Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 51
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Mol Genet Metab Rep ; 21: 100504, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31516831

RESUMO

Geleophysic dysplasia (GPHYSD1, MIM231050; GPHYSD2, MIM614185; GPHYSD3, MIM617809) is an autosomal disorder characterized by short-limb dwarfism, brachydactyly, cardiac valvular disease, and laryngotracheal stenosis. Mutations in ADAMTSL2, FBN1, and LTBP3 genes are responsible for this condition. We found that three previously described cases of GPHYSD diagnosed clinically were homozygote or compound heterozygotes for five ADAMTSL2 variants, four of which not being previously reported. By electron microscopy, skin fibroblasts available in one case homozygote for an ADAMTSL2 variant showed a defective intracellular localization of mutant ADAMTSL2 protein that did not accumulate within lysosome-like intra-cytoplasmic inclusions. Moreover, this mutant ADAMTSL2 protein was less secreted in medium and resulted in increased SMAD2 phosphorylation in transfected HEK293 cells.

2.
Cancer Lett ; 447: 86-92, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30677446

RESUMO

Somatic epigenetic inactivation of the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) is frequent in colorectal cancer (CRC); however, its involvement in CRC predisposition remains unexplored. We assessed the role and relevance of MGMT germline mutations and epimutations in familial and early-onset CRC. Mutation and promoter methylation screenings were performed in 473 familial and/or early-onset mismatch repair-proficient nonpolyposis CRC cases. No constitutional MGMT inactivation by promoter methylation was observed. Of six rare heterozygous germline variants identified, c.346C > T (p.H116Y) and c.476G > A (p.R159Q), detected in three and one families respectively, affected highly conserved residues and showed segregation with cancer in available family members. In vitro, neither p.H116Y nor p.R159Q caused statistically significant reduction of MGMT repair activity. No evidence of somatic second hits was found in the studied tumors. Case-control data showed over-representation of c.346C > T (p.H116Y) in familial CRC compared to controls, but no overall association of MGMT mutations with CRC predisposition. In conclusion, germline mutations and constitutional epimutations in MGMT are not major players in hereditary CRC. Nevertheless, the over-representation of c.346C > T (p.H116Y) in our familial CRC cohort warrants further research.


Assuntos
Neoplasias Colorretais/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Células Germinativas/fisiologia , Mutação em Linhagem Germinativa/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Metilação de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Adulto Jovem
3.
Fish Shellfish Immunol ; 86: 559-570, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30481557

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide that belongs to the secretin/glucagon superfamily, of which some members have shown antimicrobial activities. Contrasting to mammals, published studies on the action of PACAP in non-mammalian vertebrate immune system remain scarce. Some of our recent studies added this peptide to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in teleost fish. Regulation of PACAP and expression of its receptor genes has been demonstrated during an immune response mounted against acute bacterial infection in fish, though the direct effect of PACAP against fish pathogenic bacteria has never been addressed. Current work provides evidence of antimicrobial activity of Clarias gariepinus PACAP against a wide spectrum of Gram-negative and Gram-positive bacteria and fungi of interest for human medicine and aquaculture, in which computational prediction studies supported the putative PACAP therapeutic activity. Results also indicated that catfish PACAP not only exhibits inhibitory effects on pathogen growth, but also affects the proliferation of human non-small cell lung cancer cell line H460 in a dose-dependent manner. The observed cytotoxic activity of catfish PACAP against human tumor cells and pathogenic microorganisms, but not healthy fish and mammalian erythrocytes support a potential physiological role of this neuropeptide in selective microbial and cancer cell killing. All together, our findings extend the mechanisms by which PACAP could contribute to immune responses, and open up new avenues for future therapeutic application of this bioactive neuropeptide.


Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Peixes-Gato/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Aeromonas hydrophila/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Bactérias/patogenicidade , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Peixes-Gato/microbiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Eritrócitos/efeitos dos fármacos , Hemólise , Humanos , Neoplasias Pulmonares/tratamento farmacológico
4.
Genet Med ; 20(12): 1652-1662, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30008476

RESUMO

PURPOSE: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype. METHODS: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach. RESULTS: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL. CONCLUSION: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Malato Desidrogenase/genética , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Paraganglioma/patologia , Feocromocitoma/patologia , Isoformas de Proteínas
5.
Hum Mutat ; 39(9): 1214-1225, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29900613

RESUMO

The causal association of NUDT1 (=MTH1) and OGG1 with hereditary colorectal cancer (CRC) remains unclear. Here, we sought to provide additional evidence for or against the causal contribution of NUDT1 and OGG1 mutations to hereditary CRC and/or polyposis. Mutational screening was performed using pooled DNA amplification and targeted next-generation sequencing in 529 families (441 uncharacterized MMR-proficient familial nonpolyposis CRC and 88 polyposis cases). Cosegregation, in silico analyses, in vitro functional assays, and case-control associations were carried out to characterize the identified variants. Five heterozygous carriers of novel (n = 1) or rare (n = 4) NUDT1 variants were identified. In vitro deleterious effects were demonstrated for c.143G>A p.G48E (catalytic activity and protein stability) and c.403G>T p.G135W (protein stability), although cosegregation data in the carrier families were inconclusive or nonsupportive. The frequency of missense, loss-of-function, and splice-site NUDT1 variants in our familial CRC cohort was similar to the one observed in cancer-free individuals, suggesting lack of association with CRC predisposition. No OGG1 pathogenic mutations were identified. Our results suggest that the contribution of NUDT1 and OGG1 germline mutations to hereditary CRC and to polyposis is inexistent or, at most, negligible. The inclusion of these genes in routine genetic testing is not recommended.


Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , Monoéster Fosfórico Hidrolases/genética , Polipose Adenomatosa do Colo/patologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo do DNA/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética/genética , Genótipo , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação com Perda de Função/genética , Masculino , Mutação de Sentido Incorreto/genética , Estresse Oxidativo , Isoformas de Proteínas/genética
6.
Clin Cancer Res ; 24(15): 3550-3559, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29588308

RESUMO

Purpose: Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown.Experimental Design: We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts.Results: WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the KDR/VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor KDR somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our in vitro and in vivo functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the KDR hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice.Conclusions: Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. Clin Cancer Res; 24(15); 3550-9. ©2018 AACR.


Assuntos
Inibidores da Angiogênese/administração & dosagem , Neoplasias Colorretais/genética , Neovascularização Patológica/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Quinase do Linfoma Anaplásico/genética , Inibidores da Angiogênese/efeitos adversos , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias Colorretais/sangue , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Receptores ErbB/genética , Exoma/genética , Feminino , Xenoenxertos , Humanos , Camundongos , Mutação , Neovascularização Patológica/sangue , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Conformação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/sangue , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Sequenciamento Completo do Exoma
7.
Toxins (Basel) ; 10(2)2018 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-29414882

RESUMO

Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 µM), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new ß-defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae.


Assuntos
Braquiúros/efeitos dos fármacos , Neurotoxinas/toxicidade , Anêmonas-do-Mar , Animais , Gânglios Espinais/citologia , Canais Iônicos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurotoxinas/isolamento & purificação , Paralisia/induzido quimicamente , Ratos Wistar , Caramujos
8.
Mol Cancer ; 17(1): 23, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29448935

RESUMO

Germline mutations in BUB1 and BUB3 have been reported to increase the risk of developing colorectal cancer (CRC) at young age, in presence of variegated aneuploidy and reminiscent dysmorphic traits of mosaic variegated aneuploidy syndrome. We performed a mutational analysis of BUB1 and BUB3 in 456 uncharacterized mismatch repair-proficient hereditary non-polyposis CRC families and 88 polyposis cases. Four novel or rare germline variants, one splice-site and three missense, were identified in four families. Neither variegated aneuploidy nor dysmorphic traits were observed in carriers. Evident functional effects in the heterozygous form were observed for c.1965-1G>A, but not for c.2296G>A (p.E766K), in spite of the positive co-segregation in the family. BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I) remained as variants of uncertain significance. As of today, the rarity of functionally relevant mutations identified in familial and/or early onset series does not support the inclusion of BUB1 and BUB3 testing in routine genetic diagnostics of familial CRC.


