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‣ Identificação e caracterização de mutações germinativas no gene VHL em famílias com a doença de von Hippel-Lindau; Identification and characterization of germline mutations in the VHL gene in families with von Hippel-Lindau disease

Gomy, Israel
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 02/07/2008 Português
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A doença de von Hippel-Lindau (VHL) é uma síndrome de câncer familial herdada de forma autossômica dominante que predispõe ao desenvolvimento de diversos tipos de neoplasias benignas e malignas. É causada por mutações germinativas e somáticas no gene VHL e tem uma incidência aproximada de um a cada 36.000 nascimentos. O gene VHL é um supressor tumoral e codifica a proteína VHL, a qual possui, entre outras funções, uma atividade ubiquitina-ligase, responsável pela poliubiquitinização e degradação proteassômica da subunidade alfa do fator induzido por hipóxia (HIF) na presença de oxigênio. As principais características da doença de VHL são: hemangioblastomas de sistema nervoso central (SNC), principalmente do cerebelo e medula espinhal; angiomas de retina e carcinoma renal de células claras. A probabilidade de desenvolver cada um desses tumores ao longo da vida é estimada em maior que 70%, podendo manifestar-se desde a infância até a fase adulta, principalmente entre a 2ª e 3ª décadas de vida. Classifica-se a doença de VHL conforme a ausência (tipo 1) ou presença de feocromocitoma (tipo 2). A doença do tipo 2 é causada, essencialmente, por mutações missense no gene VHL. As mutações podem ser grandes deleções (20%) ou pontuais (80%) do tipo missense...

‣ Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Forbes, J R; Cox, D W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1998 Português
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Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein...

‣ X-linked dyskeratosis congenita is predominantly caused by missense mutations in the DKC1 gene.

Knight, S W; Heiss, N S; Vulliamy, T J; Greschner, S; Stavrides, G; Pai, G S; Lestringant, G; Varma, N; Mason, P J; Dokal, I; Poustka, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1999 Português
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Dyskeratosis congenita is a rare inherited bone marrow-failure syndrome characterized by abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia. More than 80% of patients develop bone-marrow failure, and this is the major cause of premature death. The X-linked form of the disease (MIM 305000) has been shown to be caused by mutations in the DKC1 gene. The gene encodes a 514-amino-acid protein, dyskerin, that is homologous to Saccharomyces cerevisiae Cbf5p and rat Nap57 proteins. By analogy to the homologues in other species, dyskerin is predicted to be a nucleolar protein with a role in both the biogenesis of ribosomes and, in particular, the pseudouridylation of rRNA precursors. We have determined the genomic structure of the DKC1 gene; it consists of 15 exons spanning a region of 15 kb. This has enabled us to screen for mutations in the genomic DNA, by using SSCP analysis. Mutations were detected in 21 of 37 additional families with dyskeratosis congenita that were analyzed. These mutations consisted of 11 different single-nucleotide substitutions, which resulted in 10 missense mutations and 1 putative splicing mutation within an intron. The missense change A353V was observed in 10 different families and was shown to be a recurring de novo event. Two polymorphisms were also detected...

‣ Functional Analysis of TBX5 Missense Mutations Associated with Holt-Oram Syndrome*

Fan, Chun; Liu, Mugen; Wang, Qing
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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TBX5 is a T-box transcription factor that plays a critical role in organogenesis. Seven missense mutations in TBX5 have been identified in patients with Holt-Oram syndrome characterized by congenital heart defects and upper limb abnormalities. However, the functional significance and molecular pathogenic mechanisms of these mutations are not clear. In this study we describe functional defects in DNA binding, transcriptional activity, protein-protein interaction, and cellular localization of mutant TBX5 with these missense mutations (Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I). Mutations G80R, R237Q, and R237W represent a group of mutations that dramatically reduce DNA-binding activity of TBX5, leading to reduced transcription activation by TBX5 and the loss of synergy in transcriptional activation between TBX5 and NKX2.5. The second group of mutations includes Q49K, I54T, G169R, and S252I, which have no or moderate effect on DNA-binding activity and the function of transcription activation of TBX5 but cause the complete loss of synergistic transcription activity between TBX5 and NKX2.5. All seven missense mutations greatly reduced the interaction of TBX5 with NKX2.5 in vivo and in vitro. Immunofluorescent staining showed that wild type TBX5 was localized completely into the nucleus...

