Clinical Chemistry and Laboratory Medicine (CCLM)

Published by De Gruyter

Volume 38 Issue 9

Issue of Clinical Chemistry and Laboratory Medicine (CCLM)

Contents
Journal Overview

July 27, 2005

The Basis of the Medicine of Tomorrow "Validating and Using Pharmacogenomics" Joint IFCC-Roche Diagnostics Conference, Kyoto, Japan, 1619 April 2000

Heiner Dreismann, Gérard Siest

Page range: 803-803

Abstract

This special issue of the Journal is entirely devoted to the papers delivered at the Second IFCC Roche Conference entitled "Validating and Using Pharmacogenomics" which was held in Kyoto, on the 1619 April 2000. Molecular biology techniques are expected to revolutionise drug discovery and drug treatment. The purpose of these conferences is to highlight the progress in molecular genetics and its potential for therapy and diagnosis. This Conference brought together 200 scientists. There were more than 40 invited speakers, selected oral presentations and 45 posters. In this issue we will present first some general reviews covering the international progress made in the human genome study, the methods used for genotyping and phenotyping and the knowledge on the genes involved in drug metabolism and drug absorption. A second group of papers deals with the stateoftheart in polymorphisms of drug metabolizing enzymes, oxidation enzymes (phase l), conjugation enzymes (phase 2) and transport proteins (phase 3). In a third part we have selected presentations on clinical pharmacology and laboratory medicine (clinical pharmacology, cancer, psychoactive drugs, neurodegenerative diseases). We regret that it was impossible to present the discussions which took place after the presentations, which were also very interesting at this very successful meeting. Some of the presentations will be published later in the Journal CCLM as specific reviews.

June 1, 2005

Diagnostics and the Future of Medicine

Jonathan K.C. Knowles

Page range: 804-804

June 1, 2005

Operomics: Molecular Analysis of Tissues from DNA to RNA to Protein

Samir M. Hanash

Page range: 805-813

Abstract

The identification of coding sequences in a number of species, including human in the near future, has ushered in the post-genome era. In this era, technologies are becoming available that allow the profiling of tissues and cell populations at the genomic, transcriptomic and proteomic levels. The molecular analysis of tissues at all three levels has been referred to as operomics. This review covers some basic technologies for operomics and their application to some lymphoid disorders. It is proposed that no one type of analysis is fully informative and that information that can be derived from the different compartments encompassed in operomics is complementary. Prospects for introducing such profiling technologies into the clinical laboratory will depend on their robustness, their user friendliness and the clinical relevance of the added information they provide, which cannot be captured through other technologies in use in the clinical laboratory.

June 1, 2005

Idiosyncratic Reactions to Drugs: Can Medicine Response Profiles Provide a Dynamic Drug Surveillance System?

Allen D. Roses

Page range: 815-818

Abstract

Over the next few years it will be possible to use advances in single nucleotide polymorphism map technology to correlate information from patients' DNA with their response to medicines. This provides significant opportunities to enhance current drug surveillance systems by collecting data that would enable rare serious adverse events to be predicted in subsequent patients before the medicine is prescribed. Reasonable discussion regarding perceived or real barriers to this surveillance concept is required to realize the significant patient benefits which will accrue from this system.

June 1, 2005

Hunting for Disease Genes in Multi-Functional Diseases

Chi-Pui Pang, Larry Baum, Dennis S. C. Lam

Page range: 819-825

Abstract

Disease genes may be identified through functional, positional, and candidate gene approaches. Although extensive and often labor-intensive studies such as family linkage analysis, functional investigation of gene products and genome database searches are usually involved, thousands of human disease genes, especially for monogenic diseases with Mendelian transmission, have been identified. However, in diseases caused by more than one gene, or by a combination of genetic and environmental factors, identification of the genes is even more difficult. Common examples include atherosclerosis, cancer, Alzheimer's disease, asthma, diabetes, glaucoma, and age-related macular degeneration. There have been conflicting reports on the roles of associated genes. Even with population-based case-control studies and new statistical methods such as the sib-ship disequilibrium test and the discordant alleles test, there is no agreement on whether α 2 -macroglobulin ( A2M ) is a gene for Alzheimer's disease. Another example is the inconsistent association between age-related macular degeneration and ATP-binding cassette transporter ( ABCR ). Ethnic variation causes further complications. In our investigation of LDL-receptor variants in familial hypercholesterolemia, and the trabecular meshwork inducible glucocorticoid response protein, or myocillin ( TIGR-MYOC ) mutation pattern in primary open angle glaucoma, we did find dissimilar results in Chinese compared to Caucasians. New information from the Human Genome Project and advancements in technologies will aid the search for and confirm identification of disease genes despite such challenges.

June 1, 2005

Familial Studies on the Genetics of Cardiovascular Diseases: the Stanislas Cohort

Sophie Visvikis, Catherine Sass, Celine Pallaud, Michael A. Grow, Faiez Zannad, Gérard Siest, Henry A. Erlich, Suzanne Cheng

Page range: 827-832

Abstract

In a given individual, the level of cardiovascular risk results from the combination of and interactions between genetic and environmental components. We choose to investigate segregation analysis of intermediate phenotypes in healthy nuclear families, belonging to the Stanislas cohort, a large familial cohort composed of 1006 families, which will be followed for 10 years. We developed a panel of 35 genetic markers including genes involved in lipid metabolism, regulation of blood pressure, thrombosis, platelet function, and endothelial cell adhesion. The allele frequencies of the studied polymorphisms were in agreement with those reported in other Caucasian populations. As an example of segregation analysis, we investigated carotid intima-media thickness (CIMT) variability in a subset sample of the Stanislas cohort. We found that about 30% of CIMT variability was attributable to genetic factors. Associations between CIMT and polymorphisms in apo CIII, cholesteryl ester transfer protein, methylene tetrahydrofolate reductase, and fibrinogen genes were observed and explained about 20% of CIMT variability in men. Furthermore, as another example of association studies, we investigated the relations between E-selectin polymorphisms and blood pressure interindividual variability and longitudinal changes in unrelated adults of this familial population. The E-selectin Phe 554 allele was found associated with lower systolic blood pressure and diastolic blood pressure.

June 1, 2005

Quantitative PCR

Roman Jung, Krishna Soondrum, Michael Neumaier

Page range: 833-836

Abstract

The classic molecular biology methods like Northern or Southern blot analyse non-amplified DNA or RNA, but need large amounts of nucleic acids, in many instances from tissues or cells that are heterogeneous. In contrast, polymerase chain reaction (PCR)-based techniques allow us to obtain genetic information through the specific amplification of nucleic acid sequences starting with a very low number of target copies. These reactions are characterized by a logarithmic amplification of the target sequences i.e. increase of PCR copies followed by a plateau phase showing a rapid decrease to zero of copy number increment per cycle. Accordingly, the amount of specific DNA product at the end of the PCR run bears no correlation to the number of target copies present in the original specimen. However, many applications in medicine or research require quantification of the number of specific targets in the specimen. This has generated a rapidly increasing need for the development of quantitative PCR techniques. Prominent examples are the determination of viral load in blood specimens for the diagnosis of HIV or HCV infections, the determination of changes in gene dosage through amplification or deletion e.g. of MDR-1, erb-B2, c-myc or the loss of heterozygosity in general. Finally, the analysis of gene expression on the mRNA level does require quantitative approaches to reverse transcriptase PCR, e.g. for studies in morphogenesis or the profiling of cancer cells. Recent advances in technology allow detection of the increment per cycle of a specifically generated PCR product in “real-time mode”. Together with the new powerful methods to dissect heterogeneous tissues or fractionate bodily fluids, this now sets the stage for a detailed analysis not only of the genes and genetic changes within a single cell, but also of the use such cell makes of its genes e.g. in pharmacogenomics. Examples of recent developments of the technology and their applications will be given.

June 1, 2005

Gene Amplification as Means for Determining Therapeutic Strategies in Human Cancers

Gregory J. Tsongalis, Richard W. Cartun, Andrew Ricci

Page range: 837-839

Abstract

Pharmacogenomic analysis aspires to identify individuals with specific genetic characteristics in order to predict a positive response or reduce a negative response to a therapeutic modality. While the search continues for the many single nucleotide polymorphisms which will be used in such genetic analyses, other genetic alterations in specific cell types have proven useful in determining the potential for response to therapy. One such genetic alteration is amplification of entire gene sequences which results in overexpression of a gene product or protein. Amplification of the HER2 ( neu , erbB-2 ) oncogene is found in up to 35% of human breast cancers and is associated with a poor prognosis. In addition, this genetic alteration may predict response to various therapeutic modalities. Assays are available to detect the HER2 protein receptor or copies of the HER2 gene sequence to determine eligibility for Herceptin treatment or adriamycin treatment in node positive patients, respectively. This model represents a somatic event used in the functional determination of a therapeutic strategy.

June 1, 2005

Apolipoprotein E Polymorphisms and Concentration in Chronic Diseases and Drug Responses

Gérard Siest, Philippe Bertrand, Bernard Herbeth, Monique Vincent-Viry, Françoise Schiele, Catherine Sass, Sophie Visvikis

Page range: 841-852

Abstract

Apolipoprotein (apo) E is an important circulating and tissue protein involved in cholesterol homeostasis and many other functions. The common polymorphism in the coding region of the gene, four polymorphisms in the promoter region, other additional single nucleotide polymorphisms, as well as several apo E variants have been identified. The common coding polymorphism strongly influences the lipid metabolism and the circulating concentration of apo E itself. This polymorphism is at the origin of the implication of apo E in cardiovascular and neurodegenerative diseases, but also of the relation of apo E with longevity. Probably due to its many metabolic and functional consequences, apo E polymorphism has been shown to influence the responses of patients to several drugs (fibrates, statins, hormone replacement therapy, anti-Alzheimer drugs) or environmental interventions (black tea, alcohol, diet). Apo E genotyping may be clinically helpful in defining the risk of patients and their responses to therapeutics. Finally, circulating apo E concentration appears to be altered in diseases and can be modulated by some of the drugs cited above. This parameter can thus also give interesting clinical information and could be a therapeutic target, providing it is validated. At the present time, we cannot exclude that apo E concentration may be the most prominent apo E parameter to be considered in health and disease, while apo E polymorphisms would represent only secondary parameters influencing apo E concentration.

June 1, 2005

Angiotensin I-Converting Enzyme Gene Polymorphism and Drug Response

Bruno Baudin

Page range: 853-856

Abstract

An insertion/deletion ( I/D ) polymorphism of the angiotensin I-converting enzyme (ACE) gene has been described in chromosome 17q23 of the human genome. Subjects with the genotype DD have markedly higher plasma ACE levels than those with genotype II ; although ACE concentration in plasma is not rate-limiting for the production of angiotensin II, it has been suggested that the renin-angiotensin system may have an enhanced role in cardiovascular homeostasis in subjects with DD genotype or D allele. Metaanalysis confirmed the association of the D allele with an increased risk of myocardial infarction and stroke, but these relations are still uncertain with longevity and renal deterioration. Otherwise, I allele seems to be related with an improved response to physical training. The I/D polymorphism of the ACE gene is not a marker for any form of hypertension, though some conflicting results have been described. Nevertheless this polymorphism may have an influence on the antihypertensive response, particularly when using ACE inhibitors (ACEI). For example, blood pressure normalization with captopril in patients suffering from cardiac failure would be more effective in II genotype; conversely, both regression in left ventricular hypertrophy and improvement in diastolic filling would be greater after long-term treatment with enalapril in patients with essential hypertension and DD genotype. Conflicting results were also described using ACEI as a renoprotective therapy. This review therefore supports the justification for further evaluation in appropriately powered studies.

June 1, 2005

Drug-Metabolizing Enzymes, Polymorphisms and Interindividual Response to Environmental Toxicants

Daniel W. Nebert

Page range: 857-861

Abstract

Individual risk of toxicity or cancer reflects the amount of exposure to environmental agents, combined with one's underlying genetic predisposition. More than six dozen human ecogenetic polymorphisms have been described; whereas some of these have been demonstrated to be associated with altered risks of toxicity or cancer, others presently remain equivocal and require further study. Thus, genetic differences in the regulation, expression and activity of “environmental susceptibility genes” can be decisive in defining susceptibility to toxicity or cancer. “Drug-metabolizing enzymes” (DMEs) are regarded as one class of environmental susceptibility genes. DME genes have actually existed on this planet for more than 2.5 billion years, and might more appropriately be named “effector-metabolizing enzymes.” Receptors controlling DME levels have been called “DME receptors.” DMEs have functioned in many critical life processes in prokaryotes and, more recently, in countless basic functions in plants and animals—events that evolved long before the existence of pharmaceutical companies and apothecaries. DME genes exist in every eukaryotic cell and probably in all prokaryotes. Virtually all environmental agents act as either agonists or antagonists—in competing with endogenous ligands that bind to DME receptors and/or competing as substrates for the DMEs. Over the past decade it has become clear that each of us has our own “individual fingerprint” of unique alleles coding for DMEs. The underlying genetic predisposition of each patient will reflect combinations of poor- and extensive-metabolizer phenotypes; if these enzymes cooperate in the same metabolic pathway for any given drug or environmental agent, such ecogenetic variability might be synergistic and lead to as much as 30- or >40-fold differences in activation or degradation. The end result can be large interindividual differences in risk of environmentally caused toxicity or cancer.

