Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
1.
J Pediatr Gastroenterol Nutr ; 74(5): e115-e121, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35129155

RESUMO

OBJECTIVES: Progressive familial intrahepatic cholestasis is an expanding group of autosomal recessive intrahepatic cholestatic disorders. Recently, next-generation sequencing allowed identifying new genes responsible for new specific disorders. Two biochemical phenotypes have been identified according to gamma-glutamyltransferase (GGT) activity. Mutations of the myosin 5B gene (MYO5B) are known to cause microvillus inclusion disease. Recently, different mutations in MYO5B gene have been reported in patients with low-GGT cholestasis. METHODS: A multicenter retrospective and prospective study was conducted in 32 children with cryptogenic intrahepatic cholestasis. Clinical, biochemical, histological, and treatment data were analyzed in these patients. DNA from peripheral blood was extracted, and all patients were studied by whole exome sequencing followed by Sanger sequencing. RESULTS: Six patients out of 32 had mutations in the MYO5B gene. Of these six patients, the median age at disease onset was 0.8 years, and the median length of follow-up was 4.2 years. The most common signs were pruritus, poor growth, hepatomegaly, jaundice, and hypocholic stools. Two patients also showed intestinal involvement. Transaminases and conjugated bilirubin were moderately increased, serum bile acids elevated, and GGT persistently normal. At anti-Myo5B immunostaining, performed in liver biopsy of two patients, coarse granules were evident within the cytoplasm of hepatocytes while bile salt export pump was normally expressed at the canalicular membrane. Six variants in homozygosity or compound heterozygosity in the MYO5B gene were identified, and three of them have never been described before. All nucleotide alterations were located on the myosin motor domain except one missense variant found in the isoleucine-glutamine calmodulin-binding motif. CONCLUSIONS: We identified causative mutations in MYO5B in 18.7% of a selected cohort of patients with intrahepatic cholestasis confirming a relevant role for the MYO5B gene in low-GGT cholestasis.


Assuntos
Colestase Intra-Hepática , Colestase , Miosina Tipo V , Colestase/genética , Colestase Intra-Hepática/diagnóstico , Humanos , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Miosinas/genética , Fenótipo , Estudos Prospectivos , Estudos Retrospectivos , gama-Glutamiltransferase/genética
3.
PLoS One ; 16(3): e0247603, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33667229

RESUMO

The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods' synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Humano , Reparo de DNA por Recombinação/genética , Fator de Necrose Tumoral alfa/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , DNA de Cadeia Simples/genética , Células HEK293 , Humanos , Mutação , Nucleotídeos/genética , RNA Guia de Cinetoplastídeos/genética , Transfecção , Repetições de Trinucleotídeos/genética
4.
Mol Med Rep ; 23(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33179092

RESUMO

Uterine leiomyoma presents the highest incidence among benign tumors of the female reproductive tract. The present study compared the proteome of leiomyoma treated with ulipristal acetate with that of untreated leiomyoma to investigate protein expression patterns in relation to oxidative stress. Paired tissue samples from seven treated and untreated leiomyomas were collected and the proteome was analyzed by two­dimensional gel electrophoresis (2­DE). Western blotting was used to validate the results of 2­DE, and mass spectrometry was used to identify proteins. The tissue expression of 30 proteins was markedly affected by treatment with ulipristal acetate. Bioinformatics analysis revealed that several of the differentially expressed proteins were involved in the degradation of hydrogen peroxide and the synthesis of reactive oxygen species. The present study suggested the involvement of oxidative stress as a novel mechanism of action of ulipristal acetate. These findings require further investigations to understand the role of ulipristal acetate in the treatment of the leiomyoma.


