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      Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development

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          Abstract

          To investigate the genetic basis of hereditary lens opacities we analyzed 31 cases of bilateral congenital cataract in Red Holstein Friesian cattle. A genome-wide association study revealed a significant association on bovine chromosome 7 at positions 6,166,179 and 12,429,691. Whole genome re-sequencing of one case and four relatives showed a nonsense mutation (g.5995966 C>T) in the PZP-like, alpha-2-macroglobulin domain containing 8 ( CPAMD8) gene leading to a premature stop codon (CPAMD8 p.Gln74*) associated with cataract development in cattle. With immunohistochemistry we confirmed a physiological expression of CPAMD8 in the ciliary body epithelium of the eye in unaffected cattle, while the protein was not detectable in the ciliary body of cattle with cataracts. RNA expression of CPAMD8 was detected in healthy adult, fetal and cataractous lenses.

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          Most cited references40

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            Congenital cataracts and their molecular genetics.

            Cataract can be defined as any opacity of the crystalline lens. Congenital cataract is particularly serious because it has the potential for inhibiting visual development, resulting in permanent blindness. Inherited cataracts represent a major contribution to congenital cataracts, especially in developed countries. While cataract represents a common end stage of mutations in a potentially large number of genes acting through varied mechanisms in practice most inherited cataracts have been associated with a subgroup of genes encoding proteins of particular importance for the maintenance of lens transparency and homeostasis. The increasing availability of more detailed information about these proteins and their functions and is making it possible to understand the pathophysiology of cataracts and the biology of the lens in general.
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              Genetics of crystallins: cataract and beyond.

              The crystallins were discovered more than 100 years ago by Mörner (1893. Untersuchungen der Proteinsubstanzen in den lichtbrechenden Medien des Auges. Z. Physiol. Chem. 18, 61-106) as the main structural proteins of the vertebrate eye lens. Since that time the major mammalian crystallins referred to as alpha-, beta-, and gamma-crystallins were characterized with respect to their genetic organization, regulation of their expression pattern and participation in several diseases. In recent years, more and more crystallins have also been identified outside the lens. Evolutionary analysis has demonstrated the relationship of crystallins to proteins involved in protection against stress. The alpha-crystallins form large complexes up to 1Mio Da; they are built up by two subunits referred to as alphaA- and alphaB-crystallins. These subunits are encoded by individual genes, Cryaa and Cryab being localized on different chromosomes and members of the small heat-shock protein family. The alphaA-crystallin is considered to be a molecular chaperone. It is expressed mainly in the lens - mutations in the Cryaa gene lead to recessive or dominant cataracts. In contrast, the alphaB-crystallin is rather ubiquitously expressed; mutations in the Cryab gene are associated with a broad variety of neurological, cardiac and muscular disorders. The beta/gamma-crystallin super family is encoded by at least 14 genes; the proteins are characterized by four Greek key motifs. In mammals, these genes are not only organized as individual genes (Cryba1, Cryba2, Crygf, Crygs, CrygN), but also in duplets (Cryba4-Crybb1 and Crybb2-Crybb3) and in one major cluster (Cryga-Cryge). The various Cryb and Cryg genes are considered to have been evolved by various duplications of the Greek key encoding units. The two main families are distinguished by the fact that each Greek key motif in the Cryb genes is encoded by one exon, whereas two motifs are encoded by one single exon in the Cryg genes. An intermediate between these subfamilies is CrygN encoding the first two Greek key motifs by individual exons, but the others by one single exon. Mutations in the Cryb/Cryg genes lead mainly to an opacification of the eye lens. In some Cryg mutants evidence was presented that the formation of large amyloid-like intranuclear inclusions containing the altered gamma-crystallins is a key event in cataract formation. Cataract formation, caused by Cryg mutations is further characterized by stopping the secondary lens fiber differentiation as indicated by the presence of remnants of cell nuclei, which are usually degraded in secondary fiber cells. Moreover, additional clinical features are being increasingly reported since these crystallins are found outside the eye: the betaB2-crystallin (previously referred to the basic principle crystallin) is also involved in neurogenesis and male infertility. For some of the beta/gamma-crystallins, Ca(2+)-binding properties have been discussed; however, it is an unsolved question whether these crystallins serve as Ca(2+) stores in vivo. Enzyme crystallins are enzymes, which have been recruited to the lens and are expressed there in high concentrations. The mu- and zeta-crystallins (gene symbols: Crym and Cryz, respectively) are discussed as examples for mammals. Mutations in the human CRYM gene lead to non-syndromic deafness, and mutations in the Cryz gene of guinea pigs cause cataracts.
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                Author and article information