‣ Heterogeneity in residual function of MeCP2 carrying missense mutations in the methyl CpG binding domain

Kudo, S; Nomura, Y; Segawa, M; Fujita, N; Nakao, M; Schanen, C; Tamura, M
Fonte: BMJ Group Publicador: BMJ Group
Tipo: Artigo de Revista Científica
Publicado em /07/2003 Português
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Rett syndrome is a neurodevelopmental disorder with severe mental retardation caused by mutations in the MECP2 gene. Mutations in the MECP2 gene are also associated with other genetic disorders, including X linked mental retardation in males. Missense mutations identified so far are present primarily in the methyl CpG binding domain (MBD) of MECP2. Here, the functional significance of 28 MBD missense mutations identified in patients were analysed by transient expression of the mutant proteins in cultured cells. The effects of mutations were evaluated by analysis of the affinity of MeCP2 to pericentromeric heterochromatin in mouse L929 cells and on transcriptional repressive activity of MeCP2 in Drosophila SL2 cells. These analyses showed that approximately one-third (9/28) of MBD missense mutations showed strong impairment of MeCP2 function. The mutation of the R111 residue, which directly interacts with the methyl group of methyl cytosine, completely abolished MeCP2 function and mutations affecting ß-sheets and a hairpin loop have substantial functional consequences. In contrast, mutations that showed marginal or mild impairment of the function fell in unstructured regions with no DNA interaction. Since each of these mutations is known to be pathogenic...

‣ Human TBX1 Missense Mutations Cause Gain of Function Resulting in the Same Phenotype as 22q11.2 Deletions

Zweier, Christiane; Sticht, Heinrich; Aydin-Yaylagül, Inci; Campbell, Christine E.; Rauch, Anita
Fonte: The American Society of Human Genetics Publicador: The American Society of Human Genetics
Tipo: Artigo de Revista Científica
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Deletion 22q11.2 syndrome is the most frequent known microdeletion syndrome and is associated with a highly variable phenotype, including DiGeorge and Shprintzen (velocardiofacial) syndromes. Although haploinsufficiency of the T-box transcription factor gene TBX1 is thought to cause the phenotype, to date, only four different point mutations in TBX1 have been reported in association with six of the major features of 22q11.2 deletion syndrome. Although, for the two truncating mutations, loss of function was previously shown, the pathomechanism of the missense mutations remains unknown. We report a novel heterozygous missense mutation, H194Q, in a familial case of Shprintzen syndrome and show that this and the two previously reported missense mutations result in gain of function, possibly through stabilization of the protein dimer DNA complex. We therefore conclude that TBX1 gain-of-function mutations can result in the same phenotypic spectrum as haploinsufficiency caused by loss-of-function mutations or deletions.

‣ Functional Characterization of Pathogenic Human MSH2 Missense Mutations in Saccharomyces cerevisiae

Gammie, Alison E.; Erdeniz, Naz; Beaver, Julia; Devlin, Barbara; Nanji, Afshan; Rose, Mark D.
Fonte: Copyright © 2007 by the Genetics Society of America Publicador: Copyright © 2007 by the Genetics Society of America
Tipo: Artigo de Revista Científica
Publicado em /10/2007 Português
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Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with defects in DNA mismatch repair. Mutations in either hMSH2 or hMLH1 underlie the majority of HNPCC cases. Approximately 25% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 934 amino acids. We engineered 54 missense mutations in the cognate positions in yeast MSH2 and tested for function. Of the human alleles, 55% conferred strong defects, 8% displayed intermediate defects, and 38% showed no defects in mismatch repair assays. Fifty percent of the defective alleles resulted in decreased steady-state levels of the variant Msh2 protein, and 49% of the Msh2 variants lost crucial protein–protein interactions. Finally, nine positions are predicted to influence the mismatch recognition complex ATPase activity. In summary, the missense mutations leading to loss of mismatch repair defined important structure–function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. Of medical relevance are 15 human alleles annotated as pathogenic in public databases that conferred no obvious defects in mismatch repair assays. This analysis underscores the importance of functional characterization of missense alleles to ensure that they are the causative factor for disease.