Assuntos
Polipose Adenomatosa do Colo/genética , Proteínas de Ciclo Celular/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação em Linhagem Germinativa , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/genética , Proteínas de Ciclo Celular/química , Humanos , Modelos Moleculares , Linhagem , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Proteínas Serina-Treonina Quinases/química
9.
Gastroenterology ; 154(1): 181-194.e20, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28912018

RESUMO

BACKGROUND & AIMS: Although there is a genetic predisposition to colorectal cancer (CRC), few of the genes that affect risk have been identified. We performed whole-exome sequence analysis of individuals in a high-risk family without mutations in genes previously associated with CRC risk to identify variants associated with inherited CRC. METHODS: We collected blood samples from 3 relatives with CRC in Spain (65, 62, and 40 years old at diagnosis) and performed whole-exome sequence analyses. Rare missense, truncating or splice-site variants shared by the 3 relatives were selected. We used targeted pooled DNA amplification followed by next generation sequencing to screen for mutations in candidate genes in 547 additional hereditary and/or early-onset CRC cases (502 additional families). We carried out protein-dependent yeast growth assays and transfection studies in the HT29 human CRC cell line to test the effects of the identified variants. RESULTS: A total of 42 unique or rare (population minor allele frequency below 1%) nonsynonymous genetic variants in 38 genes were shared by all 3 relatives. We selected the BRF1 gene, which encodes an RNA polymerase III transcription initiation factor subunit for further analysis, based on the predicted effect of the identified variant and previous association of BRF1 with cancer. Previously unreported or rare germline variants in BRF1 were identified in 11 of 503 CRC families, a significantly greater proportion than in the control population (34 of 4300). Seven of the identified variants (1 detected in 2 families) affected BRF1 mRNA splicing, protein stability, or expression and/or function. CONCLUSIONS: In an analysis of families with a history of CRC, we associated germline mutations in BRF1 with predisposition to CRC. We associated deleterious BRF1 variants with 1.4% of familial CRC cases, in individuals without mutations in high-penetrance genes previously associated with CRC. Our findings add additional evidence to the link between defects in genes that regulate ribosome synthesis and risk of CRC.


Assuntos
Neoplasias Colorretais/genética , Mutação em Linhagem Germinativa/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Adulto , Idoso , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Espanha
10.
Toxins ; 10(2): 72, 2018.
Artigo | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib14940

RESUMO

Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 mu M), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new -defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae.

11.
Nat Methods ; 14(8): 782-788, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28714987

RESUMO

Understanding genetic events that lead to cancer initiation and progression remains one of the biggest challenges in cancer biology. Traditionally, most algorithms for cancer-driver identification look for genes that have more mutations than expected from the average background mutation rate. However, there is now a wide variety of methods that look for nonrandom distribution of mutations within proteins as a signal for the driving role of mutations in cancer. Here we classify and review such subgene-resolution algorithms, compare their findings on four distinct cancer data sets from The Cancer Genome Atlas and discuss how predictions from these algorithms can be interpreted in the emerging paradigms that challenge the simple dichotomy between driver and passenger genes.


Assuntos
Algoritmos , Carcinogênese/genética , Mapeamento Cromossômico/métodos , Genes Neoplásicos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Humanos , Sensibilidade e Especificidade
12.
Int J Cancer ; 141(7): 1365-1380, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28577310

