DPSPDNT - Posters/abstracts em congressos internacionais
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- Correlation between lipid profile / cardiovascular risk and type of mutation in Portuguese patients with familial hypercholesterolemiaPublication . Alves, A.C.; Medeiros, A.M.; Bourbon, M.Familial Hypercholesterolemia (FH) is characterized by high levels of LDLc in plasma, accelerated atherosclerosis and increased risk of premature coronary heart disease. FH results from mutations in three genes involved in lipid metabolism: LDLR, APOB, PCSK9. It is known that FH patients’ phenotype is heterogeneous varying with different conditions. The aim of this study was to analyse the biochemical profile of patients with genetically diagnosed FH in accordance with the mutations in different genes and the different types of mutations identified in the LDLR gene to identify whether there is a correlation between these variables in Portuguese patients. Biochemical parameters, total cholesterol (TC), LDLc, HDLc, triglycerides, ApoB and ApoA of 325 patients with FH were analyzed by SPSS using ANOVA and Tukey. Patients with mutations in the PCSK9 have LDLc and TC levels significantly higher than patients with mutations in the remaining two genes. There is no significant difference in these parameters between patients with mutations in LDLR and APOB genes, although all values are higher in patients with mutations in the LDLR. Patients with nonsense mutations in the LDLR gene present values of TC, LDL and ApoB statistically higher than in patients with missense mutations. The comparison between the other categories showed no significant changes in biochemical parameters. Patients with mutations in the PCSK9 have a more severe phenotype than patients with mutations in LDLR and APOB genes. The type of mutation (in different genes or different mutations in LDLR) is important in determining cardiovascular risk in FH patients.
- Milder phenotype of relatives of index patients can misdiagnose Familial HypercholesterolemiaPublication . Medeiros, A.M.; Alves, A.C.; Bourbon, M.Familial Hypercholesterolemia (FH) is a genetic disorder characterized by high levels of LDLc in plasma, accelerated atherosclerosis and increased risk of premature coronary heart disease (pCHD). FH results from mutations in three genes involved in lipid metabolism: LDLR, APOB, PCSK9. It is known that FH patients’ phenotype is heterogeneous varying with different conditions, as gene and type of mutation. The present study pretends to characterize the biochemical profile of FH patients genetically identified in Portugal. The Portuguese FH Study identified 420 patients: 182 index (60 children, 122 adults) and 238 relatives (56 children, 182 adults) with a genetic defect. Biochemical parameters (total cholesterol (TC), LDLc, HDLc, triglycerides, ApoB, ApoAI) were analyzed with SPSS software using ANOVA tests. TC and LDLc levels are statistically higher in index patients than in relatives identified in cascade screening: index children, TC=315.96±61.51mg/dl and LDLc=239.61±60.43mg/dl vs TC=277.52±66.40mg/dl and LDLc=209.05±53.44mg/dl for relatives children (p=0.002, p=0.014); index adults TC=369.56±78.94mg/dl and LDLc=287.72±78.93mg/dl vs TC=332.93±75.54mg/dl and LDLc=246.26±71.79mg/dl for relatives adults (p<0.001, p=0.001). Although CT and LDLc mean values are above FH criteria a considered number of relatives have both TC and LDLc bellow these values (16%). Only 40% of relatives adults genetically identified are in treatment and 13% have pCHD vs 79% of index adults in treatment and 25% have pCHD. Genetic diagnosis of FH in Portugal allows early identification of FH patients, in particular relatives with mild phenotype that would not be identified by clinical criteria alone, allowing early implementation of therapeutic measures that will reduce their cardiovascular risk.
- Influence of APOE genotype in the phenotype of clinical diagnosed Portuguese FH patientsPublication . Medeiros, A.M.; Santos, T.; Alves, A.C.; Bourbon, M.APOE gene is polymorphic, comprises three frequent alleles (e2, e3, e4) which create six different genotypes and six protein isoforms with different affinity to LDLR. Isoform E2 has less affinity to receptor while isoform E4 have much efficient bond and compete with apoB. The present study pretends to evaluate the distribution of APOE alleles/genotype of index patients with clinical diagnosis of Familial Hypercholesterolemia (FH) and investigate if a specific allele/genotype is cause of hypercholesterolemia. APOE genotyping was performed using SnaPShot Multiplex System after PCR amplification. Biochemical parameters were determined by routine methods and results were analyzed with SPSS software using t- test. For this analysis, children and adults were divided according to their genetic diagnosis. Total of 353 patients were analyzed: 140 patients with mutation in LDLR, APOB or PCSK9 (44 children (G1), 96 adults (G2)) and 213 without a detectable mutation (70 children (G3), 143 adults (G4). Distribution of 6 genotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, E4/E4) was as following: G1, 0%, 2.3%, 4.5%, 68.2%, 20.5%, 4.5%; G2, 1.0%, 1.0%, 4.2%, 62.5%, 28.1%, 3.1%; G3, 0%, 1.4%, 5.7%, 55.7%, 35.7%, 1.4%; G4, 0%, 1.4%, 5.6%, 63.5%, 25.9%, 3.5%. Statistical analysis revealed that presence of at least one e4 is associated with high levels of LDLc in G3/G4 patients (LDLcG3=182.96±49.46mg/dl, (e4/eX, X=2,3,4) vs LDLcG3=152.65±60.60mg/dl (eY/eZ, Y,Z=2,3), p=0.025; LDLcG4=227.69±43.17mg/dl, (e4/eX, X=2,3,4) vs LDLcG4=179.70±64.78mg/dl (eY/eZ, Y,Z=2,3), p<0.001)) but not in G1/G2 patients (p=0.100, p=0.832). Presence of e4 can be the cause of hypercholesterolemia presented by patients without genetic diagnosis of FH.
- Complete sequencing by Pyrosequencing of APOB gene of patients with clinical diagnosis of Familial HypercholesterolaemiaPublication . Alves, A.C.; Bourbon, M.Familial hypercholesterolemia (FH) is a monogenic condition caused in most cases by mutations in LDLR gene, but mutations in APOB and PCSK9 genes are also cause of FH. These 3 genes are currently studied in the “Portuguese FH Study”. From the 404 families with a clinical diagnosis of FH studied, only 48% of these have a detectable mutation in the 3 genes mention above so, other mutations in these genes or other gene defects must exist to explain the cause of hypercholesterolemia in the remaining families. The main aim of this project was the whole sequencing of APOB gene, in 65 index patients with clinical diagnosis of FH, without mutations in LDLR gene or in fragments of exon 26 and 29 of APOB gene, by pyrosequencing, in order to identify the genetic cause of the hypercholesterolemia in these patients. A pool of the 65 DNAs was sequenced by pyrosequecing method and a total of 227688 nucleotide reads were obtained, corresponding to a mean coverage of 35x/fragment/individual. The results obtained included the detection of 87 alterations, being 27 previously described SNPs. All patients were re-sequenced for the fragments containing exons 26 and 29 alterations identified by this method. From the 26 alterations detected in these exons, only 12 were found and four of these had not been previously described. After family studies only one alteration did not co-segregate in the family, providing further evidence that 3 of the alterations found can be mutations causing disease but, functional studies are required to prove pathogenicity. Pyrosequencing allows the rapid sequencing of a large number of individuals, but apart from its high cost, it has some limitations and requires an improvement of technique.
