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‣ Mutator phenotypes of yeast strains heterozygous for mutations in the MSH2 gene

Drotschmann, Karin; Clark, Alan B.; Tran, Hiep T.; Resnick, Michael A.; Gordenin, Dmitry A.; Kunkel, Thomas A.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/03/1999 Português
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Heterozygosity for germ-line mutations in the DNA mismatch repair gene MSH2 predisposes humans to cancer. Here we use a highly sensitive reporter to describe a spontaneous mutator phenotype in diploid yeast cells containing a deletion of only one MSH2 allele. We also identify five MSH2 missense mutations that have dominant mutator effects in heterozygous cells when expressed at normal levels from the natural MSH2 promoter. For example, a 230-fold mutator effect is observed in an MSH2/msh2 diploid strain in which Gly693, which is invariant in MutS homologs and involved in ATP hydrolysis, is changed to alanine. DNA binding data suggest that mismatch repair is suppressed by binding of a mutant Msh2–Msh6 heterodimer to a mismatch with subsequent inability to dissociate from the mismatch in the presence of ATP. A dominant mutator effect also is observed in yeast when Gly693 is changed to serine. An early onset colorectal tumor is heterozygous for the analogous Gly → Ser mutation in hMSH2, and a second hMSH2 mutation was not found, suggesting that this missense mutation may predispose to cancer via a dominant mutator effect. The mutator effects of the deletion mutant and the Gly → Ala missense mutant in yeast MSH2 are enhanced by heterozygosity for a missense mutation in DNA polymerase δ that reduces its proofreading activity but is not a mutator in the heterozygous state. The synergistic effects of heterozygosity for mutations in two different genes that act in series to correct replication errors may be relevant to cancer predisposition.

‣ Saccharomyces cerevisiae pms2 mutations are alleles of MLH1, and pms2-2 corresponds to a hereditary nonpolyposis colorectal carcinoma-causing missense mutation.

Jeyaprakash, A; Das Gupta, R; Kolodner, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1996 Português
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A number of mutant Saccharomyces cerevisiae strains having phenotypes consistent with defects in DNA mismatch repair have been described, but not all have been extensively characterized. In this study we demonstrate that the pms2-1 and pms2-2 alleles arise from missense mutations in the MLH1 gene which inactivate MLH1. One of these alleles, pms2-2, causes the same amino acid substitution in a highly conserved region of the known MutL homologs as that caused by a proposed missense mutation observed in a Swedish hereditary nonpolyposis colorectal carcinoma kindred. This observation supports the functional significance of missense mutations found in hereditary nonpolyposis colorectal carcinoma kindreds and indicates that in some cases S. cerevisiae can serve as a useful model system for the analysis of such mutations.

‣ Precise missense and silent point mutations are fixed in the genomes of poliovirus mutants from persistently infected cells.

Borzakian, S; Pelletier, I; Calvez, V; Colbere-Garapin, F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1993 Português
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Poliovirus mutants selected in persistently infected human neuroblastoma cells have a modified cell tropism and can establish a secondary persistent infection in nonneural cells, such as HEp-2c cells. Nucleotide sequence analysis revealed that the genome of a persistent mutant, S11, differed from that of the parental lytic Sabin 1 poliovirus strain by 31 point mutations. Three mutations occurred in the noncoding regions. The other mutations resulted in 12 amino acid substitutions; 1 substitution occurred in a nonstructural protein (3A), while the other 11 substitutions were clustered in the capsid proteins VP2 and VP1. The same missense mutations, as well as many of the silent mutations that we observed in mutant S11, also accumulated in the genome of two other persistent viruses isolated from independent infections. This finding indicates that both missense and silent mutations are selected during the persistent infection of neuroblastoma cells and suggests that the secondary structure of RNA in the coding region may play a role in viral infection.

‣ Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditis elegans.