Assuntos
Redes Reguladoras de Genes/efeitos dos fármacos , Leiomioma/tratamento farmacológico , Norpregnadienos/administração & dosagem , Proteômica/métodos , Neoplasias Uterinas/tratamento farmacológico , Adulto , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Leiomioma/metabolismo , Espectrometria de Massas , Norpregnadienos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Mapas de Interação de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Uterinas/metabolismo
5.
Biotech Histochem ; 95(8): 634-640, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32551953

RESUMO

Both bacterial infections and innate oral immunity response participate in development of adeno-tonsillar hypertrophy (ATH). ATH can lead to obstructive sleep apnea. We investigated the beta-defensin 2 (hBD-2) encoding gene, DEFB4, by analyzing the copy number variations (CNVs) of the defensin gene cluster in patients with ATH and by correlating CNV with DEFB4 gene expression. We enrolled 79 patients with ATH, 21 of whom presented with only adenoid hypertrophy, while 58 exhibited hypertrophy of both adenoid and tonsil. CNVs of the defensin gene cluster, DEFB4 mRNA, and hBD-2 protein expression were assessed. Also, beta-defensin 2 was localized histologically using immunohistochemistry. The distribution of defensin gene cluster CNV was similar among the 79 subjects. DEFB4 expression analysis exhibited considerable inter-individual variability, but with neither specific differences among subjects nor correlation with the CNV number. Immunohistochemistry enabled localization of hBD-2 in the tonsil and adenoid epithelium. No differences in localization between the two ATH presentations were found. Inducible antimicrobial defensin peptides exhibited great inter-individual variability in terms of both CNV and gene expression, but no correlation with presentation of ATH was found.


Assuntos
Tonsila Faríngea/patologia , Variações do Número de Cópias de DNA , Regulação da Expressão Gênica/fisiologia , Hipertrofia/genética , Tonsila Palatina/patologia , beta-Defensinas/metabolismo , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença , Humanos , Hipertrofia/patologia , Lactente , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , beta-Defensinas/genética
6.
Oncotarget ; 8(35): 59235-59245, 2017 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-28938632

RESUMO

Ibrutinib blocks B-cell receptor signaling and interferes with leukemic cell-to-microenvironment interactions. Ibrutinib plays a key role in the management of B-CLL and is recommended for first line treatment of high-risk CLL patients with 17p deletion. Therefore, elucidating the factors governing sensitivity/resistance to Ibrutinib represents a relevant issue. For this purpose, in 3 B-CLL patient samples harboring functional TP53 mutations, the frequency of the mutated clones was monitored during in vivo Ibrutinib therapy, revealing a progressive decline of the frequency of TP53mut clones during 12 months of treatment. In parallel, the anti-leukemic activity of Ibrutinib was assessed in vitro on B-CLL patient cell cultures in combination with γ-secretase inhibitors (GSI). In the in vitro assays, the combination of Ibrutinib+GSI exhibited enhanced cytotoxicity on B-CLL cells also in the presence of stroma and it was coupled to the down-regulation of the stroma-activated NOTCH1 and c-MYC pathways. Moreover, the combined treatment was effective in reducing CXCR4 expression and functions. Therefore, the ability of GSI to enhance the Ibrutinib anti-leukemic activity in B-CLL cells, by down-regulating the NOTCH1 and c-MYC pathways, warrants further experimentation for its potential therapeutic applications.

7.
Anticancer Res ; 37(6): 3073-3083, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28551647

RESUMO

BACKGROUND: Germline mutations of the oncosuppressor gene breast cancer 1-associated protein 1 (BAP1) were recently related to an autosomal-dominant tumor predisposition syndrome (BAP1-TPDS), characterized by uveal melanoma, malignant mesothelioma (MM), cutaneous melanoma, and other malignancies. The demonstration that BAP1 mutations are strongly associated with MM has provided a real breakthrough in the study of genetic predisposition in MM, that may explain why only a fraction of asbestos-exposed individuals go on to develop MM. MATERIALS AND METHODS: To evaluate the possible role of BAP1 mutations in the epidemiology of sporadic MM, and their relationship with asbestos exposure, we determined the prevalence of germline BAP1 mutations by the Sanger method in a group of 29 asbestos-exposed patients, 21 of which were diagnosed with MM. They were residents of Trieste, a ship-building town in Northeast Italy with a very high incidence of mesothelioma. RESULTS: We identified non-obviously pathogenetic germline sequence variants of BAP1 in 3/29 patients and in 2/21 MM cases (10%). CONCLUSION: Non obviously pathogenic germline sequence variants of BAP1 were found. Nevertheless, limitations of predictive web tools allowed us to comment on some interesting peculiarities of our findings.