RESUMO

In a proportion of patients presenting mismatch repair (MMR)-deficient tumors, no germline MMR mutations are identified, the so-called Lynch-like syndrome (LLS). Recently, MMR-deficient tumors have been associated with germline mutations in POLE and MUTYH or double somatic MMR events. Our aim was to elucidate the molecular basis of MSH2-deficient LS-suspected cases using a comprehensive analysis of colorectal cancer (CRC)-associated genes at germline and somatic level. Fifty-eight probands harboring MSH2-deficient tumors were included. Germline mutational analysis of MSH2 (including EPCAM deletions) and MSH6 was performed. Pathogenicity of MSH2 variants was assessed by RNA analysis and multifactorial likelihood calculations. MSH2 cDNA and methylation of MSH2 and MSH6 promoters were studied. Matched blood and tumor DNA were analyzed using a customized next generation sequencing panel. Thirty-five individuals were carriers of pathogenic or probably pathogenic variants in MSH2 and EPCAM. Five patients harbored 4 different MSH2 variants of unknown significance (VUS) and one had 2 novel MSH6 promoter VUS. Pathogenicity assessment allowed the reclassification of the 4 MSH2 VUS and 6 probably pathogenic variants as pathogenic mutations, enabling a total of 40 LS diagnostics. Predicted pathogenic germline variants in BUB1, SETD2, FAN1 and MUTYH were identified in 5 cases. Three patients had double somatic hits in MSH2 or MSH6, and another 2 had somatic alterations in other MMR genes and/or proofreading polymerases. In conclusion, our comprehensive strategy combining germline and somatic mutational status of CRC-associated genes by means of a subexome panel allows the elucidation of up to 86% of MSH2-deficient suspected LS tumors.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , DNA Glicosilases/genética , Metilação de DNA , Análise Mutacional de DNA , Proteínas de Ligação a DNA/deficiência , Molécula de Adesão da Célula Epitelial/genética , Exodesoxirribonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Histona-Lisina N-Metiltransferase/genética , Humanos , Perda de Heterozigosidade , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética
13.
Hum Mol Genet ; 26(1): 33-43, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28013292

RESUMO

We performed whole exome sequencing in individuals from a family with autosomal dominant gastropathy resembling Ménétrier disease, a premalignant gastric disorder with epithelial hyperplasia and enhanced EGFR signalling. Ménétrier disease is believed to be an acquired disorder, but its aetiology is unknown. In affected members, we found a missense p.V742G variant in MIB2, a gene regulating NOTCH signalling that has not been previously linked to human diseases. The variant segregated with the disease in the pedigree, affected a highly conserved amino acid residue, and was predicted to be deleterious although it was found with a low frequency in control individuals. The purified protein carrying the p.V742G variant showed reduced ubiquitination activity in vitro and white blood cells from affected individuals exhibited significant reductions of HES1 and NOTCH3 expression reflecting alteration of NOTCH signalling. Because mutations of MIB1, the homolog of MIB2, have been found in patients with left ventricle non-compaction (LVNC), we investigated members of our family with Ménétrier-like disease for this cardiac abnormality. Asymptomatic left ventricular hypertrabeculation, the mildest end of the LVNC spectrum, was detected in two members carrying the MIB2 variant. Finally, we identified an additional MIB2 variant (p.V984L) affecting protein stability in an unrelated isolated case with LVNC. Expression of both MIB2 variants affected NOTCH signalling, proliferation and apoptosis in primary rat cardiomyocytes.In conclusion, we report the first example of left ventricular hypertrabeculation/LVNC with germline MIB2 variants resulting in altered NOTCH signalling that might be associated with a gastropathy clinically overlapping with Ménétrier disease.


Assuntos
Cardiomiopatias/patologia , Gastrite Hipertrófica/patologia , Mutação de Sentido Incorreto/genética , Receptores Notch/metabolismo , Gastropatias/patologia , Ubiquitina-Proteína Ligases/genética , Disfunção Ventricular Esquerda/patologia , Animais , Animais Recém-Nascidos , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Exoma/genética , Feminino , Gastrite Hipertrófica/etiologia , Gastrite Hipertrófica/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Linhagem , Fenótipo , Ratos , Receptores Notch/genética , Transdução de Sinais , Gastropatias/etiologia , Gastropatias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/metabolismo
14.
BMC Genomics ; 17 Suppl 2: 396, 2016 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-27357839

