Detection of F8 mutations in carriers and patients with severe hemophilia A: Identification of a novel mutation / Detección de mutaciones en el gen F8 en mujeres portadoras y en pacientes con hemofilia A: Identificación de una mutación nueva

The molecular diagnosis of haemophilia A (HA) patients has many benefits including diagnosis confirmation and inhibitor risk development prediction. In female carries of a mutation, the molecular diagnosis allows for genetic counseling and prenatal diagnosis, which have become part of the comprehensive care for HA in many countries. Therefore, the aim of this study was to determine the F8 mutations in severe HA (sHA) patients and female carriers. In 12 patients with sHA, the presence of the intron 22 and intron 1 inversions was investigated using an inverse and a conventional PCR method, respectively. In patients negative for the inversions, the F8 gene was screened through conformation sensitive gel electrophoresis (CSGE) and further sequencing. The causative mutation was successfully identified in 6/12 patients, including the novel mutation p.G190C. The mothers of these six patients and those of seven other sHA patients molecularly diagnosed in a previous work were investigated for the presence of the genetic alterations found in their sons. All mothers were found to be carriers. This is the first study conducted in Venezuela which directly analyzes the F8 gene in potential carrier mothers to specifically identify the presence of the mutation that was detected in their sons, and complements a previous study on sHA patients. Our findings will facilitate the implementation of regular screening of HA carriers in our country and will allow a better care of bleeding symptoms and genetic counseling.

El diagnóstico molecular de pacientes con hemofilia A (HA) tiene múltiples beneficios, incluyendo la confirmación del diagnóstico y la predicción del riesgo de desarrollar inhibidores. En mujeres portadoras de una mutación, el diagnóstico molecular permite el consejo genético y el diagnóstico prenatal, los cuales son parte de la atención integral de HA en muchos países. Así, el objetivo de este estudio fue determinar mutaciones en el gen F8 en pacientes con HA severa (HAs) y en mujeres portadoras. En 12 pacientes con HAs, la presencia de la inversión del intrón 22 y el intrón 1 fue investigada utilizando una PCR inversa y una convencional, respectivamente. En pacientes negativos para cualquiera de las inversiones, el gen del F8 fue analizado a través de la técnica de electroforesis en gel sensible a conformación (CSGE) y posterior secuenciación. La mutación causante de la enfermedad fue identificada en 6/12 pacientes, incluyendo la mutación nueva p.G190C. Las madres de estos seis pacientes y las de otros siete pacientes de HAs diagnosticados en un estudio previo y fueron investigadas para la presencia de alteraciones genéticas encontradas en sus hijos. Todas las madres resultaron ser portadoras. Éste es el primer estudio realizado en Venezuela donde se analiza directamente el gen F8 en portadoras potenciales para identificar específicamente la presencia de una mutación que fue detectada en sus hijos, y complementa un estudio previo con pacientes HAs. Nuestros hallazgos facilitarán la implementación del análisis regular de portadoras de HA en nuestro país y permitirán un mejor cuidado de los síntomas de sangrado y consejo genético.

Relación entre la ingesta de antioxidantes, factores nutricionales e indicadores bioquímicos en voluntarios sanos / Relationship between antioxidant intake, nutritional factors and biochemical indicators in healthy volunteers

El estrés oxidativo constituye un factor importante en el desarrollo de Enfermedades Cardiovasculares (ECVs) debido a los daños graves que provocan las especies reactivas de oxígeno en las biomoléculas, por lo que el consumo adecuado de vitaminas con propiedades antioxidantes podría prevenir o retrasar la aparición de estas enfermedades. El objetivo de este trabajo fue determinar la relación entre la ingesta de antioxidantes, factores nutricionales y marcadores bioquímicos en un grupo de individuos sanos de Caracas, Venezuela. El estudio incluyó 29 participantes entre 18-40 años de edad a los cuales se les realizó tres recordatorios dietéticos de 24h, mediciones antropométricas [peso, estatura, circunferencia de cintura (CC), índice cintura cadera (ICC) y % grasa corporal (% GC)] según normativa del Programa Internacional de Biología (IBP). Adicionalmente, se determinó el perfil lipídico y la concentración de 8-isoprostano como marcador de estrés oxidativo. Los participantes tomaron 1 cápsula diaria de vitaminas antioxidantes por 30 días. Posterior al tratamiento con antioxidante, no hubo cambios significativos en las concentraciones de triglicéridos (TG), colesterol total (CT), colesterol de las lipoproteínas de baja densidad (c-LDL) y colesterol de las lipoproteínas de alta densidad (c-HDL). Por su parte, el 8-isoprostano registró una correlación significativa entre antes y después del tratamiento (r=0,374; p<0,05); siendo el mayor descenso en los individuos que presentaron mayor % GC y CC. Los hallazgos sugieren que el suplemento de antioxidantes tiende a disminuir el estrés oxidativo en un corto período de tiempo, particularmente en individuos con mayor % GC, previniendo el desarrollo de ECVs(AU)

Oxidative stress is an important risk factor for the development of cardiovascular diseases (CVD) due to the serious damage caused by reactive oxygen species to biomolecules, thus, adequate intake of vitamins with antioxidant properties could prevent or delay the onset of these diseases. The aim of this study was to determine the relationship between antioxidant intake, nutritional factors and biochemical markers in a group of healthy individuals in Caracas, Venezuela. The study included 29 participants between 18-40 years of age who underwent three 24-hour dietary recalls, anthropometric measurements [weight, height, waist circumference (WC), waist-hip ratio (WHR) and % body fat (% BF)] according to the International Biology Program (IBP) methodology. In addition, the lipid profile and the concentration of 8-isoprostane as a marker of oxidative stress was determined. The participants took one daily capsule of antioxidant vitamins for 30 days. After treatment with antioxidants, no significant changes in triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDLC) levels were observed. Meanwhile, the 8-isoprostane recorded a significative correlation between before and after treatment (r=0.374; p<0.05). The decline in 8-isoprostane levels was more evident in those individuals with the highest % BF and WC. These findings suggest that antioxidant supplementation decreases oxidative stress in a short period of time, particularly in higher % BF individuals, and might help prevent CVDs(AU)

Detection of F8 mutations in carriers and patients with severe hemophilia A. Identification of a novel mutation.

The molecular diagnosis of haemophilia A (HA) patients has many benefits including diagnosis confirmation and inhibitor risk development prediction. In female carries of a mutation, the molecular diagnosis allows for genetic counseling and prenatal diagnosis, which have become part of the comprehensive care for HA in many countries. Therefore, the aim of this study was to determine the F8 mutations in severe HA (sHA) patients and female carriers. In 12 patients with sHA, the presence of the intron 22 and intron 1 inversions was investigated using an inverse and a conventional PCR method, respectively. In patients negative for the inversions, the F8 gene was screened through conformation sensitive gel electrophoresis (CSGE) and further sequencing. The causative mutation was successfully identified in 6/12 patients, including the novel mutation p.G190C. The mothers of these six patients and those of seven other sHA patients molecularly diagnosed in a previous work were investigated for the presence of the genetic alterations found in their sons. All mothers were found to be carriers. This is the first study conducted in Venezuela which directly analyzes the F8 gene in potential carrier mothers to specifically identify the presence of the mutation that was detected in their sons, and complements a previous study on sHA patients. Our findings will facilitate the implementation of regular screening of HA carriers in our country and will allow a better care of bleeding symptoms and genetic counseling.

Relationship between antioxidant in- take, nutritional factors and biochemical indicators in healthy volunteers.

Oxidative stress is an important risk factor for the development of cardiovascular ciseases (CVD) due to the serious damage caused by reactive oxygen species to biomolecules, thus, adequate intake of vitamins with antioxidant properties could prevent or delay the onset of these diseases. The aim of this study was to determine the relationship between antioxidant intake, nutritional factors and biochemical markers in a group of healthy individuals in Caracas, Venezuela. The study included 29 participants between 18-40 years of age who underwent three 24-hour dietary recalls, anthropometric measurements [weight, height, waist circumference (WC), waist-hip ratio (WHR) and % body fat (% BF)] according to the International Biology Program (IBP) methodology. Tn addition, the lipid profile and the concentration of 8-isoprostane as a marker of oxidative stress was determined. The participants took one daily capsule of antioxidant vitamins for.30 days. After treatment with antioxidants, no significant changes in triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (14DL-C) levels were observed. Meanwhile, the 8-isoprostane recorded a significative correlation between before and after treatment (r=0.374; p<0.05). The decline in 8-isoprostane levels was more evident in those individuals with the highest % BF and WC. These findings suggest that antioxidant supplementation decreases oxidative stress in a short period of time, particularly in higher % BF individuals, and might help prevent CVDs.

BRCA1 and BRCA2 mutations in breast cancer patients from Venezuela.

A sample of 58 familial breast cancer patients from Venezuela were screened for germline mutations in the coding sequences and exon-intron boundaries of BRCA1 (MIM no. 113705) and BRCA2 (MIM no. 600185) genes by using conformation-sensitive gel electrophoresis. Ashkenazi Jewish founder mutations were not found in any of the samples. We identified 6 (10.3%) and 4 (6.9%) patients carrying germline mutations in BRCA1 and BRCA2, respectively. Four pathogenic mutations were found in BRCA1, one is a novel mutation (c.951_952insA), while the other three had been previously reported (c.1129_1135insA, c.4603G>T and IVS20+1G>A). We also found 4 pathogenic mutations in BRCA2, two novel mutations (c.2732_2733insA and c.3870_3873delG) and two that have been already reported (c.3036_3039delACAA and c.6024_6025_delTA). In addition, 17 variants of unknown significance (6 BRCA1 variants and 11 BRCA2 variants), 5 BRCA2 variants with no clinical importance and 22 polymorphisms (12 in BRCA1 and 10 in BRCA2) were also identified. This is the first genetic study on BRCA gene mutations conducted in breast cancer patients from Venezuela. The ethnicity of our population, as well as the heterogeneous and broad spectrum of BRCA genes mutations, must be considered to optimize genetic counseling and disease prevention in affected families.