Assuntos
Neoplasias Pulmonares/genética , Mesotelioma/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Idoso , Idoso de 80 Anos ou mais , Amianto/efeitos adversos , Exposição Ambiental/efeitos adversos , Feminino , Mutação em Linhagem Germinativa , Humanos , Itália , Neoplasias Pulmonares/etiologia , Masculino , Mesotelioma/etiologia , Mesotelioma Maligno , Pessoa de Meia-Idade , Risco
8.
Genome ; 60(2): 183-192, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28092167

RESUMO

Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Quimeras de Transplante/genética , Alelos , Marcadores Genéticos , Humanos , Polimorfismo Genético , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Transplante Homólogo
9.
Mol Med Rep ; 14(4): 2967-74, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27499173

RESUMO

At present, the most common genetic diagnostic method for chimerism evaluation following hematopoietic stem cell transplantation is microsatellite analysis by capillary electrophoresis. The main objective was to establish, through repeated analysis over time, if a complete chimerism was present, or if the mixed chimerism was stable, increasing or decreasing over time. Considering the recent introduction of next generation sequencing (NGS) in clinical diagnostics, a detailed study evaluating an NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44­amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (<1%). The protocol followed the standard Ion AmpliSeq library preparation and Ion Torrent Personal Genome Machine guidelines. Overall, the present study added to the scientific literature, identifying novel technical details for a possible future application of NGS for chimerism quantification.


Assuntos
Quimerismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adulto , Feminino , Genômica/métodos , Genótipo , Técnicas de Genotipagem/métodos , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA/métodos , Adulto Jovem
10.
Oncol Rep ; 35(5): 3094-100, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26986808

RESUMO

Uterine leiomyomas are monoclonal tumors. Several factors are involved in the neoplastic transformation of the myometrium. In our study we focused on dysregulated cytoskeletal proteins in the leiomyoma as compared to the myometrium. Paired tissue samples of ten leiomyomas and adjacent myometria were obtained and analyzed by two­dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification, and western blotting for 2-DE data validation. The values of ten cytoskeletal proteins were found to be significantly different: eight proteins were upregulated in the leiomyoma and two proteins were downregulated. Three of the upregulated proteins (myosin regulatory light polypeptide 9, four and a half LIM domains protein 1 and LIM and SH3 domain protein 1) are involved in cell migration, while downregulated protein transgelin is involved in replicative senescence. Myosin regulatory light polypeptide 9 (MYL9) was further validated by western blotting because it is considered to be a cell migration marker in several cancers and could play a key role in leiomyoma development. Our data demonstrate significant alterations in the expression of cytoskeletal proteins involved in leiomyoma growth. A better understanding of the involvement of cytoskeletal proteins in leiomyoma pathogenesis may contribute to the identification of new therapeutic targets and the development of new pharmacological approaches.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Leiomioma/metabolismo , Neoplasias Uterinas/metabolismo , Adulto , Movimento Celular , Feminino , Humanos , Leiomioma/patologia , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Proteoma/metabolismo , Neoplasias Uterinas/patologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-25866490

