Vangelis G. Manolopoulos

Regulation of a swelling‐activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins

1 The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling‐activated Cl− current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole‐cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5 % (mild hypotonicity) or 25 % (strong hypotonicity). 2 The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited ICl,swell with IC50 values of, respectively, 9.2 ± 0.2, 61.4 ± 1.7 and 62.9 ± 1.3μM. Tyrphostin A1, a tyrphostin analogue with little effect on PTK activity, and daidzein, an inactive genistein analogue, were without effect on ICl,swell. 3 The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 μM) and dephostatin (20 μM) potentiated ICl,swell activated by mild hypotonicity by 47 ± 9 and 69 ± 15 %, respectively. 4 Intracellular perfusion with GTPγS (100 μM) transiently activated a Cl− current with an identical biophysical and pharmacological profile to ICl,swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity‐induced cell shrinkage completely inhibited the GTPγS‐activated Cl− current. 5 Intracellular perfusion with GDPβS (1 mM) caused a time‐dependent inhibition of ICl,swell, which was more pronounced when the current was activated by mild hypotonicity. 6 Our results demonstrate that the activity of endothelial swelling‐activated Cl− channels is dependent on tyrosine phosphorylation and suggest that G proteins regulate the sensitivity to cell swelling.

A randomized trial of genotype-guided dosing of acenocoumarol and phenprocoumon.

BACKGROUND Observational evidence suggests that the use of a genotype-guided dosing algorithm may increase the effectiveness and safety of acenocoumarol and phenprocoumon therapy. METHODS We conducted two single-blind, randomized trials comparing a genotype-guided dosing algorithm that included clinical variables and genotyping for CYP2C9 and VKORC1 with a dosing algorithm that included only clinical variables, for the initiation of acenocoumarol or phenprocoumon treatment in patients with atrial fibrillation or venous thromboembolism. The primary outcome was the percentage of time in the target range for the international normalized ratio (INR; target range, 2.0 to 3.0) in the 12-week period after the initiation of therapy. Owing to low enrollment, the two trials were combined for analysis. The primary outcome was assessed in patients who remained in the trial for at least 10 weeks. RESULTS A total of 548 patients were enrolled (273 patients in the genotype-guided group and 275 in the control group). The follow-up was at least 10 weeks for 239 patients in the genotype-guided group and 245 in the control group. The percentage of time in the therapeutic INR range was 61.6% for patients receiving genotype-guided dosing and 60.2% for those receiving clinically guided dosing (P=0.52). There were no significant differences between the two groups for several secondary outcomes. The percentage of time in the therapeutic range during the first 4 weeks after the initiation of treatment in the two groups was 52.8% and 47.5% (P=0.02), respectively. There were no significant differences with respect to the incidence of bleeding or thromboembolic events. CONCLUSIONS Genotype-guided dosing of acenocoumarol or phenprocoumon did not improve the percentage of time in the therapeutic INR range during the 12 weeks after the initiation of therapy. (Funded by the European Commission Seventh Framework Programme and others; EU-PACT ClinicalTrials.gov numbers, NCT01119261 and NCT01119274.).

Genotype-guided dosing of coumarin derivatives: the European pharmacogenetics of anticoagulant therapy (EU-PACT) trial design

The narrow therapeutic range and wide interpatient variability in dose requirement make anticoagulation response to coumarin derivatives unpredictable. As a result, patients require frequent monitoring to avert adverse effects and maintain therapeutic efficacy. Polymorphisms in VKORC1 and CYP2C9 jointly account for about 40% of the interindividual variability in dose requirements. To date, several pharmacogenetic-guided dosing algorithms for coumarin derivatives, predominately for warfarin, have been developed. However, the potential benefit of these dosing algorithms in terms of their safety and clinical utility has not been adequately investigated in randomized settings. The European Pharmacogenetics of Anticoagulant Therapy (EU-PACT) trial will assess, in a single-blinded and randomized controlled trial with a follow-up period of 3 months, the safety and clinical utility of genotype-guided dosing in daily practice for the three main coumarin derivatives used in Europe. The primary outcome measure is the percentage time in the therapeutic range for international normalized ratio. This report describes the design and protocol for the trial.

Realities and expectations of pharmacogenomics and personalized medicine: impact of translating genetic knowledge into clinical practice

The implementation of genetic data for a better prediction of response to medications and adverse drug reactions is becoming a reality in some clinical fields. However, to be successful, personalized medicine should take advantage of an informational structured framework of genetic, phenotypic and environmental factors in order to provide the healthcare system with useful tools that can optimize the effectiveness of specific treatment. The impact of personalized medicine is potentially enormous, but the results that have so far been gathered are often difficult to translate into clinical practice. In this article we have summarized the most relevant applications of pharmacogenomics on diseases to which they have already been applied and fields in which they are currently emerging. The article provides an overview of the opportunities and shortcomings of the implementation of genetic information into personalized medicine and its full adoption in the clinic. In the second instance, it provides readers from different fields of expertise with an accessible interpretation to the barriers and opportunities in the use/adoption of pharmacogenomic testing between the different clinical areas.

Pharmacogenomics education: International Society of Pharmacogenomics recommendations for medical, pharmaceutical, and health schools deans of education.

Pharmacogenomics would be instrumental for the realization of personalized medicine in coming decades. Efforts are evident to clarify the potential bioethical, societal, and legal implications of key pharmacogenomics-based technologies projected to be soon introduced into the core practice of medicine. In sharp contrast, a lack of sufficient attention to educational aspects of pharmacogenomics, both for professionals and for society at large, is evident. In order to contribute to this discussion, a ‘Pharmacogenomics Education Forum’ was held on October 2, 2004 during the 3rd Annual Meeting of the International Society of Pharmacogenomics (ISP) at Santorini, Greece. The participants, members of the ISP Pharmacogenomics Education Forum, after deliberate discussions, proposed a document of ‘Background Statement’ and ‘Recommendations and Call for Action’ addressed to Deans of Education at Medical, Pharmaceutical, and Health Schools globally. This document has been considered by the education committee of the International Society of Pharmacogenomics and the result is presented here. We hope that this call would be listened to, and soon followed by beneficial action, ultimately leading to enhanced implementation of personalized medicine into core medical education and practice.

Genetic polymorphisms of drug-metabolizing enzymes CYP2D6, CYP2C9, CYP2C19 and CYP3A5 in the Greek population.

The aim of the present study was to determine the prevalence of the most common allelic variants of the polymorphic cytochrome P450 (CYP) enzymes CYP2D6, CYP2C9, CYP2C19 and CYP3A5 and to predict the genotype frequency for each polymorphism in the Greek population. DNA isolated from peripheral blood samples derived from 283 non‐related Greek ethnic subjects was used to determine the frequency of CYP2D6*3, CYP2D6*4, CYP2C9*2, CYP2C9*3 and CYP3A5*3 allelic variants by the polymerase chain reaction (PCR)‐restriction fragment length polymorphism method, CYP2C19*2 and CYP2C19*3 with allelic specific amplification (PCR‐ASA), and CYP2D6*2 (gene duplications) by long PCR analysis. The allelic frequencies (out of a total of 566 alleles) for CYP2D6*3 and CYP2D6*4, were 2.3% and 17.8%, respectively, while gene duplications (CYP2D6*2) were found in 7.4% of the subjects tested. For CYP2C9*2 and CYP2C9*3 polymorphisms the allelic frequencies were 12.9% and 8.13% respectively. For CYP2C19, the *2 polymorphism was present at an allelic frequency of 13.1%, while no subjects were found carrying the CYP2C19*3 allele. Finally, the CYP3A5*3 allele was abundantly present in the Greek population with an allelic frequency of 94.4%. Overall our results show that the frequencies of the common defective allelic variants of CYP2C9, CYP2C19 and CYP3A5 in Greek subjects are similar to those reported for several other Caucasian populations. Finally, a high prevalence of CYP2D6 gene duplication among Greeks was found, a finding that strengthens the idea that a South/North gradient exists in the occurrence of CYP2D6 ultrarapid metabolizers in European populations.

Need for reassessment of reported CYP2C19 allele frequencies in various populations in view of CYP2C19*17 discovery: the case of Greece

INTRODUCTION CYP2C19*17 is a novel variant allele causing ultrarapid metabolism of CYP2C19 substrates. In the present study we investigated the CYP2C19*17 allelic frequency and recalculated previously reported frequencies of the CYP2C19*1/*1 genotype and of all genotype-derived phenotypes for CYP2C19 in the Greek population. MATERIALS & METHODS A total of 283 nonrelated healthy Greek ethnic subjects that had already been genotyped for CYP2C19*2 and *3 alleles as well as for CYP2D6 and CYP2C9 variant alleles participated in the study. The CYP2C19*17 allele was genotyped by the PCR-RFLP method. RESULTS The CYP2C19*17 allele frequency was 19.61%. The prevalence of CYP2C19*17 carriers in the Greek population was estimated at 31.80%, while the frequency of CYP2C19*1/*1 genotype was recalculated to 44.17% from 75.97% in our previous study. Several subjects possessing both CYP2C19*17 and variant alleles of CYP2D6 and CYP2C9 were also identified. DISCUSSION & CONCLUSION The CYP2C19*17 allele is present in Greeks at a high frequency similar to that found in other European populations of Caucasian origin. Our study highlights the need of reassessing and updating CYP2C19 allelic frequencies in various populations in view of the major role that CYP2C19*17 may have in predicting the clinical outcome of drugs metabolized by CYP2C19.