RESUMO

BACKGROUND: The association between aberrant signal processing by protein kinases and human diseases such as cancer was established long time ago. However, understanding the link between sequence variants in the protein kinase superfamily and the mechanistic complex traits at the molecular level remains challenging: cells tolerate most genomic alterations and only a minor fraction disrupt molecular function sufficiently and drive disease. RESULTS: KinMutRF is a novel random-forest method to automatically identify pathogenic variants in human kinases. Twenty six decision trees implemented as a random forest ponder a battery of features that characterize the variants: a) at the gene level, including membership to a Kinbase group and Gene Ontology terms; b) at the PFAM domain level; and c) at the residue level, the types of amino acids involved, changes in biochemical properties, functional annotations from UniProt, Phospho.ELM and FireDB. KinMutRF identifies disease-associated variants satisfactorily (Acc: 0.88, Prec:0.82, Rec:0.75, F-score:0.78, MCC:0.68) when trained and cross-validated with the 3689 human kinase variants from UniProt that have been annotated as neutral or pathogenic. All unclassified variants were excluded from the training set. Furthermore, KinMutRF is discussed with respect to two independent kinase-specific sets of mutations no included in the training and testing, Kin-Driver (643 variants) and Pon-BTK (1495 variants). Moreover, we provide predictions for the 848 protein kinase variants in UniProt that remained unclassified. A public implementation of KinMutRF, including documentation and examples, is available online ( http://kinmut2.bioinfo.cnio.es ). The source code for local installation is released under a GPL version 3 license, and can be downloaded from https://github.com/Rbbt-Workflows/KinMut2 . CONCLUSIONS: KinMutRF is capable of classifying kinase variation with good performance. Predictions by KinMutRF compare favorably in a benchmark with other state-of-the-art methods (i.e. SIFT, Polyphen-2, MutationAssesor, MutationTaster, LRT, CADD, FATHMM, and VEST). Kinase-specific features rank as the most elucidatory in terms of information gain and are likely the improvement in prediction performance. This advocates for the development of family-specific classifiers able to exploit the discriminatory power of features unique to individual protein families.


Assuntos
Biologia Computacional/métodos , Mutação , Proteínas Quinases/genética , Bases de Dados de Proteínas , Árvores de Decisões , Variação Genética , Humanos , Software
15.
J Struct Biol ; 195(2): 259-271, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27329566

RESUMO

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical ß-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop.


Assuntos
Ácido Aspártico Proteases/química , Serina Proteases/química , Inibidor da Tripsina de Soja de Kunitz/química , Sequência de Aminoácidos , Ácido Aspártico Proteases/ultraestrutura , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Serina/química , Serina Proteases/ultraestrutura , Inibidor da Tripsina de Soja de Kunitz/ultraestrutura
17.
Toxins (Basel) ; 8(4): 110, 2016 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-27089366

RESUMO

The bovine pancreatic trypsin inhibitor (BPTI)-Kunitz-type protein ShPI-1 (UniProt: P31713) is the major protease inhibitor from the sea anemone Stichodactyla helianthus. This molecule is used in biotechnology and has biomedical potential related to its anti-parasitic effect. A pseudo wild-type variant, rShPI-1A, with additional residues at the N- and C-terminal, has a similar three-dimensional structure and comparable trypsin inhibition strength. Further insights into the structure-function relationship of rShPI-1A are required in order to obtain a better understanding of the mechanism of action of this sea anemone peptide. Using enzyme kinetics, we now investigated its activity against other serine proteases. Considering previous reports of bifunctional Kunitz-type proteins from anemones, we also studied the effect of rShPI-1A on voltage-gated potassium (Kv) channels. rShPI-1A binds Kv1.1, Kv1.2, and Kv1.6 channels with IC50 values in the nM range. Hence, ShPI-1 is the first member of the sea anemone type 2 potassium channel toxins family with tight-binding potency against several proteases and different Kv1 channels. In depth sequence analysis and structural comparison of ShPI-1 with similar protease inhibitors and Kv channel toxins showed apparent non-sequence conservation for known key residues. However, we detected two subtle patterns of coordinated amino acid substitutions flanking the conserved cysteine residues at the N- and C-terminal ends.