RESUMO

BACKGROUND: Periodic fever syndromes (PFS) are an emerging group of autoinflammatory disorders. Clinical overlap exists and multiple genetic analyses may be needed to assist diagnosis. We evaluated the diagnostic value of a 5-gene sequencing panel (5GP) in patients with undiagnosed PFS. METHODS: Simultaneous double strand Sanger sequencing of MEFV, MVK, TNFRSF1A, NLRP3, NLRP12 genes was performed in 42 patients with unexplained PFS. Clinical features were correlated with genetic results. RESULTS: None of 42 patients analyzed displayed a causative genotype. However, single or multiple genetic variants of uncertain significance were detected in 24 subjects. Only in 5 subjects a definite diagnosis was made by taking into account both genetic and clinical data (2 TRAPS syndrome; 2 FMF; 1 FCAS). Statistical analysis showed that patients carrying genetic variants in one or more of the five selected genes displayed a significantly lower response to glucocorticoids compared with subjects who had completely negative genetic results. CONCLUSIONS: The sequencing of multiple genes is of little help in the diagnostics of PFS and can often lead to results of uncertain interpretation, thus the clinically driven sequencing of single genes should remain the recommended approach. However, the presence of single or multiple genetic variants of uncertain significance, even if not allowing any specific diagnosis, correlated with a poorer response to glucocorticoids, possibly indicating a multifactorial subgroup of PFS with differential response to pharmacological treatment.


Assuntos
Síndromes Periódicas Associadas à Criopirina/genética , Febre Familiar do Mediterrâneo/genética , Perfilação da Expressão Gênica , Genótipo , Doenças Hereditárias Autoinflamatórias/genética , Adolescente , Proteínas de Transporte/genética , Criança , Síndromes Periódicas Associadas à Criopirina/diagnóstico , Proteínas do Citoesqueleto/genética , Febre Familiar do Mediterrâneo/diagnóstico , Feminino , Febre , Doenças Hereditárias Autoinflamatórias/diagnóstico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Logísticos , Masculino , Mutação/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Estudos Prospectivos , Pirina , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Síndrome
13.
Stem Cells ; 32(9): 2373-85, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24801508

RESUMO

Cardiac stem cells (CSC) from explanted decompensated hearts (E-CSC) are, with respect to those obtained from healthy donors (D-CSC), senescent and functionally impaired. We aimed to identify alterations in signaling pathways that are associated with CSC senescence. Additionally, we investigated if pharmacological modulation of altered pathways can reduce CSC senescence in vitro and enhance their reparative ability in vivo. Measurement of secreted factors showed that E-CSC release larger amounts of proinflammatory cytokine IL1ß compared with D-CSC. Using blocking antibodies, we verified that IL1ß hampers the paracrine protective action of E-CSC on cardiomyocyte viability. IL1ß acts intracranially inducing IKKß signaling, a mechanism that via nuclear factor-κB upregulates the expression of IL1ß itself. Moreover, E-CSC show reduced levels of AMP protein kinase (AMPK) activating phosphorylation. This latter event, together with enhanced IKKß signaling, increases TORC1 activity, thereby impairing the autophagic flux and inhibiting the phosphorylation of Akt and cAMP response element-binding protein. The combined use of rapamycin and resveratrol enhanced AMPK, thereby restoring downstream signaling and reducing IL1ß secretion. These molecular corrections reduced E-CSC senescence, re-establishing their protective activity on cardiomyocytes. Moreover ex vivo treatment with rapamycin and resveratrol improved E-CSC capacity to induce cardiac repair upon injection in the mouse infarcted heart, leading to reduced cardiomyocyte senescence and apoptosis and increased abundance of endogenous c-Kit(+) CSC in the peri-infarct area. Molecular rejuvenation of patient-derived CSC by short pharmacologic conditioning boosts their in vivo reparative abilities. This approach might prove useful for refinement of CSC-based therapies.


