Browsing by Author "Bronze-da-Rocha, Elsa"
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- Bilirubin dependent on UGT1A1 polymorphisms, hemoglobin, fasting time and body mass indexPublication . Rodrigues, Carina; Costa, Elísio; Vieira, Emília; Carvalho, João; Santos, Rosário; Rocha-Pereira, Petronila; Santos-Silva, Alice; Bronze-da-Rocha, ElsaIn humans, bilirubin levels are influenced by different factors. This study aims to evaluate the influence of several nongenetic factors (hematologic data, smoking status, alcohol intake, fasting time, physical activity, oral contraceptive therapy and caloric intake) and the genetic contribution of UGT1A1 polymorphisms for the bilirubin levels, in a cohort of young women. Hematologic data, bilirubin and screening of TA duplication in the TATA box region of the UGT1A1 gene were performed in 146 young white women. Body mass index (BMI) and body fat were determined, and a questionnaire about fasting time, smoking habits, oral contraceptive therapy, caloric intake and physical activity was performed. Participants were divided into 3 groups according to the tertiles of bilirubin levels. Subjects from the second and third tertile had significant increases in hemoglobin (Hb) concentration, hematocrit, mean cell Hb and mean cell Hb concentration compared with those in the first tertile. Red blood cell count was significantly increased in subjects in the third tertile. A significant increased frequency was found for the c.-41_-40dupTA allele in homozygosity for both second and third tertiles. Multiple linear regression analysis showed that the c.-41_-40dupTA allele, Hb, BMI and fasting hours were independent variables associated with bilirubin serum levels. Hb concentration, fasting time and BMI were identified as nongenetic causes, together with the genetic UGT1A1 polymorphisms, as the main factors associated with variations in bilirubin levels in a healthy female population.
- Bilirubin levels and redox status in a young healthy populationPublication . Rodrigues, Carina; Rocha, Susana; Nascimento, Henrique; Vieira, Emília; Santos, Rosário; Santos-Silva, Alice; Costa, Elísio; Bronze-da-Rocha, ElsaThe additional TA repeat (c.-41_ -40dupTA) in the promoter of the uridine diphosphate glucuronosyltransferase (UGT1A1) gene is associated with a decrease in gene transcription, a decline in bilirubin conjugation and, therefore, with an increase in circulating unconjugated bilirubin (UCB) [1] . The TA repeat polymorphism is remarkably prevalent in the Caucasian white population and is the main cause of Gilbert’s syndrome [1] . Bilirubin, the key metabolic product of hemoglobin (Hb) catabolism, has antioxidant properties that seem to have a protective effect in oxidative stress conditions, such as in atherosclerosis, coronary heart disease, inflammation, and cancer [2] . Some studies showed that high levels of bilirubin can be toxic to neurons in newborn infants.
