A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice

Purpose To characterize an N-ethyl-N-nitrosourea-induced dominant mouse mutant, M-1156, that exhibits progressive retinal degeneration and to investigate the pathogenesis of the retinal phenotype in the mutant. Methods A positional candidate gene approach was used to identify the causative gene in the M-1156 mutant. Funduscopic examination, light microscopy, transmission electron microscopy, and electroretinography were performed to analyze the M-1156 phenotype. Real-time quantitative PCR, immunohistochemistry, and western blotting were also performed. Results Linkage analysis enabled the mutant gene to be mapped to a region of chromosome 19 containing Rom1, which encodes rod outer segment membrane protein 1. Sequence analysis demonstrated that the mutation consisted of a single base T→C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed. A putative missense mutation designated Rom1Rgsc1156 that was identified in the M-1156 mutant mouse causes a Trp to Arg substitution at position 182 in the translated protein. Rom1Rgsc1156 heterozygotes were found to have a mottled retina and narrowed arteries in the fundus oculi. Photomicrographs of the retina revealed significant differences among the genotypes in the thickness of the outer nuclear layer and in the length of the outer segments of the photoreceptors. The alterations were more marked in the homozygotes than in the heterozygotes. Electron micrographs showed that the diameters of the discs varied in the heterozygotes and that the discs were more compactly stacked than in the wild type. There were significant differences among the genotypes in the amplitude of the a-wave in single-flash electroretinograms, but there were no significant differences among the photopic electroretinograms. Real-time quantitative PCR showed that there were no significant differences among the genotypes in Rom1 or peripherin/rds (Prph2) mRNA levels relative to the rhodopsin (Rho) mRNA level. Rom1 and Prph2 immunoreactivity were decreased in the retinas of the Rom1Rgsc1156 mutants. Semiquantitative western blot analysis of retinas from 3-week-old Rom1Rgsc1156 mutants demonstrated significant decreases in Rom1, Prph2, and Rho protein levels in all of the genotypes. Conclusions The Trp182Arg substitution in Rom1Rgsc1156 mutants causes retinal degeneration. The results suggested that Trp182Arg mutant Rom1 causes a decrease in the levels of wild-type Prph2 and Rom1, which in turn cause a reduction in the level of Prph2 containing tetramers in the disc rim region and ultimately result in unstable, disorganized outer segments and photoreceptor degeneration.

[1]  Hatice Duzkale,et al.  Prevalence of disease-causing mutations in families with autosomal dominant retinitis pigmentosa: a screen of known genes in 200 families. , 2006, Investigative ophthalmology & visual science.

[2]  T. Dryja,et al.  Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci. , 1994, Science.

[3]  R. Molday,et al.  Cloning of the CDNA for a novel photoreceptor membrane protein (rom-1) identifies a disk rim protein family implicated in human retinopathies , 1992, Neuron.

[4]  Christopher J. R. Loewen,et al.  Cysteine residues of photoreceptor peripherin/rds: role in subunit assembly and autosomal dominant retinitis pigmentosa. , 1998, Biochemistry.

[5]  R. Molday,et al.  Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor cell protein peripherin in the rod outer segment disk membrane. , 1990, Biochemistry.

[6]  T. Dryja,et al.  Mutations in the human retinal degeneration slow gene in autosomal dominant retinitis pigmentosa , 1991, Nature.

[7]  J. Sutcliffe,et al.  Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds) , 1989, Nature.

[8]  V. Sheffield,et al.  Phenotypic variation including retinitis pigmentosa, pattern dystrophy, and fundus flavimaculatus in a single family with a deletion of codon 153 or 154 of the peripherin/RDS gene. , 1993, Archives of ophthalmology.

[9]  Carlo Rivolta,et al.  Retinitis pigmentosa and allied diseases: numerous diseases, genes, and inheritance patterns. , 2002, Human molecular genetics.

[10]  D. Bok,et al.  Generation and analysis of transgenic mice expressing P216L-substituted rds/peripherin in rod photoreceptors. , 1997, Investigative ophthalmology & visual science.

[11]  H. Masuya,et al.  A novel dominant-negative mutation in Gdf5 generated by ENU mutagenesis impairs joint formation and causes osteoarthritis in mice. , 2007, Human molecular genetics.

[12]  M. Naash,et al.  The Effect of Peripherin/rds Haploinsufficiency on Rod and Cone Photoreceptors , 1997, The Journal of Neuroscience.

[13]  Christopher J. R. Loewen,et al.  The role of subunit assembly in peripherin-2 targeting to rod photoreceptor disk membranes and retinitis pigmentosa. , 2003, Molecular biology of the cell.