Assuntos
Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Transplante de Células-Tronco/métodos , Animais , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos SCID , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Resveratrol , Transdução de Sinais , Sirolimo/farmacologia , Estilbenos/farmacologia
14.
Pharmacogenomics ; 15(5): 619-27, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24798719

RESUMO

AIM: In the AIEOP-BFM ALL (Associazione Italiana Ematologia Oncologia Pediatrica-Berlin Frankfurt Münster acute lymphoblastic leukemia) 2000 protocol, 70% of relapsed patients had favorable prognostic features and fell within less intensive polychemotherapeutic regimens, suggesting the need for better assessing lower risk stratification. MATERIALS & METHODS: A novel two-phase study design selected 614 children to be genotyped for TNF-α SNP rs1800629 (-308G>A). A weighted Cox model was applied to evaluate the SNP effect on hazard of relapse, adjusting for immunophenotype, risk group, age and gender and including interaction terms. RESULTS: Significant interaction was found with immunophenotypes (p = 0.0007, with minor allele genotypes being adverse genetic markers in B-cell acute lymphoblastic leukemia and protective ones in T-cell acute lymphoblastic leukemia), and also with risk protocols (p = 0.0041, with minor allele genotypes as prognostic factor of relapse for standard risk patients [only one T-cell acute lymphoblastic leukemia in the subgroup analyzed]). CONCLUSION: The presence of at least one A allele in TNF-α SNP rs1800629 should suggest a closer monitoring in B-cell acute lymphoblastic leukemia standard risk patients.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Fator de Necrose Tumoral alfa/genética , Adolescente , Antineoplásicos/uso terapêutico , Criança , Pré-Escolar , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Genótipo , Humanos , Lactente , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Fenótipo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Recidiva , Medição de Risco , Esteroides/uso terapêutico
15.
Am J Med Genet A ; 164A(1): 42-7, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24273071

RESUMO

Stickler syndrome (STL) is a clinically variable and genetically heterogeneous syndrome characterized by ophthalmic, articular, orofacial, and auditory manifestations. STL has been described with both autosomal dominant and recessive inheritance. The dominant form is caused by mutations of COL2A1 (STL 1, OMIM 108300), COL11A1 (STL 2, OMIM 604841), and COL11A2 (STL 3, OMIM 184840) genes, while recessive forms have been associated with mutations of COL9A1 (OMIM 120210) and COL9A2 (OMIM 120260) genes. Type IX collagen is a heterotrimeric molecule formed by three genetically distinct chains: α1, α2, and α3 encoded by the COL9A1, COL9A2, and COL9A3 genes. Up to this time, only heterozygous mutations of COL9A3 gene have been reported in human and related to: (1) multiple epiphyseal dysplasia type 3, (2) susceptibility to an intervertebral disc disease, and (3) hearing loss. Here, we describe the first autosomal recessive Stickler family due to loss of function mutations (c.1176_1198del, p.Gln393Cysfs*25) of COL9A3 gene. These findings extend further the role of collagen genes family in the disease pathogenesis.


Assuntos
Colágeno Tipo IX/genética , Genes Recessivos , Adolescente , Artrite , Osso e Ossos/anormalidades , Osso e Ossos/diagnóstico por imagem , Criança , Pré-Escolar , Doenças do Colágeno/diagnóstico , Doenças do Colágeno/genética , Doenças do Tecido Conjuntivo , Análise Mutacional de DNA , Fácies , Feminino , Perda Auditiva/genética , Perda Auditiva Neurossensorial , Homozigoto , Humanos , Masculino , Mutação , Linhagem , Radiografia , Descolamento Retiniano
16.
Oncotarget ; 5(24): 12635-45, 2014 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-25587027

