CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real-time PCR

The genetic polymorphism of the cytochrome P450 monooxygenase, CYP2D6, comprises at least 43 alleles giving rise to distinct drug metabolism phenotypes termed ultrarapid, extensive, intermediate, and poor metabolizers. As a consequence, drug side effects or lack of drug effect may occur if standard doses are applied. Genetic prediction of drug oxidation phenotype as a basis for dose selection requires analysis of single nucleotide polymorphisms and of alleles with duplicated or deleted genes. Here we developed a novel method to determine the CYP2D6 gene dose per genome. A TaqMan real-time PCR assay to specifically amplify genomic CYP2D6 was established by using a specific set of amplification primers and probe, located in exon 9, which effectively prevent amplification of CYP2D7 and CYP2D8 pseudogenes. Quantitative CYP2D6 amplification data were normalized to albumin as an internal reference gene which was coamplified simultaneously in a single-tube biplex assay. The assay was validated with a selection of previously genotyped DNA samples containing none, one, two, or three CYP2D6 gene copies. The results were highly reproducible and closely matched the number of genes with no overlap between the groups. Analysis of DNA samples comprising all major alleles and genotypes revealed high sensitivity and specificity of the assay, as demonstrated by agreement of the determined gene dose with the presence of CYP2D6(*)2 x 2 (gene duplication) and CYP2D6(*) 5 (gene deletion) alleles. The predictability of the new strategy was systematically evaluated. The semiautomatic TaqMan assay allows high sample throughput and will be useful for pharmacogenetic studies and in the clinical setting.     

Inhibition of cell cycle and induction of apoptosis by 2‐oxoheksyl isothiocyanate and alyssin in cell lines carrying various inherited BRCA1 mutations

An important aspect of the chemopreventive activity of isothiocyanates (ITC) is their ability to induce cell growth inhibition and apoptosis. In this study, the effect of two sulforaphane analogues, 2‐oxoheksyl isothiocyanate and alyssin, on lymphoblastoid cells, derived from people carrying four different germ‐line mutations in BRCA1 gene, was tested and compared to the effect on wild type cells. The mutations studied were: C61G; 3819del5; 4153delA, and 5382INSC. Changes in cell viability and density after 2‐oxoheksyl isothiocyanate and alyssin treatment were evaluated, as well as cell cycle progression, mitochondrial membrane potential changes, and phosphatidylserine externalization. Both isothiocyanates were shown to reduce cell viability and density in all cell lines tested, as well as the change in cell cycle phase's distribution. The response of cells to two ITC tested was various, as well as mutation type‐modulated. We found that change of cellular maintenance by chemopreventive agents can be modulated by single allele BRCA1 mutation. Drug Dev. Res. 65:84–92, 2005. © 2005 Wiley‐Liss, Inc.     

Pharmacogenetics of chronic cardiovascular drugs: applications and implications

Cardiovascular disease continues to be a tremendous worldwide problem, and drug therapy is a major modality to attenuate its burden. At present, conditions such as hypertension, dyslipidaemia and heart failure are pharmacologically managed with an empirical trial-and-error approach. However, it has been suggested that this approach fails to adequately address the therapeutic needs of many patients, and pharmacogenetics has been offered as a tool to enhance patient-specific drug therapy. This review outlines pharmacogenetic studies of common cardiovascular drugs, such as diuretics, β-blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, statins and warfarin, ultimately highlighting considerations for future research and practice.     

European Medicines Agency initiatives and perspectives on pharmacogenomics

Pharmacogenomics, the study of variations of DNA and RNA characteristics as related to drug response, has become an integral part of drug development and pharmacovigilance, as reflected by the incorporation of pharmacogenomic data in EU product information. In this short review article, we describe recent European Medicines Agency initiatives intended to support further the implementation of pharmacogenomics in drug development and surveillance so that patients and the public can benefit from advances in genomic science and technology.     

Stroke pharmacogenomics

Circulatory disease accounts for fifteen million deaths each year, of which stroke accounts for four and a half million- with an estimated nine million stroke survivors annually. The overall incidence rate of stroke is 2 to 2.5 per thousand adults with an approximate prevalence of 5 per thousand and an estimated 5-year risk of stroke recurrence of 15 to 40 percent. Conventional risk factors for stroke include: increasing age, hypertension, diabetes mellitus, smoking, increased body mass index, ischemic heart disease, heart failure, atrial fibrillation and lack of physical activity. Age is the strongest risk factor for both ischemic and haemorrhagic stroke with its incidence doubling for each successive decade after the age of fifty-five years. However, there is a substantial portion of patients with significant cerebrovascular disease who do not have any of these stroke risk-factors, leading to the speculation that there are other factors that have not been identified yet So as to improve diagnosis and treatment strategies, as well as to reduce the related public health burden, it could be helpful to successfully identify its extremely complex genetic determinants (polygenic, multiple genes play a role).

Pharmacogenetics is the field of pharmacology that deals with the influence of genetic variation on drug response by correlating gene expression and gene variants with the efficacy or toxicity of drugs. The principle drugs in stroke medicine are antithrombotics. The aim of this paper was to review the most commonly used drugs for stroke such as rtPA in the acute phase as well as antiplatelets and wafarin for secondary prophylaxis.     

Pharmacogenomics and personalized medicine: a review focused on their application in the Chinese population

The field of pharmacogenomics was initiated in the 1950s and began to thrive after the completion of the human genome project 10 years ago. Thus far, more than 100 drug labels and clinical guidelines referring to pharmacogenomic biomarkers have been published, and several key pharmacogenomic markers for either drug safety or efficacy have been identified and subsequently adopted in clinical practice as pre-treatment genetic tests. However, a tremendous variation of genetic backgrounds exists between different ethnic groups. The application of pharmacogenomics in the Chinese population is still a long way off, since the published guidelines issued by the organizations such as US Food and Drug Administration require further confirmation in the Chinese population. This review highlights important pharmacogenomic discoveries in the Chinese population and compares the Chinese population with other nations regarding the pharmacogenomics of five most commonly used drugs, ie, tacrolimus, cyclosporine A, warfarin, cyclophosphamide and azathioprine.     

Genetic Mapping and Tailored Antihypertensive Therapy

A tailored or individualized antihypertensive therapy represents the new frontier for the treatment of essential hypertension and its organ complications. Indeed, individual variation in the efficacy and tolerability of antihypertensive drugs in human essential hypertension is currently experienced by all physicians and is linked to the genetic heterogeneity of this multifactorial disease. Different approaches have been pursued in the attempt to correlate specific responsiveness to the therapy with phenotypic traits of the patients, but with poor results. More recently, the genetic approach to the study of the mechanisms underlying hypertension has led to the identification of some quantitative trait loci or genes that influence blood pressure both in animal models and in patients.But the relevance of these polymorphisms for defining and classifying patients in terms of therapy responsiveness must be analyzed in a more complex context that takes into account the crucial aspects of environmental influences, stage of disease, previous treatments, efficacy, tolerance, and duration of the treatment. Only a few examples of a pharmacogenomic approach to hypertension therapy are now available. In particular, the association of different variants of ACE, angiotensinogen, and G-protein genes with the blood pressure response to drugs interfering with RAS or -adrenergic receptor has been studied. However, the results of these studies cannot be considered conclusive, since not all the criteria have been fully applied for proper assessment of an association between genetic polymorphism and drug response. Our group has identified a polymorphism of the genes coding for the cytoskeletal protein, adducin, which is associated with both rat and human hypertension, sodium sensitivity, and the antihypertensive effects of diuretics. A modification of the renal Na–KATPase leading to an increase of tubular sodium reabsorption seems to be the most likely underlying mechanism. A new antihypertensive compound has been developed that can correct the abnormality of the renal Na–KATPase and the blood pressure increase associated with adducin polymorphism in the rat. At present, the antihypertensive activity of this compound is under evaluation in patients with different adducin genotypes.