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Chaykovska L.,Charite University Hospital | Chaykovska L.,University of Zürich | Zientara A.,City Hospital Triemli | Reser D.,University of Zürich | And 4 more authors.
Clinical Laboratory | Year: 2014

Background: Cardiovascular diseases are the leading cause of death in developed countries. The underlying mechanism is often atherosclerotic remodeling of blood vessels in organs such as heart, kidney, brain, and large arteries in case of peripheral arterial disease. Beside environmental and behavioral factors such as smoking or lack of physical activity, genetic variants in genes involved in lipid metabolism, blood pressure regulation, oxidative stress, and coagulation play a prominent role in the pathogenesis of atherosclerosis. Methods: Thus, we developed and validated for clinical use and research a macroarray system for the simultaneous detection of key genetic variants in genes involved in lipid metabolism, blood pressure regulation, oxidative stress, and coagulation. Results: When compared with standard PCR technologies to determine all these genetic variants in parallel, the macroarray system (MutaCHIP® ARTERO) was as accurate but faster, cheaper, and easier to handle compared to classical real time PCR based technologies. Conclusions: MutaCHIP® ARTERO is a gene chip for diagnostics of a complex genetic panel involved in the pathogenesis of atherosclerosis. This method is as sensitive and precise as real time PCR and is able to replicate real time PCR data previously validated in evaluation studies.


Chaudhrya S.R.,University of Bonn | Muhammada S.,University of Bonn | Eidensb M.,PharmGenomics GmbH | Klemmb M.,PharmGenomics GmbH | And 3 more authors.
Current Drug Metabolism | Year: 2014

Interindividual variability in drug response depends on a number of genetic and environmental factors. The metabolic enzymes are well known for their contribution to this variability due to drug-drug interactions and genetic polymorphisms. The phase I drug metabolism is highly dependent upon the cytochrome P450 mono-oxygenases (CYP) and their genetic polymorphism leads to the variable internal drug exposures. The highly polymorphic CYP2C9, CYP2C19 and CYP2D6 isozymes are responsible for metabolizing a large portion of routinely prescribed drugs and contribute significantly to adverse drug reactions and therapeutic failures. In this review, two attractive and easily implementable approaches are highlighted to recommend drug doses ensuring similar internal exposures in the face of these polymorphisms. The first approach relies on subpopulation-based dose recommendations that consider the original population dose as an average of the doses recommended in genetically polymorphic subpopulations. By using bioequivalence principles and assuming linear gene-dose effect, dose recommendations can be made for different metabolic phenotypes. The second approach relates area under the curve to two characteristic parameters; the contribution ratio (CR), computes for the contribution of the metabolic enzyme and the fractional activity (FA), considers the impact of the genetic polymorphism. This approach provides valid and error free internal drug exposure predictions and can take into consideration genetic polymorphisms and drug interactions and/ or both simultaneously. Despite certain advantages and limitations, both approaches provide a good initial frame-work for devising models to predict internal exposure and individualize drug therapy, one of the promises from human genome project. © 2014 Bentham Science Publishers.


Lorenz M.,PharmGenomics GmbH | Lorenz M.,University of Bonn | Weise A.,PharmGenomics GmbH | Prause S.,PharmGenomics GmbH | And 9 more authors.
Clinical Laboratory | Year: 2012

Background: It is widely accepted that many medications exhibit inter-individual variability in their efficacy and toxicity due to polymorphisms in genes encoding drug-metabolising enzymes. One of the most often cited examples in this context is thiopurine S-methyltransferase (TPMT) polymorphism. TPMT is a phase 2 detoxification enzyme that catalyzes the S-methylation of thiopurine drugs such as thioguanine and 6-mercaptopurine. Approximately 11% of the Caucasian population carry a heterozygous deficiency of this enzyme causing intermediate enzyme activity, whereas 0.3% show a homozygous deficiency. In both cases, severe myelosuppression can develop upon treatment with thiopurines. These are commonly used in the treatment of leukemia. Therefore, genotyping of patients before treatment is absolutely necessary. Development of a fast and reliable real-time PCR application for TPMT genotyping would greatly improve thiopurine treatment regimens and allow the avoidance of adverse drug reactions. Methods: Blood was obtained from a Caucasian cohort of 143 individuals. After extraction of DNA, all samples were genotyped for TPMT polymorphisms *2, *3A, *3B, and *3C by real-time PCR as well as by PCR-RFLP as the reference method, in order to validate the new method. Results: Four different genotypes were found in the population studied. Of the 143 individuals investigated, 1 was heterozygous for TPMT*2 (0.70%), 2 were heterozygous for TPMT*3B (1.40%), and 8 heterozygous for TPMT-*3C (5.60%). No homozygous genotype could be identified. In total, 7.7% of the individuals carried mutations. Results from the newly developed real-time PCR were 100% concordant with those obtained using standard PCR-RFLP analysis, leading to 100% sensitivity and specificity. The hands-on time is approximately one third of the time needed for standard PCR-RFLP methods. Conclusions: A new high-throughput genotyping method could be successfully established and optimised for the commonly found mutant alleles TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A), and TPMT*3C (A719G) via real-time PCR on the LightCycler®(Roche) instrument and using the standard PCR-RFLP as reference method.


Weise A.,IKFE Institute for Clinical Research and Development | Weise A.,PharmGenomics GmbH | Prause S.,PharmGenomics GmbH | Eidens M.,PharmGenomics GmbH | And 6 more authors.
Clinical Laboratory | Year: 2010

Background: Drug degradation in the human organism is driven by detoxification mechanisms that can be affected in their efficiency by genetic mutations. The purpose of this pilot investigation was to investigate whether Type 2 diabetes is associated with mutations in prominent members of the CYP 450 isoenzyme family. Methods: Genomic DNA was isolated from EDTA blood samples of 203 Caucasian subjects (101 patients with Type 2 diabetes and 102 non-diabetic subjects, age (mean ± STD): 49 ± 16 years) was analyzed. Genomic DNA was isolated from EDTA blood. Mutation analysis for CYP2C8 (*2/*3/*4), CYP2C9 (*2/*3), CYP2C19 (*2/*3), CYP2D6 (*3/*4/*5/*6) and PPARγ (P12A) was performed by means of real-time PCR methods (Light-Cycler, Roche Diagnostics, Indianapolis, IN, USA). Results: The genotyping revealed the following allele frequency distributions for the two investigated groups: CYP2C8:*2(type 2 diabetes 3% vs. 1%, n.s.),*3(16% vs. 3%, n.s.),*4(15% vs. 2%, p < 0.05), CYP2C9:*2(20% vs. 24%, n.s.),*3(22% vs. 21%, n.s.), CYP2C19:*2(23% s. 33%, n.s.),*3(0% vs. 0%, n.s.), CYP2D6:*3(3% vs. 4%, n.s.),*4(40% vs. 37%, n.s.),*5(3% vs. 2%, n.s.),*6(0% vs. 0%, n.s.), PPARγ P12A(15% vs. 21%, n.s.), i.e. all but one mutation (CYP2C8*4) were found with equal prevalence in the two cohorts. Conclusions: In this pilot investigation, we found an increased prevalence of the CYP2C8*4 mutation in the Type 2 diabetic patient group. This may result in a modification of drug degradation and drug efficacy in these patients and may have an influence, e.g. on the choice of anti-diabetic drugs. However, further trials are necessary in order to confirm our findings.


Klemm M.,PharmGenomics GmbH | Eidens M.,PharmGenomics GmbH | Lorenz M.,PharmGenomics GmbH | Prause S.,PharmGenomics GmbH | And 7 more authors.
Clinical Laboratory | Year: 2010

Background: The cytochrome P450 1A2 (CYP1A2) gene encodes one of the most important enzymes of the Phase I drug metabolism, which is involved in the metabolism of many lipophilic xenobiotics, such as haloperidol, theophylline, phenacetine, and others. The recently discovered single nucleotide polymorphisms CYP1A2*1C (-3860G→A) in the 5′ flanking region of the gene and CYP1A2*1F (-163C→A) in intron 1 seem to interfere with the expression rate or catalytic function of the enzyme. Polymorphism carriers may either have a risk of reduced drug degradation and side effects, or may present with an increased induction of enzymatic activity resulting in clinical non-response to the prescribed therapy. We investigated two populations, a mental disease group and a healthy control group, to identify whether these two genetic variants are correlated with the general development of a mental disorder and if they could potentially be used as predictive markers for manifestation of the same. Methods: Using specifically designed primers, we established a high-throughput multiplex screening realtime PCR method for the two polymorphisms on the CYP1A2 gene with the Roche LightCycler® instrument. Results: We analysed the two cohorts to identify whether one of the two described genetic variants may be associated with the manifestation of a mental disorder in general. For the CYP1A2*1C variant, we identified an allele frequency of 1.7 % for both cohorts. For the CYP1A*1F polymorphism, we found an allele frequency of 74.5 % for the mental disease group and 68.6 % for the healthy control group. Conclusions: This new diagnostic method of multiplex detection may be helpful to routinely identify carriers of CYP1A2 variants and to improve the therapeutic effectiveness by selection of the most appropriate therapeutic regimen. As a result of this pilot study, there appeared to be no significant correlation between the existence of one of the investigated genetic variants and the development of a mental disorder.


Eidens M.,PharmGenomics GmbH | Weise A.,PharmGenomics GmbH | Klemm M.,PharmGenomics GmbH | Fleischer M.,PharmGenomics GmbH | Prause S.,PharmGenomics GmbH
Clinical Laboratory | Year: 2015

Background: A prominent example for inter-individual differences in drug-metabolizing enzymes is the cytochrome P450 family. These monooxygenases comprise enzymes responsible for metabolism of about 90% of common medications. CYP3A5 and CYP3A4 account for 50% of hepatic cytochrome P450 and conversion of about half of all their substrates. CYP3A5 is the predominant extra-hepatic CYP enzyme and shows varying inter-individual expression attributable to genetic variations in the corresponding gene. CYP3A5∗2 and ∗3 are the most common among Caucasian populations. Among CYP3A5 substrates are cyclosporine and tacrolimus prescribed after organ transplantations. A high incidence of nephrotoxic events after administering these drugs is related to low expression of the CYP3A5 enzyme. A fast and reliable genotyping method for the CYP3A5 gene would help avoid unwanted adverse drug reactions. Methods: Blood samples from 143 Caucasian subjects were genotyped by means of a real-time PCR multiplex assay testing the two CYP3A5 variations, CYP3AS∗2 and ∗5. This assay was validated against RFLP-PCR. Results: Both mutations examined could be found in the study. No sample was homozygous for CYP3A5∗2, but 2 out of 143 showed heterozygosity (allele frequency: 0.7%). For the CYP3A5∗3 variant 17 samples were heterozygous and 115 were homozygous (allele frequency 86.4%). The multiplex real-time PCR yields shorter hands-on time and reduced cost compared to RFLP-PCR. Conclusions: Establishment of a multiplex real-time PCR has been successful as could be proven by correctly identifying the desired mutations CYP3A5∗2 and CYP3A5∗3 against a standard reference method.


Lorenz M.,PharmGenomics GmbH | Lorenz M.,Nurnberg University of Applied Sciences | Klemm M.,PharmGenomics GmbH | Eidens M.,PharmGenomics GmbH | And 10 more authors.
Current Pharmacogenomics and Personalized Medicine | Year: 2013

Nutrigenomics and nutrigenetics are receiving growing attention from a diverse range of stakeholders including health care professionals, citizens, governments, insurers and industry. Currently there is special focus on research on how our food influences us and might cause discomfort or even symptoms of disease, but also the fact that several food intolerances are caused by genetic alterations. The strong interest expressed by certain segments of the general population for predictive and preventive diagnostic testing in relation to diet and ways in which this can improve overall health led to a fast growing market of nutrigenetic based tests. This puts pressures and challenges on governments and insurers for how best to reimburse new genetic tests. These discussions are best informed by a sound understanding of nutrigenetics science and technology, its promises and challenges, which are addressed in this paper. For example, some of the most common food intolerances caused by genetic variations are lactose intolerance, inherited fructose intolerance, congenital sucrase isomaltase deficiency (sucrose intolerance), celiac disease, glucose-6-phosphate deficiency (favism), alcohol intolerance and hemochromatosis. The increasing understanding of molecular mechanisms associated with these conditions is stimulating the development of a broad range of diagnostics allowing any person with adequate resources to have their genetic predispositions determined. However, many of the currently available tests cover only one of the above mentioned diseases or a small set of responsible mutations, which is in strong contrast to the evolution of medicine towards a more holistic approach as, for example, P4 medicine. Additionally, available tests are often not based on evidence or other guidelines for genetic test development as recommended by the ACCE evidentiary framework. In this paper we discuss the most common nutrigenetic diseases and their potential and demonstrated impacts on public health, as well as ways to devise personalized diet informed by human genomics variation in the future. © 2013 Bentham Science Publishers.


Lorenz M.,PharmGenomics GmbH | Prause S.,PharmGenomics GmbH | Prause S.,University Hospital | Eidens M.,PharmGenomics GmbH | And 9 more authors.
Current Pharmacogenomics and Personalized Medicine | Year: 2010

The polymorphic cytochrome P450 2D6 (CYP2D6) enzyme, a member of the cytochrome P450 mixed-function oxidase system, is one of the clinically most important enzymes involved in the metabolism of drugs and other xenobiotics. This highly polymorphic enzyme exhibits vastly different phenotypes: poor metabolizers show no enzyme activity, whilst ultrarapid metabolizers exhibit a significantly higher activity. Between these two extremes, there are the extensive and intermediate metabolizers, two heterogeneous groups with overlapping boundaries concerning CYP2D6 activity. These different phenotype groups can be correlated partly to the genotype of an individual. More than 70 different CYP2D6 alleles have been described to date, which encode for null alleles, have a decreased activity or carry gene duplications. However, there is a need for guidelines to translate different CYP2D6 allele combinations into phenotypes. We hereby summarize the current state of the knowledge concerning the relationship between CYP2D6 genotype and pheno- type. Clinical relevance of CYP2D6 variation is highlighted with respect to both drugs and putative endogenous ligands. Looking forward, we present a practical genotype interpretation tool, which may help to implement CYP2D6 pharmacogenetics in medical practice. Additionally, we discuss CYP2D6 activity measurement in the context of the recent efforts for multiplexed phenotyping of drug metabolism in vivo. © 2010 Bentham Science Publishers Ltd.


PubMed | PharmGenomics GmbH
Type: Journal Article | Journal: Clinical laboratory | Year: 2010

The human dopamine receptor D2 (DRD2) is known to be involved in several mental disorders. A series of studies revealed that the polymorphisms *TaqIA and *C957T are linked to these disorders and might influence drug response.Screening methods were established for the *TaqIA and *C957T polymorphisms. In the past, the technique of choice has been the time and resource consuming PCR with restriction fragment length polymorphism (PCR-RFLP). It served as the reference method for this investigation.A protocol was successfully developed for detecting *C957T and *TaqIA on the Roche LightCycler system. In addition, an allele frequency study was conducted comparing 98 samples from healthy and 86 samples from mentally disordered subjects for the *C957T and *TaqIA polymorphisms. Allele frequencies in the healthy population turned out to be 50% for the *C957T and 18% for the *TaqIA polymorphism. In the mentally disordered group, allele frequencies occurred with 54% for the *C957T and with 21% for the *TaqIA polymorphism.The protocol developed for identifying the *C957T and *TaqIA polymorphisms is suitable for high-throughput screening. Findings of the allele frequency study did not reveal any significant difference in the prevalence of the investigated polymorphisms.


The cytochrome P450 1A2 (CYP1A2) gene encodes one of the most important enzymes of the Phase I drug metabolism, which is involved in the metabolism of many lipophilic xenobiotics, such as haloperidol, theophylline, phenacetine, and others. The recently discovered single nucleotide polymorphisms CYP1A2*1C (-3860G-->A) in the 5 flanking region of the gene and CYP1A2*1F (-163C-->A) in intron 1 seem to interfere with the expression rate or catalytic function of the enzyme. Polymorphism carriers may either have a risk of reduced drug degradation and side effects, or may present with an increased induction of enzymatic activity resulting in clinical non-response to the prescribed therapy. We investigated two populations, a mental disease group and a healthy control group, to identify whether these two genetic variants are correlated with the general development of a mental disorder and if they could potentially be used as predictive markers for manifestation of the same.Using specifically designed primers, we established a high-throughput multiplex screening realtime PCR method for the two polymorphisms on the CYP1A2 gene with the Roche LightCycler instrument.We analysed the two cohorts to identify whether one of the two described genetic variants may be associated with the manifestation of a mental disorder in general. For the CYP1A2*1C variant, we identified an allele frequency of 1.7% for both cohorts. For the CYP1A*1F polymorphism, we found an allele frequency of 74.5% for the mental disease group and 68.6% for the healthy control group.This new diagnostic method of multiplex detection may be helpful to routinely identify carriers of CYP1A2 variants and to improve the therapeutic effectiveness by selection of the most appropriate therapeutic regimen. As a result of this pilot study, there appeared to be no significant correlation between the existence of one of the investigated genetic variants and the development of a mental disorder.

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