Kodoyianni, V; Maine, E M; Kimble, J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1992 Português
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The glp-1 gene encodes a membrane protein required for inductive cell interactions during development of the nematode Caenorhabditis elegans. Here we report the molecular characterization of 15 loss-of-function (lf) mutations of glp-1. Two nonsense mutations appear to eliminate glp-1 activity; both truncate the glp-1 protein in its extracellular domain and have a strong loss-of-function phenotype. Twelve missense mutations and one in-frame deletion map to sites within the repeated motifs of the glp-1 protein (10 epidermal growth factor [EGF]-like and 3 LNG repeats extracellularly and 6 cdc10/SWI6, or ankyrin, repeats intracellularly). We find that all three types of repeated motifs are critical to glp-1 function, and two individual EGF-like repeats may have distinct functions. Intriguingly, all four missense mutations in one phenotypic class map to the N-terminal EGF-like repeats and all six missense mutations in a second phenotypic class reside in the intracellular cdc10/SWI6 repeats. These two clusters of mutations may identify functional domains within the glp-1 protein.

‣ Human MutL homolog (MLH1) function in DNA mismatch repair: a prospective screen for missense mutations in the ATPase domain

Ellison, Aaron R.; Lofing, Joan; Bitter, Grant A.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1 are responsible for the majority of hereditary non-polyposis colorectal cancer (HNPCC), an autosomal-dominant early-onset cancer syndrome. Genetic testing of both MSH2 and MLH1 from individuals suspected of HNPCC has revealed a considerable number of missense codons, which are difficult to classify as either pathogenic mutations or silent polymorphisms. To identify novel MLH1 missense codons that impair MMR activity, a prospective genetic screen in the yeast Saccharomyces cerevisiae was developed. The screen utilized hybrid human-yeast MLH1 genes that encode proteins having regions of the yeast ATPase domain replaced by homologous regions from the human protein. These hybrid MLH1 proteins are functional in MMR in vivo in yeast. Mutagenized MLH1 fragments of the human coding region were synthesized by error-prone PCR and cloned directly in yeast by in vivo gap repair. The resulting yeast colonies, which constitute a library of hybrid MLH1 gene variants, were initially screened by semi-quantitative in vivo MMR assays. The hybrid MLH1 genes were recovered from yeast clones that exhibited a MMR defect and sequenced to identify alterations in the mutagenized region. This investigation identified 117 missense codons that conferred a 2-fold or greater decreased efficiency of MMR in subsequent quantitative MMR assays. Notably...

‣ Mutations in the Transmembrane Natriuretic Peptide Receptor NPR-B Impair Skeletal Growth and Cause Acromesomelic Dysplasia, Type Maroteaux

Bartels, Cynthia F.; Bükülmez, Hülya; Padayatti, Pius; Rhee, David K.; van Ravenswaaij-Arts, Conny; Pauli, Richard M.; Mundlos, Stefan; Chitayat, David; Shih, Ling-Yu; Al-Gazali, Lihadh I.; Kant, Sarina; Cole, Trevor; Morton, Jenny; Cormier-Daire, Val
Fonte: The American Society of Human Genetics Publicador: The American Society of Human Genetics
Tipo: Artigo de Revista Científica
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The homodimeric transmembrane receptor natriuretic peptide receptor B (NPR-B [also known as guanylate cyclase B, GC-B, and GUC2B]; gene name NPR2) produces cytoplasmic cyclic GMP from GTP on binding its extracellular ligand, C-type natriuretic peptide (CNP). CNP has previously been implicated in the regulation of skeletal growth in transgenic and knockout mice. The autosomal recessive skeletal dysplasia known as “acromesomelic dysplasia, type Maroteaux” (AMDM) maps to an interval that contains NPR2. We sequenced DNA from 21 families affected by AMDM and found 4 nonsense mutations, 4 frameshift mutations, 2 splice-site mutations, and 11 missense mutations. Molecular modeling was used to examine the putative protein change brought about by each missense mutation. Three missense mutations were tested in a functional assay and were found to have markedly deficient guanylyl cyclase activity. We also found that obligate carriers of NPR2 mutations have heights that are below the mean for matched controls. We conclude that, although NPR-B is expressed in a number of tissues, its major role is in the regulation of skeletal growth.