RESUMO

By using next generation sequencing, we have analyzed 108 B chronic lymphocytic leukemia (B-CLL) patients. Among genes involved in the TP53 pathway, we found frequent mutations in ATM (n=18), TP53 (n=10) and NOTCH1 (n=10) genes, rare mutations of NOTCH2 (n=2) and CDKN1A/p21 (n=1) and no mutations in BAX, MDM2, TNFRSF10A and TNFRSF10B genes. The in vitro treatment of primary B-CLL cells with the activator of p53 Nutlin-3 induced the transcription of p53 target genes, without significant differences between the B-CLL without mutations and those harboring either ATM or NOTCH1mutations. On the other hand, the subgroup of TP53mutated B-CLL exhibited a significantly lower induction of the p53 target genes in response to Nutlin-3 as compared to the other B-CLL samples. However, among the TP53mutated B-CLL, those showing mutations in the high hot spot region of the DNA binding domain [273-280 aa] maintained a significantly higher p53-dependent transcriptional activity as compared to the other TP53mutated B-CLL samples. Since the ability to elicit a p53-dependent transcriptional activity in vitro has a positive prognostic significance, our data suggest that ATMmutated, NOTCH1mutated and surprisingly, also a subset of TP53mutated B-CLL patients might benefit from therapeutic combinations including small molecule activator of the p53 pathway.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Genes p53 , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Receptor Notch1/genética , Idoso , Idoso de 80 Anos ou mais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Receptor Notch1/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
17.
Gene ; 521(1): 160-5, 2013 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-23506826

RESUMO

Congenital hyperinsulinism (CHI) is a genetic disorder characterized by profound hypoglycemia related to an inappropriate insulin secretion. It is a heterogeneous disease classified into two major subgroups: "channelopathies" due to defects in ATP-sensitive potassium channel, encoded by ABCC8 and KCNJ11 genes, and "metabolopathies" caused by mutation of several genes (GLUD1, GCK, HADH, SLC16A1, HNF4A and HNF1A) and involved in different metabolic pathways. To elucidate the genetic etiology of CHI in the Italian population, we conducted an extensive sequencing analysis of the CHI-related genes in a large cohort of 36 patients: Twenty-nine suffering from classic hyperinsulinism (HI) and seven from hyperinsulinism-hyperammonemia (HI/HA). Seventeen mutations have been found in fifteen HI patients and five mutations in five HI/HA patients. Our data confirm the major role of ATP-sensitive potassium channel in the pathogenesis of Italian cases (~70%) while the remaining percentage should be attributed to other. A better knowledge of molecular basis of CHI would lead to improve strategies for genetic screening and prenatal diagnosis. Moreover, genetic analysis might also help to distinguish the two histopathological forms of CHI, which would lead to a clear improvement in the treatment and in genetic counseling.


Assuntos
Simulação por Computador , Hiperinsulinismo Congênito/genética , Mutação , Transportadores de Cassetes de Ligação de ATP/genética , Estudos de Coortes , Feminino , Quinases do Centro Germinativo , Glutamato Desidrogenase/genética , Fator 4 Nuclear de Hepatócito/genética , Humanos , Hiperamonemia/genética , Lactente , Itália , Masculino , Proteínas Mitocondriais/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteínas Serina-Treonina Quinases/genética , Receptores de Droga/genética , Sirtuínas/genética , Receptores de Sulfonilureias
18.
Gene ; 516(1): 122-5, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23266803

RESUMO

Congenital hyperinsulinism (CHI) occurs as a consequence of unregulated insulin secretion from the pancreatic beta-cells. Severe recessive mutations and milder dominant mutations have been described in the ABCC8 and KCNJ11 genes encoding SUR1 and Kir6.2 subunits of the beta-cell ATP-sensitive K(+) channel. Here we report two patients with CHI unresponsive to medical therapy with diazoxide. Sequencing analysis identified a compound heterozygous mutation in ABCC8 in both patients. The first one is a carrier for the known mild dominant mutation p.Glu1506Lys jointly with the novel mutation p.Glu1323Lys. The second carries the p.Glu1323Lys mutation and a second novel mutation, p.Met1394Arg. Functional studies of both novel alleles showed reduced or null cell surface expression, typical of recessive mutations. Compound heterozygous mutations in congenital hyperinsulinism result in complex interactions. Studying these mechanisms can improve the knowledge of this disease and modify its therapy.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Hiperinsulinismo Congênito/genética , Mutação , Canais de Potássio Corretores do Fluxo de Internalização/genética , Receptores de Droga/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Pré-Escolar , Hiperinsulinismo Congênito/tratamento farmacológico , Diazóxido/uso terapêutico , Genes Recessivos , Heterozigoto , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Linhagem , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/metabolismo , Análise de Sequência de DNA , Receptores de Sulfonilureias
19.
Ophthalmic Genet ; 34(1-2): 115-7, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22846113

