cancer patients and healthy volunteers. The results will be further used to explore whether the genetic polymorphism of these metabolic enzymes affects the response to a certain drug substance. The study population consisted of 996 Finnish breast cancer patients. Common genetic variants affecting the enzymatic activity of CYP2D6, CYP3A4 and CYP3A5 were studied. In addition to gene copy number, ten single nucleotide polymorphisms (SNP) of the CYP2D6 gene were genotyped. For CYP3A4 gene, genotyping was done for intron 6 SNP rs35599367 shown to decrease CYP3A4 gene expression. CYP3A5 SNP 6986A>G leading to splicing defect and premature STOP codon was also genotyped. Genotyping and copy number determination was done using PCR-based TaqMan® 5'-nuclease method. CYP2D6 haplotype analysis and phenotype predictions were derived based on genotype data. According to CYP2D6 enzyme activity individuals are commonly classified as poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) or ultra-rapid metabolizers (UM). The frequencies of CYP2D6 phenotypic classes in our study population were the following: PM, 2.8%; IM 2.0 %; EM 87.7% and UM 7.6%. The haplotype and phenotype frequencies determined for breast cancer patients coincide with the values observed earlier for Finnish healthy volunteers. In our study population, the minor allele frequency (MAF) of the CYP3A4 rs35599367 SNP was 2.7% and the MAF of the CYP3A5 6986G>A SNP 7.6%. The MAF of CYP3A5 6986G>A SNP found in our study is in line with the previous findings for Finnish healthy volunteers. There are no previous publications on the frequency of CYP3A4 rs35599367 SNP in Finnish population. In conclusion, no differences were detected in the frequency of the studied CYP2D6 and CYP3A5 genetic variations between Finnish breast cancer patients and healthy volunteers. Frequency of CYP3A4 rs35599367 SNP in Finnish healthy volunteers should be determined in order to compare it with our findings in the population comprising of breast cancer patients. The results of this study can be further used to explore the effects of CYP2D6, CYP3A4 and CYP3A5 genetic polymorphism on drug response.

The effect of genes on drug response is studied in the field of pharmacogenetics. Genetic polymorphism occurs in several genes that code drug metabolizing enzymes or drug transporters. A protein coded by a variant gene may be dysfunctional, which can affect the efficiency and safety of the substrate drug individually. The common polymorphisms of the gene ABCG2 coding the efflux transporter BCRP and the gene SLCO1B1 coding the influx transporter OATP1B1 are associated with the interindividual variation in the effectiveness and tolerability of the cholesterol-lowering statins. In this study, the effects of the polymorphisms ABCG2 c.421C>A and SLCO1B1 c.521T>C on rosuvastatin concentration in plasma and the liver were studied with two different pharmacokinetic models. The developed liver model illustrating the enterohepatic circulation of drugs was compared to a commercial Simcyp model. According to the simulations with both models, the effect of the polymorphisms of OATP1B1 and BCRP on the plasma concentration of rosuvastatin is additive. The plasma concentration increases up to fourfold if the same individual has homozygous polymorphic forms of both the OATP1B1 and the BCRP. Based on the modellings, the change of the rosuvastatin concentration in the liver owing to polymorphism does not follow the same pattern as in plasma. In consequence of the polymorphism of the BCRP, the rosuvastatin concentration rises two to three times larger in the liver, which is the site of action of the statins. The polymorphism of the OATP1B1 instead causes the liver concentration to decrease little compared to the wild type. In conclusion, the efflux transporter BCRP seems to have a greater significance on regulating the concentration of rosuvastatin in the liver than the influx transporter OATP1B1. Computer modelling is worth exploiting as a supportive method of other study methods in the pharmacogenetic research, for example when the relative significance of separate transporter proteins is evaluated.

Purpose of reviewThe review describes recent advances in genetics and genomics of postoperative pain, the association between genetic variants and the efficacy of analgesics, and the role of pharmacogenomics in the selection of appropriate analgesic treatments for postoperative pain.Recent findingsRecent genetic studies have reported associations of genetic variants in catechol-O-methyltransferase (COMT), brain-derived neurotrophic factor (BDNF), voltage-gated channel alpha subunit 11 (SCN11A) and -opioid receptor (OPRM1) genes with postoperative pain. The recent pharmacogenetics studies revealed an association of the organic cation transporter 1 (OCT1) and ATP-binding cassette C3 (ABCC3) polymorphisms with morphine-related adverse effects, an effect of polymorphisms in cytochrome P450 gene CYP2D6 on the analgesic efficacy of tramadol and no effect of CYP2C8 and CYP2C9 variants on efficacy of piroxicam.SummaryGenetic variants associate with inter-individual variability in drug responses and they can affect pain sensitivity and intensity of postoperative pain. Despite the recent progress in genetics and genomics of postoperative pain, it is still not possible to precisely predict the patients who are genetically predisposed to have severe postoperative pain or who develop chronic postoperative pain.