‣ Disease-causing missense mutations in actin binding domain 1 of dystrophin induce thermodynamic instability and protein aggregation

Henderson, Davin M.; Lee, Ann; Ervasti, James M.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD) most commonly through loss of protein expression. In a small subpopulation of patients, missense mutations can cause DMD, Becker muscular dystrophy, or X-linked cardiomyopathy. Nearly one-half of disease-causing missense mutations are located in actin-binding domain 1 (ABD1) of dystrophin. To test the hypothesis that ABD1 missense mutations cause disease by impairing actin-binding activity, we engineered the K18N, L54R, D165V, A168D, L172H, and Y231N mutations into the full-length dystrophin cDNA and characterized the biochemical properties of each mutant protein. The K18N and L54R mutations are associated with the most severe diseases in humans and each caused a small but significant 4-fold decrease in actin-binding affinity, while the affinities of the other four mutant proteins were not significantly different from WT dystrophin. More interestingly, WT dystrophin was observed to unfold in a single-step, highly cooperative manner. In contrast, all six mutant proteins were significantly more prone to thermal denaturation and aggregation. Our results suggest that missense mutations in ABD1 may all cause loss of dystrophin function via protein instability and aggregation rather than through loss of ligand binding function. However...

‣ Disease Risk of Missense Mutations Using Structural Inference from Predicted Function

Horst, Jeremy A.; Wang, Kai; Horst, Orapin V.; Cunningham, Michael L.; Samudrala, Ram
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/2010 Português
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Advancements in sequencing techniques place personalized genomic medicine upon the horizon, bringing along the responsibility of clinicians to understand the likelihood for a mutation to cause disease, and of scientists to separate etiology from nonpathologic variability. Pathogenicity is discernable from patterns of interactions between a missense mutation, the surrounding protein structure, and intermolecular interactions. Physicochemical stability calculations are not accessible without structures, as is the case for the vast majority of human proteins, so diagnostic accuracy remains in infancy. To model the effects of missense mutations on functional stability without structure, we combine novel protein sequence analysis algorithms to discern spatial distributions of sequence, evolutionary, and physicochemical conservation, through a new approach to optimize component selection. Novel components include a combinatory substitution matrix and two heuristic algorithms that detect positions which confer structural support to interaction interfaces. The method reaches 0.91 AUC in ten-fold cross-validation to predict alteration of function for 6,392 in vitro mutations. For clinical utility we trained the method on 7,022 disease associated missense mutations within the Online Mendelian inheritance in man amongst a larger randomized set. In a blinded prospective test to delineate mutations unique to 186 patients with craniosynostosis from those in the 95 highly variant Coriell controls and 1000 age matched controls...

‣ Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

Zampieri, Stefania; Bembi, Bruno; Rosso, Natalia; Filocamo, Mirella; Dardis, Andrea
Fonte: Springer Berlin Heidelberg Publicador: Springer Berlin Heidelberg
Tipo: Artigo de Revista Científica
Publicado em 06/09/2011 Português
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Niemann Pick type C (NPC) disease is an autosomal recessive disorder characterized by the lysosomal/late endosomal (LE) accumulation of unesterified cholesterol and other lipids due to a defect in the intracellular lipid trafficking. About 95% of patients present mutations in the NPC1 gene. Among the 290 mutations reported in the NPC1 gene, about 70% are missense. However, little information is available regarding the impact of missense mutations on NPC1 protein stability and function. In this study, we in vitro characterized the pathogenic effect of 7 NPC1 missense mutations. In all cases, the basal levels of mutant NPC1 expression were reduced with respect to wild type. Treatment of fibroblasts carrying NPC1 missense mutations in homo or hemizygosity, with the proteasome inhibitor MG132 or glycerol 10%, a chemical chaperone known to stabilize misfolded proteins, resulted in a significant increase of NPC1 protein levels in all cell lines, indicating that these mutants are subjected to proteasomal degradation due to protein misfolding The increment of NPC1 mutant protein induced by the proteasome inhibitor was associated with a localization of NPC1 protein within lysosomal/LE compartment. In cell lines carrying mutations p.N1156S, p.L1191F...

‣ The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

Figueiredo, Joana; Söderberg, Ola; Simões-Correia, Joana; Grannas, Karin; Suriano, Gianpaolo; Seruca, Raquel
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
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In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, β-catenin, PIPKIγ and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work...

‣ Missense mutations of MLH1 and MSH2 genes detected in patients with gastrointestinal cancer are associated with exonic splicing enhancers and silencers

ZHU, MING; CHEN, HUI-MEI; WANG, YA-PING
Fonte: D.A. Spandidos Publicador: D.A. Spandidos
Tipo: Artigo de Revista Científica
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The MLH1 and MSH2 genes in DNA mismatch repair are important in the pathogenesis of gastrointestinal cancer. Recent studies of normal and alternative splicing suggest that the deleterious effects of missense mutations may in fact be splicing-related when they are located in exonic splicing enhancers (ESEs) or exonic splicing silencers (ESSs). In this study, we used ESE-finder and FAS-ESS software to analyze the potential ESE/ESS motifs of the 114 missense mutations detected in the two genes in East Asian gastrointestinal cancer patients. In addition, we used the SIFT tool to functionally analyze these mutations. The amount of the ESE losses (68) was 51.1% higher than the ESE gains (45) of all the mutations. However, the amount of the ESS gains (27) was 107.7% higher than the ESS losses (13). In total, 56 (49.1%) mutations possessed a potential exonic splicing regulator (ESR) error. Eighty-one mutations (71.1%) were predicted to be deleterious with a lower tolerance index as detected by the Sorting Intolerant from Tolerant (SIFT) tool. Among these, 38 (33.3%) mutations were predicted to be functionally deleterious and possess one potential ESR error, while 18 (15.8%) mutations were predicted to be functionally deleterious and exhibit two potential ESR errors. These may be more likely to affect exon splicing. Our results indicated that there is a strong correlation between missense mutations in MLH1 and MSH2 genes detected in East Asian gastrointestinal cancer patients and ESR motifs. In order to correctly understand the molecular nature of mutations...

‣ Cancer Missense Mutations Alter Binding Properties of Proteins and Their Interaction Networks

Nishi, Hafumi; Tyagi, Manoj; Teng, Shaolei; Shoemaker, Benjamin A.; Hashimoto, Kosuke; Alexov, Emil; Wuchty, Stefan; Panchenko, Anna R.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 14/06/2013 Português
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Many studies have shown that missense mutations might play an important role in carcinogenesis. However, the extent to which cancer mutations might affect biomolecular interactions remains unclear. Here, we map glioblastoma missense mutations on the human protein interactome, model the structures of affected protein complexes and decipher the effect of mutations on protein-protein, protein-nucleic acid and protein-ion binding interfaces. Although some missense mutations over-stabilize protein complexes, we found that the overall effect of mutations is destabilizing, mostly affecting the electrostatic component of binding energy. We also showed that mutations on interfaces resulted in more drastic changes of amino acid physico-chemical properties than mutations occurring outside the interfaces. Analysis of glioblastoma mutations on interfaces allowed us to stratify cancer-related interactions, identify potential driver genes, and propose two dozen additional cancer biomarkers, including those specific to functions of the nervous system. Such an analysis also offered insight into the molecular mechanism of the phenotypic outcomes of mutations, including effects on complex stability, activity, binding and turnover rate. As a result of mutated protein and gene network analysis...

‣ Functional Characterization and Categorization of Missense Mutations that Cause Methylmalonyl-CoA Mutase (MUT) Deficiency

Forny, Patrick; Froese, D. Sean; Suormala, Terttu; Yue, Wyatt W.; Baumgartner, Matthias R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/2014 Português
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Methylmalonyl-CoA mutase (MUT) is an essential enzyme in propionate catabolism that requires adenosylcobalamin as a cofactor. Almost 250 inherited mutations in the MUT gene are known to cause the devastating disorder methylmalonic aciduria; however, the mechanism of dysfunction of these mutations, more than half of which are missense changes, has not been thoroughly investigated. Here, we examined 23 patient missense mutations covering a spectrum of exonic/structural regions, clinical phenotypes, and ethnic populations in order to determine their influence on protein stability, using two recombinant expression systems and a thermostability assay, and enzymatic function by measuring MUT activity and affinity for its cofactor and substrate. Our data stratify MUT missense mutations into categories of biochemical defects, including (1) reduced protein level due to misfolding, (2) increased thermolability, (3) impaired enzyme activity, and (4) reduced cofactor response in substrate turnover. We further demonstrate the stabilization of wild-type and thermolabile mutants by chemical chaperones in vitro and in bacterial cells. This in-depth mutation study illustrates the tools available for MUT enzyme characterization, guides future categorization of further missense mutations...

‣ Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human

Alur, Ramakrishna P.; Vijayasarathy, Camasamudram; Brown, Jacob D.; Mehtani, Mohit; Onojafe, Ighovie F.; Sergeev, Yuri V.; Boobalan, Elangovan; Jones, MaryPat; Tang, Ke; Liu, Haiquan; Xia, Chun-hong; Gong, Xiaohua; Brooks, Brian P.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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Papillorenal syndrome (PRS, also known as renal-coloboma syndrome) is an autosomal dominant disease characterized by potentially-blinding congenital optic nerve excavation and congenital kidney abnormalities. Many patients with PRS have mutations in the paired box transcription factor gene, PAX2. Although most mutations in PAX2 are predicted to result in complete loss of one allele's function, three missense mutations have been reported, raising the possibility that more subtle alterations in PAX2 function may be disease-causing. To date, the molecular behaviors of these mutations have not been explored. We describe a novel mouse model of PRS due to a missense mutation in a highly-conserved threonine residue in the paired domain of Pax2 (p.T74A) that recapitulates the ocular and kidney findings of patients. This mutation is in the Pax2 paired domain at the same location as two human missense mutations. We show that all three missense mutations disrupt potentially critical hydrogen bonds in atomic models and result in reduced Pax2 transactivation, but do not affect nuclear localization, steady state mRNA levels, or the ability of Pax2 to bind its DNA consensus sequence. Moreover, these mutations show reduced steady-state levels of Pax2 protein in vitro and (for p.T74A) in vivo...

‣ Computational analysis of missense mutations causing Snyder-Robinson Syndrome

Zhang, Zhe; Teng, Shaolei; Wang, Liangjiang; Schwartz, Charles E.; Alexov, Emil
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/2010 Português
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The Snyder-Robinson syndrome is caused by missense mutations in the spermine sythase gene that encodes a protein (SMS) of 529 amino acids. Here we investigate, in silico, the molecular effect of three missense mutations, c.267G>A (p.G56S), c.496T>G (p.V132G) and c.550T>C (p.I150T) in SMS that were clinically identified to cause the disease. Single-point energy calculations, molecular dynamics simulations and pKa calculations revealed the effects of these mutations on SMS's stability, flexibility and interactions. It was predicted that the catalytic residue, Asp276, should be protonated prior binding the substrates. The pKa calculations indicated the p.I150T mutation causes pKa changes with respect to the wild type SMS which involve titratable residues interacting with the S-methyl-5'-thioadenosine (MTA) substrate. The p.I150T missense mutation was also found to decrease the stability of the C-terminal domain and to induce structural changes in the vicinity of the MTA binding site. The other two missense mutations, p.G56S and p.V132G, are away from active site and do not perturb its wild type properties, but affect the stability of both the monomers and the dimer. Specifically, the p.G56S mutation is predicted to greatly reduce the affinity of monomers to form a dimer and therefore should have a dramatic effect on SMS function since dimerization is essential for SMS activity.

‣ Underexpression and abnormal localization of ATM products in ataxia telangiectasia patients bearing ATM missense mutations

Jacquemin, Virginie; Rieunier, Guillaume; Jacob, Sandrine; Bellanger, Dorine; d'Enghien, Catherine Dubois; Laugé, Anthony; Stoppa-Lyonnet, Dominique; Stern, Marc-Henri
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
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Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immune defects and predisposition to malignancies. A-T is caused by biallelic inactivation of the ATM gene, in most cases by frameshift or nonsense mutations. More rarely, ATM missense mutations with unknown consequences on ATM function are found, making definitive diagnosis more challenging. In this study, a series of 15 missense mutations, including 11 not previously reported, were identified in 16 patients with clinical diagnosis of A-T belonging to 14 families and 1 patient with atypical clinical features. ATM function was evaluated in patient lymphoblastoid cell lines by measuring H2AX and KAP1 phosphorylation in response to ionizing radiation, confirming the A-T diagnosis for 16 cases. In accordance with previous studies, we showed that missense mutations associated with A-T often lead to ATM protein underexpression (15 out of 16 cases). In addition, we demonstrated that most missense mutations lead to an abnormal cytoplasmic localization of ATM, correlated with its decreased expression. This new finding highlights ATM mislocalization as a new mechanism of ATM dysfunction, which may lead to therapeutic strategies for missense mutation associated A-T.

‣ Predicting non-neutral missense mutations and their biochemical consequences using genome-scale homology modeling of human protein complexes

Bordner, Andrew J.; Zorman, Barry
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 20/08/2013 Português
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Computational methods are needed to differentiate the small fraction of missense mutations that contribute to disease by disrupting protein function from neutral variants. We describe several complementary methods using large-scale homology modeling of human protein complexes to detect non-neutral mutations. Importantly, unlike sequence conservation-based methods, this structure-based approach provides experimentally testable biochemical mechanisms for mutations in disease. Specifically, we infer metal ion, small molecule, protein-protein, and nucleic acid binding sites by homology and find that disease-associated missense mutations are more prevalent in each class of binding site than are neutral mutations. Importantly, our approach identifies considerably more binding sites than those annotated in the RefSeq database. Furthermore, an analysis of metal ion and protein-protein binding sites predicted by machine learning shows a similar preponderance of disease-associated mutations in these sites. We also derive a statistical score for predicting how mutations affect metal ion binding and find many dbSNP mutations that likely disrupt ion binding but were not previously considered deleterious. We also cluster mutations in the protein structure to discover putative functional regions. Finally...

‣ An integrated computational approach can classify VHL missense mutations according to risk of clear cell Renal carcinoma

Gossage, Lucy; Pires, Douglas E. V.; Olivera-Nappa, ?lvaro; Asenjo, Juan A.; Bycroft, Mark; Blundell, Tom L.; Eisen, Tim
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Article; published version
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This is the final published version, also available from the publisher website at at: http://hmg.oxfordjournals.org/content/early/2014/06/26/hmg.ddu321.long; Mutations?in?the?von?Hippel?Lindau?(VHL)?gene?are?pathogenic?in?VHL?disease,?congenital? polycythaemia?and?clear?cell?renal?carcinoma.?pVHL?forms?a?ternary?complex?with?Elongin?C? and?Elongin?B,?critical?for?pVHL?stability?and?function,?which?interacts?with?Cullin?2?and? RING?box?protein?1?to?target?Hypoxia?inducible?factor?for?polyubiquitination?and? proteasomal?degradation.?We?describe?a?comprehensive?database?of?missense?VHL? mutations?linked?to?experimental?and?clinical?data.?We?use?predictions?from?in?silico?tools?to? link?the?functional?effects?of?missense?VHL?mutations?to?phenotype.?The?risk?of?clear?cell? renal?carcinoma?in?VHL?disease?is?linked?to?the?degree?of?destabilisation?resulting?from? missense?mutations.?An?optimised?binary?classification?system?(Symphony),?which?integrates? predictions?from?five?in?silico?methods,?can?predict?the?risk?of?ccRCC?associated?with?VHL? missense?mutations?with?high?sensitivity?and?specificity.?We?use?Symphony?to?generate? predictions?for?risk?of?ccRCC?for?all?possible?VHL?missense?mutations?and?present?these? predictions...

‣ Comparison of predicted and actual consequences of missense mutations

Miosge, Lisa; Field, Matthew; Sontani, Yovina; Cho, Eun; Johnson, Simon; Palkova, Anna; Balakishnan, Bhavani; Liang, Rong; Zhang, Yafei; Lyon, Stephen; Beutler, Bruce; Whittle, Belinda; Bertram, Edward; Enders, Anselm; Goodnow, Christopher; Andrews, Thoma
Fonte: National Academy of Sciences (USA) Publicador: National Academy of Sciences (USA)
Tipo: Artigo de Revista Científica
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Each person's genome sequence has thousands of missense variants. Practical interpretation of their functional significance must rely on computational inferences in the absence of exhaustive experimental measurements. Here we analyzed the efficacy of these inferences in 33 de novo missense mutations revealed by sequencing in first-generation progeny of N-ethyl-N-nitrosourea- treated mice, involving 23 essential immune system genes. Poly- Phen2, SIFT, MutationAssessor, Panther, CADD, and Condel were used to predict each mutation's functional importance, whereas the actual effect was measured by breeding and testing homozygotes for the expected in vivo loss-of-function phenotype. Only 20% of mutations predicted to be deleterious by PolyPhen2 (and 15% by CADD) showed a discernible phenotype in individual homozygotes. Half of all possible missense mutations in the same 23 immune genes were predicted to be deleterious, and most of these appear to become subject to purifying selection because few persist between separate mouse substrains, rodents, or primates. Because defects in immune genes could be phenotypically masked in vivo by compensation and environment, we compared inferences by the same tools with the in vitro phenotype of all 2...