The association of adipokine levels in plasma and synovial fluid with the severity of knee osteoarthritis

OBJECTIVE We sought to determine the association between plasma and SF levels of leptin and adiponectin in patients with knee OA. METHODS Plasma and SF levels of adipokines and soluble leptin receptor (sOB-R) were determined by ELISA in 96 patients with knee OA at different stages, according to Ahlbacks classification. RESULTS Levels of adiponectin, leptin, sOB-R and free leptin in plasma and SF did not differ significantly across categories of OA severity. However, the ratio of SF to plasma leptin was significantly lower in the advanced OA stage compared with early stages of the disease (P = 0.02). After adjustment for sex and BMI, plasma leptin positively correlated with categories of OA severity (r = 0.23, P = 0.02), whereas SF/plasma leptin negatively correlated with OA stage (r = -0.27, P = 0.01). Cluster analysis showed that all men were included in one cluster and distributed in different stages of OA, whereas women formed three clusters with similar BMI, but those who were older and had the highest plasma leptin levels suffered from advanced OA. CONCLUSION Plasma leptin positively correlated with the severity of knee OA. The ratio of SF to plasma leptin might be a marker related to the severity of knee OA. Further studies should investigate whether similar associations exist in other joints affected by OA.

A global view of the OCA2-HERC2 region and pigmentation

Mutations in the gene OCA2 are responsible for oculocutaneous albinism type 2, but polymorphisms in and around OCA2 have also been associated with normal pigment variation. In Europeans, three haplotypes in the region have been shown to be associated with eye pigmentation and a missense SNP (rs1800407) has been associated with green/hazel eyes (Branicki et al. in Ann Hum Genet 73:160–170, 2009). In addition, a missense mutation (rs1800414) is a candidate for light skin pigmentation in East Asia (Yuasa et al. in Biochem Genet 45:535–542, 2007; Anno et al. in Int J Biol Sci 4, 2008). We have genotyped 3,432 individuals from 72 populations for 21 SNPs in the OCA2-HERC2 region including those previously associated with eye or skin pigmentation. We report that the blue-eye associated alleles at all three haplotypes were found at high frequencies in Europe; however, one is restricted to Europe and surrounding regions, while the other two are found at moderate to high frequencies throughout the world. We also observed that the derived allele of rs1800414 is essentially limited to East Asia where it is found at high frequencies. Long-range haplotype tests provide evidence of selection for the blue-eye allele at the three haplotyped systems but not for the green/hazel eye SNP allele. We also saw evidence of selection at the derived allele of rs1800414 in East Asia. Our data suggest that the haplotype restricted to Europe is the strongest marker for blue eyes globally and add further inferential evidence that the derived allele of rs1800414 is an East Asian skin pigmentation allele.

Pharmacogenomics of oral antidiabetic medications: current data and pharmacoepigenomic perspective.

Type 2 diabetes mellitus (T2DM) is an increasingly prevalent disease. Several classes of drugs are currently available to treat T2DM patients; however, clinical response to these drugs often exhibits significant variation among individuals. For the oral antidiabetic drug classes of sulfonylureas, nonsulfonylurea insulin secretagogs, biguanides and thiazolidinediones, pharmacogenomic evidence has accumulated demonstrating an association between specific gene polymorphisms and interindividual variability in their therapeutic and adverse reaction effects. These polymorphisms are in genes of molecules involved in metabolism, transport and therapeutic mechanisms of the aforementioned drugs. Overall, it appears that pharmacogenomics has the potential to improve the management of T2DM and help clinicians in the effective prescribing of oral antidiabetic medications. Although pharmacogenomics can explain some of the heterogeneity in dose requirements, response and incidence of adverse effects of drugs between individuals, it is now clearly understood that much of the diversity in drug effects cannot be solely explained by studying the genomic diversity. Epigenomics, the field that focuses on nongenomic modifications that influence gene expression, may expand the scope of pharmacogenomics towards optimization of drug therapy. Therefore, pharmacoepigenomics, the combined analysis of genetic variations and epigenetic modifications, holds promise for the realization of personalized medicine. Although pharmacoepigenomics has so far been evaluated mainly in cancer pharmacotherapy, studies on epigenomic modifications during T2DM development provide useful data on the potential of pharmacoepigenomics to elucidate the mechanisms underlying interindividual response to oral antidiabetic treatment. In summary, the present article focuses on available data from pharmacogenomic studies of oral antidiabetic drugs and also provides an overview of T2DM epigenomic research, which has the potential to boost the development of pharmacoepigenomics in antidiabetic treatment.