- Update of the biochemical and molecular results of Portuguese patients with Familial Combined HyperlipidaemiaPublication . Santos, T.; Rato, Q.; Gaspar, I.M.; Rico, M.T.; Silva, J.M.; Bourbon, M.FCHL is a complex disorder with a highly atherogenic profile. The aim of this study is the biochemical/molecular characterization of FCHL patients. Molecular study of LPL, APOAIV, APOAV, APOCIII and USF1 (s1,s2) was performed in 35 index patients by PCR amplification and sequencing. Total cholesterol (TC), HDL-c, sdLDL, triglycerides (TG), apoB and apoCIII were measured in automated analysers. sdLDL was also analysed by electrophoresis with Lipoprint®. ApoAIV and ApoAV were measured by ELISA. For all patients, biochemical characterization of apolipoproteins and sdLDL before treatment was not possible to determine. Prior to medication the levels of TC (305±62mg/dL) and TG (380±269mg/dL) were high but HDL-c was normal (44±11mg/dL). Even under medication these patients presented high levels of CT (205±58mg/dL), TG (233±109mg/dL) and ApoCIII (15±4mg/dL) and normal levels of HDL-c (44±9mg/dL). ApoAIV (17±10mg/dL), ApoAV (156±140ng/mL) and ApoB (89±41mg/dL) levels were within normal range in majority of cases. Lipoprint® analysis of 8 patients under medication revealed an atherogenic pattern. Two new alterations were found. One patient with TC=271mg/dl and TG=275mg/dL carried APOAIV Q359_E362 (values without medication). Another patient with TC=419mg/dL and TG=1095mg/dL presented APOAV D332fsX336 and, even under medication, had high sdLDL (39 mg/dL, cut-off value 35mg/dl) and an atherogenic pattern with Lipoprint® analysis (pattern B). Some patients had a novel alteration APOCIII 3269C>A that was proven later that was a polymorphism. Patients with FCHL have increased cardiovascular risk that can be prevented with an early genetic identification and an extensive biochemical characterization that can be important to evaluate the efficacy of treatment.
- Determination of sdLDL particles in patients with Familial Hyercholesterolaemia and Familial Combined HyperlipidaemiaPublication . Gomes, A.; Santos, T.; Bourbon, M.Several studies demonstrated that sdLDL are an emerging cardiovascular (CV) risk factor. The objective of this study was sdLDL measurement in patients with genetic diagnosis of Familial Hypercholesterolaemia (FH) and clinical diagnosis of Familial Combined Hyperlipidaemia (FCHL) to establish a relation between sdLDL, CV risk and the efficacy of therapeutics. Lipid profile was determined using a polyacrylamide gel electrophoresis system that separates the particles in serum that contain cholesterol. The lipidogram obtained classifies the patients as being profile A (low CV risk) or B (high CV risk) depending on the sdLDL concentration. The lipid profile was obtained from 43 FH adults and 46 FCHL adults, index and relatives. FH and FCHL patients without medication and with high sdLDL (>6mg/dl) have significant higher levels of total cholesterol, LDL and ApoB and FCHL patients also have significant higher triglycerides, compared to FH and FCHL patients with sdLDL levels under recommended values. Under medication FH patients have significant higher ApoB levels and lower HDL, and FCHL patients have significant higher total cholesterol, LDL and ApoB levels. Interestingly, 71,4% of FCHL patients under medication presented high CV risk profile, showing that statins seem not to decrease sdLDL levels and neither CV risk. Also FCHL patients are not well medicated or do not respond to usual medication to decrease cholesterol. These preliminary results indicate that sdLDL could be a good biomarker for treatment control but further studies are needed to evaluate the effect of medication in sdLDL levels in FH and FCHL patients.
- Cardiovascular Risk of Children With Clinical Diagnosis of Familial HypercholesterolemiaPublication . Alves, A.C.; Medeiros, A.M.; Bourbon, M.Familial hypercholesterolemia (FH) is an inherited disorder of cholesterol metabolism, clinically characterized by high levels of LDL-associated cholesterol in plasma leading to accelerated atherosclerosis and increased risk of premature coronary heart disease (CHD). FH results from mutations in three genes involved in lipid metabolism: LDLR, APOB, PCSK9. Molecular identification of these patients can reduce the burden of mortality from cardiovascular disorders simply by the correct identification of the disease early in life, followed by counselling on appropriate lifestyle modifications and therapeutic measures when required. The aim of the Portuguese FH Study (PFHS) is to identify FH patients in order to prevent the development of premature CHD. From 563 index patients sent for the study, 153 are children. The aim of this study was to analyse the data from children with and without genetically diagnosis of FH recruited for the PFHS concerning their BMI.
