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      A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation‐prone Ano5 peptide

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          Mutations in ANO5 cause several human diseases including gnathodiaphyseal dysplasia 1 (GDD1), limb‐girdle muscular dystrophy 2L (LGMD2L), and Miyoshi myopathy 3 (MMD3). Previous work showed that complete genetic disruption of Ano5 in mice did not recapitulate human muscular dystrophy, while residual expression of mutant Ano5 in a gene trapped mouse developed muscular dystrophy with defective membrane repair. This suggests that truncated Ano5 expression may be pathogenic. Here, we screened a panel of commercial anti‐Ano5 antibodies using a recombinant adenovirus expressing human Ano5 with FLAG and YFP at the N‐ and C‐terminus, respectively. The monoclonal antibody (mAb) N421A/85 was found to specifically detect human Ano5 by immunoblotting and immunofluorescence staining. The antigen epitope was mapped to a region of 28 residues within the N‐terminus. Immunofluorescence staining of muscle cryosections from healthy control subjects showed that Ano5 is localized at the sarcoplasmic reticulum. The muscle biopsy from a LGMD2L patient homozygous for the c.191dupA mutation showed no Ano5 signal, confirming the specificity of the N421A/85 antibody. Surprisingly, strong Ano5 signal was detected in a patient with compound heterozygous mutations (c.191dupA and a novel splice donor site variant c.363 + 4A > G at the exon 6–intron 6 junction). Interestingly, insertion of the mutant intron 6, but not the wild‐type intron 6, into human ANO5 cDNA resulted in a major transcript that carried the first 158‐bp of intron 6. Transfection of the construct encoding the first 121 amino acids into C2C12 cells resulted in protein aggregate formation, suggesting that aggregate‐forming Ano5 peptide may contribute to the pathogenesis of muscular dystrophy.

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          Most cited references 31

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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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            Prediction of human mRNA donor and acceptor sites from the DNA sequence.

            Artificial neural networks have been applied to the prediction of splice site location in human pre-mRNA. A joint prediction scheme where prediction of transition regions between introns and exons regulates a cutoff level for splice site assignment was able to predict splice site locations with confidence levels far better than previously reported in the literature. The problem of predicting donor and acceptor sites in human genes is hampered by the presence of numerous amounts of false positives: here, the distribution of these false splice sites is examined and linked to a possible scenario for the splicing mechanism in vivo. When the presented method detects 95% of the true donor and acceptor sites, it makes less than 0.1% false donor site assignments and less than 0.4% false acceptor site assignments. For the large data set used in this study, this means that on average there are one and a half false donor sites per true donor site and six false acceptor sites per true acceptor site. With the joint assignment method, more than a fifth of the true donor sites and around one fourth of the true acceptor sites could be detected without accompaniment of any false positive predictions. Highly confident splice sites could not be isolated with a widely used weight matrix method or by separate splice site networks. A complementary relation between the confidence levels of the coding/non-coding and the separate splice site networks was observed, with many weak splice sites having sharp transitions in the coding/non-coding signal and many stronger splice sites having more ill-defined transitions between coding and non-coding.
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              X-ray structure of a calcium-activated TMEM16 lipid scramblase.

              The TMEM16 family of proteins, also known as anoctamins, features a remarkable functional diversity. This family contains the long sought-after Ca(2+)-activated chloride channels as well as lipid scramblases and cation channels. Here we present the crystal structure of a TMEM16 family member from the fungus Nectria haematococca that operates as a Ca(2+)-activated lipid scramblase. Each subunit of the homodimeric protein contains ten transmembrane helices and a hydrophilic membrane-traversing cavity that is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbours a conserved Ca(2+)-binding site located within the hydrophobic core of the membrane. Mutations of residues involved in Ca(2+) coordination affect both lipid scrambling in N. haematococca TMEM16 and ion conduction in the Cl(-) channel TMEM16A. The structure reveals the general architecture of the family and its mode of Ca(2+) activation. It also provides insight into potential scrambling mechanisms and serves as a framework to unravel the conduction of ions in certain TMEM16 proteins.

                Author and article information

                J Pathol Clin Res
                J Pathol Clin Res
                The Journal of Pathology: Clinical Research
                John Wiley and Sons Inc. (Hoboken )
                01 March 2018
                April 2018
                : 4
                : 2 ( doiID: 10.1002/cjp2.v4.2 )
                : 135-145
                [ 1 ] Division of Cardiovascular Medicine, Department of Cardiac Surgery, Dorothy M. Davis Heart and Lung Research Institute The Ohio State University Wexner Medical Center Columbus OH USA
                [ 2 ] Department of Pathology, Carver College of Medicine University of Iowa Iowa City IA USA
                Author notes
                [* ]Correspondence to: Renzhi Han, Department of Cardiac Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Biomedical Research Tower 316, Columbus, OH 43210, USA. E‐mail: renzhi.han@ 123456osumc.edu

                These authors contributed equally to this work.

                © 2018 The Authors The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd

                This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

                Page count
                Figures: 6, Tables: 1, Pages: 11, Words: 6053
                Funded by: US National Institutes of Health
                Award ID: HL116546
                Award ID: AR064241
                Award ID: U54
                Award ID: NS053672
                Funded by: Iowa Wellstone Muscular Dystrophy Cooperative Research Center
                Original Article
                Original Articles
                Custom metadata
                April 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.3.4 mode:remove_FC converted:17.04.2018

                aggregate, anoctamin, ano5, lgmd2l, muscular dystrophy, tmem16


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