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      Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

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          Abstract

          Purpose

          To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation.

          Methods

          Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c.3277C>T, a nonsense mutation, and c.3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays.

          Results

          We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components.

          Conclusions

          COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

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          Most cited references 36

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          Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.

          We previously identified the angiogenesis inhibitor angiostatin. Using a similar strategy, we have identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma. Endostatin is a 20 kDa C-terminal fragment of collagen XVIII. Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. By a novel method of sustained release, E. coli-derived endostatin was administered as a nonrefolded suspension. Primary tumors were regressed to dormant microscopic lesions. Immunohistochemistry revealed blocked angiogenesis accompanied by high proliferation balanced by apoptosis in tumor cells. There was no toxicity. Together with angiostatin data, these findings validate a strategy for identifying endogenous angiogenesis inhibitors, suggest a theme of fragments of proteins as angiogenesis inhibitors, and demonstrate dormancy therapy.
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            Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons.

            We demonstrate in this work that the surface tension, water-organic solvent, transfer-free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions. We first develop a model for the curvature dependence of the hydrophobic effect and find that the macroscopic concept of interfacial free energy is applicable at the molecular level. Application of a well-known relationship involving surface tension and adhesion energies reveals that dispersion forces play little or no net role in hydrophobic interactions; rather, the standard model of disruption of water structure (entropically driven at 25 degrees C) is correct. The hydrophobic interaction is found, in agreement with the classical picture, to provide a major driving force for protein folding. Analysis of the melting behavior of hydrocarbons reveals that close packing of the protein interior makes only a small free energy contribution to folding because the enthalpic gain resulting from increased dispersion interactions (relative to the liquid) is countered by the freezing of side chain motion. The identical effect should occur in association reactions, which may provide an enormous simplification in the evaluation of binding energies. Protein binding reactions, even between nearly planar or concave/convex interfaces, are found to have effective hydrophobicities considerably smaller than the prediction based on macroscopic surface tension. This is due to the formation of a concave collar region that usually accompanies complex formation. This effect may preclude the formation of complexes between convex surfaces.
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              Improved splice site detection in Genie.

              We present an improved splice site predictor for the genefinding program Genie. Genie is based on a generalized Hidden Markov Model (GHMM) that describes the grammar of a legal parse of a multi-exon gene in a DNA sequence. In Genie, probabilities are estimated for gene features by using dynamic programming to combine information from multiple content and signal sensors, including sensors that integrate matches to homologous sequences from a database. One of the hardest problems in genefinding is to determine the complete gene structure correctly. The splice site sensors are the key signal sensors that address this problem. We replaced the existing splice site sensors in Genie with two novel neural networks based on dinucleotide frequencies. Using these novel sensors, Genie shows significant improvements in the sensitivity and specificity of gene structure identification. Experimental results in tests using a standard set of annotated genes showed that Genie identified 86% of coding nucleotides correctly with a specificity of 85%, versus 80% and 84% in the older system. In further splice site experiments, we also looked at correlations between splice site scores and intron and exon lengths, as well as at the effect of distance to the nearest splice site on false positive rates.
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                Author and article information

                Journal
                Mol Vis
                MV
                Molecular Vision
                Molecular Vision
                1090-0535
                2009
                23 April 2009
                : 15
                : 801-809
                Affiliations
                [1 ]Centro de Estudos do Genoma Humano, Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil
                [2 ]Collagen Research Unit, Biocenter and Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland
                [3 ]Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
                [4 ]Departamento de Física e Informática, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, Brazil
                [5 ]Departamento de Físico-Química, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, Brazil
                Author notes
                Correspondence to: Dr. Marita-Rita Passos-Bueno, Centro de Estudos do Genoma Humano, Instituto de Biociências, Universidade de São Paulo, Departamento de Genética e Biologia Evolutiva, Rua do Matão, 277, Sala 200, São Paulo, SP 05508-090, Brazil; Phone: 55-11-3091-9910; FAX: 55-11-3091-7419; email: passos@ 123456ib.usp.br
                Article
                82 2008MOLVIS363
                2671584
                19390655

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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