[14]  Klaus Schughart,et al.  Genome-wide, large-scale production of mutant mice by ENU mutagenesis , 2000, Nature Genetics.

[15]  Hiroshi Masuya,et al.  Implementation of the modified-SHIRPA protocol for screening of dominant phenotypes in a large-scale ENU mutagenesis program , 2005, Mammalian Genome.

[16]  Janet Rossant,et al.  Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis , 2000, Nature Genetics.

[17]  G. Travis,et al.  Analysis of the rds/peripherin·rom1 Complex in Transgenic Photoreceptors That Express a Chimeric Protein* , 1999, The Journal of Biological Chemistry.

[18]  K. Fujiki,et al.  A heterozygous putative null mutation in ROM1 without a mutation in peripherin/RDS in a family with retinitis pigmentosa. , 1995, Genomics.

[19]  R. Mcinnes,et al.  Photoreceptor peripherin is the normal product of the gene responsible for retinal degeneration in the rds mouse. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. Sutcliffe,et al.  The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated glycoprotein , 1991, Neuron.

[21]  Steve D. M. Brown,et al.  A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse , 2000, Nature Genetics.

[22]  R. Rosenfeld.  Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.

[23]  Christopher J. R. Loewen,et al.  Disulfide-mediated Oligomerization of Peripherin/Rds and Rom-1 in Photoreceptor Disk Membranes , 2000, The Journal of Biological Chemistry.

[24]  Alan Bird,et al.  Mutations in the human retinal degeneration slow (RDS) gene can cause either retinitis pigmentosa or macular dystrophy , 1993, Nature Genetics.

[25]  T. Magnuson,et al.  Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells , 2000, Nature Genetics.

[26]  Tetsuro Toyoda,et al.  Statistical search on the Semantic Web , 2008, Bioinform..

[27]  T. Dryja,et al.  Dominant and digenic mutations in the peripherin/RDS and ROM1 genes in retinitis pigmentosa. , 1997, Investigative ophthalmology & visual science.

[28]  F. Zindy,et al.  Pathogenesis of persistent hyperplastic primary vitreous in mice lacking the arf tumor suppressor gene. , 2004, Investigative ophthalmology & visual science.

[29]  H. Jansen,et al.  Absence of receptor outer segments in the retina of rds mutant mice , 1981, Neuroscience Letters.

[30]  Christopher J. R. Loewen,et al.  Molecular Characterization of Peripherin-2 and Rom-1 Mutants Responsible for Digenic Retinitis Pigmentosa* , 2001, The Journal of Biological Chemistry.

[31]  S. Tilghman,et al.  An engineered 800 kilobase deletion of Uchl3 and Lmo7 on mouse chromosome 14 causes defects in viability, postnatal growth and degeneration of muscle and retina. , 2003, Human molecular genetics.

[32]  R. Molday,et al.  Defective subunit assembly underlies a digenic form of retinitis pigmentosa linked to mutations in peripherin/rds and rom-1. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[33]  H. Jansen,et al.  Development and degeneration of retina in rds mutant mice: photoreceptor abnormalities in the heterozygotes. , 1985, Experimental eye research.

[34]  Hiroshi Masuya,et al.  Enamelin (Enam) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI). , 2005, Human molecular genetics.

[35]  Hiroshi Masuya,et al.  A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud. , 2007, Genomics.

[36]  M. Bayés,et al.  Putative association of a mutant ROM1 allele with retinitis pigmentosa , 1997, Human Genetics.

[37]  R. Mcinnes,et al.  Mutation analysis of the ROM1 gene in retinitis pigmentosa. , 1995, Human molecular genetics.

[38]  Yoshiyuki Sakaki,et al.  OmicBrowse: a browser of multidimensional omics annotations , 2007, Bioinform..

[39]  Hiroshi Masuya,et al.  A series of maturity onset diabetes of the young, type 2 (MODY2) mouse models generated by a large-scale ENU mutagenesis program. , 2004, Human molecular genetics.

[40]  D. Bok,et al.  Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) mice , 1992, Neuron.

[41]  J. Heckenlively,et al.  Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes. , 1999, Molecular vision.

[42]  R. Mcinnes,et al.  Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[43]  R. Bronson,et al.  Calorie restriction modulates age-dependent changes in the retinas of Brown Norway rats , 2000, Mechanisms of Ageing and Development.

[44]  P. Humphries,et al.  A three-base-pair deletion in the peripherin–RDS gene in one form of retinitis pigmentosa , 1991, Nature.