Mutational screening in the LDLR gene among patients presenting familial hypercholesterolemia in the Southeast of Brazil

Zanette, Dalila Lucíola. “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-03-27T12:36:58Z No. of bitstreams: 1 Molfetta GA Mutational screening in the LDLR gene....pdf: 342859 bytes, checksum: 9e8d92035643cdd905392bba319d042d (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-03-27T12:59:07Z (GMT) No. of bitstreams: 1 Molfetta GA Mutational screening in the LDLR gene....pdf: 342859 bytes, checksum: 9e8d92035643cdd905392bba319d042d (MD5)Made available in DSpace on 2018-03-27T12:59:07Z (GMT). No. of bitstreams: 1 Molfetta GA Mutational screening in the LDLR gene....pdf: 342859 bytes, checksum: 9e8d92035643cdd905392bba319d042d (MD5) Previous issue date: 2017Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Universidade de São Paulo. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. Centro de Medicina Genômica. Ribeirão Preto, SP, Brasil / Universidade de São Paulo. Instituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular. Fundação Hemocentro de Ribeirão Preto. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Universidade de São Paulo. Instituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular. Fundação Hemocentro de Ribeirão Preto. Ribeirão Preto, SP,Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Clínica Médica. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Universidade de São Paulo. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto. Centro de Medicina Genômica. Ribeirão Preto, SP, Brasil / Universidade de São Paulo. Instituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular. Fundação Hemocentro de Ribeirão Preto. Ribeirão Preto, SP, BrasilFamilial hypercholesterolemia (FH) is a dominant, autosomal disease characterized by high LDL levels in blood plasma, and is caused by a defect in the gene encoding the LDL receptor (LDLR). The clinical diagnosis is based on personal and familial history, physical examination findings, and measures of high LDL cholesterol concentrations. LDLR is a cell-surface glycoprotein that controls the level of blood plasma cholesterol and triglyceride by LDLR-mediated endocytosis. Here we sequenced the entire LDLR gene-coding region to screen for mutations in 32 patients diagnosed with FH, and we have found 20 mutations including synonymous, missense, and intronic mutations. Six of them were characterized as pathogenic mutations (D178Y, C184Y, S326C, C681X, IVS7+10G>C, and IVS11-10G>A). We have also found one intronic mutation not described so far (IVS11-63C>A). Our study corroborates the broad spectrum of mutations distributed along the entire LDLR gene, and we suggest that the genes APOB and PCSK9 should also be screened for mutations when considering the diagnosis of FH. It is already known that different types of mutations are directly associated with the phenotype heterogeneity presented by patients. Considering that Brazilian population is highly admixed, it is important to determine the geographic spectrum of LDLR mutations to provide information on the prognosis and treatment of each FH patient

Heme changes HIF-α, eNOS and nitrite production in HUVECs after simvastatin, HU, and ascorbic acid therapies

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2016-05-10T15:54:44Z No. of bitstreams: 1 Guarda CC Heme changes....pdf: 517853 bytes, checksum: 521387a5f13e3781b0af22ec40769e20 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2016-05-10T16:03:23Z (GMT) No. of bitstreams: 1 Guarda CC Heme changes....pdf: 517853 bytes, checksum: 521387a5f13e3781b0af22ec40769e20 (MD5)Made available in DSpace on 2016-05-10T16:03:23Z (GMT). No. of bitstreams: 1 Guarda CC Heme changes....pdf: 517853 bytes, checksum: 521387a5f13e3781b0af22ec40769e20 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Farmácia. Departamento de Análises Clínicas e Toxicológicas. Salvador, BA, BrasilThe sickle cell disease (SCD) is a hemolytic genetic anemia characterized by free heme and hemoglobin release into intravascular spaces, with endothelial activation. Heme is a proinflammatory molecule able to directly activate vascular endothelium, thus, endothelial dysfunction and vascular disease are major chronic events described in SCD. The aim of this study was to evaluate the production of endothelial nitric oxide synthase (eNOS), nitrite and hypoxia inducible factor alpha (HIF-α) in HUVECs (human umbilical vein endothelial cells) activated by heme in response to simvastatin, hydroxyurea (HU), and ascorbic acid therapies. eNOS and HIF-α production were evaluated by ELISA and nitrite was measured by the Griess technique. The production of HIF-α increased when the cells were stimulated by heme (p b 0.01), while treatment with HU and simvastatin reduced the production (p b 0.01), and treatment with ascorbic acid increased HIF-1a production by the cells (p b 0.01). Hemeincreased eNOS production, (p b 0.01) but showed a heterogeneous pattern, and the lowest concentrations of all the treatments reduced the enzyme production (p b 0.01). The nitrite production by HUVECs was enhanced by stimulation with heme (p b 0.001) andwas reduced by treatment with HU (p b 0.001), ascorbic acid (p b 0.001) and simvastatin (p b 0.01). In summary, our results suggest that the hemolytic vascular microenvironment in SCD requires different therapeutic approaches to promote clinical improvement, and that a combination of therapies may be a viable strategy for treating patients

Comparison of microRNA expression in high-count monoclonal B-cell lymphocytosis and Binet A chronic lymphocytic leukemia

Abstract Background Evidence suggests that monoclonal B-cell lymphocytosis precedes all chronic lymphocytic leukemia cases, although the molecular mechanisms responsible for disease progression are not understood. Aberrant miRNA expression may contribute to the pathogenesis of chronic lymphocytic leukemia. The objective of this study was to compare miRNA expression profiles of patients with Binet A chronic lymphocytic leukemia with those of subjects with high-count monoclonal B-cell lymphocytosis and healthy volunteers (controls). Methods Twenty-one chronic lymphocytic leukemia patients, 12 subjects with monoclonal B-cell lymphocytosis and ten healthy volunteers were enrolled in this study. Flow cytometry CD19+CD5+-based cell sorting was performed for the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups and CD19+ cells were sorted to analyze the control group. The expressions of miRNAs (miR-15a, miR-16-1, miR-29b, miR-34a, miR-181a, miR-181b and miR-155) were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Results Significant differences between the expressions in the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups were restricted to the expression of miR-155, which was higher in the former group. A comparison between healthy controls and monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia patients revealed higher miR-155 and miR-34a levels and lower miR-15a, miR-16-1, miR-181a and miR-181b in the latter group. Conclusions Our results show a progressive increase of miR-155 expression from controls to monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia. The role of miR-155 in the development of overt chronic lymphocytic leukemia in individuals with monoclonal B-cell lymphocytosis must be further analyzed

Microarray profiles of ex vivo expanded hematopoietic stem cells show induction of genes involved in noncanonical Wnt signaling

Zanette, Dalila Lucíola. “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-04-13T17:59:31Z No. of bitstreams: 1 Zanette DL Microarray profiles of ex vivo expanded....pdf: 429718 bytes, checksum: 44af439761969d8585f1abad49511734 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-04-13T18:12:41Z (GMT) No. of bitstreams: 1 Zanette DL Microarray profiles of ex vivo expanded....pdf: 429718 bytes, checksum: 44af439761969d8585f1abad49511734 (MD5)Made available in DSpace on 2018-04-13T18:12:41Z (GMT). No. of bitstreams: 1 Zanette DL Microarray profiles of ex vivo expanded....pdf: 429718 bytes, checksum: 44af439761969d8585f1abad49511734 (MD5) Previous issue date: 2013FAPESP and CNPqUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilCentro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, Brasil / Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Clínica Médica Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilCentro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, Brasil / Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Clínica Médica Ribeirão Preto, SP, BrasilThe low number of hematopoietic stem cells (HSC) in umbilical cord blood (UCB) is directly related to increased risk of transplant failure. Effective ex vivo expansion of HSC has been tried for many years, with conflicting results because of the inability to reproduce in vitro HSC proliferation in the same way it occurs in vivo. We compared freshly isolated HSC with their expanded counterparts by microarray analysis and detected activation of the noncanonical Wnt (wingless-type MMTV integration site family) pathway. Study of early alterations during ex vivo UCB-HSC expansion could contribute to improvement of ex vivo expansion systems

Simvastatin Modulates Mesenchymal Stromal Cell Proliferation and Gene Expression

<div><p>Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC) are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate <i>in vitro</i> into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy) minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester) staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR). These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells) proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages <i>in vitro</i>. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential.</p></div

CFSE proliferation assays of MSC.

<p>The values correspond to the median percentage of viable, CFSE+ cells. S_1uM: MSC treated with simvastatin in the concentration of 1μM. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M). S+GGPP, MSC treated with 5μM of simvastatin and 5μM of GGPP (20-carbon geranylgeranyl pyrophosphate).</p

Median values of CFSE+ PBMC to assess the ability of simvastatin-treated MSC to inhibit PBMC proliferation.

<p>PBMC alone correspond to the proliferation rate of PBMC alone. Control refers to co-cultures of PBMC with MSC samples exposed only to the vehicle (absolute ethanol). S_1uM: MSC treated with simvastatin in the concentration of 1μM. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M).</p

MSC subpopulations by size (FSC) and complexity (SSC).

<p>The different subsets are surrounded by a blue line drawn around them and the values correspond to the percentage of each in this example. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M).</p

Analysis of MSC proliferation for the quadrant of small (FSC^lo) and low complexity (SSC^lo) cells.

<p>S_1uM: MSC treated with simvastatin in the concentration of 1μM. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M). S+GGPP, MSC treated with 5μM of simvastatin and 5μM of GGPP (20-carbon geranylgeranyl pyrophosphate). A) gating strategy to remove debril and dead cells; B) FSC and SSC quadrants definition; C) Percentages of cells with FSC and SSC< 200; D) Percentages of cells with FSC and SSC> 200.</p

Analysis of MSC proliferation for A) the quadrant of small (FSC^lo) and proliferative (CFSE^hi) MSC and for B) quadrant of large (FSC^hi) and non-proliferative (CFSE^lo) MSC.

<p>S_1uM: MSC treated with simvastatin in the concentration of 1μM. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M). S+GGPP, MSC treated with 5μM of simvastatin and 5μM of GGPP (20-carbon geranylgeranyl pyrophosphate).</p