Caracterização de autoanticorpos produzidos por ratinhos portadores de lupus eritematoso sistémico : Análise citológica e molecular dos respectivos antigénios

Dissertação de Doutoramento em Farmácia, área de especialização em Bioquímica, apresentada à Faculdade de Farmácia da Universidade do Porto, área de especialização em Bioquímic

DNA Damage in End-Stage Renal Disease Patients. Assessment by In Vitro Comet Assay and by Cell-Free DNA Quantification

Inflammation is a common feature in end stage renal disease (ESRD) that might contribute to increase DNA damage. ESRD patients present increased circulating cell-free DNA (cfDNA) and different types of DNA injury. The underlying inflammatory process in ESRD may be associated with increased genomic damage and cfDNA contributing to further enhance inflammation. We analyzed the degree of genomic damage in ESRD patients under hemodialysis therapy, using the comet assay and cfDNA quantification. ESRD patients presented significantly higher C-reactive protein (CRP) and cell damaged DNA. The cfDNA correlated with age and inflammatory stage. Nine out of 39 patients died during the one year follow-up period and presented significantly higher cfDNA, than those who persisted alive. At lower CRP values, the increased DNA damage is still within the cell, and at higher CRP the damaged DNA is released in to plasma. The higher degree of genomic damage in ESRD might be a consequence of inflammation and aging, and may contribute to increase cancer and cardiovascular mortality risk. Our data suggest that the comet assay is more sensitive for low-grade inflammatory conditions, while cfDNA appears as a good biomarker for more severe inflammatory conditions, and as a biomarker for the outcome of ESRD patients

Performance of in silico tools for the evaluation of UGT1A1 missense variants

Variations 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 nonsynonymous 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 16 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 Sorting Intolerant from Tolerant (SIFT). The prediction measures varied significantly when predictors such us SIFT, polyphen-2, and Prediction of Pathological Mutations on Proteins 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. Our results showed that the prediction performance of some methods based on sequence conservation analysis can be negatively affected when nsSNPs are positioned at the hypervariable or constant regions of UGT1A1 ortholog sequences

Desempenho de ferramentas in silico: Avaliação de variantes de mutações missense do gene UGT1A1

Variations 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.info:eu-repo/semantics/publishedVersio

Bilirubin dependent on UGT1A1 polymorphisms, hemoglobin, fasting time and body mass index

In PressIn 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.Bolsa de doutoramento SFRH/BD/42791/2007 da Fundação para a Ciência e Tecnologia (FCT) e Fundo Social Europeu

Impact of UGT1A1 gene variants on total bilirubin levels in Gilbert syndrome patients and in healthy subjects

A 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.A PhD grant (SFRH/BD/42791/2007) attributed to Carina Rodrigues, from Fundação para a Ciência e Tecnologia (FCT) and Fundo Social Europeu (FSE), supported this work

UGT1A1 gene variations in individuals with and without clinical diagnosis of Gilbert Syndrome

UGT1A1 gene variations in individuals with and without clinical diagnosis of Gilbert Syndrome Bilirubin is a non-polar metabolite, results from catabolism of haemoglobin and is bound to glucuronic acid in the liver by the uridine diphosphate glucuronosyltransferase (UGT1A1) activity. Molecular studies showed that the presence of two extra bases (TA duplication) in the promoter region of the UGT1A1 gene is responsible for the reduced UGT1A1 glucuronization activity and is the main cause of unconjugated hyperbilirubinemia observed in patients with Gilbert Syndrome (GS). However, individuals with normal bilirubin levels and no clinical symptoms of SG may also present this polymorphism in homozygosity 1,2,3. Consequently, the aim of this work is to determine the presence of other mutations in the UGT1A1gene, downstream of the TA duplication, and how they may contribute towards the inter-individual variation of serum bilirubin levels. This study was carried out in two groups: one comprising 36 individuals without clinical diagnosis of GS (14 with 6/6TA, 11 with 6/7TA, and 11 with 7/7TA repeats); the other group consisting of 36 patients clinically diagnosed with GS. In both, bilirubin levels were determined and direct sequencing of the UGT1A1 was performed. Among the individuals without clinical diagnosis of GS, two new sequence variants were found in heterozygosity (c.643A>G, and c.1156G>A), in the 6/6TA group. No additional mutations were detected in the 6/7 and 7/7 TA groups. In patients clinically diagnosed with GS, 28 were homozygous and 7 heterozygous for the TA duplication, and one with a normal number of repeats. Molecular analysis showed that one (3,6%) of the 7/7TA patients had another mutation in the UGT1A1 gene (c.674T>G). In the 6/7TA group, one additional mutation was also found in three patients (43%), two of which had been previously described (c.674T>G and c.923G>A) and a new one (c.1423C>T). No further mutations were detected in the 6/6TA group. Additionally, 4 polymorphisms were found (c.864+89C>T; c.997-37T>C; c.997-82A>C; c.997-87A>C). In conclusion, we can infer that homozygosity for the TA duplication is associated with GS. In the group without GS, no further mutations were detected in the 6/7 and 7/7 clusters, but in the 6/6 group, two new mutations were found in heterozygosity. These mutations are not associated with increased bilirubin levels. However, they could be associated with GS in the presence of other UGT1A1 mutations. Furthermore, in the GS group with heterozygosity for the TA duplication, we found mutations in 43% of the patients, emphasizing the importance of complete UGT1A1 analysis

Genetic and acquired factors that modulate serum bilirubin levels

The 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.Bolsa de Doutoramento(SFRH/BD/42791/2007) Fundação para a Ciência e Tecnologia (FCT) attributed to Carina Rodrigues and Fundo Social Europeu (FSE

Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytes

Two COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.info:eu-repo/semantics/acceptedVersio

Ugt1a1 gene variants and total bilirubin levels in healthy subjects and in gilbert syndrome patients

Gilbert syndrome (GS, OMIM 606785) is an autosomal recessive condition characterized by unconjugated hiperbilirubinemia in the absence of hemolysis or underlying liver disease due to the reduced activity of the uridine diphosphate-glucuronosyltransferase (UGT1A1). This enzyme is mainly expressed in the liver and has an important role in the glucuronidation of bilirubin, 17β-estradiol, some therapeutic drugs and mutagenic xenobiotics. Absence or severe reductions of UGT1A1 activity are associated with Crigler-Najjar syndrome type I and type II, respectively. Heterozygous carriers of Crigler-Najjar syndrome also present a high incidence of mild hyperbilirubinemia, a feature of GS. Aim: This work investigated the effect of UGT1A1 variants on total bilirubin levels in Gilbert patients (n=45) and healthy controls (n=161). Total bilirubin levels were determined using a colorimetric method. Molecular analysis of exons 1-5 and two UGT1A1 promoter regions were performed by direct sequencing and automatic analysis of fragments. Five in silico methods predicted the effect of new identified variants. A 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. We demonstrated that total bilirubin levels are manly 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