- Contribution of red cell mass and UGT1A1 alleles in serum bilirubin levels of the portuguese populationPublication . Rodrigues, Carina; Costa, Elísio; Santos-Silva, Alice; Santos, Rosário; Bronze-da-Rocha, ElsaHepatic glucuronization of insoluble bilirubin is catalyzed by isoenzyme 1A1 of UDP-glucuronosyltransferase (UGT1A1), which is essential for efficient biliary excretion of bilirubin. The main cause of Gilbert syndrome (GS) in all populations studied to date is a TA duplication [(TA)7 allele] in the repetitive TATA-box sequence of the gene promoter, which normally consists of six TA repeats. However, this genetic polymorphism is not sufficient for the clinical phenotype of GS. By this reason, some studies have been performed to provide information about additional factors that could contribute to the pathogenesis of this disease. Recently, it was described that increased red cell mass probably plays a role in the pathogenesis of GS (Buyukasik et al. 2008 Am J Med Sci. 335,115-119). The aim of this work is to investigate the putative role of increased red cell mass and the (TA)7 allele in bilirubin serum levels, in the Portuguese population. This study was performed in 109 volunteer healthy young adults (20.3±1.9 years) without liver and/or hematological disorders, chronic infection, recent inflammation, malignancy, hemorrhage and medication. Blood samples were collected and processed in order to determine bilirubin serum levels, complete blood cells count, and DNA extraction. The TATA-box region was analyzed by PCR amplification followed by subsequent analysis by automated capillary electrophoresis. Among our population, 6 were homozygous for the (TA)7 allele, 55 were heterozygous and 48 were homozygous for the normal allele. One of the subjects was a compound heterozygous for the (TA)5 and (TA)7 alleles. Comparing the blood cells counts and the bilirubin serum levels according to the UGT1A1 genotype, we found statistically differences only in bilirubin levels [(TA)6/(TA)6: 0.49±0.20 mg/dL; (TA)6/(TA)7: 0.70±0.32 mg/dL; (TA)7/(TA)7: 1.10±0.74 mg/dL, p<0.05]. A positive statistically significant correlation (p<0.05) were found between bilirubin serum levels and haematocrit and mean cell volume. Our work showed that higher bilirubin serum levels are correlated with an increase red blood mass. However, no association was found between higher red blood mass and abnormal number of TA repeats in the promoter of UGT1A1 gene. This data suggests that in our population the presence of abnormal number of TA repeats in the UGT1A1 gene is associated with increased bilirubin levels but not with higher red blood mass, as previously described for GS patients.
- Contribution of red cell mass and UGT1A1 alleles in serum bilirubin levels of the portuguese populationPublication . Rodrigues, Carina; Costa, Elísio; Santos-Silva, Alice; Santos, M.R.; Bronze-da-Rocha, ElsaHepatic glucuronization of insoluble bilirubin is catalyzed by isoenzyme 1A1 of UDP-glucuronosyltransferase (UGT1A1), which is essential for efficient biliary excretion of bilirubin. The main cause of Gilbert syndrome (GS) in all populations studied to date is a TA duplication [(TA)7 allele] in the repetitive TATA-box sequence of the gene promoter, which normally consists of six TA repeats. However, this genetic polymorphism is not sufficient for the clinical phenotype of GS. By this reason, some studies have been performed to provide information about additional factors that could contribute to the pathogenesis of this disease. Recently, it was described that increased red cell mass probably plays a role in the pathogenesis of GS. The aim of this work is to investigate the putative role of increased red cell mass and the (TA)7 allele in bilirubin serum levels, in the Portuguese population. This study was performed in 109 volunteer healthy young adults (20.3 ± 1.9 years) without liver and/or hematological disorders, chronic infection, recent inflammation, malignancy, hemorrhage and medication. Blood samples were collected and processed in order to determine bilirubin serum levels, complete blood cells count, and DNA extraction. The TATA-box region was analyzed by PCR amplification followed by subsequent analysis by automated capillary electrophoresis. Among our population, 6 were homozygous for the (TA)7 allele, 55 were heterozygous and 48 were homozygous for the normal allele. One of the subjects was a compound heterozygous for the (TA)5 and (TA)7 alleles. Comparing the blood cells counts and the bilirubin serum levels according to the UGT1A1 genotype, we found statistically differences only in bilirubin levels [(TA)6/(TA)6: 0.49 ± 0.20 mg/dL; (TA)6/(TA)7: 0.70 ± 0.32 mg/dL; (TA)7/(TA)7: 1.10 ± 0.74 mg/dL, p<0.05]. A positive statistically significant correlation (p<0.05) were found between bilirubin serum levels and haematocrit and mean cell volume. Our work showed that higher bilirubin serum levels are correlated with an increase red blood mass. However, no association was found between higher red blood mass and the presence of abnormal number of TA repeats in the promoter of UGT1A1 gene. This data suggests that in our population the presence of abnormal number of TA repeats in the UGT1A1 gene is associated with increased bilirubin levels and that increased haematocrit and mean cell volume could contribute for this phenotype. Further studies comparing a larger group of GS patients, homozygous for the (TA)7) allele, are required to better understand the contribution of the red blood mass in the hiperlirubinemia.
- Contribution of red cell mass and ugt1a1 alleles in serum bilirubin levels of the portuguese populationPublication . Rodrigues, Carina; Costa, Elísio; Santos-Silva, Alice; Bronze-da-Rocha, ElsaHepatic glucuronization of insoluble bilirubin is catalyzed by isoenzyme 1A1 of UDP-glucuronosyltransferase (UGT1A1), which is essential for efficient biliary excretion of bilirubin. The main cause of Gilbert syndrome (GS) in all populations studied to date is a TA duplication [(TA)7 allele] in the repetitive TATA-box sequence of the gene promoter, which normally consists of six TA repeats. However, this genetic polymorphism is not sufficient for the clinical phenotype of GS. By this reason, some studies have been performed to provide information about additional factors that could contribute to the pathogenesis of this disease. Recently, it was described that increased red cell mass probably plays a role in the pathogenesis of GS. The aim of this work is to investigate the putative role of increased red cell mass and the (TA)7 allele in bilirubin serum levels, in the Portuguese population. This study was performed in 109 volunteer healthy young adults (20.3 ± 1.9 years) without liver and/or hematological disorders, chronic infection, recent inflammation, malignancy, hemorrhage and medication. Blood samples were collected and processed in order to determine bilirubin serum levels, complete blood cells count, and DNA extraction. The TATA-box region was analyzed by PCR amplification followed by subsequent analysis by automated capillary electrophoresis. Among our population, 6 were homozygous for the (TA)7 allele, 55 were heterozygous and 48 were homozygous for the normal allele. One of the subjects was a compound heterozygous for the (TA)5 and (TA)7 alleles. Comparing the blood cells counts and the bilirubin serum levels according to the UGT1A1 genotype, we found statistically differences only in bilirubin levels [(TA)6/(TA)6: 0.49 ± 0.20 mg/dL; (TA)6/(TA)7: 0.70 ± 0.32 mg/dL; (TA)7/(TA)7: 1.10 ± 0.74 mg/dL, p<0.05]. A positive statistically significant correlation (p<0.05) were found between bilirubin serum levels and haematocrit and mean cell volume. Our work showed that higher bilirubin serum levels are correlated with an increase red blood mass. However, no association was found between higher red blood mass and the presence of abnormal number of TA repeats in the promoter of UGT1A1 gene. This data suggests that in our population the presence of abnormal number of TA repeats in the UGT1A1 gene is associated with increased bilirubin levels and that increased haematocrit and mean cell volume could contribute for this phenotype. Further studies comparing a larger group of GS patients, homozygous for the (TA)7) allele, are required to better understand the contribution of the red blood mass in the hiperlirubinemia.
- Desempenho de ferramentas in silico: Avaliação de variantes de mutações missense do gene UGT1A1Publication . Rodrigues, Carina; Santos-Silva, Alice; Costa, Elísio; Bronze-da-Rocha, ElsaVariations in the gene encoding uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) are particularly important because they have been associated with hyperbilirubinemia in Gilbert’s and Crigler-Najjar syndromes as well as with changes in drug metabolism. Several variants associated with these phenotypes are non-synonymous single nucleotide polymorphisms (nsSNPs). Bioinformatics approaches have gained increasing importance in predicting the functional significance of these variants. This study was focused on the predictive ability of bioinformatics approaches to determine the pathogenicity of human UGT1A1 nsSNPs, which were previously characterized at the protein level by in vivo and in vitro studies. Using sixteen web algorithms, we evaluated 48 nsSNPs described in the literature and databases. Eight of these algorithms reached or exceeded 90% sensitivity and six presented a Matthews correlation coefficient above 0.46. The best performing method was MutPred, followed by SIFT. The prediction measures varied significantly when predictors such us SIFT, Polyphen-2 and PMUT were run with their native alignment generated by the tool, or with an input alignment that was strictly built with UGT1A1 orthologs and manually curated. The capacity of the SNP prediction methods can vary according to the available structural information and the MSA employed. We verified that methods primarily based on protein structural information such as SNPeffect failed to give reliable information for the UGT1A1 variants. There are numerous available evolutionary tools that do not allow the user to select the alignments. One of the concepts that our data suggest is that the selection of user inputs improves the performance of these methods. Our data also suggest that whenever possible, the user should consider optimizing the sequence alignment employed. The performance study of SNP predictors using a set of functionally well-characterized variants is essential to help redirect the in silico analysis of a particular gene.
- Factores genéticos e ambientais que afectam os níveis séricos de bilirrubina na população portuguesaPublication . Rodrigues, Carina; Costa, Elísio; Santos, Rosário; Santos-Silva, Alice; Bronze-da-Rocha, ElsaA bilirrubina é um marcador bioquímico largamente utilizado no diagnóstico e monitorização das doenças hepáticas e doenças hematológicas. A bilirrubina é um produto tóxico que resulta sobretudo do metabolismo do grupo heme da hemoglobina libertada durante o processo de senescência dos eritrócitos mas pode, igualmente, ter outras origens tais como a eritropoiese ineficaz e o metabolismo de outras hemoproteínas. No fígado, a isoenzima UDP-glucoroniltransferase 1A1 (UGT1A1) catalisa a glucuronidação da bilirrubina, essencial para a sua excreção a nível biliar. A presença de uma duplicação de dois nucleotídeos (TA) na região TATAA do promotor do gene UGT1A1 resulta numa redução da actividade da enzima em cerca de 30%, por diminuição da eficiência de transcrição do gene. Esta alteração está muitas vezes associada a uma hiperbilirrubinémia não conjugada hereditária designada por Síndrome de Gilbert (SG). Embora a frequência do alelo com a duplicação (TA7) na população caucasiana seja elevada (38,7%), esta não explica na totalidade a presença desta síndrome. Há variações inter-individuais que parecem estar associadas ao sexo, idade, consumo de tabaco, uso de contraceptivos orais, exercício físico, stress, doenças intercorrentes e tempo de jejum nocturno. Mais recentemente, colocou-se a hipótese de que uma maior massa eritrocitária e respectivos índices hematimétricos poderiam explicar a presença de hiperbilirrubinémia. O objectivo deste trabalho foi avaliar o efeito dos polimorfismos genéticos no gene UGT1A1, o efeito de alguns factores ambientais e da massa eritrocitária nas variações da concentração da bilirrubina na população Portuguesa. Este estudo foi realizado num grupo controlo de 165 jovens adultos (19.5 ± 2.1 anos) ao qual foi aplicado um instrumento de recolha de dados que incluiu questões sobre hábitos tabágicos, terapêutica com contraceptivos orais, ingestão calórica, jejum nocturno e actividade física. Foram colhidas amostras de sangue para a realização de hemogramas, determinação dos níveis séricos de bilirrubina e extracção de DNA. Foi feita a avaliação dos níveis séricos de bilirrubina (método colorimétrico) e a detecção de polimorfismos no promotor do gene UGT1A1 (por PCR e pesquisa dos fragmentos num analisador automático). A genotipagem do gene UGT1A1 evidenciou que 15 indivíduos eram homozigóticos para o alelo com a duplicação (TA7/TA7), 79 heterozigóticos (TA6/TA7) e 71 homozigóticos para o alelo normal (TA6/TA6). A frequência do alelo TA7 obtida foi de 33%, um valor próximo do que está descrito para a população caucasiana. A análise da associação do polimorfismo com os níveis de bilirrubina demonstrou diferenças significativas entre os 3 genótipos (p=0.001). Foram igualmente observadas diferenças estatisticamente significativas de acordo com o sexo (p=0,09) e uma tendência de valores mais baixos de bilirrubina total, sem contudo alcançar significância estatística (p<0,05), em indivíduos fumadores e em mulheres que não tomam contraceptivos orais. Identificou-se uma correlação positiva entre a concentração sérica de bilirrubina total e o tempo de jejum nocturno, aquando da realização da colheita (p=0,01); a ingestão calórica (p=0,03); a hemoglobina (p=0,001); o volume globular (p=0,04); hemoglobina globular média (p=0,006). Verificou-se uma correlação negativa entre os níveis de bilirrubina total sérica e número de leucócitos (p=0,021) e plaquetas (p=0,001). Os resultados deste trabalho evidenciaram que a bilirrubina sérica na nossa população é condicionada pela presença do polimorfismo do UGT1A1 e verificou-se que um intervalo maior de jejum nocturno e uma maior massa eritrocitária, dentro dos valores considerados normais para a população em geral, estão associados a valores mais elevados de bilirrubina.
- Genetic and acquired factors that modulate serum bilirubin levelsPublication . Rodrigues, Carina; Costa, Elísio; Santos-Silva, Alice; Santos, Rosário; Bronze-da-Rocha, ElsaThe isoenzyme UDP-glucuronosyltransferase 1A1 (UGT1A1) catalyzes bilirubin glucuronidation. Molecular studies suggest that the presence of two extra bases in the repetitive promoter TATA box region of the UGT1A1 gene, described as (TA)7 allele, is responsible for the reduced UGT1A1 activity that leads to hyperbilirubinemia. In fact, patients with Gilbert’s syndrome (GS), a recessive disorder characterized by a mild unconjugated hyperbilirubinemia, are often homozygous for the TA duplication. The “major” recessive gene (UGT1A1) and other non-genetic factors are also associated with the inter-individual variation of bilirubin concentration. To establish the influence of genetic and non-genetic variables in serum bilirubin concentration, we recruit 81 young adults (62 females and 19 males with average age 20,2 ±1,7 years) that give their written informed consent. A standardized questionnaire inquiring about smoking habits, oral contraceptive therapy, caloric intake, fasting time and physical activity was performed to select the participants without liver and/or haematological disorders. After an overnight fasting, venous blood samples were collected to determine total and direct-reacting bilirubin and to analyze the UGT1A1 promoter region in genomic DNA. From UGT1A1 genotyping, we identified 6 homozygous for the (TA)7 allele, 40 were heterozygous and 35 were homozygous for the normal allele. Mean (± SD) serum bilirubin levels were 10.60 ± 4.46 μmol/L, but trend to higher bilirubin levels was found in males than in females (12.7 ± 6.33 μmol/L vs. 10,3 ± 5.5 μmol/L). Higher bilirubin concentrations were found in non-smoking subjects (11.2 ± 6.07 μmol/L vs. 9.7 ± 2,6 μmol/L) and in females taken oral contraceptives (11.9 ± 7.9 μmol/L vs. 9.5 ± 3,3 μmol/L). Statistically significant correlations were found between bilirubin serum levels and fasting time (r=0,421, p=0.001), as well as caloric intake (r=-0.255; p=0.021). Multiple regression analysis identified fasting time (β=0.36; p=0.01), under oral contraceptive therapy (β=0.232; p=0.024) and TA polymorphism (β=0.480; p=0.001) as independent variables that account for 41,9% of total serum bilirubin levels variation (R2=0.419). No significant association was found between bilirrubin concentrations and physical activity. Our results suggest that beyond the genetic information, the caloric intake, fasting time, smoking status and oral contraceptive therapy also contribute to the interindividual variation of serum bilirubin levels.
- Genetic and acquired factors that modulate serum bilirubin levelsPublication . Rodrigues, Carina; Costa, Elísio; Santos-Silva, Alice; Santos, Rosário; Bronze-da-Rocha, ElsaThe isoenzyme UDP-glucuronosyltransferase 1A1 (UGT1A1) catalyzes bilirubin glucuronidation. Molecular studies suggest that the presence of two extra bases in the repetitive promoter TATA box region of the UGT1A1 gene, described as (TA)7 allele, is responsible for the reduced UGT1A1 activity that leads to hyperbilirubinemia. In fact, patients with Gilbert’s syndrome (GS), a recessive disorder characterized by a mild unconjugated hyperbilirubinemia, are often homozygous for the TA duplication. The “major” recessive gene (UGT1A1) and other non-genetic factors are also associated with the inter-individual variation of bilirubin concentration. To establish the influence of genetic and non-genetic variables in serum bilirubin concentration, we recruit 81 young adults (62 females and 19 males with average age 20,2 ±1,7 years) that give their written informed consent. A standardized questionnaire inquiring about smoking habits, oral contraceptive therapy, caloric intake, fasting time and physical activity was performed to select the participants without liver and/or haematological disorders. After an overnight fasting, venous blood samples were collected to determine total and direct-reacting bilirubin and to analyze the UGT1A1 promoter region in genomic DNA. From UGT1A1 genotyping, we identified 6 homozygous for the (TA)7 allele, 40 were heterozygous and 35 were homozygous for the normal allele. Mean (± SD) serum bilirubin levels were 10.60 ± 4.46 μmol/L, but trend to higher bilirubin levels was found in males than in females (12.7 ± 6.33 μmol/L vs. 10,3 ± 5.5 μmol/L). Higher bilirubin concentrations were found in non-smoking subjects (11.2 ± 6.07 μmol/L vs. 9.7 ± 2,6 μmol/L) and in females taken oral contraceptives (11.9 ± 7.9 μmol/L vs. 9.5 ± 3,3 μmol/L). Statistically significant correlations were found between bilirubin serum levels and fasting time (r=0,421, p=0.001), as well as caloric intake (r=-0.255; p=0.021). Multiple regression analysis identified fasting time (β=0.36; p=0.01), under oral contraceptive therapy (β=0.232; p=0.024) and TA polymorphism (β=0.480; p=0.001) as independent variables that account for 41,9% of total serum bilirubin levels variation (R2=0.419). No significant association was found between bilirrubin concentrations and physical activity. Our results suggest that beyond the genetic information, the caloric intake, fasting time, smoking status and oral contraceptive therapy also contribute to the interindividual variation of serum bilirubin levels.
- Impact of UGT1A1 gene variants on total bilirubin levels in Gilbert syndrome patients and in healthy subjectsPublication . Rodrigues, Carina; Vieira, Emília; Santos, Rosário; Carvalho, João; Santos-Silva, Alice; Costa, Elísio; Bronze-da-Rocha, ElsaA significant different allelic distribution, in Gilbert patients and in controls, was found for two promoter polymorphisms. Among patients, 82.2% were homozygous and 17.8% heterozygous for the c.− 41_ − 40dupTA allele; in control group, 9.9% were homozygous and 43.5% heterozygous for this promoter variant, while 46.6% (n = 75) presented the [A(TA)6TAA]. For the T>G transition at c.− 3279 promoter region, in patients, 86.7% were homozygous and 13.3% heterozygous; in control group, 33.5% were homozygous for the wild type allele, 44.1% were heterozygous and 22.4% homozygous for the mutated allele. The two polymorphisms were in Hardy–Weinberg equilibrium in both groups. Sequencing of UGT1A1 coding region identified nine novel variants, five in patients and four in controls. In silico analysis of these amino acids replacements predicted four of them as benign and three as damaging. In conclusion, we demonstrated that total bilirubin levels are mainly determined by the TA duplication in the TATA-box promoter and by the c.− 3279T>G variant. Alterations in the UGT1A1 coding region seem to be associated with increased bilirubin levels, and, therefore, with Gilbert syndrome.
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