‣ Complete Loss of P/Q Calcium Channel Activity Caused by a CACNA1A Missense Mutation Carried by Patients with Episodic Ataxia Type 2

Guida, Serena; Trettel, Flavia; Pagnutti, Stefano; Mantuano, Elide; Tottene, Angelita; Veneziano, Liana; Fellin, Tommaso; Spadaro, Maria; Stauderman, Kenneth A.; Williams, Mark E.; Volsen, Stephen; Ophoff, Roel A.; Frants, Rune R.; Jodice, Carla; Frontali
Fonte: The American Society of Human Genetics Publicador: The American Society of Human Genetics
Tipo: Artigo de Revista Científica
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Familial hemiplegic migraine, episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 are allelic disorders of the CACNA1A gene (coding for the α1A subunit of P/Q calcium channels), usually associated with different types of mutations (missense, protein truncating, and expansion, respectively). However, the finding of expansion and missense mutations in patients with EA2 has blurred this genotype-phenotype correlation. We report the first functional analysis of a new missense mutation, associated with an EA2 phenotype—that is, T→C transition of nt 4747 in exon 28, predicted to change a highly conserved phenylalanine residue to a serine at codon 1491, located in the putative transmembrane segment S6 of domain III. Patch-clamp recording in HEK 293 cells, coexpressing the mutagenized human α1A-2 subunit, together with human β4 and α2δ subunits, showed that channel activity was completely abolished, although the mutated protein is expressed in the cell. These results indicate that a complete loss of P/Q channel function is the mechanism underlying EA2, whether due to truncating or to missense mutations.

‣ Missense and nonsense mutations in the lysosomal alpha-mannosidase gene (MANB) in severe and mild forms of alpha-mannosidosis.

Gotoda, Y; Wakamatsu, N; Kawai, H; Nishida, Y; Matsumoto, T
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1998 Português
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alpha-Mannosidosis is an autosomal recessive lysosomal-storage disorder caused by a deficiency of lysosomal alpha-mannosidase activity. This disease shows a wide range of clinical phenotypes, from a severe, infantile form (type I), which is fatal at <3-8 years of age, to a less severe, late-onset form (type II), which ultimately may involve hearing loss, coarse face, mental retardation, and hepatosplenomegaly. To elucidate the molecular mechanism underlying this disease in both types of patients, we have used PCR, followed by either SSCP analysis or direct sequencing, to analyze the 24 exons and intron/exon boundaries of the alpha-mannosidase gene (MANB) from five patients. Two amino acid substitutions-H72L and R750W, in exons 2 and 18, respectively-and two nonsense mutations-Q639X and R760X, in exons 15 and 19, respectively-were identified in four type II patients. One amino acid substitution, P356R, was identified in exon 8 from a type I patient. This patient and three of the type II patients were homozygous for their mutations (H72L, P356R, R750W, and R760X) and one type II patient was heterozygous for the Q639X and R750W mutations. Transfection experiments of COS 7 cells, using the alpha-mannosidase cDNA containing one of the missense mutations-H72L...

‣ Three different frameshift mutations of the tyrosinase gene in type IA oculocutaneous albinism.

Oetting, W S; Mentink, M M; Summers, C G; Lewis, R A; White, J G; King, R A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1991 Português
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Mutations in the gene for the pigment-producing enzyme tyrosinase are responsible for type IA (tyrosinase-negative) oculocutaneous albinism (OCA). Most reported mutations have been single base substitutions. We now report three different frameshift mutations in three unrelated individuals with type IA OCA. The first individual has a single base deletion within a series of five guanidines, resulting in a premature stop codon in exon I on one allele and a missense mutation at codon 382 in exon III on the homologous allele. The second individual is a genetic compound of two separate frameshift mutations, including both the same exon I single base deletion found in the first individual and a deletion of a thymidine-guanidine pair, within the sequence GTGTG, forming a termination codon (TAG) in exon I on the homologous allele. The third individual has a single base insertion in exon I on one allele and a missense mutation at codon 373 in exon III on the homologous allele. The two missense mutations occur within the copper Bbinding region and may interfere with either copper binding to the enzyme or oxygen binding to the copper. These five different mutations disrupt tyrosinase function and are associated with a total lack of melanin biosynthesis.

‣ Defective cellular trafficking of missense NPR-B mutants is the major mechanism underlying acromesomelic dysplasia-type Maroteaux

Hume, Alistair N.; Buttgereit, Jens; Al-Awadhi, Aydah M.; Al-Suwaidi, Sarah S.; John, Anne; Bader, Michael; Seabra, Miguel C.; Al-Gazali, Lihadh; Ali, Bassam R.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Natriuretic peptides (NPs) comprise a family of structurally related but genetically distinct hormones that regulate a variety of physiological processes such as cardiac growth, blood pressure, axonal pathfinding and endochondral ossification leading to the formation of vertebrae and long bones. The biological actions of NPs are mediated by natriuretic peptide receptors (NPRs) A, B and C that are located on the cell surface. Mutations in NPR-B have been shown to cause acromesomelic dysplasia-type Maroteaux (AMDM), a growth disorder in humans and severe dwarfism in mice. We hypothesized that missense mutations of NPR-B associated with AMDM primarily affect NPR-B function by the arrest of receptor trafficking at the endoplasmic reticulum (ER), due to conformational change, rather than an impairment of ligand binding, transmission of signal through the membrane or catalytic activity. Twelve missense mutations found in AMDM patients and cn/cn mice were generated by site-directed mutagenesis and transiently overexpressed in HeLa cells. Confocal microscopy revealed that 11 out of 12 mutants were retained in the ER. Determination of the ligand-dependent cGMP response confirmed that ER-retained NPR-B mutants are non-functional. Meanwhile, the only cell surface-targeted NPR-B missense mutant (D176E) displayed greatly reduced enzymatic activity due to impaired ligand binding. Thus...

‣ Infection-Triggered Familial or Recurrent Cases of Acute Necrotizing Encephalopathy Caused by Mutations in a Component of the Nuclear Pore, RANBP2

Neilson, Derek E.; Adams, Mark D.; Orr, Caitlin M.D.; Schelling, Deborah K.; Eiben, Robert M.; Kerr, Douglas S.; Anderson, Jane; Bassuk, Alexander G.; Bye, Ann M.; Childs, Anne-Marie; Clarke, Antonia; Crow, Yanick J.; Di Rocco, Maja; Dohna-Schwake, Christ
Fonte: American Society of Human Genetics Publicador: American Society of Human Genetics
Tipo: Artigo de Revista Científica
Publicado em 09/01/2009 Português
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Acute necrotizing encephalopathy (ANE) is a rapidly progressive encephalopathy that can occur in otherwise healthy children after common viral infections such as influenza and parainfluenza. Most ANE is sporadic and nonrecurrent (isolated ANE). However, we identified a 7 Mb interval containing a susceptibility locus (ANE1) in a family segregating recurrent ANE as an incompletely penetrant, autosomal-dominant trait. We now report that all affected individuals and obligate carriers in this family are heterozygous for a missense mutation (c.1880C→T, p.Thr585Met) in the gene encoding the nuclear pore protein Ran Binding Protein 2 (RANBP2). To determine whether this mutation is the susceptibility allele, we screened controls and other patients with ANE who are unrelated to the index family. Patients from 9 of 15 additional kindreds with familial or recurrent ANE had the identical mutation. It arose de novo in two families and independently in several other families. Two other patients with familial ANE had different RANBP2 missense mutations that altered conserved residues. None of the three RANBP2 missense mutations were found in 19 patients with isolated ANE or in unaffected controls. We conclude that missense mutations in RANBP2 are susceptibility alleles for familial and recurrent cases of ANE.

‣ Effects of a chemical chaperone on genetic mutations in α-galactosidase A in Korean patients with Fabry disease

Park, Jung-Young; Kim, Gu-Hwan; Kim, Sung-Su; Ko, Jung Min; Lee, Jin-Joo; Yoo, Han-Wook
Fonte: Korean Society of Medical Biochemistry and Molecular Biology Publicador: Korean Society of Medical Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Fabry disease is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the gene encoding the α-galactosidase A (GLA) enzyme. We have identified 15 distinct mutations in the GLA gene in 13 unrelated patients with classic Fabry disease and 2 unrelated patients with atypical Fabry disease. Two of the identified mutations were novel (i.e., the D231G missense mutation and the L268delfsX1 deletion mutation). This study evaluated the effects of the chemical chaperones 1-deoxygalactonojirimycin (DGJ) on the function of GLA in vitro, in cells containing missense mutations in the GLA gene. Nine missense and a nonsense mutations, including one novel mutation were cloned into mammalian expression vectors. After transient expression in COS-7 cells, GLA enzyme activity and protein expression were analyzed using fluorescence spectrophotometry and Western blot analysis, respectively. DGJ enhanced GLA enzyme activity in the M42V, I91T, R112C and F113L mutants. Interestingly, the I91T and F113L mutations are associated with the atypical form of Fabry disease. However, DGJ treatment did not have any significant effect on the GLA enzyme activity and protein expression of other mutants, including C142W, D231G, D266N, and S297F. Of note...

‣ Lynch Syndrome-associated Mutations in MSH2 Alter DNA Repair and Checkpoint Response Functions In Vivo

Mastrocola, Adam S; Heinen, Christopher D
Fonte: Wiley Subscription Services, Inc., A Wiley Company Publicador: Wiley Subscription Services, Inc., A Wiley Company
Tipo: Artigo de Revista Científica
Publicado em /10/2010 Português
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The DNA mismatch repair (MMR) pathway is essential in maintaining genomic stability through its role in DNA repair and the checkpoint response. Loss of DNA MMR underlies the hereditary cancer disease Lynch Syndrome (LS). Germline mutations in MSH2 account for approximately 40% of LS patients and of these, 18% are missense variants. One important clinical challenge has been discriminating between missense variants that are pathogenic and those that are not. Current analysis of missense mutations in MSH2 is performed using a combination of clinical, biochemical, and functional data; however, suitable cell culture models to test the various functions of the DNA MMR proteins are lacking. Here, we have generated human cell lines stably expressing a subset of MSH2 missense mutants and tested their effect on DNA repair and checkpoint response functions. We have expanded on previous biochemical and functional analyses performed in non-human systems to further understand defects conferred by this subset of single amino acid alterations. The functional characterization of MSH2 missense mutants combined with clinical and biochemical data is essential for appropriate patient management and genetic counseling decisions. ©2010 Wiley-Liss, Inc.

‣ Fibulin-5 mutations link inherited neuropathies, age-related macular degeneration and hyperelastic skin

