Warfarin genetic biomarkers in VKORC1 and CYP2C9*2 genes: Advancing personalized anticoagulant therapy using electrochemical genosensors

The genetic variants of vitamin K epoxide reductase complex (VKORC1) and in the cytochrome CYP2C9*2 genes have been identified to influence the anticoagulant warfarin and influence its plasmatic levels. Therefore, the pharmacogenetic information on these genes is useful for reducing warfarin adverse reaction. This work addresses the development of disposable electrochemical genosensors able of detecting single nucleotide polymorphism (SNP) in the VKORC1 and CYP2C9*2 genes. The genosensor methodology implied the immobilization of a mixed self-assembled monolayer (SAM) linear DNA-capture probe and mercaptohexanol (MCH) onto screen-printed gold electrodes (SPGE). To improve the genosensor’s selectivity and avoid strong secondary structures, that could hinder the hybridization efficiency, a sandwich format of the DNA allele was designed using a complementary fluorescein isothiocyanate-labelled signaling DNA probe and enzymatic amplification of the electrochemical signal. The developed electrochemical genosensors were able to discriminate between the two synthetic target DNA targets in both SNPs, as well as the targeted denatured genomic DNA. Several analytical parameters, such as DNA capture probe, 6-mercaptohexanol (as spacer) and antibody concentrations, as well as hybridization temperature and incubation time, were optimized. Using the best analytical conditions calibration curves employing increasing DNA target concentractions were ploted. Polymerase Chain Reaction (PCR), will be used for further validation of the electrochemical genosensor. Disposable electrochemical genosensors capable of detecting and distinguishing between two synthetic CYP2C9*2 and VKORC1 polymorphic sequences, with high selectivity and sensibility and in various concentrations, was developed. The functionality of these analytical approaches as alternative to the conventional genotyping methodologies can relieve the public health-care systems and, hopefully, prevent ADRs related to CDV episodes.info:eu-repo/semantics/publishedVersio

Gene expression analysis of aberrant signaling pathways in meningiomas

Examining aberrant pathway alterations is one method for understanding the abnormal signals that are involved in tumorigenesis and tumor progression. In the present study, expression arrays were performed on tumor-related genes in meningiomas. The GE Array Q Series HS-006 was used to determine the expression levels of 96 genes that corresponded to six primary biological regulatory pathways in a series of 42 meningiomas, including 32 grade I, four recurrent grade I and six grade II tumors, in addition to three normal tissue controls. Results showed that 25 genes that were primarily associated with apoptosis and angiogenesis functions were downregulated and 13 genes frequently involving DNA damage repair functions were upregulated. In addition to the inactivation of the neurofibromin gene, NF2, which is considered to be an early step in tumorigenesis, variations of other biological regulatory pathways may play a significant role in the development of meningiomaThis study was partially supported by Fondo de Investigaciones Sanitarias, Ministerio de Ciencia e Innovación, Spain, Grants PI‑08/1849 and PI‑10/1972; and by grant PI‑10‑045 from the Fundación Sociosanitaria de Castilla‑La Mancha, Spai

Development of electrochemical genosensors for the CYPC*2 gene polymorphism detection

Pharmacogenetic studies search for heritable genetic polymorphisms that influence responses to drug therapy. Pharmacogenetics has many possible applications in cardiovascular pharmacotherapy including screening for polymorphisms to choose agents with the greatest potential for efficacy and least risk of toxicity. Pharmacogenetics also informs dose adaptations for specific drugs in patients with aberrant metabolism. Cardiovascular diseases (CVD) are considered one of the leading causes of death worldwide. To prevent cardiovascular complications and further loss of life oral anticoagulants (e.g., warfarin) are frequently prescribed to patients. Nevertheless, warfarin therapeutic agent presents narrow therapeutic windows with well-documented health risks. Some of these dose-responses are a result of specific single-nucleotide polymorphism (SNP) genetic variations present in a patient´s DNA. Among them, determined SNP in the cytochrome P4502C9 (CYP2C9), namely the CYP2C9*2, gene has been identified as dose-response altering SNP. Therefore, the need for a rapid, selective, low-cost and in real time detection device is crucial before prescribing any anticoagulant. In this work an analytical approach based on electrochemical genosensor technique is under development to create a low-cost genotyping platform able to genotype SNPs related with the therapeutic response of warfarin. Analyzing public databases, two specific 71 bp DNA probes, one with adenine (TA) and other with guanine (TG) SNP genetic variation were selected and designed. The design of this electrochemical genosensor consists of ssDNA immobilization onto gold surfaces that act as the SNPs complementary probes. The hybridization reaction is performed in a sandwich format of the complementary ssDNA, using an enzymatic scheme to amplify the electrochemical signal. The electrochemical signal was performed by using chronoamperometric technique.info:eu-repo/semantics/publishedVersio

VKORC1 gene polymorphism as cardiovascular biomarker: Detection by electrochemical genosensors

Warfarin is an anticoagulant generally used to prevent cardiovascular diseases. Since of the low therapeutic index of warfarin and frequent complications of prevention or treatment, significant differences in individual doses of warfarin are needed to achieve prophylactic and therapeutic ranges. Recent studies have been reporting that genetic variants of vitamin K epoxide reductase complex (VKORC1) influence the response to warfarin and doses [9]. So, the genetic and pharmacogenetic information of the major cardiovascular diseases plays an important role in the identification of the cardiovascular risk factors and in the diagnosis and treatment of these conditions. This work addresses the development of a disposable electrochemical genosensor able of detecting single nucleotide polymorphism (SNP) in the VKORC1 gene. Analysing public databases, two specific 52 bp DNA probes, one with adenine (TA) and another with guanine (TG) SNP genetic variation were selected and selected and designed. The genosensor methodology implied the immobilization of a mixed self-assembled monolayer (SAM) linear VKORC1 DNA-capture probe and mercaptohexanol (MCH) onto screen-printed gold electrodes (SPGE). To improve the genosensor´s selectivity and avoid strong secondary structures, that could hinder the hybridization efficiency, a sandwich format of the VKORC1 allele was designed using a complementary fluorescein isothiocyanate-labelled signaling DNA probe and enzymatic amplification of the electrochemical signal. Preliminary studies indicate that differences in the electrochemical answers were obtained depending of the hybridization reaction format. In fact, higher electrochemical intensities were measured when the hybridization reaction was performed with a complementary DNA (without SNPs). These results suggested that the sensor is able to discriminate between the complementary DNA and single base mismatch targets having a great potential for the DNA polymorphism analysis.info:eu-repo/semantics/publishedVersio

Microarray analysis of gene expression in vestibular schwannomas reveals SPP1/MET signaling pathway and androgen receptor deregulation

Vestibular schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of neurofibromin 2 (NF2). Transcriptomic alterations, such as the neuregulin 1 (NRG1)/ErbB2 pathway, have been described in schwannomas. In this study, we performed a whole transcriptome analysis in 31 vestibular schwannomas and 9 control nerves in the Affymetrix Gene 1.0 ST platform, validated by quantitative real-time PCR (qRT-PCR) using TaqMan Low Density arrays. We performed a mutational analysis of NF2 by PCR̸denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA), as well as a microsatellite marker analysis of the loss of heterozygosity (LOH) of chromosome 22q. The microarray analysis demonstrated that 1,516 genes were deregulated and 48 of the genes were validated by qRT-PCR. At least 2 genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed 1 hit and 8 tumors showed no NF2 alteration. MET and associated genes, such as integrin, alpha 4 (ITGA4)̸B6, PLEXNB3/SEMA5 and caveolin-1 (CAV1) showed a clear deregulation in vestibular schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in schwannoma merlin depletion. Finally, no major differences were observed among tumors of different size, histological type or NF2 status, which suggests that, at the mRNA level, all schwannomas, regardless of their molecular and clinical characteristics, may share common features that can be used in their treatmentThis study was supported by grants PI07/0577, PI08/1849 and PI10/1972 from Fondo de Investigaciones Sanitarias, Ministerio de Ciencia e Innovación, Spain and PI10-045, and from the Fundación Sociosanitaria de Castilla-La Mancha, Spai

WRN Cys1367Arg SNP is not associated with risk and prognosis of gliomas in Southeast Brazil

Werner syndrome (WS) is a premature aging disorder characterized by early onset of symptoms related to normal aging and by a high predisposition to various types of cancer, including gliomas. WS is caused by inherited recessive mutations in the WRN gene, which encodes a helicase considered a caretaker of the genome. Aiming to study the role of WRN Cys1367Arg in glioma susceptibility and oncologic prognosis of patients, we investigated the genotype distribution of this single nucleotide polymorphism in 94 glioma patients and 100 healthy subjects. Comparisons of genotype distributions and allele frequencies did not reveal any significant difference between the groups. Overall and disease-free survival rates were calculated, but no statistically significant difference was observed. Our data suggest that WRN Cys1367Arg SNP is not involved either in susceptibility to developing gliomas or in patient survival, at least in the Brazilian population.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)[308256/2006-9]Financiadora de Estudos e Projetos (FINEP CT-INFRA/FADESP)[1017-01

Association study of an epidermal growth factor gene functional polymorphism with the risk and prognosis of gliomas in Brazil

Epidermal growth factor (EGF) plays an important role in cancer. A functional single nucleotide polymorphism (SNP) in the 5`-untranslated region of the EGF gene (+61 A>G) may influence its expression and contribute to cancer predisposition and aggressiveness. Aiming to investigate the role of EGF +61 A>G in the susceptibility to glioma and its prognosis, we performed a case-control study with 165 patients and 200 healthy controls from Brazil. Comparisons of genotype distributions and allele frequencies did not reveal any significant differences between the groups. The mean overall survival was 9.2 months for A/A, 8.2 months for A/G, and 7.7 months for GIG. When survival curves were plotted we found that the +61G allele is associated with poor overall survival (p=0.023) but not with disease-free survival (p=0.527). Our data suggest that, although there is no association between the EGF +61 A>G genotype and glioma susceptibility, this SNP is associated with shorter overall survival of glioma patients in the Brazilian population. Nevertheless, future studies utilizing a larger series are essential for a definitive conclusion. (Int J Biol Markers 2009; 24: 277-81)Conselho Nacional de Desenvolvimento Cientifco e Tecnologico (CNPq

Cytochrome P450 polymorphisms with impact in cardiovascular drugs metabolisms in European populations

The cytochrome P450 (CYP) enzymes constitute a large polymorphic family that play a huge role in the metabolism of endogenous compounds and in the metabolization of 70–80% of all clinically prescribed medications. Among them, the CYP2C9, CYP2C19, CYP2D6 and CYP4F2 genes are of clinical relevance, as they are highly polymorphic and implicated in the metabolism of several drugs. These genetic polymorphisms which induce variability in CYPs expression present qualitative and quantitative differences between ethnic groups and geographic regions. This review aims to evaluate the allele frequencies, genotypic distribution and predicted CYP2C9, CYP2C19, CYP2D6 and CYP4F2 genetic variants in the European countries. Therefore, a PubMed and a Web of Science search from 1989 to 2021 on the data on the polymorphic prevalence among European countries of the CYP2C9, CYP2C19, CYP2D6 and CYP4F2 genes was performed. After excluding the duplicates, a total of 1179 studies were found. The results were structured and presented in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The present paper is an overview on the frequency CYP genetic variations, facilitating the prediction of a patient's response to medication and, consequently, enabling the selection of personalized medicineThis work was funded by the Bilateral Cooperation FCT/CAPES 2018/2019 Processo 4.4.1.00 CAPES (CAPES-FCT2017484310P 200.137.174.210). This work was also supported by UIDB/50006/2020 and UIDP/50006/2020 by the Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through national funds. M. F. Barroso thanks FCT for the FCT Investigator (ref.2020.03107.CEECIND)info:eu-repo/semantics/publishedVersio

Development of an electrochemical DNA-based biosensor for the detection of the cardiovascular pharmacogenetic-altering SNP CYP2C9*3

Cardiovascular diseases are among the major causes of mortality and morbidity. Warfarin is often prescribed for these disorders, an anticoagulant with inter and intra-dosage variability dose required to achieve the target international normalized ratio. Warfarin presents a narrow therapeutic index, and due to its variability, it can often be associated with the risk of hemorrhage, or in other patients, thromboembolism. Single-nucleotide polymorphisms are included in the causes that contribute to this variability. The Cytochrome P450 (CYP) 2C9*3 genetic polymorphism modifies its enzymatic activity, and hence warfarin's plasmatic concentration. Thus, the need for a selective, rapid, low-cost, and real-time detection device is crucial before prescribing warfarin. In this work, a disposable electrochemical DNA-based biosensor capable of detecting CYP2C9*3 polymorphism was developed. By analyzing genomic databases, two specific 78 base pairs DNA probes; one with the wild-type adenine (Target-A) and another with the cytosine (Target-C) single-nucleotide genetic variation were designed. The biosensor implied the immobilization on screen-printed gold electrodes of a self-assembled monolayer composed by mercaptohexanol and a linear CYP2C9*3 DNA-capture probe. To improve the selectivity and avoid secondary structures a sandwich format of the CYP2C9*3 allele was designed using complementary fluorescein isothiocyanate-labeled signaling DNA probe and enzymatic amplification of the electrochemical signal. Chronoamperometric measurements were performed at a range of 0.015–1.00 nM for both DNA targets achieving limit of detection of 42 p.m. The developed DNA-based biosensor was able to discriminate between the two synthetic target DNA targets, as well as the targeted denatured genomic DNA, extracted from volunteers genotyped as non-variant homozygous (A/A) and heterozygous (A/C) of the CYP2C9*3 polymorphism.info:eu-repo/semantics/publishedVersio