BRCA1 and BRCA2mutations in breast cancer patients from Venezuela

A sample of 58 familial breast cancer patients from Venezuela were screened for germline mutations in the coding sequences and exon-intron boundaries of BRCA1 (MIM no. 113705) and BRCA2 (MIM no. 600185) genes by using conformation-sensitive gel electrophoresis. Ashkenazi Jewish founder mutations were not found in any of the samples. We identified 6 (10.3%) and 4 (6.9%) patients carrying germline mutations in BRCA1 and BRCA2, respectively. Four pathogenic mutations were found in BRCA1, one is a novel mutation (c.951_952insA), while the other three had been previously reported (c.1129_1135insA, c.4603G>T and IVS20+1G>A). We also found 4 pathogenic mutations in BRCA2, two novel mutations (c.2732_2733insA and c.3870_3873delG) and two that have been already reported (c.3036_3039delACAA and c.6024_6025_delTA). In addition, 17 variants of unknown significance (6 BRCA1 variants and 11 BRCA2 variants), 5 BRCA2 variants with no clinical importance and 22 polymorphisms (12 in BRCA1 and10 in BRCA2) were also identified. This is the first genetic study on BRCA gene mutations conducted in breast cancer patients from Venezuela. The ethnicity of our population, as well as the heterogeneous and broad spectrum of BRCA genes mutations, must be considered to optimize genetic counseling and disease prevention in affected families.

Factor V Leiden and the risk of venous thrombosis, myocardial infarction, and stroke: a case-control study in Venezuela.

The most common genetic defect associated with deep vein thrombosis (DVT) is a mutation in the Factor V gene (G1691A), known as Factor V Leiden (FVL). We investigated the genotypes for FVL in 571 individuals in Venezuela: 208 patients with DVT, 175 patients with acute myocardial infarction, 54 patients with stroke, and 134 control subjects. Our results showed in the population analyzed here that the FVL was associated with a fourfold increase in the risk for DVT (odds ratio, 4.24; 95% confidence interval, 1.35-14.79); particularly, women carriers showed a 6.5-fold increase in the risk for DVT. No relation was observed between the presence of FVL and the risk for acute myocardial infarction or stroke. In conclusion, a clear association between the FVL mutation and DVT was observed in the population analyzed in Venezuela. These results are in agreement with those found in other populations with different ethnic backgrounds.

Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: cloning and expression of gntP and gntK in Escherichia coli.

Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral medium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constitutively expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli M1-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: Cloning and expression of gntP and gntK in Escherichia coli

Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral médium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constituvely expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli Ml-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

The metabolism of gluconate in Escherichia coli: Physiological evidences of a regulatory effect of idnR on the expression of the gntR regulon operons

Uptake and phosphorylation initiate the catabolism of gluconate in E. coli. Such activities conform two systems,GntI and GntII, encoded by two sets of genes differently located on the E. coli chromosome and under different regulation. gntT, gntU and gntK (minute 76) encode for high and low affinity gluconate transports and for a thermoresistant gluconokinase respectively, that conform GntI; the mentioned genes and those of the edd-eda operon (minute 41) are negatively regulated by the gntR gene product conforming the gntR regulon. idnT and gntV (minute 96), encode for another gluconate transport and a thermosensitive gluconokinase, conforming GntII. These genes are presumably positively controlled by IdnR. IdnT also functions as a permease for idonate; the corresponding gene is included in the idnDOTR operon, responsible for idonate metabolism, in which gluconate is an intermediary. Here we report a regulatory action of IdnR on the operons of the gntR regulon; i.e., gntT, gntKU and edd-eda. The expression of these operons, was diminished in a gntR mutant complemented with a clone of idnR and also in E. coli mutants in which the idnDOTR operon is expressed in a gluconate dependent inducibility. This is the first report of a regulatory effect of IdnR on edd-eda operon expression.

El transporte y la fosforilación inician el catabolismo del gluconato en E. coli. Estas actividades conforman dossistemas, GntI y GntII, codificados por dos grupos de genes, diferentemente regulados y ubicados en distintos sitios del cromosoma. Los genes gntT, gntU y gntK (minuto 76), codifican distintas proteínas para transportes de alta y baja afinidad para gluconato y una gluconoquinasa termoresistente respectivamente, que forman el sistema GntI; los genes respectivos junto con los del operón edd-eda (minuto 41), son regulados negativamente por el producto de gntR (minuto76) constituyendo el regulón gntR. Los genes IdnT y gntV (minuto 96), codifican otra permeasa para gluconato y una gluconoquinasa termosensible que forman GntII. IdnT funciona también como permeasa para idonato; el gen correspondiente es parte del operón idnDOTR, regulado positivamente por IdnR y responsable del metabolismo del idonato en el que gluconato es un intermediario. Se reporta un efecto regulatorio de IdnR sobre los operones del regulón gntR; i.e., gntT, gntKU and edd-eda. La expresión de estos operones resultó disminuida en una mutante gntR complementada con un clon de idnR y también en mutantes de E. coli en la que la expresión del operón idnDOTR se induce en presencia de gluconato. Este es el primer reporte de la acción regulatoria de IdnR sobre la expresión del operón eddeda.