Assuntos
Aprotinina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Inibidores de Serino Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Aprotinina/química , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Serina Proteases/fisiologia , Xenopus laevis
18.
Genet Med ; 18(4): 325-32, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26133394

RESUMO

PURPOSE: Germ-line mutations in the exonuclease domains of POLE and POLD1 have been recently associated with polyposis and colorectal cancer (CRC) predisposition. Here, we aimed to gain a better understanding of the phenotypic characteristics of this syndrome to establish specific criteria for POLE and POLD1 mutation screening and to help define the clinical management of mutation carriers. METHODS: The exonuclease domains of POLE and POLD1 were studied in 529 kindred, 441 with familial nonpolyposis CRC and 88 with polyposis, by using pooled DNA amplification and massively parallel sequencing. RESULTS: Seven novel or rare genetic variants were identified. In addition to the POLE p.L424V recurrent mutation in a patient with polyposis, CRC and oligodendroglioma, six novel or rare POLD1 variants (four of them, p.D316H, p.D316G, p.R409W, and p.L474P, with strong evidence for pathogenicity) were identified in nonpolyposis CRC families. Phenotypic data from these and previously reported POLE/POLD1 carriers point to an associated phenotype characterized by attenuated or oligo-adenomatous colorectal polyposis, CRC, and probably brain tumors. In addition, POLD1 mutations predispose to endometrial and breast tumors. CONCLUSION: Our results widen the phenotypic spectrum of the POLE/POLD1-associated syndrome and identify novel pathogenic variants. We propose guidelines for genetic testing and surveillance recommendations.Genet Med 18 4, 325-332.


Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , DNA Polimerase III/genética , DNA Polimerase II/genética , Mutação , Polipose Adenomatosa do Colo/diagnóstico , Alelos , Neoplasias Colorretais/diagnóstico , DNA Polimerase II/química , DNA Polimerase III/química , Feminino , Estudos de Associação Genética , Testes Genéticos , Genótipo , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linhagem , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose , Domínios e Motivos de Interação entre Proteínas/genética
19.
Hum Mutat ; 37(1): 36-42, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26443060

RESUMO

Most genomic alterations are tolerated while only a minor fraction disrupts molecular function sufficiently to drive disease. Protein kinases play a central biological function and the functional consequences of their variants are abundantly characterized. However, this heterogeneous information is often scattered across different sources, which makes the integrative analysis complex and laborious. wKinMut-2 constitutes a solution to facilitate the interpretation of the consequences of human protein kinase variation. Nine methods predict their pathogenicity, including a kinase-specific random forest approach. To understand the biological mechanisms causative of human diseases and cancer, information from pertinent reference knowledge bases and the literature is automatically mined, digested, and homogenized. Variants are visualized in their structural contexts and residues affecting catalytic and drug binding are identified. Known protein-protein interactions are reported. Altogether, this information is intended to assist the generation of new working hypothesis to be corroborated with ulterior experimental work. The wKinMut-2 system, along with a user manual and examples, is freely accessible at http://kinmut2.bioinfo.cnio.es, the code for local installations can be downloaded from https://github.com/Rbbt-Workflows/KinMut2.


Assuntos
Biologia Computacional/métodos , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Variação Genética , Genômica/métodos , Proteínas Quinases/genética , Software , Mineração de Dados , Bases de Dados Genéticas , Fator 1 de Crescimento de Fibroblastos/química , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/metabolismo , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Navegador
20.
Gastroenterology ; 149(3): 563-6, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26052075

RESUMO

Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Exodesoxirribonucleases/genética , Mutação em Linhagem Germinativa , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Pré-Escolar , Neoplasias Colorretais Hereditárias sem Polipose/enzimologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Exodesoxirribonucleases/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Adulto Jovem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA