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Ibrahim, Daniel Murad. "ChIP-seq reveals mutation-specific pathomechanisms of HOXD13 missense mutations." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2015. http://dx.doi.org/10.18452/17102.
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Mutationen von Transkriptionsfaktoren (TF) betreffen nicht nur die Funktion des TFs, sondern auch die Expression seiner Zielgene und liegen häufig angeborenen Entwicklungsdefekten zugrunde. Über 20 Mutationen in HOXD13, einem TF der die Entwicklung der Extremitäten kontrolliert, sind bisher als Ursache verschiedenartiger Extremitätenfehlbildungen entdeckt worden. Eine molekularbiologische Grundlage für die Vielgestaltigkeit der HOXD13-Mutationen ist jedoch unbekannt. Die bisherigen Methoden zur funktionellen Charakterisierung von TF-Mutationen ermöglichten eine lediglich eingeschränkte Interpretation der molekularen Pathomechanismen. Die kürzlich entwickelte ChIP-seq Methode ermöglicht eine umfassende, funktionelle Charakterisierung eines TFs. In dieser Arbeit wurde eine Methode etabliert, um eine Vielzahl von Transkriptionsfaktoren und TF-Mutationen systematisch zu untersuchen. Zur Validierung wurden zwei neue Punktmutationen in HOXD13, p.Q317K und p.R298Q, charakterisiert. Beide Mutationen betreffen die DNA-bindende Domäne von HOXD13, rufen aber stark unterschiedliche Fehlbildungen hervor. Die Ergebnisse zeigen, dass die HOXD13Q317K Mutante eine veränderte Sequenzspezifität aufweist, welche nun jener eines anderen TFs, PITX1, ähnelt. Auch genomweit zeigt HOXD13Q317K ein Bindungsprofil, welches eher PITX1 als HOXD13wt entspricht. Durch weitere, unabhängige Analysen und Experimente wurde bestätigt, dass die p.Q317K Mutation HOXD13 in einen TF mit PITX1-ähnlichen Eigenschaften verändert. Die HOXD13R298Q-Mutante zeigt eine weitgehend unveränderte Bindungssequenz gegenüber HOXD13wt, jedoch eine veränderte Zusammensetzung der genomischen Bindestellen. Dies weist, in Kombination mit dem humanen Phänotyp auf einen dominant-negativen Pathomechanismus dieser Mutanten hin. Zusammengenommen zeigt diese Arbeit durch die Erhebung von experimentellen Daten, dass klar unterscheidbare molekularbiologische Mechanismen den HOXD13Q317K- und HOXD13R298Q-Mutationen zugrunde liegen.<br>Mutations in transcription factors (TF) do not only affect the function of the TF, but also the expression of its target genes and are frequently underlying congenital malformations. More than 20 distinct pathogenic mutations in HOXD13, a TF controlling limb development, have been associated with a broad range of limb malformations. However, a molecular basis underlying the variability of HOXD13-associated phenotypes remains elusive. To date, the experimental methods used to functionally characters TF mutations have allowed only limited insights into the underlying molecular pathomechanisms. The recently developed ChIP-seq technology has proven to be a powerful method to profile the binding characteristics of TFs; however a number of technical hurdles hinder its application for functional characterization of mutant TFs. This work describes the establishment of a ChIP-seq approach to investigate a wide spectrum of TFs and TF mutations. The approach was applied to characterize two previously unknown missense mutations in HOXD13, p.Q317K and p.R298Q, which both alter the DNA-binding domain of HOXD13 but cause very different disease phenotypes. The results show that the HOXD13Q317K mutant has an altered sequence specificity that resembles the recognition sequence of another TF, PITX1. Further, the genome-wide binding pattern of HOXD13Q317K shifts towards a more PITX1-like binding pattern. Even further analysis and viral overexpression in chicken limb buds confirm that the mutation partially converts HOXD13Q317K into a TF with PITX1-like properties. The HOXD13R298Q has a largely unchanged sequence specificity, but an altered composition of genomic binding sites. This, in combination with the human phenotype, indicates that the mutant might act in a dominant-negative manner. Collectively, this work shows through generation of direct experimental evidence, that clearly distinct molecular mechanisms underlie the pathogenicity of HOXD13Q317K and HOXD13R298Q mutations.
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Zerey, Marc. "Functional analysis of human MLH1 missense mutations using Saccharomyces cerevisiae." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79210.
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Hereditary nonpolyposis colorectal carcinoma (HNPCC) is linked to inherited defects in human genes (hMLH1, hMSH2, hMSH6, hPMS1, and hPMS2) that are involved in the repair of mismatched bases that may occur during DNA replication. Germline missense mutations in human MLH1 (hMLH1) are frequently detected and their functional characterization is critical to the development of genetic testing for HNPCC. We used several functional assays to characterize two hMLH1 mutations: T117M and R182G. Saccharomyces cerevisiae were transformed with hMLH1 cDNA expression vectors containing either mutation. The presence of functional hMLH1 produces an accumulation of mutations in mismatch repair (MSM)-proficient yeast due to MLH1 protein homology and interference with normal MMR. The transformants were tested for increased mutation rates using three assays: mutation of the gene for canavanine resistance (CAN1 ), reversion of the hom 3--10 allele, and insertion-deletions at a dinucleotide repeat regulating expression of beta-galactosidase. Based on the results of these assays T117M is likely a functional mutation while R182G may be a polymorphism. We conclude that this assay may be applicable in genetic testing for HNPCC.
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Maxwell, Megan Amanda, and n/a. "PEX1 Mutations in Australasian Patients with Disorders of Peroxisome Biogenesis." Griffith University. School of Biomolecular and Biomedical Science, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040219.100649.
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The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed 137 T>C and 53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.
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Maxwell, Megan Amanda. "PEX1 Mutations in Australasian Patients with Disorders of Peroxisome Biogenesis." Thesis, Griffith University, 2004. http://hdl.handle.net/10072/366184.
Texto completoResumen
The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed –137 T>C and –53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Biomolecular and Biomedical Sciences<br>Full Text
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岡田, 浩美, H. Okada, T. Yamazaki, A. Takagi, T. Murate, K. Yamamoto, J. Takamatsu, et al. "In vitro characterization of missense mutations associated with quantitative protein Sdeficiency." Thesis, Schattauer, 2006. http://hdl.handle.net/2237/11695.
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名古屋大学博士学位論文 学位の種類:博士（医療技術学）（課程）学位授与年月日:平成19年3月23日<br>"In vitro characterization of missense mutations associated with quantitative protein Sdeficiency" Schattauer, v.4, iss.9, pp.2003-2009を、博士論文として提出したもの。
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Drozdova, Tetyana. "Nephrin missense mutations altez cellular trafficking and induce endoplasmic retioulum stress." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=106541.
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Nephrin, a key component of the filtration slit diaphragm, undergoes post-translational modifications in the endoplasmic reticulum (ER). Mutations in nephrin lead to proteinuria. We examined the effects of missense mutations in nephrin on protein folding in the ER, cellular trafficking, and induction of the unfolded protein response (UPR). Wild type (WT) nephrin and the I171N, G270C, S366R, S724C and R743C mutant cDNAs were expressed in 293T cells or glomerular epithelial cells (GECs) by transient transfection. Association of nephrin with the ER chaperone, calnexin, was studied by co-immunoprecipitation. Activation of the UPR was assessed by monitoring expression of the ER chaperone, Grp94, phosphorylation of eukaryotic translation initiation factor-2α subunit (eIF2α), and induction of C/EBP homologous protein-10 (CHOP), as well as activating transcription factor-6 (ATF6)-luciferase reporter activity. All nephrin mutants showed increased association with calnexin, compared with WT nephrin. The I171N and G270C mutants increased expression of Grp94 in 293T cells, and stimulated ATF6-luciferase activity in both 293T cells and GECs. Nephrin S366R and S724C tended to induce the UPR, but changes in Grp94 and ATF6-luciferase activity were less consistent. The R743C mutant did not enhance Grp94 expression, nor ATF6-luciferase activity. All nephrin mutants did not increase eIF2α phosphorylation, nor CHOP expression. Immunofluorescence microscopy showed WT nephrin at the plasma membrane, while the I171N and S366R mutants were perinuclear, colocalized with calnexin. Moreover, the two nephrin mutants induced aggregation of the ER chaperone, calreticulin, compared with WT. Treatment of cells with castanospermine (which reduces the interaction of nephrin with calnexin) resulted in a portion of nephrin I171N and S366R appearing at the plasma membrane. Thus, certain nephrin mutants show impaired folding in the ER, and activate the ATF6 branch of the UPR. Induction of ER chaperones may represent a cytoprotective response, allowing cells to withstand proteotoxic injury. Blocking the interaction of nephrin with calnexin results in a partial rescue of certain nephrin mutants to the plasma membrane.<br>La néphrine, un composant clé du diaphragme de fente, subit des modifications post-traductionnelles dans le réticulum endoplasmique (RE). Des mutations de la néphrine provoquent une protéinurie. Nous avons examiné les effets des mutations faux-sens de la néphrine sur le repliement de cette protéine dans RE, sur son trafic cellulaire et sur l'induction de réponse déplié protéines (UPR). Le type sauvage (TS) de la néphrine et les mutants d'ADNc, I171N, G270C, S366R, S724C et R743C ont été exprimés dans des cellules 293T ou cellules glomérulaires épithéliales (GECs) par une transfection transitoire. Association de néphrine avec le chaperon de RE, la calnexine, a été étudiée par la co-immunoprécipitation. Activation de l'UPR a été évaluée par l'étude de l'expression du chaperon du RE, Grp94, la phosphorylation de la sous-unité (eIF2α) du facteur 2α d'initiation de la traduction eucaryote, et l'induction de C/EBP homologue de la protéine-10 (CHOP), ainsi que l'activation du facteur-6 de la transcription (ATF6)- l'activité luciférase du gène rapporteur. Tous les mutants de la néphrine ont montré l'association accrue avec la calnexine, par rapport au TS de la néphrine. Les mutants I171N et le G270C ont augmenté l'expression du Grp94 dans les cellules 293T, ont stimulé l'ATF6-activité luciférase dans les deux cellules 293T et GECs. Néphrine S366R et S724C ont tendance à induire l'UPR, mais les changements dans le Grp94 et l'activité ATF6-luciférase ont été moins cohérents. Le mutant R743C n'a pas amélioré l'expression de Grp94, ni l'ATF6-activité luciférase. Tous les mutants de la néphrine n'ont pas augmenté ni la phosphorylation d'eIF2α, ni l'expression de CHOP. La microscopie en immunofluorescence a montré la localisation du TS néphrine à la membrane plasmique, tandis que les mutants I171N et S366R à la partie périnucléaire, colocalisés avec la calnexine. Par ailleurs, les deux mutants de néphrine ont provoqué l'agrégation du chaperon du RE, la calréticuline, par rapport au TS. Le traitement des cellules avec la castanospermine (qui réduit l'interaction de la néphrine avec la calnexine) a entraîné la localisation d'une partie des mutants I171N et S366R de la néphrine à la membrane plasmique. Ainsi, certains mutants de néphrine montrent une déficience du repliment de la protéine dans RE et activent la branche ATF6 de l'UPR. L'induction de chaperons du RE peut représenter une réponse cytoprotectrice, permettant aux cellules de résister aux lesions protéotoxique. Le blocage de l'interaction de la néphrine avec la calnexine resulte à un retour partiel au TS de certains mutants de néphrine, et donc à la localisation de néphrine à la membrane plasmique.
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Ibrahim, Daniel Murad [Verfasser], Stefan [Akademischer Betreuer] Mundlos, and Petra [Akademischer Betreuer] Seemann. "ChIP-seq reveals mutation-specific pathomechanisms of HOXD13 missense mutations / Daniel Murad Ibrahim. Gutachter: Stefan Mundlos ; Petra Seemann." Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2015. http://d-nb.info/1065301065/34.
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Ibrahim, Daniel [Verfasser], Stefan [Akademischer Betreuer] Mundlos, and Petra [Akademischer Betreuer] Seemann. "ChIP-seq reveals mutation-specific pathomechanisms of HOXD13 missense mutations / Daniel Murad Ibrahim. Gutachter: Stefan Mundlos ; Petra Seemann." Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2015. http://nbn-resolving.de/urn:nbn:de:kobv:11-100225655.
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Hasselbacher, Katrin. "Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2 associated disorders /." Erlangen, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000252715.
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Sabbagh, Yves. "Impact of disease-causing missense mutations on the structure and function of PHEX." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38517.
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X-linked hypophosphatemia (XLH), the most prevalent form of inherited rickets in humans, is caused by mutations in the PHEX gene, which encodes a protein with high homology to the M13 family of type-II integral membrane zinc metallopeptidases. We created an online mutation database, PHEXdb (http://data.mch.mcgill.ca/phexdb), to catalogue PHEX mutations identified in XLH patients, and found that missense mutations account for 22% of the 157 mutations reported to date. We also undertook to examine the effects of eight missense mutations (C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y) on synthesis, glycosylation, cellular trafficking, and catalytic activity of the recombinant proteins using several approaches. The wild-type protein was resistant to endoglycosidase H (endo H), indicating that it is fully glycosylated. In addition, biotinylation and immunofluorescence studies revealed that the wild-type protein resides at the cell surface. The D237G, Y317F and F731Y mutant PHEX proteins were also endo H resistant and thus terminally glycosylated. In contrast, endo H digestion demonstrated that C85R, G579R, G579V, S711R and A720T were not terminally glycosylated. Furthermore, immunofluorescence showed that C85R, G579R and S711R were sequestered in the endoplasmic reticulum (ER). A secreted form of wild-type and mutant PHEX (secPHEX) proteins was generated to examine catalytic activity, using a synthetic fluorogenic peptide substrate. For this purpose, rescue of ER-trapped mutant proteins was attempted by growing transfected cells at 26°C. Low temperature was able to rescue three of the five trapped mutant proteins (G579V, S711R and A720T). Residual catalytic activity was observed with four mutant proteins (D237G, Y317F, A720T and F731Y) relative to the wild-type. However, the rescued S711R mutant was devoid of catalytic activity. Finally, limited proteolysis with trypsin and endoproteinase Glu-c revealed that the mutations D237G and F731Y induce conform
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Madabusi, Narasimhan Kandaye. "Characterization of three SMN missense mutations using mouse models of Spinal Muscular Atrophy." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1339442849.
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Dias, Henriques Sara. "Towards pharmacological strategies for missense mutations in two genes linked to muscular dystrophies." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLE015.
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La pathogénicité de nombreuses maladies génétiques humaines peut être liée à la reconnaissance de la protéine mutante mal repliée par le système de contrôle de la qualité des cellules (CQ), conduisant à leur dégradation. Néanmoins, un certain nombre de ces protéines mutées ont conservé une activité biologique, suggérant que le sauvetage de la dégradation peut réduire la pathologie et ouvrir la porte à des stratégies thérapeutiques pour traiter ces maladies.Les dystrophies musculaires des ceintures (LGMD) sont des maladies musculaires caractérisés par une atrophie progressive de la ceinture pelvienne et scapulaire. Des études menées par nous et par d'autres groupes ont montré que le mécanisme pathologique de certaines LGMD (sarcoglycanopathies et dystroglycanopathies) est associé à une dégradation prématurée des protéines mal repliées par le CQ. Les sarcoglycanopathies (LGMD2C-F) sont causées par des mutations dans l'un des 4 sarcoglycanes (SG). Ces protéines transmembranaires font partie du complexe dystrophine-glycoprotéine (DGC), qui lie le cytosquelette à la matrice extracellulaire (ECM) et est crucial pour la résistance mécanique des fibres musculaires. Les dystroglycanopathies sont un groupe de maladies associées à l'hypoglycosylation de l'alpha-dystroglycane (a-DG). Au DGC, a-DG est en contact direct avec l'ECM par une glycosylation complexe générée par l'action de plusieurs enzymes. Notre laboratoire se concentre sur l'une de ces enzymes, la protéine liée à la fukutine (FKRP), dont les mutations mènent à LGMD2I.Dans le but d'identifier des molécules candidates capables de sauver les protéines mutantes sarcoglycanes et FKRP, un criblage à haute-débit de composés pharmacologiques validés a été envisagé. Pour ce type de criblage, des modèles cellulaires in vitro appropriés ont été générés. Grâce à une approche candidate et à une criblage, nous avons identifié plusieurs molécules capables de sauver et de localiser correctement les mutants alpha-SG à la membrane cellulaire. Dans le but de tester l'efficacité et la sécurité des molécules in vivo, nous avons généré un nouveau modèle de souris portant une mutation T151R b-SG, car le modèle précédent portant une mutation correspondant à la mutation humaine R77C ne présentait pas de pathologie. Le nouveau modèle ne présentait pas non plus de phénotype dystrophique, la protéine bêta-SG mutée étant correctement présente à la membrane de la fibre musculaire, ce qui indique que le système de CQ est différent entre les deux espèces.En ce qui concerne FKRP, nous avons caractérisé certaines protéines mutantes in vitro et identifié deux différentes classes de mutants: des mutants retenus dans l'ER mais néanmoins capables de trafiquer vers le Golgi où ils ont montré une fonctionnalité. D'autres mutations FKRP correctement adressées au Golgi ont cependant perdu leur fonctionnalité. Les patients affectés par ces mutations ne peuvent pas bénéficier de stratégies pharmacologiques ciblant le CQ et pourraient être des candidats pour des approches de thérapie génique. En utilisant les mutants FKRP qui pourraient être sauvés de la dégradation et être fonctionnels, nous avons généré de nouveaux modèles cellulaires pour les criblages à haut-débit qui sont actuellement en cours de validation.Globalement, ce projet a permis la génération de modèles in vitro pertinents pour le test de médicaments pour le sauvetage de protéines mutantes faux-sens menant à deux maladies musculaires pour lesquelles aucun traitement curatif actuel n'est disponible<br>The pathogenicity of many human genetic diseases can be related to the recognition of the mutant misfolded protein by the cell quality control (QC) system, leading to their degradation. Interestingly, a number of these mutated proteins have nevertheless retained biological activity, suggesting that rescue from degradation can reduce the pathology and open the door for therapeutic strategies to treat these diseases.Limb-girdle muscular dystrophies (LGMDs) are muscular disorders characterized by progressive wasting of the pelvic and shoulder muscles. Previously studies by us and other groups showed that the pathological mechanism of some LGMDs (sarcoglycanopathies and dystroglycanopathies) is associated with premature degradation of misfolded proteins through QC. Sarcoglycanopathies (LGMD2C-F) are caused by mutations in any of the 4 sarcoglycans (SGs). These transmembrane proteins are part of the dystrophin-glycoprotein complex (DGC), which connects the cytoskeleton to the extracellular matrix (ECM) and is crucial for the mechanical resistance of the muscle fibers. Dystroglycanopathies are a group of diseases associated with hypoglycosylation of alpha-dystroglycan (a-DG). At the DGC, a-DG is in direct contact with the ECM through a complex glycosylation generated by the action of several enzymes. Our lab focuses on one of these enzymes, Fukutin-related protein (FKRP), whose mutations lead to LGMD2I.With the aim of identifying candidate molecules able to rescue the sarcoglycan and FKRP mutant proteins, a high-content screen of approved and validated pharmacological compounds was envisaged. For this type of screen, appropriate in vitro cellular models were generated. Through a candidate approach and high-content screen, we identified several molecules able to rescue and correctly localize alpha-SG mutants to the cell membrane. For the purpose of testing the efficacy and safety of the molecules in vivo, we generated a new mouse model carrying a T151R b-SG mutation, as the previous model carrying a mutation corresponding to the human R77C mutation failed to reproduce the pathology. The new model also did not present a dystrophic phenotype, with the mutated beta-SG protein correctly present at the muscle fiber membrane, indicating that the QC system is different between the two species.As for FKRP, we characterized a number of mutant proteins in vitro showing two different classes of mutants: some mutants were retained in the ER but could nonetheless overcome this retention and be allowed to traffic to the Golgi where they showed functionality. Other FKRP mutations are correctly addressed to the Golgi but are functionally impaired. Patients affected by these mutations may not benefit from QC-targeting pharmacological strategies and could be candidates for gene therapy approaches. Utilizing the FKRP mutants that could be rescued of degradation and be functional, we have generated new cellular models for high-content screens which are currently being validated.Altogether, this project allowed the generation of relevant in vitro models for identification of drugs allowing the rescue of missense mutant proteins leading to two muscular diseases for which no current curative treatment is available
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Thornburg, Adrienne. "Resolving the molecular mechanisms of inherited deafness caused by missense mutations in cadherin 23." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461284758.
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Essawy, Nada. "Characterization of emerin LEM-domain missense mutations present in patients with exclusive atrial cardiac defects." Electronic Thesis or Diss., Sorbonne université, 2018. http://www.theses.fr/2018SORUS299.
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La dystrophie musculaire d’Emery-Dreifuss (DMED) est l'une des dystrophies musculaires génétiques humaines les plus répandues. L'implication cardiaque dans la maladie est le symptôme qui met le plus la vie en danger et la principale cause de mortalité. La majorité des cas de son type liée à l’X sont dus à des mutations dans un gène codant pour une protéine de l'enveloppe nucléaire, l'émérine. Malgré les progrès considérables qui ont été réalisés en termes de caractérisation de la structure de l'émerine, ses différents partenaires de liaison, et ses fonctions dans le corps humain, le tableau est encore assez incomplet. Cinquante ans après que la DMED ait été documentée pour la première fois, les chercheurs n'ont toujours pas compris la pathophysiologie de la maladie. Il n'est donc pas surprenant qu'à ce jour, il n'existe pas de traitement décrit pour la DMED. Cette thèse est une première tentative pour caractériser trois mutations faux-sens du domaine LEM de l'émerine (P22L, ΔK37, et T43I) présentes chez des patients présentant uniquement des symptômes cardiaques. L'objectif principal de cette thèse est d'étudier l'effet des trois mutations sur la structure de l'émerine, son auto-assemblage, et les interactions avec certains de ses partenaires de liaison bien décrits. Le travail présenté souligne que malgré la présence des trois mutations dans la seule région repliée de l'émerine, les variants ne présentent aucun défaut global dans leur structure, à l'exception de la déstabilisation du domaine LEM du variant ΔK37. Il est important de noter que les mutants sont capables de s'auto-assembler, mais avec une cinétique de polymérisation rapide. En outre, l’étude a montré que les trois variants, bien que mutés dans le domaine de liaison à BAF, sont étonnamment capables de se lier à la protéine BAF. De plus, l'analyse ne révèle aucune différence dans les interactions des variants avec l’Ig-fold de la lamine A/C. De plus, il n'y a pas de défaut dans la phosphorylation du variant ΔK37 par la kinase Src. La caractérisation préliminaire de la mutation ΔK37 dans des fibroblastes humains immortalisés n'a pas montré de défauts manifestes en mécanobiologie et dans l'expression des protéines de l'enveloppe nucléaire ou du cytosquelette. Pris dans son ensemble, le travail présenté souligne que les trois mutations faux-sens de l’émerine ne provoquent aucun défaut dans plusieurs propriétés importantes de l'émerine, qui sont testées dans cette thèse. Sur la base des résultats de la recherche menée, nous avons acquis des connaissances considérables en ce qui concerne les conséquences des mutations d'intérêt. En d'autres termes, le travail présenté montre qu’il faut continuer les recherches sur l’émerine, afin d'explorer d'autres propriétés ou fonctions de cette protéine qui pourraient être associées à la physiopathologie de la DMED<br>Emery-Dreifuss Muscular Dystrophy (EDMD) is among the most widely common human genetic muscular dystrophies. The cardiac involvement in the disease is the most life-threatening symptom and the major cause of mortality. The majority of cases of its X-linked type are due to mutations in a gene encoding for the nuclear envelope protein, emerin. Despite the considerable advances that have been achieved in terms of the characterization of emerin structure, various binding partners, and functions in the human body, the picture is still rather incomplete. Fifty years now after EDMD had been first documented, researchers still fall short of understanding the pathophysiology of the disease. Thereby, it comes as no surprise that, to date, there is no described treatment of EDMD. Accordingly, this thesis is an initial attempt to characterize three emerin LEM-domain missense mutations (P22L, ΔK37, and T43I) present in patients with exclusive cardiac defects. The main objective of this thesis is to investigate the effect of the three mutations on: emerin structure, its self-assembly, and interactions with some of its well-described binding partners. The presented work highlights that albeit the localization of the three mutations in the only folded region of emerin, the variants show no global defect in their structure, except for the destabilization of the LEM-domain of the variant ΔK37. Importantly, the mutants are able to self-assemble, yet with astonishing fast polymerization kinetics. In addition, the investigations have illustrated that the three variants, despite the presence of the mutations in the BAF-binding domain, are surprisingly capable of binding BAF. The analysis did not reveal any differences in the mutants binding to Ig-fold domain of lamin A/C. Further, there is no defect in ΔK37 phosphorylation by Src kinase. Also, preliminary characterization of the ΔK37 mutation in immortalized human fibroblasts has featured no overt defects in mechanobiology, and in the expression of nuclear envelope or cytoskeletal proteins. Taken all together, the presented work outlines that the three emerin missense mutations display no defects in several prominent emerin properties, which are questioned in this thesis. On the basis of the results of the conducted research, considerable insight has been gained with regard to the consequences of the mutations of interest. In other words, the presented work lends support to following investigations in order to explore other unquestioned properties or functions of emerin that might be associated with the pathophysiology of EDMD
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Pan, Yingzhou Edward [Verfasser], and Hans-Jürgen [Akademischer Betreuer] Kreienkamp. "Functional analysis of disease-associated CASK missense mutations / Yingzhou Edward Pan ; Betreuer: Hans-Jürgen Kreienkamp." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1210647087/34.
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Ozaki, Norio, Nakao Iwata, Kozo Kaibuchi, Masatoshi Takeda, Ryota Hashimoto, Toshiya Inada, Michio Suzuki, et al. "Resequencing and Association Analysis of the KALRN and EPHB1 Genes And Their Contribution to Schizophrenia Susceptibility." Thesis, Oxford University Press, 2012. http://hdl.handle.net/2237/14925.
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Torrieri, Érico. "Análise Estrutural de Mutações na Enzima GALNS associadas à Mucopolissacaridose IVA utilizando a Técnica de Modelagem Comparativa." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-28072015-113748/.
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As Mucopolissacaridoses (MPS) são um grupo de doenças de armazenamento lisossômico causadas por deficiência de enzimas que catalisam a degradação gradual das glicosaminoglicanas (GAGs). GAGs (anteriormente chamadas de mucopolissacarídeos) são produtos de degradação das proteoglicanas que existem na matriz extracelular e tem efeito proteolítico. A classificação das MPS é baseada na deficiência enzimática específica. A MPS IVA é causada por mutações no gene que codifica a enzima GALNS (Nacetilgalactosamina-6-sulfatase), a qual desempenha um papel crucial na degradação do sulfato de queratano e condroitina-6-sulfatase. As mutações na enzima se resumem em três categorias: interrupção do sítio ativo, alterações no núcleo hidrofóbico e exposição da superfície, onde mutações missense na estrutura podem afetar gravemente a atividade da proteína GALNS, alterando seu núcleo hidrofóbico ou modificando seu enovelamento (folding). Com a falta de tratamentos efetivos, sendo em sua maioria paliativos, e tendo como base a estrutura já resolvida da GLANS selvagem, este trabalho teve como objetivo modelar 3 variantes da enzima GALNs, sendo uma mutação no sítio ativo, uma no núcleo hidrofóbico e uma na superfície. Foi usado o software MODELLER 9.12 para a modelagem comparativa, os softwares Prochek, PROSA II, ERRATv2, Verify3d, ProQ para a avaliação dos modelos, o software NAND 2.10, para simulação de dinâmica molecular e o software Chimera 1.10.1 para cálculo de superfícies eletrostáticas e hidrofobicidade da superfície. Os modelos apresentaram bons resultados segundo os softwares de avaliação e análise visual. Apresentaram poucas diferenças estruturais em relação à estrutura da GALNS selvagem, demonstraram estabilidade em simulação de dinâmica molecular. Entretanto, algumas diferenças foram observadas com relação à distribuição de cargas e hidrofobicidade no sítio ativo do modelo da variante com mutação no sítio ativo. Pôde ser concluído que as 3 mutações analisadas não causaram alterações estruturais significativas, não interferiram na estabilidade estrutural em simulação de dinâmica molecular, entretanto, foi demonstrado que mutações na região do sítio ativo podem interferir na função da enzima.<br>The Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by deficiencies in enzymes that catalyze the gradual glycosaminoglycans (GAGs) degradation. GAGs (formerly called mucopolysaccharides) are products of proteoglycan degradation that exist in the extracellular matrix and have proteolytic effect. The classification of MPS is based on the specific enzyme deficiency. MPS IVA is caused by mutations in the gene that encodes the GALNS enzyme (Nacetilgalactosamina-6-sulfatase), which plays a crucial role in the degradation of keratan sulfate and chondroitin-6-sulfatase. Mutations in the enzyme can be summarized in three categories: interruption of the active site, changes in the hydrophobic core and display surface, where missense mutations in the structure can seriously affect the activity of GALNS protein, changing its hydrophobic core or modifying its folding. With the lack of effective treatments, in its most palliative, and based on the wild GALNS structure already determined, this study aimed to model 3 variants of GALNS enzyme, a mutation in the active site, one in the hydrophobic core and a on the surface. 9.12 MODELLER was used for comparative modeling software, the software Prochek, Prose II, ERRATv2, Verify3d, ProQ models for the evaluation of the NAND 2.10 software, for molecular dynamics simulation and software Chimera 1.10.1 calculates electrostatic and hydrophobic surface. The models showed good results according to the evaluation software and visual analysis. Presented few structural differences from the wild GALNS structure and showed stability in molecular dynamics simulation. However, some differences were observed with respect to the charge distribution and hydrophobicity in the active site of the variants of the model with a mutation in the active site. It might be concluded that the three mutations analyzed did not cause significant structural changes and did not affect the structural stability in molecular dynamics simulation, however, it has been shown that mutations in the active site region may interfere with the function of this enzyme.
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Bidshahri, Arezoo (Roza). "Novel ultra-sensitive digital PCR assays for screening and detection of rare missense mutations in (proto)-oncogenes." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62151.
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Somatic mutations can lead to cancer, often by altering the activity of kinases within signaling pathways that control cell growth and proliferation. Targeted cancer therapeutics are designed and used to regulate these aberrant signaling pathways in cases where somatic mutations within kinase genes predict a positive patient response to those treatments. For example, the V600E mutation in BRAF, the gene coding for the BRAF serine threonine kinase, predicts the effectiveness of vemurafenib in treating metastatic melanoma, while the mutational status of codons G12/G13 in the KRAS gene predicts likely colorectal cancer patient response to the monoclonal antibody (mAb) cetuximab.¹-³ However, FDA approved assays currently used to detect missense mutations in BRAF V600 and KRAS G12/G13 are not capable of detecting clinically actionable mutations at mutational frequencies low enough to permit their robust application to early disease detection or minimal residual disease monitoring. Moreover, detection of all clinically actionable missense mutations is not certain or generally achieved, in part due to limitations to assay specificities and the inability to unequivocally discriminate missense mutations from synonymous germline sequence variations.
This thesis addresses that limitation through the development and validation of a novel platform for creating highly sensitive assays against all possible missense mutations in an oncogenic hotspot codon or adjacent set of hotspot codons that ameliorates the known limitations to current FDA-approved assays. The platform is designed to enable development of assays against all possible missense mutations in oncogenic hotspots and, if required, unequivocally differentiate them from synonymous germline alleles. It utilizes droplet digital PCR (ddPCR) technology and chimeric wild-type specific LNA/DNA probes to create a novel “WT-negative” screening paradigm. The platform is applied to the creation of two new assays of potential clinical use in cancer diagnostics and theranostics. The first provides a reliable and sensitive screening and detection of all known clinically actionable mutations in BRAF V600, and the second achieves the same for KRAS G12/G13. Both assays show complete diagnostic accuracy when applied to formalin-fixed paraffin-embedded (FFPE) tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.<br>Applied Science, Faculty of<br>Graduate
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Essawy, Nada [Verfasser]. "Characterization of emerin LEM-domain missense mutations present in patients with exclusive atrial cardiac defects / Nada Essawy." Berlin : Freie Universität Berlin, 2019. http://d-nb.info/1179277864/34.
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Jeromson, Sarah Joy. "Development of a yeast model to distinguish missense mutations from polymorphisms in the Wilson's disease gene ATP7B." Thesis, University of Huddersfield, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288501.
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Sanjurjo, Soriano Carla. "Functional characterisation of FEVR-related LGR4 missense mutations : implications in Norrin-β-Catenin signalling pathway and angiogenesis". Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/17046/.
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Preeprem, Thanawadee. "Functional assessments of amino acid variation in human genomes." Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/51869.
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The Human Genome Project, initiated in 1990, creates an enormous amount of excitement in human genetics—a field of study that seeks answers to the understanding of human evolution, diseases and development, gene therapy, and preventive medicine. The first completion of a human genome in 2003 and the breakthroughs of sequencing technologies in the past few years deliver the promised benefits of genome studies, especially in the roles of genomic variability and human health. However, intensive resource requirements and the associated costs make it infeasible to experimentally verify the effect of every genetic variation. At this stage of genome studies, in silico predictions play an important role in identifying putative functional variants. The most common practice for genome variant evaluation is based on the evolutionary conservation at the mutation site. Nonetheless, sequence conservation is not the absolute predictor for deleteriousness since phylogenetic diversity of aligned sequences used to construct the prediction algorithm has substantial effects on the analysis. This dissertation aims at overcoming the weaknesses of the conservation-based assumption for predicting the variant effects. The dissertation describes three different integrative computational approaches to identify a subset of high-priority amino acid mutations, derived from human genome data. The methods investigate variant-function relationships in three aspects of genome studies—personal genomics, genomics of epilepsy disorders, and genomics of variable drug responses.
For genetic variants found in genomes of healthy individuals, an eight-level variant classification scheme is implemented to rank variants that are important towards individualized health profiles. For candidate genetic variants of epilepsy disorders, a novel 3-dimensional structure-based assessment protocol for amino acid mutations is established to improve discrimination between neutral and causal variants at less conserved sites, and to facilitate variant prioritization for experimental validations. For genomic variants that may affect inter-individual variability in drug responses, an explicit structure-based predictor for structural disturbances is developed to efficiently evaluate unknown variants in pharmacogenes. Overall, the three integrative approaches provide an opportunity for examining the effects of genomic variants from multiple perspectives of genome studies. They also introduce an efficient way to catalog amino acid variants on a large scale genome data.
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Kamat, M. A. "The involvement of non-B DNA forming sequences in mediating missense mutations, micro-deletions and micro-insertions in human inherited disease." Thesis, Nottingham Trent University, 2014. http://irep.ntu.ac.uk/id/eprint/3366/.
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The involvement of the local DNA sequence features (repetitive elements capable of adopting non-B structures, hotspot motifs, mononucleotide runs and tandem repeats) and epigenetic marks in mediating germline missense and nonsense mutations, micro-deletions and micro-insertions causing human inherited disease and obtained from Human Gene Mutation Database (HGMD; http://www.hgmd.org) was studied in silico. A novel algorithm with a linear running time has been designed to detect the non-B DNA forming repeats in DNA sequences. The distributions of these repeats in the vicinity of mutations were analysed. We found that ~15% of missense mutations, ~12% of nonsense mutations, ~28% of micro-deletions and ~21% of micro-insertions occurred within direct repeats and is explicable by the formation/resolution/correction of non-B slipped structures. Several novel mutational mechanisms such as slipped strand mispairing/non-B slipped structure formation/DNA repair, non-B triplex formation/DNA repair and hairpin loop formation/DNA repair mechanisms have been proposed to explain single basepair substitutions leading to the formation of respectively exact direct, mirror and inverted repeats from inexact repeats. The role of CpG dinucleotides, CpHpG trinucleotides in mediating single base-pair substitutions and 83 known hotspot motifs together with other repetitive elements was studied in the context of micro-deletions and micro-insertions. We found that the number of single basepair missense and nonsense mutations (C>T and G>A) in CpG or CpHpG oligonucleotides was significantly (Fisher’s Exact test; p<2.37×10-120 higher than would have occurred by chance. Three DNA hotspot motifs, Chinese hamster scaffold attachment region motif WWWAAHAWWA, DNA polymerase α frameshift hotspot motif TCCCCC and its mirror image motif, were found to be overrepresented in both ± 20bp and ±2bp vicinity of micro-deletions and micro-insertions. Enrichment in the post-translational histone modification marks and binding sites (H3k4me3, H3k36me3, CTCF binding sites, DHSs) was found in the vicinity of all types of mutations analysed whereas histone modification mark H3k27me3 was overrepresented in the vicinity of missense and micro-lesion mutations. Thus, this study is a first systematic ascertainment of the involvement of non-B DNA forming sequences and epigenetic marks in mediating subtle mutations causing or associated with human inherited disease. The results shed light on the plausible underlying mutational mechanisms that involve non-B DNA forming repeats.
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Zein, Aiman. "Structure-Function Relationship of the Sterol Transporter ABCG5/G8: Expression, Purification and Enzymatic Characterization of ABCG5/G8 Missense Loss of Function Mutations." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/40742.
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The heterodimeric ATP-binding cassette (ABC) transporter, ABCG5/G8, is responsible for direct secretion of cholesterol and dietary sterols into the gut lumen and the bile. Inactivating mutations of ABCG5/G8 cause sitosterolemia, a rare autosomal recessive disease characterized by the accumulation of plant sterols in plasma, hypercholesterolemia and development of premature coronary heart disease. Functional and structural characterization of ABCG5/G8 is necessary to understand its mechanism and how the genetic defects impact its function. In this thesis, I expressed seventeen constructs of various disease-causing or catalytically deficient missense mutations in Pichia pastoris yeast. This establishes reagents for in vitro functional and structural studies. Secondly, I focused on two disease mutants (ABCG5-E146Q and ABCG8-R543S) and a sterol binding mutation (ABCG5-A540F) and established large-scale purification of these mutants. Using a cholesterol hemisuccinate (CHS)-dependent ATPase assay, I determined ATP hydrolysis by these three mutants and analyze their kinetic parameters. All missense mutants showed a significantly impaired ATPase activity, but the ability of ATP binding appeared unchanged between the WT and the mutants. This work demonstrates an intimate structure-function relationship in ABCG5/G8 and sheds some light on the mechanistic details of this important cholesterol-regulating ABC transporter.
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Zhao, Wenchao [Verfasser]. "Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations : The establishment of translating ribosome affinity purification / Wenchao Zhao." Bonn : Universitäts- und Landesbibliothek Bonn, 2020. http://d-nb.info/1222588757/34.
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Hedrich, Christian Michael, Agnieszka Zachurzok-Buczynska, Aneta Gawlik, Susanne Russ, Gabriele Hahn, Katrin Köhler, Ewa Malecka-Tendera, and Angela Hübner. "Autosomal Dominant Neurohypophyseal Diabetes Insipidus in Two Families: Molecular Analysis of the Vasopressin-Neurophysin II Gene and Functional Studies of Three Missense Mutations." Karger, 2009. https://tud.qucosa.de/id/qucosa%3A27572.
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Background: Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease with symptoms of polydipsia, polyuria and dehydration caused by arginine vasopressin deficiency. Disease onset is within infancy or adolescence. A variety of disease-causing mutations of the arginine vasopressin neurophysin II gene (AVP) on chromosome 20p13 have been described. Methods: Two Polish families with adFNDI were screened for mutations. Processing of wild-type (WT) and mutant AVP was monitored using immunocytochemical methods in stably transfected Neuro2A cells. AVP secretion into the cell culture supernatant was investigated with an enzyme immunoassay. Results: In the first family a heterozygous p.G96D mutation was identified. Some patients additionally carried a novel heterozygous mutation p.A159T. The second family presented with a heterozygous mutation p.C98G. Confocal laser microscopy unveiled accumulation of p.G96D and p.C98G prohormones in the cellular bodies, whereas WT and p.A159T prohormones and/or processed products were located in the tips of cellular processes. Reduced levels of AVP in supernatant culture medium of p.G96D and p.C98G transfected cells in comparison to p.A159T and WT cells were found. Conclusions: We conclude that the p.G96D and p.C98G mutations cause adFNDI in the two reported families. The sequence variant p.A159T does not seem to have disease-causing effects.<br>Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Amzal, Rachida. "Pharmacothérapie ciblée dans la cholestase intrahépatique familiale progressive de type 2 (PFIC2)." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS187.
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ABCB11/BSEP est le transporteur des acides biliaires, localisé au niveau du pôle canaliculaire des hépatocytes. Les mutations de ce gène sont responsables de la cholestase familiale intrahépatique progressive de type 2.Au cours de ma thèse, j’ai évalué la capacité des aminoglycosides et du PTC124 à induire la translecture de codons stop prématurés, l’adressage et la fonction de mutants non-sens et faux sens de Bsep ainsi que l’effet d’une bithérapie (translecture+chaperone).Dans nos modèles cellulaires, la gentamicine était capable d’induire la translecture du codon-stop prématuré du mutant non-sens BsepR1090X dans les lignées NIH3T3, HEK293 et Can 10. La protéine entière générée était partiellement détectée aux membranes plasmiques des cellules HEK293 et canaliculaires des cellules Can 10 et était partiellement fonctionnelle puisqu’elle était responsable d’une augmentation de l’activité de transport de 3H-taurocholate (3H-TC) dans les clones MDCK. Ces effets étaient potentialisés par l’addition de drogues chaperones telles que le 4-phenylbutyrate (4-PB).J’ai également mis en évidence la capacité de nouveaux composés dérivés du 4-PB (MHMPB, OTNC et HMPB) à corriger l’adressage et à augmenter le transport de 3H-TC du mutant faux sens BsepR1128C à des concentrations plus faibles que le 4-PB. Enfin, j’ai pu montrer que d'autres drogues chaperones (GPB, PA, SAHA et C18), pouvaient corriger l’adressage canaliculaire de BsepR1128C et augmenter son activité de transport de 3H-TC dans les clones MDCK<br>ABCB11/BSEP is the main bile acids transporter located at the canalicular pole of hepatocytes. Mutations of ABCB11 are responsible for progressive familial intrahepatic cholestasis type 2.During my phD, I evaluated the ability of aminoglycosides and PTC124 to induce readthrough of premature termination codons, targeting and function of nonsense and missense mutants of Bsep and also the effect of combined therapy (readthrough + chaperone).In our expermental models, gentamicin increased readthrough of p.R1090X mutation NIH3T3, HEK293 and Can 10 lines. The resulting full-length protein was detected at the plasma membrane of HEK293 and at the canalicular membrane of Can 10 cells; and was partially functional since it was responsible for increasing the transport activity of 3H-taurocholate (3H-TC) in MDCK clones. These effects were potentiated by the addition of chaperone drugs such as 4-phenylbutyrate (4-PB).I have also demonstrated the ability of new 4-PB derived compounds (MHMPB, OTNC and HMPB) to correct mistrafficking and to increase 3H-TC transport of BsepR1128C missense mutant at lower concentrations than 4-PB. Finally, I showed that other chaperone drugs (GPB, PA, SAHA, and C18) were able to correct mistrafiking of BsepR1128C and to increase its 3H-TC transport activity in MDCK clones
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Tervasmäki, A. (Anna). "Hereditary predisposition to breast cancer:evaluating the role of rare copy number variant, protein-truncating and missense candidate alleles." Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526220826.
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Abstract Breast cancer is the most common cancer among women, and inherited predisposition is one of the major recognized causes of increased breast cancer risk. Only about half of the hereditary cases are explained by mutations in the known susceptibility genes, including the DNA damage response genes BRCA1, BRCA2 and PALB2, leaving the majority still uncovered. Identification of the missing genetic predisposing factors is important for more effective diagnostics and counseling of the risk families, and also for better understanding of the etiology and cellular characteristics of breast cancer. The first aim of this study was to investigate the cancer associations of six rare germline copy number variant (CNV) deletions, which were previously identified in breast cancer patients by a genome-wide microarray approach. The second aim was to identify novel susceptibility alleles, both protein-truncating variants and missense mutations, by next-generation sequencing (NGS) of nearly 800 DNA damage response genes in 189 hereditary breast cancer patients. The cancer-associations of all selected candidate alleles (6 CNVs, 39 protein-truncating variants and 35 missense mutations) were studied by case-control approach using DNA samples from several hundred breast cancer patients and healthy controls. The prevalence of the studied CNVs did not significantly differ between the cases and controls, but when studying the associations with specific clinical parameters, deletion in the CYP2C19 gene showed enrichment in the breast cancer patients with hormonally triple-negative tumors (p=0.021). As CYP2C19 functions in estrogen metabolism, the results indicate that disturbance of hormonal balance due to enzyme defects may predispose specifically to the estrogen receptor-negative subtype of breast cancer. Two protein truncating-variants, TEX15 c.7253dupT and FANCD2 c.2715+1G>A showed significant breast cancer association in the Northern Finnish cohort (p=0.018 and p=0.036, respectively). Similarly, two of the studied missense variants, RECQL p.Ile156Met (p=0.043) and POLG p.Leu392Val (p=0.010), were enriched in the breast cancer cases. Thus, this study provided novel connections between increased breast cancer risk and inherited mutations in TEX15, FANCD2 and POLG genes, and further supported the recently established role of RECQL as a breast cancer susceptibility gene<br>Tiivistelmä Rintasyöpä on naisten yleisin syöpä, ja perinnöllinen alttius on yksi merkittävimmistä sairastumisriskiin vaikuttavista tekijöistä. Tunnetuimpia alttiustekijöitä ovat mutaatiot BRCA1-, BRCA2- ja PALB2-DNA-vauriovastegeeneissä, mutta ne yhdessä muiden altistavien geenimutaatioiden kanssa selittävät kuitenkin vain noin puolet perinnöllisistä rintasyöpätapauksista. Uusien alttiusgeenien löytäminen mahdollistaa tehokkaamman diagnostiikan ja korkeassa syöpäriskissä olevien sukujen perinnöllisyysneuvonnan, sekä auttaa ymmärtämään syvemmin rintasyövän etiologiaa ja syntymekanismeja solutasolla. Tämän väitöskirjan ensimmäisenä päämääränä oli tutkia tarkemmin aiemmin genominlaajuisella mikrosirumenetelmällä rintasyöpäpotilailta tunnistettujen harvinaisten perinnöllisten DNA-kopiolukuvariaatioiden (CNV) yhteyttä rintasyöpäriskiin. Toisena tavoitteena oli tunnistaa uusia rintasyöpäalttiusalleeleja, sekä proteiinitrunkaatioita että missense-mutaatioita, hyödyntämällä uuden sukupolven sekvensointitekniikkaa, jonka avulla tutkittiin mutaatioita lähes 800 DNA-vauriovastegeenistä 189 pohjoissuomalaiselta rintasyöpäpotilaalta. Valittujen kandidaattialleelien (6 deleetion aiheuttavaa CNV:tä, 39 proteiinitrunkaatiota ja 35 missense-mutaatiota) yhteyttä rintasyöpään tutkittiin tapaus-verrokkimenetelmällä käyttäen DNA-näytteitä usealta sadalta rintasyöpäpotilaalta ja terveeltä kontrollihenkilöltä. Tutkittujen CNV:iden esiintyvyydessä ei ollut merkitseviä eroja potilaiden ja kontrollien välillä, mutta tarkasteltaessa yhteyttä potilaiden kasvaimista saatuihin kliinisiin parametreihin, deleetio CYP2C19-geenissä oli yleisempi hormonaalisesti kolmoisnegatiivisissa rintatuumoreissa kuin muissa tuumorityypeissä (p=0.021). Koska CYP2C19 on estrogeenimetaboliaan osallistuva entsyymi, sen viallinen toiminta voi mahdollisesti altistaa erityisesti estrogeenireseptorinegatiiviselle rintasyövälle. Kaksi tutkituista proteiinitrunkaatioista, TEX15 c.7253dupT ja FANCD2 c.2715+1G>A, olivat rikastuneet perinnöllisessä rintasyöpäpotilasaineistossa verrattuna kontrolleihin (p=0.018 ja p=0.036). Myös kaksi missense-alleelia, RECQL p.Ile156Met (p=0.043) ja POLG p.Leu392Val (p=0.010), olivat yleisempiä rintasyöpäpotilailla. Tulokset osoittivat uuden yhteyden kohonneen rintasyöpäriskin ja perinnöllisten muutosten TEX15-, FANCD2- ja POLG-geenien välillä, sekä tukivat aiempia tutkimustuloksia, joiden mukaan RECQL on kohtalaisen riskin rintasyöpäalttiusgeeni
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Thomas, Anne [Verfasser]. "Characterization of the structural-functional impact of heterozygous missense mutations in genes of the blood coagulation factor XIII that cause mild Factor XIII deficiency / Anne Thomas." Bonn : Universitäts- und Landesbibliothek Bonn, 2018. http://d-nb.info/1153467062/34.
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Chen, Xuhua. "A missense mutation in Atf2 in standard poodles with fatal neonatal encephalopathy." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/6042.
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Thesis (M.S.)--University of Missouri-Columbia, 2007.<br>"May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.
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Flemming, Gunter. "Funktionelle Charakterisierung heterozygoter GLI2 missense Mutationen bei Patienten mit multiplem hypophysären Hormonmangel." Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-130953.
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Der GLI2-Transkriptionsfaktor ist eines der Haupt Effektor-Proteine des Sonic Hedgehog (SHH)-Signalweges und hat vermutlich eine Schlüsselfunktion in der Entwicklung der Hypophyse. Genomische GLI2-Veränderungen welche zu abgeschnittenen Proteinen führten, wurden beschrieben als Ursache für Holoprosenzephalie (HPE) oder HPE-ähnliche Veränderungen, teilweise in Verbindung mit einer Hypophysenunterfunktion.
Ziel dieser Arbeit war die Ermittlung der Frequenz von GLI2-Mutationen in Patienten mit multiplem hypophysärem Hormonausfall (multiple pituitary hormone deficiency, MPHD) und eine funktionelle Untersuchung der gefunden Mutationen mittels Transkriptionsaktivitäts-Untersuchungen durch funktionelle Luciferase assays.
Hierfür wählten wir Teilnehmer der GeNeSIS (Genetics and Neuroendocrinology of Short Stature International Study)-Studie. Patienten bei denen bereits Mutationen eines der
etablierten Gene für MPHD nachgewiesen wurde, wurden ausgeschlossen. Insgesamt haben wir 168 Patienten mit MPHD untersucht. Bei allen Patienten waren mindestens ein GH- und ein TSH-Mangel dokumentiert, Auffälligkeiten in der zentralen Bildgebung mittels cMRT wurden bei 96 Patienten angegeben.
In fünf Studienteilnehmern wurden vier verschiedene heterozygote missense Varianten nachgewiesen, hiervon wurden zwei bislang noch nicht in der Literatur beschrieben. Eine Variante, pR516P, führte in den in-vitro Experimenten zu einem kompletten Verlust der Proteinaktivität. Zusätzlich zu einem Wachstumshormonmangel hatte der Träger dieser Mutation einen Mangel an TSH und der Gonadotropine, sowie einen nichtdeszendierten Hypophysenhinterlappen und eine Polydaktylie, aber keine ersichtlichen Mittelliniendefekte.
Anhand der funktionellen Untersuchung konnten wir erstmalig nachweisen, dass ein heterozygoter Aminosäuren-Austausch im GLI2-Protein zu einer möglichen Funktionseinschränkung der Transkriptionsaktivität führen kann und somit die Ursache für MPHD mit milden extrahypophysären Auffälligkeiten sein könnte. Der Phänotyp von GLI2-Mutationen ist variabel und die Penetranz ist unvollständig. GLI2-Mutationen sind assoziiert mit einer Hypoplasie des Hypophysenvorderlappens und treten gehäuft mit einem ektopen Hypophysenhinterlappen auf.
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Schindlbeck, Ulrike [Verfasser], and Matthias [Akademischer Betreuer] Griese. "Charakterisierung neuer Missense Mutationen im Lipidtransporter ABCA3 / Ulrike Schindlbeck ; Betreuer: Matthias Griese." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/119981640X/34.
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Ramirez, Christina J. "BRCA genes : conserved regions and the potential effect of missense changes /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/5052.
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Yu, Yanan. "NF1 Patient Missense Variants Predict a Role for ATM in Modifying Neurofibroma Initiation." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1592395217393569.
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Kobayashi, Hiromasa. "A novel homozygous missense mutation of melanocortin-4 receptor (MC4R) in a Japanese woman with severe obesity." Kyoto University, 2004. http://hdl.handle.net/2433/148274.
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Yoshida, Hidetada. "Characterization of a novel missense mutation in the pore of HERG in a patient with long QTsyndrome." Kyoto University, 2001. http://hdl.handle.net/2433/150536.
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Robins, Tiina. "Functional and structural studies on CYP21 mutants in congenital adrenal hyperplasia /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-529-1/.
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Saito, Hidehiko, Shigeru Shirakawa, Katsumi Deguchi, Hideo Wada, Eiichi Iwasaki, Junki Takamatsu, Isamu Sugiura, Tadashi Matsushita, and Koji Yamamoto. "Homozygous protein C deficiency: identification of a novel missense mutation that causes impaired secretion of the mutant protein C." Thesis, Elsevier, 1992. http://hdl.handle.net/2237/16344.
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Tanaka, Naoto. "A MISSENSE MUTATION IN CONE PHOTORECEPTOR CYCLIC NUCLEOTIDE-GATED CHANNELS ASSOCIATED WITH CANINE DAYLIGHT BLINDNESS OFFERS INSIGHT INTO CHANNEL STRUCTURE AND FUNCTION." Diss., Temple University Libraries, 2013. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/246634.
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Biology<br>Ph.D.<br>Cone cyclic nucleotide-gated (CNG) channels are located in the retinal outer segments, mediating daylight color vision. The channel is a tetramer of A-type (CNGA3) and B-type (CNGB3) subunits. CNGA3 subunits are able to form homotetrameric channels, but CNGB3 exhibits channel function only when co-expressed with CNGA3. Mutations in the genes encoding these cone CNG subunits are associated with achromatopsia, an autosomal recessive genetic disorder which causes incomplete or complete loss of daylight and color vision. A missense mutation, aspartatic acid (Asp) to asparagine (Asn) at position 262 in the canine CNGB3 subunit (cB3-D262N), results in loss of cone function and therefore daylight blindness, highlighting the crucial role of this aspartic acid residue for proper channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. We exploit the conservation of these residues in CNGA3 subunits to examine the motif using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in a loss of nucleotide-activated currents and mislocalization in heterologous expression. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 tetramers. This failure to record heteromeric currents implies that Asp/Asn mutations impact negatively both CNGA3 and CNGB3 subunits. A homology model of canine CNGA3 relaxed in a membrane using molecular dynamics simulations suggests that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3 - S4. We propose that the CNGB3-D262N mutation in daylight blind dogs results in the loss of these interactions and leads to an alteration of the electrostatic equilibrium in the S1 - S4 bundle. Because residues analogous to Tri-Asp residues in the voltage-gated Shaker K+ channel superfamily were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions might impair the monomer folding state in D262N mutant CNG channels during biogenesis. Another missesnse sense mutation, Arginine (Arg) to tryptophan (Trp) at position 424 in the canine CNGA3 subunit (cA3-R424W), also results in loss of cone function. An amino acid sequence alignment with Shaker K+ channel superfamily indicates that this R424 residue is located in the C-terminal end of the sixth transmembrane segment. A3-R424W mutant channels resulted in no cyclic nucleotide-activated currents and mislocalization with intracellular aggregates. However, the localization of cA3-R424W mutant channels was not affected as severely as the Asp/Asn mutation in S2 Tri-Asp motif, showing a lot of cells with the proper localization of Golgi-like and membrane fluorescence. Moreover, the substitution of Arg 424 to Lysine (Lys), conserving the positive charge, preserved channel function in some cells, which is different from the results of the S2 Tri-Asp motif in which the Asp/Glu substitutions, conserving the negative charge, leads to loss of cyclic nucleotide-activated currents. Even though these missense mutations are both associated with canine daylight blindness, the Arg 424 residue might not be as critical for folding as the Tri-Asp residues in the S2 Tri-Asp motif and might be more of a problem in channel structure and function. The cA3 model relaxed with MD simulations indicated a possible interaction of Arg 424 with the Glu 304 residue in the S4-S5 linker. This hypothesis is supported by electrophysiological data in which the double mutation of reversing these residues, Glu 306 to Arg and Arg 424 to Glu (E306R-R424E) preserves channel function. In the model, this salt bridge appears to contribute to stabilization of the open pore state. The R424W mutation might disrupt the salt bridge formation, leading to deforming and closing the pore region.<br>Temple University--Theses
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Bogomolovas, Julius, Jennifer R. Fleming, Brian R. Anderson, Rhys Williams, Stephan Lange, Bernd Simon, Muzamil M. Khan, et al. "Exploration of pathomechanisms triggered by a single-nucleotide polymorphism in titin's I-band: the cardiomyopathy-linked mutation T2580I." ROYAL SOC, 2016. http://hdl.handle.net/10150/621990.
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Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.
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Bengtson, Per. "Carbohydrate dependent adhesion of leukocytes and the role of fucosyltransferase VII /." Linköping : Univ, 2003. http://www.bibl.liu.se/liupubl/disp/disp2003/med762s.pdf.
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Cotta, Doné Stefania. "Nephrin - intracellular trafficking and podocyte maturation /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-411-2/.
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Jackisch, Elisa [Verfasser], Jeanette [Akademischer Betreuer] Erdmann, and Joachim [Gutachter] Weil. "Bedeutung einer Missense-Mutation im ADCY8-Gen auf die Genregulation in einer Myokardinfarkt-Großfamilie / Elisa Jackisch ; Gutachter: Joachim Weil ; Akademischer Betreuer: Jeanette Erdmann." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2020. http://d-nb.info/1208539701/34.
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Liu, Xiao Li. "Nephrin: cellular trafficking and intracellular interactions /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-899-8/.
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Flemming, Gunter [Verfasser], Roland [Akademischer Betreuer] Pfäffle, Jürgen [Akademischer Betreuer] Klammt, Thomas [Gutachter] Kapellen, and Michael [Gutachter] Stumvoll. "Funktionelle Charakterisierung heterozygoter GLI2 missense Mutationen bei Patienten mit multiplem hypophysären Hormonmangel / Gunter Flemming ; Gutachter: Thomas Kapellen, Michael Stumvoll ; Roland Pfäffle, Jürgen Klammt." Leipzig : Universitätsbibliothek Leipzig, 2014. http://d-nb.info/1238599737/34.
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Tokuda, Satoko. "The ataxic groggy rat has a missense mutation in the P/Q-type voltage-gated Ca[2+] channel α1A subunit gene and exhibits absence seizures". Kyoto University, 2007. http://hdl.handle.net/2433/135666.
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VITALE, Alessandra Maria. "GENETIC NEUROCHAPERONOPATHIES ASSOCIATED WITH CCT5 AND HSP60 VARIANTS: ANALYSIS OF THEIR MOLECULAR ANATOMY AND POSSIBLE PATHOGENIC IMPLICATIONS." Doctoral thesis, Università degli Studi di Palermo, 2022. https://hdl.handle.net/10447/563680.
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Lohmöller, Benjamin [Verfasser], and Chris [Akademischer Betreuer] Mühlhausen. "Glutarazidurie Typ 1 : Untersuchungen zu Auswirkungen von „missense“-Mutationen auf Enzymexpression, Enzymsortierung, Enzymstabilität und den Abbau der Glutaryl-Coenzym A Dehydrogenase in der Zellkultur / Benjamin Lohmöller ; Betreuer: Chris Mühlhausen." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2018. http://d-nb.info/1161530339/34.
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Decaudin, Camille. "Impacts fonctionnels et conséquences sur la différenciation hématopoïétique d’une mutation somatique récurrente du gène PU.1/SPI1 identifiée dans la macroglobulinémie de Waldenström A Recurrent Activating Missense Mutation in Waldenström Macroglobulinemia Affects the DNA Binding of the ETS Transcription Factor SPI1 and Enhances Proliferation." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL004.
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Le facteur de transcription PU.1/SPI1 de la famille ETS est un régulateur majeur de l'hématopoïèse, et de la différenciation des cellules souches, myéloïdes et lymphoïdes. Précédemment identifié comme un suppresseur de tumeur dans les malignités myéloïdes humaines, nous avons identifié une mutation somatique récurrente faux sens (Q226E) du gène PU.1 dans la macroglobulinémie de Waldenström, un syndrome lymphoprolifératif à cellules B. La mutation affecte la liaison à l'ADN de la protéine et permet au mutant de se lier et d'activer plus fréquemment les régions promotrices par rapport à la protéine de type sauvage. La liaison du mutant aux promoteurs active la transcription de gènes généralement activés par d'autres facteurs ETS, comme Ets1, entraînant une prolifération accrue dans les modèles de lignées cellulaires et les lymphocytes B primaires de souris. Pour analyser les propriétés du mutant dans des conditions physiologiques, nous avons développé un modèle de souris portant un allèle conditionnel Pu.1 Q226E Knock-In. L'utilisation d'un transgène CD19-Cre induit l'expression de la protéine mutée dans la lignée lymphoïde B. L'analyse du développement précoce des cellules B montre une augmentation de la réponse des cellules pré-B à l'IL-7, entraînant l'amplification de cette population in vivo. La protéine mutante stimule la prolifération cellulaire des lymphocytes B et augmente la différentiation des lymphocytes B en plasmocytes (CD138+). Plus précisément, ce mutant de Pu.1 semble augmenter précocement l'expression des facteurs de transcription spécifiques des plasmocytes, comme Blimp1 et Xbp1, et augmente l'activation de la voie UPR, permettant une différenciation plus importante des cellules B matures en plasmocytes. Ces résultats décrivent un mécanisme de subversion oncogénique de la fonction d’un facteur de transcription suite à la modification subtile de la spécificité de liaison à l'ADN de la protéine, qui affecte la prolifération et la différenciation<br>The ETS transcription factor PU.1/SPI1 is a major regulator of hematopoiesis. It is implicated in HSC, myeloid but also lymphoid biology and has been described as a tumor suppressor in human myeloid malignancies. We identified a PU.1 activating missense mutation in Waldenström macroglobulinemia (Q226E), a B-cell lympho-proliferative neoplasm, highlighting new oncogenic features for this transcription factor. This mutation affects the DNA-binding affinity of the protein and allows the mutant to more frequently bind to promoter regions with respect to wild-type protein, resulting in transcriptional activation of gene sets typically regulated by other ETS factors, such as ETS1, resulting in enhanced proliferation in model cell lines and murine primary B-cells. To analyze the properties of the mutant protein in physiological conditions, I developed mouse model carrying a Pu.1 Q226E Knock-In conditional allele. The use of a CD19-Cre transgene triggers the expression of the mutated protein in the B lymphoid lineage. Analysis of the early B cells development shows an increase response of pre-B cells to IL-7, associated to the accumulation of this population in vivo. I confirmed that the mutant protein stimulates B cell proliferation and showed that it also increases the differentiation of B cells into plasma cells (CD138+). Specifically, Pu.1 mutant induce an early increase of the expression of plasma-specific transcription factors, such as Blimp1 or Xbp1, and increases activation of the UPR pathway, which likely increases differentiation of mature B cells into plasma cells. These results describe a mechanism of oncogenic subversion of the function of a transcription factor as a result of the subtle modification of the DNA binding specificity of the protein, which affects proliferation and differentiation
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Borges, Luciana Moreira. "VALOR PREDITIVO DA MUTAÇÃO R337H DO GENE TP53 COMO UM MARCADOR CLÍNICO EM PACIENTES COM CÂNCER." Pontifícia Universidade Católica de Goiás, 2014. http://localhost:8080/tede/handle/tede/2379.
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Previous issue date: 2014-04-07<br>Introduction: the R337H mutation of the TP53 gene was originated in the Brazilian population through a founder effect and is considered a molecular defect of low penetrance. In combination with some polymorphisms, the R337H mutation can increase the susceptibility to tumor. The frequency of the R337H mutation in Brazilian families is considered high when compared to the observed frequencies in other countries. The unambiguous association between the mutation, the emergence of different tumor types and the high number of individual that carry the mutation makes the R337H a relevant factor in public health, particularly in the prediction of cancer. Objective: This study aimed to investigate the predictive value of the R337H mutation of the TP53 gene as a clinical marker in cancer patients. Method: a systematic literature review (SLR) was carefully performed, by searching electronic scientific literature in LILACS, IBECS, MEDLINE, Pubmed and SciELO. Twelve articles, published in English between the years 2006 to 2013, were selected by performed the relevance tests I and II. Extraction of detailed data was independently performed by two investigators, following on extraction data protocol. Results: the R337H mutation was found in 287 of 1,548 patients with cancer, two of 750 women considered healthy, 200 of 887 family members of patients with adrenocortical tumor (ACT) carrying the R337H mutation, 12 of 647 health controls and in 442 of 171 630 newborns. Eight of the twelve selected references associated the R337H mutation with family history of 411 patients with the mutation. Four studies associated the R337H mutation prognosis. Conclusion: the frequency of the R337H mutation of the TP53 gene is considerably higher in the south and southeast regions of Brazil compared to other countries. The mutation was associated with family history of cancer, the increase of the positive predictive value and the decreased of negative predictive value at diagnosis, and poor prognosis in ACT and CPC patients with with the mutation.<br>Introdução: a mutação R337H do gene TP53 foi originada na população brasileira por efeito fundador e é considerada um defeito molecular de baixa penetrância. Em combinação com alguns polimorfismos, a mutação R337H, pode aumentar a susceptibilidade ao desenvolvimento do tumor. A frequência da mutação R337H em famílias brasileiras é considerada elevada, quando comparada com as frequências observadas em outros países. A inequívoca associação entre a mutação, o surgimento de diferentes tipos tumorais e o alto número de indivíduos portadores da mutação fazem da R337H um relevante fator de saúde pública, em especial na predição do câncer. Objetivos: este estudo objetiva de investiga o valor preditivo da mutação R337H do gene TP53 como um marcador clínico em pacientes com câncer. Método: revisão bibliográfica sistemática (RBS) criteriosa foi realizada, através de busca eletrônica de artigos científicos nas bases de dados LILACS, IBECS, MEDLINE, Scielo e Pubmed. Doze artigos selecionados foram publicados em língua inglesa entre os anos de 2006 à 2013, foram selecionados para aplicação dos testes de relevância I e II. Extração de dados detalhada foi realizada de forma independente por dois investigadores seguindo o protocolo para extração de dados. Resultados: a mutação R337H foi encontrada em 287 dos 1.548 portadores de câncer, duas das 750 mulheres consideradas saudáveis, 200 dos 887 familiares de pacientes portadores de tumor do córtex adrenal (TCA) com a mutação R337H, doze dos 647 controles e 442 dos 171.630 recém-nascidos. Oito das doze referências selecionadas associaram a mutação R337H com histórico familiar de 411 pacientes com a mutação. Quatro estudos associaram a mutação R337H com o prognóstico. Conclusão: a frequência da mutação R337H do gene TP53 é consideravelmente mais elevada no sul e sudeste do Brasil quando comparada com os demais países do mundo. A mutação foi associada com: histórico familiar de câncer, aumento do valor preditivo positivo e diminuição do valor preditivo negativo no diagnóstico e mal prognóstico em pacientes com ACT e CPC com a mutação.