RESUMO

Congenital cataract is a leading cause of visual impairment in children and brings approximately 10% of childhood blindness worldwide. Molecular analysis revealed ~60 loci to be associated with several phenotypes of childhood cataracts. Until now, more than 30 loci and 18 genes on different chromosomes have been associated with autosomal dominant congenital cataract (ADCC). Here, we present a three-generation Italian family with a non syndromic ADCC. A linkage analysis carried out using HumanCytoSNP-12 DNA Analysis BeadChip led us to identify ten genomic regions virtually involved in the disease. All the genes located in these regions were scored for possible relationship with ADCC and, according to a strict clinical and genetic selection, 4 genes have been analyzed. A novel sequence variant was found in the CRYBB2 gene (p.Ser143Phe). This variant affects a conserved aminoacid in the third Greek key motif of the protein, cosegregates with the disease phenotype in all affected individuals and is not present both in the unaffected family members and 100 healthy control subjects. Finally, we identified the first CRYBB2 mutation in an Italian family causing a clinical picture of ADCC.


Assuntos
Catarata/congênito , Catarata/genética , Mutação de Sentido Incorreto , Cadeia B de beta-Cristalina/genética , Sequência de Aminoácidos , Análise Mutacional de DNA , Feminino , Genes Dominantes , Ligação Genética , Genótipo , Humanos , Itália , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo
20.
J Immunol ; 186(8): 4946-58, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21411730

RESUMO

Respiratory insufficiency is the major cause of morbidity and mortality in patients affected by cystic fibrosis (CF). An excessive neutrophilic inflammation, mainly orchestrated by the release of IL-8 from bronchial epithelial cells and amplified by chronic bacterial infection with Pseudomonas aeruginosa, leads to progressive tissue destruction. The anti-inflammatory drugs presently used in CF patients have several limitations, indicating the need for identifying novel molecular targets. To address this issue, we preliminarily studied the association of 721 single nucleotide polymorphisms from 135 genes potentially involved in signal transduction implicated in neutrophil recruitment in a cohort of F508del homozygous CF patients with either severe or mild progression of lung disease. The top ranking association was found for a nonsynonymous polymorphism of the phospholipase C-ß3 (PLCB3) gene. Studies in bronchial epithelial cells exposed to P. aeruginosa revealed that PLCB3 is implicated in extracellular nucleotide-dependent intracellular calcium signaling, leading to activation of the protein kinase Cα and Cß and of the nuclear transcription factor NF-κB p65. The proinflammatory pathway regulated by PLCB3 acts by potentiating the Toll-like Receptors' signaling cascade and represents an interesting molecular target to attenuate the excessive recruitment of neutrophils without completely abolishing the inflammatory response.


Assuntos
Fibrose Cística/genética , Células Epiteliais/metabolismo , Interleucina-8/genética , Fosfolipase C beta/genética , Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Linhagem Celular Transformada , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Ativação Enzimática , Células Epiteliais/microbiologia , Expressão Gênica/efeitos dos fármacos , Frequência do Gene , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Interleucina-8/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Pneumopatias/genética , Pneumopatias/metabolismo , Pneumopatias/patologia , Microscopia de Fluorescência , Fosfolipase C beta/metabolismo , Polimorfismo de Nucleotídeo Único , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Pseudomonas aeruginosa/fisiologia , Interferência de RNA , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA