Transcriptional study of genes involved in the passage from teliospore to hyphae stage in the fungus Thecaphora frezii, the causal agent of peanut smut.

Peanuts (Arachis hypogaea L.) are among the most important leguminous crops in Argentina. During the growing season, they are frequently attacked by fungal diseases, including Thecaphora frezii. The spores of T. frezii are structures that confer resistance to this phytopathogen. The transition from teliospore to hypha is a characteristic process of some fungi, which is essential for completing their life cycle. Using the transcriptomes of teliospores and hyphae of T. frezii, we aimed to identify genes that were differentially expressed during this transition, and we found 134 up-regulated and 66 down-regulated genes, which would participate in different cellular processes such as: (a) cell cycle and DNA processing; (b) cell fate; (c) rescue, defense and cellular virulence; (d) detoxification by CYP450; (e) energy; (f) nutrient interaction and nutritional adaptation; (g) metabolism; (g) proteins with binding functions or cofactor requirements; (h) stress, cell differentiation and biogenesis of cell components; and (i) transport, cell communication and transcription. The identification of genes in T. frezii and their expression levels during different stages of differentiation could contribute to our understanding of the biological mechanisms in this fungus.

Changes of lipids composition in different ontogenetic stages of Thecaphora frezii: expression of key enzymes for lipid biosynthetic pathways.

AIMS: It is known that Thecaphora frezii produces peanut smut that generates numerous economic losses. For this reason, it is a priority to search for control strategies. In this sense, we investigated the lipid profile of this pathogen, as possible antifungal targets, regarding polar lipid composition, fatty acid profile, and transcriptional regulation of genes involved in each stage of the development. METHOD AND RESULTS: Lipids from T. frezii teliospores, basidiospores, and hyphae were analyzed by HPLC/CAD and CG/FID. We found differences in the unsaturation levels as well as in the long-chain fatty acids along the stages. Phosphatidylcholine was the main component in the three development stages, followed by cardiolipins. Phosphatidylinositol, phosphatidylethanolamine, and lyso-phosphatidylethanolamine were found in similar amounts in all stages. Although ergosterol was not detected, we found two unsaponifiable lipids. In addition, we found transcripts that encode 28 enzymes involved in the biosynthesis of three lipids by RNA-Seq. CONCLUSIONS: Thecaphora frezii shows changes in the composition of membrane lipids in different ontogenetic stages as well as in the expression of transcripts for enzymes involved in lipid biosynthesis.

Expression and localization of tubulin isotypes and its mRNAs during Thecaphora frezii developments.

Thecaphora frezii is a phytopathogenic fungus that infects Arachys hypogaea L. and produces peanut smut. It has three ontological stages teliospores, basidiospores, and hyphae. Microtubules are cellular structures that participate in various important cellular processes. In this work, we analyzed the presence and location of α-tubulin isotypes and enzymes that participate in tyrosination-detyrosination in the three stages of T. frezii. Although both tyrosinated and detyrosinated tubulin seem to be associated with a membrane fraction component that gives it a similar behavior to integral proteins, in the soluble cytosolic fraction, only detyrosinated tubulin was detected, not tyrosinated tubulin. The presence of α-tubulin was not detected using the monoclonal antibody DM1A as neither acetylated tubulin. The RNA-Seq analysis showed the presence of α, ß, and γ-tubulins and the genes that codes for tyrosine-tubulin ligase and cytosolic carboxypeptidase 1, enzymes that are involved in post-translational modification processes. These sequences showed a high percentage of identity and homology with Ustilago maydis, Thecaphora thlaspeos, and Anthracocystis flocculosa. This is the first report for tubulins subpopulations and the cellular distribution in T. frezii, which together with the data obtained by RNA-Seq contribute to the knowledge of the pathogen, which will allow the development of control strategies.

Draft Genome Sequence of Gordonia sp. Strain Campus, a Bacterium Isolated from Diesel-Contaminated Soil with Potential Use in Phytoremediation Systems.

We present the draft genome sequence of Gordonia sp. strain Campus, which was extracted from diesel-contaminated soil in Córdoba, Argentina. It was observed that this strain, in conjunction with alfalfa and poplar, has the ability to decompose diesel-contaminated soils. The data may be important for the phytoremediation of hydrocarbon-contaminated soils.

Pharmacogenomics, biomarker network, and allele frequencies in colorectal cancer.

Colorectal cancer is one of the leading causes of cancer death worldwide. Over the last decades, several studies have shown that tumor-related genomic alterations predict tumor prognosis, drug response, and toxicity. These observations have led to the development of several therapies based on individual genomic profiles. As part of these approaches, pharmacogenomics analyses genomic alterations which may predict an efficient therapeutic response. Studying these mutations as biomarkers for predicting drug response is of a great interest to improve precision medicine. We conduct a comprehensive review of the main pharmacogenomics biomarkers and genomic alterations affecting enzyme activity, transporter capacity, channels, and receptors; and therefore the new advances in CRC precision medicine to select the best therapeutic strategy in populations worldwide, with a focus on Latin America.

Loss of the tRNALysCUU encoding gene, Chr-11 tRNA-Lys-CUU, is not associated with Type 2 diabetes mellitus.

AIM: To investigate the presence/absence of the Chr-11 tRNA-Lys-CUU gene as a marker for genetic predisposition to Type 2 diabetes mellitus (T2DM). METHODS: We enrolled 122 patients diagnosed with T2DM and 77 non-diabetic individuals. We evaluated clinical and biochemical parameters (body mass index, hypertension, cholesterol levels, glycosylated hemoglobin, triglycerides, etc.), and performed a genotypic profiling of Chr-11 tRNA-Lys-CUU by polymerase chain reaction analyses. RESULTS: Approximately one third of the population lacked Chr-11 tRNA-Lys-CUU. We did not observe a statistically significant association between the presence/absence of Chr-11 tRNA-Lys-CUU and T2DM. CONCLUSION: The genotypic distribution of Chr-11 tRNA-Lys-CUU in our population was consistent to that reported by others. This gene failed as a marker for T2DM predisposition.

Rol de variantes genéticas en el desarrollo de Enfermedad Renal Crónica en pacientes con Diabetes Mellitus Tipo 2 / Role of genetic variants to the development of Chronic Kidney Disease in patients with Type 2 Diabetes Mellitus

La Enfermedad Renal Crónica (ERC) es una afección que perjudica a un gran número de pacientes. Una de las causas es la Nefropatía en pacientes con Diabetes Mellitus Tipo 2 (DM2). OBJETIVO: Indagar si las presencias de variantes genéticas contribuyen al desarrollo de ERC en pacientes con DM2. Materiales y métodos: se evaluaron criterios clínicos, bioquímicos y moleculares en 25 pacientes con DM2. Los polimorfismos se analizaron mediante PCR-RFLP para ECA (rs4646994); CDKAL1 (rs7756992); e-NOS (rs1799983) y SLC12A3 (rs11643718). RESULTADOS: El análisis estadístico mediante modelo dominante arrojaron para: ECA (rs4646994) (OR=1,33 (IC 95%) 0,25-7,01; p=0,73); CDKAL1 (rs7756992) (OR= 1 (IC 95%) 0,14-7,39; p=NA); e-NOS (rs1799983) (OR= 0,29 (IC 95%) 0,05-1,57; p=0,14) y SLC12A3 (rs11643718 (OR= 1,62 (IC 95%) 0,19-13,93; p=0,66). CONCLUSIONES: ninguna de las variantes evaluadas en los genes ECA, CDKAL1, e-NOS y SLC12A3 mostraron asociaciones positivas o negativas con el riesgo a desarrollar ERC en pacientes con DM2. (AU)

Chronic Kidney Disease (CKD) is a condition that affects a large number of patients. One cause is the Nephropathy in Type 2 Diabetes Mellitus (DM2).patients. OBJECTIVE: Evaluate if some polymorphisms contribute to CKD risk in DM2 patients. Materials and methods: clinical, biochemical and molecular parameters from 25 patients with DM2 were evaluated. The polymorphisms were analyzed by PCR-RFLP for the ECA (rs4646994); CDKAL1 (rs7756992); e-NOS (rs1799983) and SLC12A3 (rs11643718) genes. RESULTS: Statistical analysis using a dominant model showed: ACE (rs4646994) (OR = 1.33 (95% CI) 0.25-7.01, p = 0.73); CDKAL1 (rs7756992) (OR = 1 (95% CI) 0.14-7.39; p = NA); e-NOS (rs1799983) (OR = 0.29 (95% CI) 0.05- 1.57; p = 0.14) and SLC12A3 (rs11643718 (OR = 1.62 (95% CI) 0.19-) 13.93, p = 0.66). CONCLUSIONS: none of the evaluated variants in ACE, CDKAL1, e-NOS and SLC12A3 genes showed positive or negative associations with CKD risk in DM2 patients.

Dynamics of expression of two vitellogenin genes in the Chagas' disease vector Triatoma infestans: Analysis throughout pre-vitellogenesis and vitellogenesis.

The reproductive success of all oviparous species depends on vitellogenin (Vg) biosynthesis and its accumulation in the developing oocytes. The expression levels of two Vg genes (Vg1 and Vg2) were analyzed by qPCR and western blot in fat body and ovaries of adult females, at different times after ecdysis (pre-vitellogenic phase) and after blood feeding of females (vitellogenic phase). Vg genes were also evaluated in fat bodies of adult males as well as in female fifth instar nymphs. No trace of Vg mRNA was detected in adult males or in nymphs. Vg1 and Vg2 were expressed in the fat bodies and ovaries of adult females. The Vg genes start to be expressed slightly in both tissues of adult females during pre-vitellogenesis. After blood feeding, Vg1 and Vg2 were up regulated and significant levels of Vg transcripts as well as protein expression were observed in fat bodies sampled throughout vitellogenesis. During this period however, the distribution patterns of Vg1 and Vg2 transcripts showed two peaks around early and advanced vitellogenesis (days 4 and 12 post-feeding, respectively). In the ovaries, levels of mRNAs increased from the day 10 post-blood feeding onwards. In addition, the immunofluorescence assays showed a strong signal for vitellin in the yolk bodies of terminal follicles of vitellogenic females. The involvement of fat body and ovary in the synthesis of Vg suggests different roles of Vgs in supporting the growth of oocytes.

Efectividad de la metformina en pacientes con diabetes tipo II según variantes en el gen SLC22A1 / Effectiveness of Metformin in patients with type II diabetes related to variants in the SLC22A1 gene / Eficácia de Metformina em doentes com diabetes tipo II, relacionado com variantes do gene SLC22A1

La diabetes mellitus tipo II (DM II) es una enfermedad que afecta una gran cantidad de individuos. Un medicamento empleado en el tratamiento de los pacientes es la metformina. Este medicamento es transportado al interior de los hepatocitos por un transportador codificado por el gen SLC22A1. Variantes en el gen con actividad reducida pueden disminuir la cantidad de metformina disponible en el hígado y reducir la respuesta terapéutica. Se propuso evaluar diferentes parámetros bioquímicos en relación a la dosis de metformina y la presencia de variantes en el transportador. Se estudiaron 103 pacientes mayores de 18 años con diagnóstico de DM II, tratados con 1700 mg/día de metformina por más de 6 meses. Se analizaron 5 polimorfismos en el gen SLC22A1, glucemia, HbA1c, función hepática, perfil lipídico y renal. Los niveles de HbA1c y de glucemia fueron más elevados en los pacientes que presentaban los polimorfismos R61C, G401S, M420del y G465R aunque la diferencia fue estadísticamente significativa sólo para la HbA1c en los pacientes que presentaban las variantes M420del y G465R (p=0,0273 y 0,0018, respectivamente). La presencia de polimorfismos con actividad reducida en el gen SLC22A1 afecta los niveles de glucemia y de HbA1c en pacientes con DM II cuando son tratados con metformina.

Diabetes mellitus type II (DM II) is a disease that affects a large number of individuals. One of the drugs used for the treatment is metformin. Metformin is delivered into hepatocytes by a transporter encoded by the SLC22A1 gene. Gene variants with reduced activity may decrease the amount of metformin available in the liver and reduce the therapeutic response. Various biochemical parameters were evaluated in relation to the metformin dose and the presence of transporter variants. A total of 103 patients older than 18 diagnosed with DM II who were treated with 1700 mg/day of metformin for more than six months were studied. Five polymorphisms in the SLC22A1 gene were analyzed as well as glycemia, HbA1c level, liver function, and lipid and kidney profiles. HbA1c and glycemia levels were higher in patients with the R61C, G401S, M420del and G465R polymorphisms; although the difference was statistically significant only for HbA1c in patients with the M420del and G465R variants (p=0.0273 and 0.0018, respectively). Polymorphisms with reduced activity in the SLC22A1 gene affect blood glucose levels and HbA1c in patients with DM II when they are treated with metformin.

O diabetes mellitus tipo II (DM II) é uma doença que afeta uma grande quantidade de indivíduos. Um medicamento utilizado no tratamento dos doentes é a metformina. Esse medicamento é transportado no interior dos hepatócitos por um transportador codificado pelo gene SLC22A1. Variantes no gene com atividade reduzida podem diminuir a quantidade de Metformina disponível no fígado e reduzir a resposta terapêutica. Propôs-se avaliar diferentes parâmetros bioquímicos em relação à dose da metformina e à presença de variantes no transportador. Foram estudados 103 pacientes maiores de 18 anos com diagnóstico de DM II tratados com 1700 mg/dia de metformina por mais de 6 meses. Foram analisados 5 polimorfismos no gene SLC22A1; glicemia, HbA1c, função hepática, perfil lipídico e renal. Os níveis de HbA1c e de glicemia foram superiores em doentes que apresentavam os polimorfismos R61C, G401S, M420del e G465R; embora a diferença seja estatisticamente significativa apenas para o HbA1c nos doentes que apresentavam as variantes M420del e G465R (p=0,0273 e 0,0018; respectivamente). A presença de polimorfismos com atividade reduzida no gene SLC22A1 afeta os níveis da glicemia e do HbA1c em doentes com DM II quando são tratados com metformina.

Efectividad de la metformina en pacientes con diabetes tipo II según variantes en el gen SLC22A1 / Effectiveness of Metformin in patients with type II diabetes related to variants in the SLC22A1 gene / Eficácia de Metformina em doentes com diabetes tipo II, relacionado com variantes do gene SLC22A1

La diabetes mellitus tipo II (DM II) es una enfermedad que afecta una gran cantidad de individuos. Un medicamento empleado en el tratamiento de los pacientes es la metformina. Este medicamento es transportado al interior de los hepatocitos por un transportador codificado por el gen SLC22A1. Variantes en el gen con actividad reducida pueden disminuir la cantidad de metformina disponible en el hígado y reducir la respuesta terapéutica. Se propuso evaluar diferentes parámetros bioquímicos en relación a la dosis de metformina y la presencia de variantes en el transportador. Se estudiaron 103 pacientes mayores de 18 años con diagnóstico de DM II, tratados con 1700 mg/día de metformina por más de 6 meses. Se analizaron 5 polimorfismos en el gen SLC22A1, glucemia, HbA1c, función hepática, perfil lipídico y renal. Los niveles de HbA1c y de glucemia fueron más elevados en los pacientes que presentaban los polimorfismos R61C, G401S, M420del y G465R aunque la diferencia fue estadísticamente significativa sólo para la HbA1c en los pacientes que presentaban las variantes M420del y G465R (p=0,0273 y 0,0018, respectivamente). La presencia de polimorfismos con actividad reducida en el gen SLC22A1 afecta los niveles de glucemia y de HbA1c en pacientes con DM II cuando son tratados con metformina.(AU)

Diabetes mellitus type II (DM II) is a disease that affects a large number of individuals. One of the drugs used for the treatment is metformin. Metformin is delivered into hepatocytes by a transporter encoded by the SLC22A1 gene. Gene variants with reduced activity may decrease the amount of metformin available in the liver and reduce the therapeutic response. Various biochemical parameters were evaluated in relation to the metformin dose and the presence of transporter variants. A total of 103 patients older than 18 diagnosed with DM II who were treated with 1700 mg/day of metformin for more than six months were studied. Five polymorphisms in the SLC22A1 gene were analyzed as well as glycemia, HbA1c level, liver function, and lipid and kidney profiles. HbA1c and glycemia levels were higher in patients with the R61C, G401S, M420del and G465R polymorphisms; although the difference was statistically significant only for HbA1c in patients with the M420del and G465R variants (p=0.0273 and 0.0018, respectively). Polymorphisms with reduced activity in the SLC22A1 gene affect blood glucose levels and HbA1c in patients with DM II when they are treated with metformin.(AU)

O diabetes mellitus tipo II (DM II) é uma doenþa que afeta uma grande quantidade de indivíduos. Um medicamento utilizado no tratamento dos doentes é a metformina. Esse medicamento é transportado no interior dos hepatócitos por um transportador codificado pelo gene SLC22A1. Variantes no gene com atividade reduzida podem diminuir a quantidade de Metformina disponível no fígado e reduzir a resposta terapÛutica. Prop¶s-se avaliar diferentes parÔmetros bioquímicos em relaþÒo O dose da metformina e O presenþa de variantes no transportador. Foram estudados 103 pacientes maiores de 18 anos com diagnóstico de DM II tratados com 1700 mg/dia de metformina por mais de 6 meses. Foram analisados 5 polimorfismos no gene SLC22A1; glicemia, HbA1c, funþÒo hepática, perfil lipídico e renal. Os níveis de HbA1c e de glicemia foram superiores em doentes que apresentavam os polimorfismos R61C, G401S, M420del e G465R; embora a diferenþa seja estatisticamente significativa apenas para o HbA1c nos doentes que apresentavam as variantes M420del e G465R (p=0,0273 e 0,0018; respectivamente). A presenþa de polimorfismos com atividade reduzida no gene SLC22A1 afeta os níveis da glicemia e do HbA1c em doentes com DM II quando sÒo tratados com metformina.(AU)

cDNA isolation and characterization of two vitellogenin genes in the Chagas' disease vector Triatoma infestans (Hemiptera, Reduviidae).

Two vitellogenin genes (Vg1 and Vg2) were identified in the Chagas' disease vector Triatoma infestans. The putative coding sequence corresponding to Vg2 was found to be 5553bp long, encoding 1851 amino acids in a single open reading frame. The comparative analysis of the deduced amino acid sequences from Vg1 and Vg2 cDNA fragments of T. infestans revealed 58.94% of identity with 76.43% of homology. The phylogenetic tree based on the complete Vg amino acid sequences of hemimetabolous insects unambiguously supported two clusters, one consisting of Vg sequences from dictyopteran and the other containing Vg sequences of hemipteran. The Vg1 and Vg2 mRNAs were detected in fat bodies and ovaries of adult females with the highest levels of both Vg transcripts in the first tissue. Quantitative PCR showed low expression of Vg2 in head and muscle of adult females, while the Vg1 transcript was not present in these organs. Neither Vg1 nor Vg2 was expressed in fifth instar nymph fat bodies or in adult male fat bodies, heads, and muscles.

Perception of the usefulness of drug/gene pairs and barriers for pharmacogenomics in Latin America.

Pharmacogenetics and Pharmacogenomics areas are currently emerging fields focused to manage pharmacotherapy that may prevent undertreatment while avoiding associated drug toxicity in patients. Large international differences in the awareness and in the use of pharmacogenomic testing are presumed, but not well assessed to date. In the present study we review the awareness of Latin American scientific community about pharmacogenomic testing and the perceived barriers for their clinical application. In order to that, we have compiled information from 9 countries of the region using a structured survey which is compared with surveys previously performed in USA and Spain. The most relevant group of barriers was related to the need for clear guidelines for the use of pharmacogenomics in clinical practice, followed by insufficient awareness about pharmacogenomics among clinicians and the absence of regulatory institutions that facilitate the use of pharmacogenetic tests. The higher ranked pairs were TPMT/thioguanine, TPMT/azathioprine, CYP2C9/warfarin, UGT1A1/irinotecan, CYP2D6/amitriptiline, CYP2C19/citalopram and CYP2D6/clozapine. The lower ranked pairs were SLCO1B1/simvastatin, CYP2D6/metoprolol and GP6D/chloroquine. Compared with USA and Spanish surveys, 25 pairs were of lower importance for Latin American respondents. Only CYP2C19/esomeprazole, CYP2C19/omeprazole, CYP2C19/celecoxib and G6PD/dapsone were ranked higher or similarly to the USA and Spanish surveys. Integration of pharmacogenomics in clinical practice needs training of healthcare professionals and citizens, but in addition legal and regulatory guidelines and safeguards will be needed. We propose that the approach offered by pharmacogenomics should be incorporated into the decision-making plans in Latin America.

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.

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; rs3745274; 516G) 0.618 (95% confidence interval [CI]: 0.523, 0.711), chemokine coreceptor 5 (CCR5; rs333) 0.961 (95% CI: 0.942, 0.98), histocompatibility complex P5 (HCP5; rs2395029; 335T) 0.971 (95% CI: 0.937, 1), and interleukin 28B (IL28B; rs12979860; 12007005C) 0.656 (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 (12007005T) 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.

Prevalence of Janus Kinase 2 mutations in patients with unusual site venous thrombosis.

We aimed to study patients with splanchnic vein thrombosis (SVT) and cerebral vein thrombosis (CVT) searching for JAK2 mutations. We evaluated 14 patients (median age: 41.5 years) with portal vein thrombosis (PVT) = 7; mesenteric vein thrombosis (MVT) = 3; and CVT = 4. JAK2 V617F was assessed by allele specific PCR of peripheral blood DNA. In addition, DNA was sequenced for other JAK2 mutations. Other inherited and acquired thrombophilia risk factors were evaluated. JAK2 V617F was positive in four out of seven patients with PVT and in one CVT patient. These five patients had a diagnosis of myelo-proliferative disorder (MPD) at the moment of the occurrence of thrombosis (n = 2) or later (n = 2). Patients with MVT and CVT were negative for JAK2 V617F, except one patient with CVT and a diagnosis of essential thrombocythemia. No other JAK2 mutations were found in this cohort. Besides MPD, other thrombophilia risk factors were identified in five patients. One patient had MPD as well as thrombophilia risk factor. In this group, 4 out of 7 of the patients with PVT carried the JAK2 V617F mutation with or without overt MPD. However, the investigation of other JAK2 mutations may not be necessary in patients with thrombosis at unusual sites.

Prevalence of Janus kinase 2 mutations in patients with unusual site venous thrombosis / Prevalencia de las mutaciones del gen quinasa Janus 2 en pacientes con trombosis venosa en sitios poco frecuentes

We aimed to study patients with splanchnic vein thrombosis (SVT) and cerebral vein thrombosis (CVT) searching for JAK2 mutations. We evaluated 14 patients (median age: 41.5 years) with portal vein thrombosis (PVT) = 7; mesenteric vein thrombosis (MVT) = 3; and CVT = 4. JAK2 V617F was assessed by allele specific PCR of peripheral blood DNA. In addition, DNA was sequenced for other JAK2 mutations. Other inherited and acquired thrombophilia risk factors were evaluated. JAK2 V617F was positive in four out of seven patients with PVT and in one CVT patient. These five patients had a diagnosis of myeloproliferative disorder (MPD) at the moment of the occurrence of thrombosis (n = 2) or later (n = 2). Patients with MVT and CVT were negative for JAK2 V617F, except one patient with CVT and a diagnosis of essential thrombocythemia. No other JAK2 mutations were found in this cohort. Besides MPD, other thrombophilia risk factors were identified in five patients. One patient had MPD as well as thrombophilia risk factor. In this group, 4 out of 7 of the patients with PVT carried the JAK2 V617F mutation with or without overt MPD. However, the investigation of other JAK2 mutations may not be necessary in patients with thrombosis at unusual sites.

Nuestro objetivo fue estudiar pacientes con trombosis de las venas esplácnicas (TVE) o trombosis de las venas cerebrales (TVC) en búsqueda de mutaciones del gen quinasa Janus 2 (JAK2). Se estudiaron 14 pacientes (media de edad: 41.5 años) con trombosis de la vena porta (TVP n = 7), trombosis de la vena mesentérica (TVM, n = 3) y TVC (n = 4). La mutación V617F del gen JAK2 fue evaluada por reacción en cadena de la polimerasa (PCR) alelo-específica en muestras de sangre periférica. Además, se realizó secuenciación de ADN en búsqueda de otras mutaciones del gen JAK2 distintas de V617F. También se investigaron factores genéticos y adquiridos para trombofilia. JAK2 V617F fue positiva en 4 de 7 pacientes con TVP y en un paciente con TVC. Estos 5 pacientes con la mutación tuvieron diagnóstico de síndrome mieloproliferativo (SMP) en el momento de la detección de la trombosis (n = 2) o después (n = 3). Un paciente con TVP sufrió el episodio trombótico 18 años después del diagnóstico del SMP y la mutación JAK2 V617F fue negativa. No se encontraron otras mutaciones del gen JAK2 en este grupo d e pacientes. Además del diagnóstico de SMP, se identificaron otros factores de riesgo para trombofilia en 4 pacientes. Un paciente tuvo un factor de riesgo para trombofilia además del diagnóstico de SMP. La mutación JAK2 V617F se presentó en 4/7 de los pacientes con TVP con o sin un diagnóstico obvio de SMP. La investigación de otras mutaciones podría no ser necesaria en pacientes con trombosis en sitios poco frecuentes.

Genetic profiling of GSTP1, DPYD, FCGR2A, FCGR3A and CCND1 genes in an Argentinian population.

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 (rs3918290), FCGR2A (rs1801274) 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.

Development of a method to control the RNA extraction and reverse transcription steps for the detection of dengue virus present in human blood samples.

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 (ß-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, ß-Act, and the dengue virus genome were performed. cDNA fragments for ß-Act and G6PDH were amplified for all samples, regardless of the presence or absence of viral RNA. CONCLUSIONS: Amplification of ß-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.

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

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 rs2395029 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 (rs2395029) previous to administration of Abacavir in patients with HIV infection.

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.

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.

Clinical significance of V617F mutation of the JAK2 gene in patients with chronic myeloproliferative disorders.

OBJECTIVE: To determine the prevalence of JAK2 V617F mutation and its clinical correlation in patients with chronic myeloproliferative disorders (CMD): polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). MATERIALS AND METHODS: Detection of JAK2 V617F mutation by allele specific-PCR. RESULTS: One hundred and three patients with CMD were included in the study. JAK2 V617F distribution was PV 40/45 (89%), ET 30/43 (69%), and IMF 7/15 (47%). In PV and ET patients only, 18 had thrombosis at diagnosis and 12 during follow-up (these were microvascular: 11, venous: 7 and arterial: 12); of these 28/70 (40%) were JAK2pos versus 2/18 (11%) JAK2neg; P=0.02. In a median of 4 years, two patients with PV JAK2pos evolved to myelofibrosis and one patient with PV presented in leukemic transformation (JAK2pos before and after transformation); six patients died: four patients with IMF and two patients with PV. CONCLUSIONS: We found an association between JAK2 V617F and thrombotic events in patients with PV and ET.