- Lifestyle Habits and Cardiovascular Risk Factors in a Young PopulationPublication . Medeiros, A.M.; Rocha, T.M.; Francisco, V.; Alves, A.C.; Santos, T.; Louro, M.H.; Gonçalves, C.; Bourbon, M.Although CVD is mostly present in adult life, atherosclerosis starts in childhood. Health promotion and cardiovascular prevention strategies can generate health gains and, if implemented in childhood, those gains will be superior. In pathologies with multifactorial etiology like CVD, the preventive approach should be addressed to risk factors, specially the modifiable related to unhealthy lifestyle habits. The aims of this work are: to characterize the lifestyle habits and determine the cardiovascular risk factors of a young population, and to co-relate these factors.
- Inflammatory stimuli modulates the expression of ceruloplasmin at surface of human peripheral blood mononuclear cellsPublication . Bispo, C.; Villares, A.; Banha, J.; Marques, L.; Costa, L.Atherosclerosis (ATH) is recognized as a chronic inflammatory condition and it is the leading cause of cardiovascular disease. Atherogenesis is characterized by the passage of LDL through the endothelial layer, and the early infiltration and activation of peripheral blood lymphocytes (PBL) and monocytes (PBMN) that contribute to a pro-inflammatory state in specific locations. However, the functional interaction between immune cells and the oxidation of LDL are still not fully understood. One hypothesis for the etiopathogeny of ATH may be associated with an ongoing inflammatory process caused by an pro-oxidant/antioxidant imbalance induced by metals such as iron (Fe) or copper (Cu). Ceruloplasmin (Cp) is an acute-phase protein but also a multicopper oxidase with a relevant role in Fe metabolism mainly due to its ferroxidase activity. Herein, we aim to study the effect of putative pro-atherogenic immune stimuli on the expression of Cp at the surface of peripheral blood mononuclear cells (PBMC). In order to achieve this goal, PBMC were isolated from human peripheral blood, cultured in different inflammatory and/or altered Fe/Cu status conditions and analysis of Cp expression at cell surface was performed using flow cytometry. Cell surface expression of Cp was shown to be differently modulated by several tested treatments. Importantly, PBMN incubated with IL-1β showed a significant increase of Cp expression compared to untreated cells. Similar results were found using phorbol-12-myristate-13-acetate. Also, higher cell surface expression of Cp was consistently observed in PBMN compared to PBL and in activated vs non-activated cells. These results demonstrate that inflammatory mediators could be, at least in part, involved in the modulation of Cp expression at surface of PBMC and thus, have a putative role in atherogenesis.
- Differential Regulation of Ceruloplasmin Isoforms Expression in Macrophages and HepatocytesPublication . Marques, L.; Auriac, A.; Willemetz, A.; Banha, J.; Silva, B.; Canonne-Hergaux, F.; Costa, L.Ceruloplasmin (Cp) is an acute-phase protein that has been implicated in iron metabolism due to its ferroxidase activity, assisting ferroportin (Fpn) on cellular iron efflux. However, Cp exhibits both anti- and pro-oxidant activities and its physiological functions remain unclear. Cp can be expressed as a secreted or as a membrane glycosylphosphatidylinositol-anchored protein (GPI-Cp), this latter one being mostly expressed in the brain. Herein, we studied the expression of both Cp isoforms in human peripheral blood lymphocytes, monocytes, mouse macrophages and human hepatocarcinoma cell line HepG2, using immunofluorescence and immunoblotting techniques. Co-localization of Cp and Fpn was also investigated by immunofluorescence in mouse macrophages. Cp was detected by immunoblotting and immunofluorescence in membrane and cytosol of all cells types studied. The Cp detected at cell surface was identified as the GPI-isoform by PI-PLC test and shown to localize in lipid rafts in monocytes, macrophages and HepG2 cells. In macrophages, increased expression levels and co-localization of Fpn and GPI-Cp at cell surface lipid rafts were observed after iron treatment. Such upregulation of Cp by iron was not observed in HepG2 cells. Our results revealed an unexpected ubiquitous expression of the GPI-Cp isoform in immune and hepatic cells. A differential regulation of Cp in these cells may reflect distinct physiological functions of this oxidase according to cell-type specificity. In macrophages, GPI-Cp and Fpn likely interact in lipid rafts to export iron. A better insight into the expression of both Cp isoforms in different cell types will help to clarify its role in many diseases related to iron metabolism, inflammation and oxidative biology.