June 1, 2005

Database Analysis and Gene Discovery in Pharmacogenetics

Philip Board, Natasha Tetlow, Anneke Blackburn, Gareth Chelvanayagam

Page range: 863-867

Abstract

The global genome research effort has resulted in the creation of extensive DNA and protein sequence databases that are a valuable resource for the identification of new genes and polymorphic variants of enzymes of pharmacogenetic interest. Previously undescribed members of gene families with novel functions and substrate specificities can be identified by database searching and sequence alignment strategies. Since the expressed sequence tag (EST) database contains sequences from many individuals, it can be searched for evidence of polymorphisms that can significantly influence enzyme function. The different approaches to these forms of analysis are reviewed and illustrated with examples from the glutathione transferase gene family.

June 1, 2005

How to Manage Individualized Drug Therapy: Application of Pharmacogenetic Knowledge of Drug Metabolism and Transport

Christian Meisel, Ivar Roots, Ingolf Cascorbi, Ulrich Brinkmann, Jürgen Brockmöller

Page range: 869-876

Abstract

Significant fractions of health budgets must be spent for treatment of drug side effects and for inefficient drug therapy. Hereditary variants in drug metabolizing enzymes, drug transporters, and drug targets are important determinants of drug response and toxicity and may therefore aid in selection and dosage of drugs. Today there is extensive knowledge of genetic polymorphisms of cytochrome P450 (CYP) enzymes 2A6, 2C9, 2C19, and 2D6; of phase-2 enzymes such as thiopurine S-methyltransferase; and more recently of drug transporters such as the MDR-1 gene-product P-glycoprotein, affecting a significant share of currently used drugs. However, application of pharmacogenetic knowledge to clinical routine is limited in current practice. To promote the application of pharmacogenetic knowledge in clinical routine, research on genotype-based dose adjustments is still necessary—as is the promotion of faster and cheaper genotype analyses. Furthermore, the benefits of CYP genotype-directed drug therapy should be evaluated in properly designed prospective studies. Once such steps have been successfully taken, drug therapy could well become more prevention-directed and patient-tailored than it is possible today, replacing the current “one drug in one dose for one disease” strategy by a more individualized approach.

June 1, 2005

P-Glycoprotein and Bioavailability-Implication of Polymorphism

Yan Liu, Ming Hu

Page range: 877-881

Abstract

P-Glycoprotein (P-gp) may have a significant impact on systemic and tissue/cellular bioavailability of drugs because it functions as an “anti-absorption” mechanism that effluxes drug molecules out of the lipid bilayer and cytoplasm. The ability to reduce bioavailability at the tissue/cellular level was first discovered during the investigation of the causes of multidrug resistance (MDR) in cancer chemotherapy. Initially, it was thought that MDR is only caused by P-gp. Recently, many other transporters such as multidrug resistance-related protein (MRP) have also been identified. The ability of P-gp to impact systemic drug bioavailability was only recently recognized. Dr. Alfred Schinkel's group was first to show a significant improvement in the systemic bioavailability of several drugs in the MDR1 knockout mice. The same group also discovered that the blood-brain barrier (BBB) has a very high expression level of P-gp, and that this protein is necessary to restrict the entrance of various drug molecules into the central nervous system (CNS). Polymorphism in the normal human cells has not been reported, but it has been discovered in human cancer cells. Functional implication of P-gp polymorphism in changing the tissue bioavailability has been studied in rodents. These studies strongly support the role of P-gp in restricting tissue bioavailability of anticancer drugs. These studies also support the effectiveness of P-gp in limiting CNS toxicity of the cytotoxic drugs. The functional implication of P-gp on systemic bioavailability is much less well defined in humans, although it appears to be quite obvious in MDR1 knockout mice. Pharmacokinetic models clearly suggest that a change in the absorption rate will have a significant impact on systemic blood level of a drug. However, whether functionally significant polymorphisms of P-gp exist in humans has not been determined. If they do exist, they will surely impact on both systemic bioavailability and drug interaction potentials of many drugs. In the drug development process, several models may be used to select a lead compound that may or may not interact with P-gp, depending on whether the interaction is desirable. Several inhibitors of P-gp are currently on the clinical trial stage. Natural inhibitors of P-gp have also been discovered. There is no doubt that these new developments will have significant impact on the bioavailability of a variety of anticancer and CNS drugs in the next decades.

June 1, 2005

Cancer Therapy and Polymorphisms of Cytochromes P450

Stewart L. MacLeod, Susan Nowell, Joyce Massengill, Abdul Jazieh, Gail McClure, Jason Plaxco, Fred F. Kadlubar, Nicholas P. Lang

Page range: 883-887

Abstract

Cytochrome P450 (CYP) enzymes are important in the metabolism of some endogenous compounds, environmental and dietary xenobiotics and many drugs. Many of these enzymes have genetic polymorphisms that produce significant changes in metabolic activity, however the function of other polymorphisms is unknown. Genetic polymorphisms have important influences on variability in human pharmacokinetics, including intra-individual differences in drug toxicity, drug interactions and response to chemotherapy. Other factors that influence drug metabolism include differences in enzyme expression due to differences in age, gender, smoking status, exposure to dietary or environmental xenobiotics or co-administration of other drugs. In addition, some xenobiotics and drugs can directly inhibit or induce the activity of CYPs. All of these factors can produce differences in metabolic capacities among individuals which can produce toxicity in some patients and sub-effective dosing in others. Maximum clinical benefit will require a more complete understanding of the influence of these polymorphisms on allele function and their interaction with inducers and inhibitors of enzyme expression or activity. This effort will permit the pharmacogenetic screening of patients before the administration of drugs and result in the identification of individuals who are prone to adverse reactions or poor response, resulting in more effective individualized therapy.

June 1, 2005

Polymorphisms in UDP Glucuronosyltransferase Genes: Functional Consequences and Clinical Relevance

Peter I. Mackenzie, John O. Miners, Ross A. McKinnon

Page range: 889-892

Abstract

As glucuronidation is a major process for the metabolism and removal of lipophilic chemicals, polymorphic variations in genes encoding the enzymes involved in this process, the UDP glucuronosyltransferases (UGT), may have a significant impact on our capacity to detoxify and eliminate drugs and toxins. Although 24 human UGT genes have been identified to date, only polymorphisms in five UGTs, viz. UGT1A1, UGT1A6, UGT2B4, UGT2B7 and UGT2B15 have been described. Polymorphisms in UGT1A1, the major bilirubin-glucuronidating form, often result in a decreased capacity to glucuronidate bilirubin, such as observed in Gilbert Syndrome and some forms of perinatal jaundice. The frequencies of individual UGT1A1 polymorphisms show extensive variability across ethnic groups. Two variants of UGT1A6 and UGT2B4 and one variant of UGT2B7 and UGT2B15 have been identified. However, the clinical significance of these variants is unclear. More UGT polymorphisms will undoubtedly be discovered when the human genome is sequenced. However, unless the UGT in question is responsible for the exclusive metabolism of a particular drug or chemical ( e.g. UGT1A1 and bilirubin) or is the predominant or only UGT present in the cell, it is unlikely that these polymorphisms will be of major clinical significance.

June 1, 2005

The Human Multidrug Resistance-Associated Protein (MRP) Gene Family: From Biological Function to Drug Molecular Design

Toshihisa Ishikawa, M. Tien Kuo, Kyoji Furuta, Masaaki Suzuki

Page range: 893-897

Abstract

The ATP-binding cassette transmembrane proteins play an important role in transport of drugs as well as of biologically active endogenous substances. The human multidrug resistance-associated protein (MRP) subfamily consists of at least six members, exhibiting a wide spectrum of biological functions. MRP1 operates as an ATP-dependent primary active transporter for substrates conjugated with glucuronide, sulfate or glutathione. Leukotriene C 4 is an important endogenous substrate for MRP1. Glutathione serves as a cofactor in MRP1-mediated drug transport as well. Genes encoding both MRP1 and the catalytic subunit of γ-glutamylcysteine synthetase (γ-GCS) are coordinately regulated in cultured cancer cell lines as well as colorectal cancer tissues from colon cancer patients. The induction of MRP1 and γ-GCS expression by oxidative stress varies among different cell lines, and p53 mutations are associated with elevated levels of induction. To modulate the transport function of MRP1, we have synthesized novel glutathione derivatives as photoreactive biochemical probes targeting the transporter protein. GIF-0019 restored the cellular sensitivity of MRP1-overexpressing drug-resistant cancer cells to anticancer prostaglandins in vitro , which was characterized by enhanced mRNA levels of the cyclin-dependent kinase inhibitor p21, suppressed c-myc expression and G1 arrest.

June 1, 2005

Ethnic Differences in Drug Metabolism

Hong-Hao Zhou, Zhao-Qian Liu

Page range: 899-903

Abstract

Ethnic differences in drug metabolism are well documented for a number of drugs. The molecular mechanisms responsible for ethnic differences in drug metabolism have been partly clarified because of the advances in molecular biology in recent years. Gene dosage determines the drug metabolism as demonstrated for S-mephenytoin and diazepam metabolism. Genotype analysis indicates a different frequency for the mutant alleles in different ethnic populations, which results in variations in the frequency of subjects who are homozygous for the mutant allele among the extensive metabolizers in different ethnic populations. Ethnic differences in drug metabolism may result from differences in distribution of a polymorphic trait and mutations which code for enzymes with abnormal activity which occur with altered frequency in different ethnic groups.

June 1, 2005

Hypervariable Region 1 of Hepatitis C Virus Genome and Response to Interferon Therapy

Blaženka Grahovac, Jasna Bingulac-Popović, Boris Vucelić, Irena Hrstić, Rajko Ostojić, Vesna Dražić, Melita Balija, Damir Grgičević

Page range: 905-910

Abstract

The relationship between the complexity of the hypervariable region 1 (HVR1) quasispecies of hepatitis C virus (HCV) and responsiveness to interferon-α (IFN) therapy was studied in patients with chronic hepatitis C. Twelve HCV-RNA-positive patients were treated daily with high dose IFN and ribavirin for 4 weeks, and then with IFN 3 MIU (Million International Units) TIW (three times per week) and ribavirin for 6 months. The HVR1 quasispecies complexity was analyzed by nested polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP). The baseline HCV-RNA levels in the study group ranged from 10 6 to 10 7 copies/ml. All patients exhibited HCV genotype 1 b. Initial SSCP analysis revealed four (33.3%) patients with a low complexity pattern (SSCP bands ≤4) and eight (66.6%) patients with high complexity pattern (SSCP bands >4). After 4 weeks of IFN therapy, one patient became HCV negative, and among those remaining positive, the HCV-RNA levels decreased by 2 to 3 logs and the number of SSCP decreased by 2 to 3 bands per sample. After 6 months of IFN therapy, five (41.7%) patients became HCV-RNA-negative. Seven (58.3%) patients did not respond to IFN therapy with sustained viral load from 10 3 to 10 5 copies/ml, and high complexity SSCP patterns. Our data support the HVR quasispecies complexity to be an independent predictive factor for IFN responsiveness in patients infected with HCV.

June 1, 2005

A Functional Genomic Study of the Effects of Antipsychotic Agent Chlorpromazine in PC12 Cells

Outi Kontkanen, Merja Lakso, Garry Wong, Eero Castrén

Page range: 911-915

Abstract

Expression profiling using methods of functional genomics can be used to investigate changes in gene transcription induced by drug treatment, which may lead to discovery of new potential drug targets. Antipsychotic agents alleviate symptoms of schizophrenia but the mechanism behind their clinical efficacy is unclear. We have used the PC12 cell line as a model to characterize effects of the antipsychotic drug chlorpromazine on gene expression using high-density complementary DNA array filters prepared from a rat brain entorhinal cortex complementary DNA library. Chlorpromazine treatment positively regulated the expression of several clones, five of which were selected for further characterization. Northern blotting experiments confirmed the increased expression of these genes after chlorpromazine treatment. Sequencing revealed that two clones were cytochrome c oxidase and three were novel genes. Characterization of the function of these genes could increase our understanding of the mechanisms of action of antipsychotic drugs, and might be beneficial for the development of more effective agents.

June 1, 2005

Influence of Glutathione S-Transferase M1 and T1 Genotypes on Larynx Cancer Risk among Korean Smokers

Young-Joon Hong, Jin-Kyung Lee, Guk-Haeng Lee, Seok-Il Hong

Page range: 917-919

Abstract

Glutathione S-transferase (GST) isoenzymes are involved in the detoxification of major carcinogens present in tobacco smoke. It is thus conceivable that deficiency in GST activity due to homozygous deletions of the GSTM1 and GSTT1 genes (the null genotypes) may modulate susceptibility to smoking-induced cancers. The influence of the GSTM1 and GSTT1 null genotypes on larynx cancer risk among the Korean population were evaluated using peripheral blood DNA from 82 larynx cancer patients and 63 healthy controls, all of whom were male current smokers. Increased larynx cancer risk was related to the GSTM1 null genotype (odds ratio (OR)=3.53, 95% confidence interval (CI)=1.27−9.83). The OR associated with the GSTT1 null genotype was also increased, but did not reach statistical significance (OR=1.83, 95% CI=0.70−4.79). Individuals lacking both the GSTM1 and GSTT1 genes were at a significantly higher risk for larynx cancer than individuals with both genes present (OR=4.04, 95% CI=1.33−12.30). These data confirm that the GSTM1 null genotype is an important risk modifier for larynx cancer among Korean smokers and combined GSTM1 and GSTT1 null genotypes could be a useful predictor of genetic susceptibility to larynx cancer.

June 1, 2005

CYP2D6 Genotyping in Patients on Psychoactive Drug Therapy

Elizabeta Topić, Mario Štefanović, Ana Maria Ivanišević, Franciska Blazinić, Jadranka Čulav, Željko Skočilić

Page range: 921-927

Abstract

The polymorphic isoenzyme CYP2D6 has a major role in the oxidative metabolism of many deal of psychoactive drugs. Its six mutant alleles (null alleles *3, *4, *5, *6, *7 and *8 ) encode for inactive enzyme molecules. A carrier of two mutant alleles is considered a poor metabolizer phenotype, while a carrier of only one damaged allele is considered an intermediate metabolizer phenotype. The aim of the study was to assess the prevalence of null alleles in a group of psychiatric patients suffering from depression (n=49) and schizophrenia (n=86) in comparison with healthy individuals (n=145) by the method of multiplex allele specific PCR. Only CYP2D6*3,*4 and *6 mutant alleles were found in the study subjects. No significant difference between the depression and control groups was found for allele prevalence, genotype or phenotype distribution (p>0.05). However, a significant difference was observed between schizophrenic patients and controls for allele frequency (p=0.002), genotype distribution (p=0.016), and phenotype prevalence (p=0.018). The odds ratio of 2.542 for 2D6*4 suggested a significant association between this allele and schizophrenia, significantly contributing to poor metabolizer phenotype (odds ratio=5.020). The relationship between CYP2D6 gene polymorphism and side effects in schizophrenic patients undergoing long-term psychoactive drug therapy was investigated. A significant difference was obtained for allele prevalence (p=0.002), genotype (p=0.029), and phenotype (p=0.002) distribution between patients without and with side effects. A relative risk of 2.626 and 5.333 for 2D6*4 and 2D6*6 , respectively, and of 7.08 for poor metabolizer phenotype suggested a significant association between the hereditary susceptibility for a particular type of drug metabolism (defect alleles) and side effects. These preliminary r e s u l t s suggest that the CYP2D6 genotyping appears to be useful for predicting risks for side effects of psychoactive drugs in schizophrenic patients, but their usefulness should be further explored.

June 1, 2005

Genotyping of CYP2D6 in Parkinsons's Disease

Mario Štefanovic, Elizabeta Topić, Ana Maria Ivanišević, Maja Relja, Marta Koršić

Page range: 929-934

Abstract

Parkinson's disease is characterized by progressive degradation of dopaminergic neurons. Cytochrome P450 CYP2D6 enzyme is one of the most investigated and highly polymorphic isoforms, which metabolizes many drugs and is also involved in the metabolism of dopamine. Using allele-specific multiplex PCR, we genotyped 186 subjects for CYP2D6 *3, *4, *6, *7, and *8 alleles in order to estimate allelic, genotype and predicted phenotype frequencies in the control and patient groups, and to investigate the possible statistical difference between Parkinson's disease patients (n=41) and healthy controls (n=145). Parkinson's disease patients were further divided into two subgroups according to Hoehn and Yahr staging of the disease (HY), i.e. groups with HY stage less than 2.5 (HY <2.5; n=27) and more than 2.5 (HY >2.5; n=14). A subgroup of Parkinson's disease patients exhibiting side effects such as “on-off” phenomenon and dyskinesia (both suggesting favorable response to therapy) were compared with a subgroup of patients showing no such response. The preliminary results of this study showed that only the prevalence of CYP2D6 *4 allele differed significantly between the PD patients and control group (20.7% vs. 11.0%; p=0.027; RR=2.1, 95%CI 1.113−3.994). In the HY >2.5 subgroup, the CYP2D6*4 allelic difference was even greater (25.0% vs. 11.0% in controls; p=0.062, RR=2.69, 95%CI 1.090−6.624). Genotype frequencies differed only in the HY >2.5 subgroup, however with a level of significance of p=0.095.

June 1, 2005

Rapid Analysis of CGG Repeat Length in the FMR1 Gene

Priscilla M.K. Poon, Zheng Zhao, Xiang-qian Wu, Yu-xing Ni, Chi-Pui Pang

Page range: 935-938

Abstract

The number of trinucleotide CGG repeats at the 5′ untranslated region of the FMR1 gene is associated with the fragile X syndrome of mental retardation. We screened for the CGG repeat length in the FMR1 gene of the X-chromosomes from unrelated normal Chinese subjects recruited in Hong Kong and Dalian, a southern and a northern Chinese city respectively. These cities are about 3000 km apart and the residents have few historical interactions. Genomic DNA was analysed by PCR and detected by Southern hybridisation with a radiolabelled (CGG) 5 probe for the CGG repeat number. A different distribution pattern of CGG allele size from the Caucasians is observed. It is a bimodal pattern with the most common CGG repeats allele at 29 against 30 in the Caucasians. Among the Hong Kong subjects, five alleles of more than 50 CGG repeats were detected, and four of those were in heterozygous females. There was no difference in the repeat patterns in subjects from the two cities, suggesting no genotypic variation in FMR1 between northern and southern Chinese.

June 1, 2005

Multiplex In-cell Reverse Transcription-Polymerase Chain Reaction for the Simultaneous Detection of p210 and p190 BCR-ABL mRNAs in Chronic Myeloid Leukemia and Philadelphia-Positive Acute Lymphoblastic Leukemia Cell Lines

Soon Pal Suh, Seung Jung Kee, Woo Hyun Lim, Jeong Won Song, Sang Khoo Lee, Jong Phil Kim, Jong Hee Shin, Dong Wook Ryang

Page range: 939-944

Abstract

We designed a novel multiplex in-cell reverse transcription- polymerase chain reaction method for the simultaneous detection and differentiation of p190 and p210 BCR-ABL mRNAs within single cells from the human chronic myeloid leukemia and Philadelphia positive acute lymphoblastic leukemia. Human K562 chronic myeloid leukemia and SUP B-15 Ph+ acute lymphoblastic leukemia cell lines were used as positive controls for p210 and p190 BCR-ABL mRNAs, respectively. HL60 cell line was used as a negative control. After the leukemia cells were fixed and permeabilized, without extracting nucleic acids, the mRNAs were reverse transcribed to cDNAs, and the cDNAs were amplified by multiplex polymerase chain reaction with fluorescent primers specific for p190 and p210 BCR-ABL mRNAs. After transfer onto glass slides by cytospin, the amplified cells were detected by fluorescence microscopy. Fluorescence microscopy after propidium iodide or 4′,6-diamidino-2-phenylindone counterstaining showed that the positive K562 cells exhibited a yellow-green fluorescent cytoplasm around a red nucleus, and that the positive SUP B-15 cells exhibited an orange cytoplasm around a blue nucleus. Only the red or blue nucleus was visible in respective negative HL60 cells. The specificity of amplification was confirmed by the absence of a signal when control experiments were performed either with RNase digestion of mRNA or without reverse transcriptase/ Taq polymerase. We conclude that the multiplex in-cell reverse transcription-polymerase chain reaction method is capable of simultaneously detecting and differentiating the p210 and p190 BCR-ABL mRNAs of chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia cells, and that it may be useful in quantitatively monitoring the minimal residual disease during therapy.

About this journal

Objective
Clinical Chemistry and Laboratory Medicine (CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. It is focused on basic and applied research and cutting-edge clinical laboratory medicine. CCLM is one of the leading journals in the field, with an impact factor over 8. All contributions submitted for publication in CCLM are single-blind peer reviewed by at least two experts in the field. CCLM is led by a multi-institutional editorial board. It is issued monthly, both in print and electronically. Letters to the Editor and Congress Abstracts are published online only.

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Topics

clinical biochemistry
clinical genomics and molecular biology
clinical haematology and coagulation
clinical immunology and autoimmunity
clinical microbiology
drug monitoring and analysis
evaluation of diagnostic biomarkers
disease-oriented topics (cardiovascular disease, cancer diagnostics, diabetes)
new reagents, instrumentation and technologies
new methodologies
reference materials and methods
reference values and decision limits
quality and safety in laboratory medicine
translational laboratory medicine
clinical metrology

Article formats
Research Articles, Reviews, Mini Reviews and Opinion Papers on all aspects of clinical chemistry and laboratory medicine, Guidelines and Recommendations, Point/Counterpoint Articles, Letters to the Editor, Editorials

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