                Contributors
                Role: Formal analysisRole: InvestigationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: InvestigationRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: ValidationRole: Writing – review & editing
                Role: InvestigationRole: ValidationRole: Writing – review & editing
                Role: InvestigationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: InvestigationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                6 July 2017
                2017
                : 12
                : 7
                : e0180665
                Affiliations
                [1 ]University of Goettingen, Institute of Veterinary Medicine, Goettingen, Germany
                [2 ]University Medical Center Goettingen, Department of Neuropathology, Prion and Dementia Research Unit, Goettingen, Germany
                [3 ]University of the Saarland, Institute of Neuropathology, Homburg, Germany
                [4 ]University of Veterinary Medicine Hannover, Foundation, Department of Small Animal Medicine and Surgery, Hannover, Germany
                [5 ]Chronix Biomedical, Goettingen, Germany
                Rush University Medical Center, UNITED STATES
                Author notes

                Competing Interests: JB was affiliated with Chronix Biomedical. Chronix Biomedical was neither funder of the project nor had any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in form of JB´s salary. The specific roles of JB are articulated in the author contributions´ section. JB´s affiliation with Chronix Biomedical does not alter our adherence to all PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0001-8384-0205
                http://orcid.org/0000-0002-4143-1363
                http://orcid.org/0000-0002-7635-9656
                Article
                PONE-D-17-08508
                10.1371/journal.pone.0180665
                5500361
                28683140
                07cc51c5-5eb8-4579-ab6f-9387277d9d8c
                © 2017 Hollmann et al

                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 author and source are credited.

                History
                : 28 March 2017
                : 19 June 2017
                Page count
                Figures: 4, Tables: 2, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Funded by: funder-id http://dx.doi.org/10.13039/501100003385, Georg-August-Universität Göttingen;
                We acknowledge support by the German Research Foundation and the Open Access Publication Funds of the University of Goettingen. The German Research Foundation and the Open Access Publication Funds of the University of Goettingen only cover the publication costs and did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine and Health Sciences
                Ophthalmology
                Lens Disorders
                Cataracts
                Biology and Life Sciences
                Anatomy
                Ocular System
                Ocular Anatomy
                Lens (Anatomy)
                Medicine and Health Sciences
                Anatomy
                Ocular System
                Ocular Anatomy
                Lens (Anatomy)
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Bovines
                Cattle
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Ruminants
                Cattle
                Biology and Life Sciences
                Anatomy
                Head
                Eyes
                Medicine and Health Sciences
                Anatomy
                Head
                Eyes
                Biology and Life Sciences
                Anatomy
                Ocular System
                Eyes
                Medicine and Health Sciences
                Anatomy
                Ocular System
                Eyes
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Research and Analysis Methods
                Histochemistry and Cytochemistry Techniques
                Immunohistochemistry Techniques
                Research and Analysis Methods
                Immunologic Techniques
                Immunohistochemistry Techniques
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Genome-Wide Association Studies
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Genome-Wide Association Studies
                Biology and Life Sciences
                Genetics
                Human Genetics
                Genome-Wide Association Studies
                Custom metadata
                All relevant data are within the paper and its Supporting Information files. Next Generation Sequences were deposited with the European Nucleotide Archive (ENA) under accession number PRJEB20549 ( http://www.ebi.uk/ena/data/view/PRJEB20549).

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