Auer-Grumbach, Michaela; Weger, Martin; Fink-Puches, Regina; Papić, Lea; Fröhlich, Eleonore; Auer-Grumbach, Piet; El Shabrawi-Caelen, Laila; Schabhüttl, Maria; Windpassinger, Christian; Senderek, Jan; Budka, Herbert; Trajanoski, Slave; Janecke, Andreas
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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To identify the disease-causing gene responsible for an autosomal dominantly inherited Charcot–Marie–Tooth neuropathy subtype in a family excluded for mutations in the common Charcot–Marie–Tooth genes, we used array-based sequence capture to simultaneously analyse the disease-linked protein coding exome at chromosome 14q32. A missense mutation in fibulin-5, encoding a widely expressed constituent of the extracellular matrix that has an essential role in elastic fibre assembly and has been shown to cause cutis laxa, was detected as the only novel non-synonymous sequence variant within the disease interval. Screening of 112 index probands with unclassified Charcot–Marie–Tooth neuropathies detected two further fibulin-5 missense mutations in two families with Charcot–Marie–Tooth disease and hyperextensible skin. Since fibulin-5 mutations have been described in patients with age-related macular degeneration, an additional 300 probands with exudative age-related macular degeneration were included in this study. Two further fibulin-5 missense mutations were identified in six patients. A mild to severe peripheral neuropathy was detected in the majority of patients with age-related macular degeneration carrying mutations in fibulin-5. This study identifies fibulin-5 as a gene involved in Charcot–Marie–Tooth neuropathies and reveals heterozygous fibulin-5 mutations in 2% of our patients with age-related macular degeneration. Furthermore...

‣ Identification of Two Novel Mutations of the HOMEZ Gene in Chinese Patients with Isolated Ventricular Septal Defect

Xuan, Chao; Jia, Ke-Gang; Wang, Bin-Bin; Bai, Xiao-Yan; Gao, Ge; Yang, Qin; Wang, Xiu-Li; Liu, Xiao-Cheng; Ma, Xu; He, Guo-Wei
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica
Publicado em /05/2013 Português
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Objectives: Ventricular septal defect (VSD) is the most common congenital heart disease (CHD). Genome-wide linkage analysis revealed a potential CHD susceptibility locus in the homeodomain leucine zipper-encoding (HOMEZ) gene in a South Indian population. The present study aimed to identify potential pathogenic mutations for HOMEZ and to provide insights into the etiology of isolated VSD in the Chinese population. Methods: Case–control mutational analysis was performed in 400 patients with isolated VSD and 400 healthy controls. Protein-coding exton of HOMEZ and their flanking sequences were amplified by polymerase chain reaction and sequenced on an ABI3730 Automated Sequencer. CLC workbench software was used to compare the conservatism of the HOMEZ protein with other multiple species. The ExPASy-ProtScale online tool was used to predicate the alignment of the hydrophobic features. Results: Two novel heterozygous missense mutations (c.116 C>T; c. 630T>A) were identified in HOMEZ gene exon-2. The two mutations lead to alanine to valine substitution at position 39 and serine to arginine at position 210, which are highly conserved among many species. The hydropathicity of the valine and arginine residue at the position 39 and 210 were significantly different from the wild type. Conclusions: We have identified two novel heterozygous missense mutations in HOMEZ gene exon-2 in isolated VSD patients in the Chinese population and have found that these two mutations resulted in alteration of the hydropathicity of the HOMEZ protein. Therefore...

‣ Investigation of Endoglin Wild-Type and Missense Mutant Protein Heterodimerisation Using Fluorescence Microscopy Based IF, BiFC and FRET Analyses

Förg, Tassilo; Hafner, Mathias; Lux, Andreas
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 31/07/2014 Português
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The homodimeric transmembrane receptor endoglin (CD105) plays an important role in angiogenesis. This is highlighted by mutations in its gene, causing the vascular disorder HHT1. The main role of endoglin function has been assigned to the modulation of transforming growth factor β and bone morphogenetic protein signalling in endothelial cells. Nevertheless, other functions of endoglin have been revealed to be involved in different cellular functions and in other cell types than endothelial cells. Compared to the exploration of its natural function, little experimental data have been gathered about the mode of action of endoglin HHT mutations at the cellular level, especially missense mutations, and to what degree these might interfere with normal endoglin function. In this paper, we have used fluorescence-based microscopic techniques, such as bimolecular fluorescence complementation (BiFC), immunofluorescence staining with the endoglin specific monoclonal antibody SN6, and protein interaction studies by Förster Resonance Energy Transfer (FRET) to investigate the formation and cellular localisation of possible homo- and heterodimers composed of endoglin wild-type and endoglin missense mutant proteins. The results show that all of the investigated missense mutants dimerise with themselves...