Orexin/hypocretin peptide mutations are rare in humans. Even though human narcolepsy is associated with orexin deficiency, this is only extremely rarely due to mutations in the gene coding prepro-orexin, the precursor for both orexin peptides. In contrast, coding and non-coding variants of the OX1 and OX2 orexin receptors have been identified in many human populations; sometimes, these have been associated with disease phenotype, although most confer a relatively low risk. In most cases, these studies have been based on a candidate gene hypothesis that predicts the involvement of orexins in the relevant pathophysiological processes. In the current review, the known human OX1/HCRTR1 and OX2/HCRTR2 genetic variants/polymorphisms as well as studies concerning their involvement in disorders such as narcolepsy, excessive daytime sleepiness, cluster headache, polydipsia-hyponatremia in schizophrenia, and affective disorders are discussed. In most cases, the functional cellular or pharmacological correlates of orexin variants have not been investigated-with the exception of the possible impact of an amino acid 10 Pro/Ser variant of OX2 on orexin potency-leaving conclusions on the nature of the receptor variant effects speculative. Nevertheless, we present perspectives that could shape the basis for further studies. The pharmacology and other properties of the orexin receptor variants are discussed in the context of GPCR signaling. Since orexinergic therapeutics are emerging, the impact of receptor variants on the affinity or potency of ligands deserves consideration. This perspective (pharmacogenetics) is also discussed in the review.

Haemorrhagic transformation is a complication of recombinant tissue-plasminogen activator treatment. The most severe form, parenchymal haematoma, can result in neurological deterioration, disability, and death. Our objective was to identify single nucleotide variations associated with a risk of parenchymal haematoma following thrombolytic therapy in patients with acute ischaemic stroke. A fixed-effect genome-wide meta-analysis was performed combining two-stage genome-wide association studies (n = 1904). The discovery stage (three cohorts) comprised 1324 ischaemic stroke individuals, 5.4% of whom had a parenchymal haematoma. Genetic variants yielding a P-value < 0.05 1 x 10(-5) were analysed in the validation stage (six cohorts), formed by 580 ischaemic stroke patients with 12.1% haemorrhagic events. All participants received recombinant tissue-plasminogen activator; cases were parenchymal haematoma type 1 or 2 as defined by the European Cooperative Acute Stroke Study (ECASS) criteria. Genome-wide significant findings (P < 5 x 10(-8)) were characterized by in silica functional annotation, gene expression, and DNA regulatory elements. We analysed 7 989 272 single nucleotide polymorphisms and identified a genome-wide association locus on chromosome 20 in the discovery cohort; functional annotation indicated that the ZBTB46 gene was driving the association for chromosome 20. The top single nucleotide polymorphism was rs76484331 in the ZBTB46 gene [P = 2.49 x 10(-8); odds ratio (OR): 11.21; 95% confidence interval (CI): 4.82-26.55]. In the replication cohort (n = 580), the rs76484331 polymorphism was associated with parenchymal haematoma (P = 0.01), and the overall association after meta-analysis increased (P = 1.61 x 10(-8), OR: 5.84; 95% CI: 3.16-10.76). ZBTB46 codes the zinc finger and BTB domain-containing protein 46 that acts as a transcription factor. In silica studies indicated that ZBTB46 is expressed in brain tissue by neurons and endothelial cells. Moreover, rs76484331 interacts with the promoter sites located at 20q13. In conclusion, we identified single nucleotide variants in the ZBTB46 gene associated with a higher risk of parenchymal haematoma following recombinant tissue-plasminogen activator treatment.

To study the function and expression of nine naturally occurring single-nucleotide polymorphisms (G406R, F431L, S441N, P480L, F489L, M515R, L525R, A528T and T542A) that are predicted to reside in the transmembrane regions of the ABC transporter ABCG2. The transport activity of the variants was tested in inside-out membrane vesicles from Sf9 insect and human derived HEK293 cells overexpressing ABCG2. Lucifer Yellow and estrone sulfate were used as probe substrates of activity. The expression levels and cellular localization of the variants was compared to the wild-type ABCG2 by western blotting and immunofluorescence microscopy. All studied variants of ABCG2 displayed markedly decreased transport in both Sf9-ABCG2 and HEK293-ABCG2 vesicles. Impaired transport could be explained for some variants by altered expression levels and cellular localization. Moreover, the destructive effect on transport activity of variants G406R, P480L, M515R and T542A is, to our knowledge, reported for the first time. These results indicate that the transmembrane region of ABCG2 is sensitive to amino acid substitution and that patients harboring these ABCG2 variant forms could suffer from unexpected pharmacokinetic events of ABCG2 substrate drugs or have an increased risk for diseases such as gout where ABCG2 is implicated.