Rapid HCP5 single-nucleotide polymorphism genotyping: a simple allele-specific PCR method for prediction of hypersensitivity reaction to Abacavir.

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1 A simple allele-specific polymerase chain reaction method to detect the Gly143Glu polymorphism in the human carboxylesterase 1 gene: importance of genotyping for pharmacogenetic treatment. Walter Soria N, Belaus A, Galván C, Ana Pasquali M, Velez P, Del Carmen Montes C, Beltramo DM. Cátedra de Biotecnología, Facultad de Ciencias Químicas, Universidad Católica de Córdoba, Córdoba, Argentina. Genet Test Mol Biomarkers Dec;14(6): Epub 2010 Sep 21. Human carboxylesterases 1 and 2 (CES1 and CES2) catalyze the hydrolysis of many exogenous compounds. Alterations in CES sequences could lead to variability in both the inactivation of drugs and the activation of prodrugs. The human CES1 gene encodes for the enzyme carboxylesterase 1, a serine esterase governing both metabolic deactivation and activation of numerous therapeutic agents. Some of theses drugs are the antiviral oseltamivir used to treat some types of influenza infections and the methylphenidate employed in the treatment of patients with attention deficit. The Gly143Glu polymorphism in CES1 gene has been shown to reduce enzyme activity. The aim of the present study was to develop an easy and cheap method to detect this polymorphism. For this, we studied a group of people from Córdoba, a Mediterranean area from Argentina. Our results show that our methodology could detect the presence of this polymorphism with a frequency around 1.8%, only in the heterozygote form. These results could be relevant to patients before the treatment with some drugs where the CES1 enzyme is involved.

2 Rapid HCP5 single-nucleotide polymorphism genotyping: a simple allele-specific PCR method for prediction of hypersensitivity reaction to Abacavir. Clin Chim Acta Jul 15;412(15-16): Epub 2011 Apr 14. BACKGROUND: The most important factor limiting the success of an antiretroviral therapy is toxicity. The HLA-B*5701 allele is predictive of hypersensitivity reaction to Abacavir, and this gene is in a perfect linkage disequilibrium with the rs SNP present in the HCP5 gene. METHODS: Genomic DNA was extracted from blood obtained from 201 unrelated healthy Argentinean volunteers. The DNA was subjected to an allele-specific PCR method. Sequencing was performed to validate the test results. RESULTS: We were successful to amplify specific fragment of interest from the DNA samples. The method is easy, specific and reproducible. CONCLUSIONS: The application of this methodology is a rapid and simple method to detect the HCP5 polymorphism (rs ) previous to administration of Abacavir in patients with HIV infection.

3 Genetic profiling of GSTP1, DPYD, FCGR2A, FCGR3A and CCND1 genes in an Argentinian population. Clin Biochem Sep;44(13): Epub 2011 Jun 24. OBJECTIVES: To determine the frequencies of relevant allelic variants in oncology for the GSTP1, DPYD, FCGR2A, FCGR3A and CCND1 genes in a population from Central Argentina. To compare the allelic distribution found with the frequencies reported for other ethnic groups. DESIGN AND METHODS: Genotyping was carried out in a total of 102 unrelated Argentinian subjects. FCGR3A (rs396991) was detected using allele specific polymerase chain reaction (PCR) assay, while GSTP1 (rs1695), DPYD (rs ), FCGR2A (rs ) and CCND1 (rs9344) variants were assessed by PCR-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The allele frequencies for GSTP*1B, DPYD*2A, FCGR2A (131R), FCGR3A (158F) and CCND1 (870G) in Argentinians were 0.35, 0.005, 0.41, 0.77 and 0.47, respectively. CONCLUSIONS: We found that the Argentinian population tested resembles other Caucasians populations, especially Spaniards; yet the differences in allele distribution with other Caucasian groups, uncover population admixture with native Amerindian and other ethnic groups, consistent with the well documented immigration flows landing Argentina from several countries.

4 Development of a method to control the RNA extraction and reverse transcription steps for the detection of dengue virus present in human blood samples. Genet Test Mol Biomarkers Dec;15(12): Epub 2011 Jun 20. AIMS: Molecular biology techniques based on the detection of genomic sequences by reverse transcription combined with polymerase chain reaction (PCR) have enabled the detection of different RNA viruses in serum or plasma samples. Since the dengue epidemic outbreak declared in Argentina in 2009, numerous patients' samples were analyzed for the acute phase of infection. One of the main methodological drawbacks is the lack of internal control to measure the effectiveness of the viral extraction and reverse transcription process. In this article, we propose to standardize a molecular method to detect beta actin (β-act) and glucose 6 phosphate dehydrogenase (G6PDH) complementary DNAs (cdnas) present in patient's plasma/serum, as a control process. RESULTS: RNA extraction, reverse transcription, and PCRs for human G6PDH, β- Act, and the dengue virus genome were performed. cdna fragments for β-act and G6PDH were amplified for all samples, regardless of the presence or absence of viral RNA. CONCLUSIONS: Amplification of β-act and G6PDH cdnas can be used as a control for the extraction and reverse transcription processes during dengue virus detection. This could also be a useful method for controlling the above steps when infections caused by other RNA viruses are studied, even if another methodology is employed, such as real-time PCR.

5 Human Immunodefieciency Virus: Pharmacogenetics of Antiretroviral Treatment. Susana A Pesoa 1, Cristian A Galván 1,2, Dante M Beltramo 2,3,4 and Néstor W Soria 1,2 1Laboratorio de Análisis Clínicos Especializados (LACE), Córdoba, Argentina 2Cátedra de Biotecnología, Facultad de Ciencias Químicas, Universidad Católica de Córdoba, Córdoba, Argentina 3Centro de Excelencia en Productos y Procesos de Córdoba (CEPROCOR), Santa María de Punilla, Córdoba, Argentina 4Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Pharmacogenomics & Pharmacoproteomics. Pesoa et al., J Pharmacogenom Pharmacoproteomics 2011, S6 The introduction of highly active antiretroviral therapy as standard of care has considerably enhanced the life expectancy among HIV-infected individuals. Although this combination of drugs virtually suppresses viral replication, the therapeutic effect may be limited by differing rates of adverse events and responses in terms of efficacy and toxicity. These differences arise from complex interactions between biological and environmental factors. Pharmacogenetic studies are contributing to our understanding of the inter-individual differences in the response to antirretroviral drugs. Several studies have provided a relevant number of associations between human genetic variants and predisposition to adverse events and for some antiretroviral drugs clear and causal genotype phenotype correlation has been established. These findings make the idea of personalized medicine in this field increasingly attractive. We will discuss here current achievements on pharmacogenetics of HIV treatment with special emphasis on the genetic polymorphisms underlying toxic effects and/or those already implemented in the clinical setting

6 Distribution of polymorphisms in cytochrome P450 2B6, histocompatibility complex P5, chemokine coreceptor 5, and interleukin 28B genes in inhabitants from the central area of Argentina. Genet Test Mol Biomarkers Feb;16(2): Epub 2011 Aug 19. AIMS: The selection of the most appropriate treatment for several diseases relies on a number of factors such as environment, age, gender, and nutrition. Additionally, the contribution of different genetic polymorphisms to treatment efficacy has been largely recognized. The lack of information on the pharmacogenetic profile of our population prompted us to analyze the frequency of polymorphisms known to be relevant to achieve treatment efficacy with different therapeutic agents in viral infectious diseases, such as Hepatitis C and AIDS. RESULTS: The allelic frequencies for the wild-type variant of the genes analyzed were cytochrome P450 2B6 (CYP2B6; rs ; 516G) (95% confidence interval [CI]: 0.523, 0.711), chemokine coreceptor 5 (CCR5; rs333) (95% CI: 0.942, 0.98), histocompatibility complex P5 (HCP5; rs ; 335T) (95% CI: 0.937, 1), and interleukin 28B (IL28B; rs ; C) (95% CI: 0.564, 0.747), respectively. CONCLUSIONS: Our data indicate that the genetic profile of the population studied is similar to that reported for other Caucasian populations, with only slight differences for CYP2B6. Noteworthy, the considerable number of patients carrying CYP2B6 (516T) and IL28B ( T) alleles underlies the importance of considering pharmacogenetic testing before starting drug therapy protocols to prevent toxicity and/or lack of effectiveness in AIDS or hepatitis C virus infections.

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