‣ Naturally Occurring Mutations Alter the Stability of Polycystin-1 Polycystic Kidney Disease (PKD) Domains*

Ma, Liang; Xu, Meixiang; Forman, Julia R.; Clarke, Jane; Oberhauser, Andres F.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Mutations in polycystin-1 (PC1) can cause autosomal dominant polycystic kidney disease, which is a leading cause of renal failure. The available evidence suggests that PC1 acts as a mechanosensor, receiving signals from the primary cilia, neighboring cells, and extracellular matrix. PC1 is a large membrane protein that has a long N-terminal extracellular region (about 3000 amino acids) with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains, which are targeted by many naturally occurring missense mutations. Nothing is known about the effects of these mutations on the biophysical properties of PKD domains. Here we investigate the effects of several naturally occurring mutations on the mechanical stability of the first PKD domain of human PC1 (HuPKDd1). We found that several missense mutations alter the mechanical unfolding pathways of HuPKDd1, resulting in distinct mechanical phenotypes. Moreover, we found that these mutations also alter the thermodynamic stability of a structurally homologous archaeal PKD domain. Based on these findings, we hypothesize that missense mutations may cause autosomal dominant polycystic kidney disease by altering the stability of the PC1 ectodomain, thereby perturbing its ability to sense mechanical signals.

‣ Toward Classification of BRCA1 Missense Variants Using a Biophysical Approach

Rowling, Pamela J. E.; Cook, Rebecca; Itzhaki, Laura S.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Carriers of germ line mutations in breast cancer susceptibility gene BRCA1 have an increased risk of developing breast and ovarian cancers; missense mutations have, however, been difficult to assess for disease association. Here we have used a biophysical approach to classify these variants. We established an assay for measuring the thermodynamic stability of the BRCA1 BRCT domains and investigated the effects of 36 missense mutations. The mutations show a range of effects. Some do not change the stability, whereas others destabilize the protein by as much as 6 kcal mol−1; one-third of the mutants could not be expressed in soluble form in Escherichia coli, and we conclude that these destabilize the protein by an even greater amount. We tested several computer algorithms for their ability to predict the mutant effects and found that by grouping them into two classes (destabilizing by less than or more than 2.2 kcal mol−1), the algorithms could predict the stability changes. Importantly, with the exception of the few mutants located in the binding site, none showed a significant reduction in affinity for phosphorylated substrate. These results indicate that despite very large losses in stability, the integrity of the structure is not compromised by the mutations. Thus...

‣ Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency.

Goyette, P; Frosst, P; Rosenblatt, D S; Rozen, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1995 Português
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5-Methyltetrahydrofolate, the major form of folate in plasma, is a carbon donor for the remethylation of homocysteine to methionine. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. Patients with an autosomal recessive severe deficiency of MTHFR have homocystinuria and a wide range of neurological and vascular disturbances. We have recently described the isolation of a cDNA for MTHFR and the identification of two mutations in patients with severe MTHFR deficiency. We report here the characterization of seven novel mutations in this gene: six missense mutations and a 5' splice-site defect that activates a cryptic splice site in the coding sequence. We also present a preliminary analysis of the relationship between genotype and phenotype for all nine mutations identified thus far in this gene. A nonsense mutation and two missense mutations (proline to leucine and threonine to methionine) in the homozygous state are associated with extremely low activity (0%-3%) and onset of symptoms within the 1st year of age. Other missense mutations (arginine to cysteine and arginine to glutamine) are associated with higher enzyme activity and later onset of symptoms.

‣ Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Parvin, Jeffrey; Chiba, Natsuko; Ransburgh, Derek
Fonte: MyJove Corporation Publicador: MyJove Corporation
Tipo: Artigo de Revista Científica
Publicado em 17/02/2011 Português
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The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break...