Identical mutation in a novel retinal gene causes progressive rod-cone degeneration in dogs and retinitis pigmentosa in humans

Progressive rod-cone degeneration (prcd) is a late-onset, autosomal recessive photoreceptor degeneration of dogs and a homolog for some forms of human retinitis pigmentosa (RP). Previously, the disease-relevant interval was reduced to a 106-kb region on CFA9, and a common phenotype-specific haplotype was identified in all affected dogs from several different breeds and breed varieties. Screening of a canine retinal EST library identified partial cDNAs for novel candidate genes in the disease-relevant interval. The complete cDNA of one of these, PRCD, was cloned in dog, human, and mouse. The gene codes for a 54-amino-acid (aa) protein in dog and human and a 53-aa protein in the mouse; the first 24 aa, coded for by exon 1, are highly conserved in 14 vertebrate species. A homozygous mutation (TGC --> TAC) in the second codon shows complete concordance with the disorder in 18 different dog breeds/breed varieties tested. The same homozygous mutation was identified in a human patient from Bangladesh with autosomal recessive RP. Expression studies support the predominant expression of this gene in the retina, with equal expression in the retinal pigment epithelium, photoreceptor, and ganglion cell layers. This study provides strong evidence that a mutation in the novel gene PRCD is the cause of autosomal recessive retinal degeneration in both dogs and humans.     

Linkage disequilibrium mapping in domestic dog breeds narrows the progressive rod-cone degeneration interval and identifies ancestral disease-transmitting chromosome

Canine progressive rod-cone degeneration (prcd) is a retinal disease previously mapped to a broad, gene-rich centromeric region of canine chromosome 9. As allelic disorders are present in multiple breeds, we used linkage disequilibrium (LD) to narrow the approximately 6.4-Mb interval candidate region. Multiple dog breeds, each representing genetically isolated populations, were typed for SNPs and other polymorphisms identified from BACs. The candidate region was initially localized to a 1.5-Mb zero recombination interval between growth factor receptor-bound protein 2 (GRB2) and SEC14-like 1 (SEC14L). A fine-scale haplotype of the region was developed, which reduced the LD interval to 106 kb and identified a conserved haplotype of 98 polymorphisms present in all prcd-affected chromosomes from 14 different dog breeds. The findings strongly suggest that a common ancestor transmitted the prcd disease allele to many of the modern dog breeds and demonstrate the power of the LD approach in the canine model.     

Cloning and characterization of human homologue of Drosophila retinal degeneration B: A candidate gene for degenerative retinal diseases

Mutations in the Drosophila retinal degeneration B (D-rdgB) gene cause light-enhanced retinal degeneration. Here, we report the isolation of the cDNA encoding human homologue of the D-rdgB and initial characterization of the gene products. Like D-rdgB, the human rdgB homologue (H-rdgB) is a transmembrane protein with the N-terminus sharing high homology to two closely related cytosolic proteins, phosphatidylinositol transfer protein (PITP) α and β, indicating that rdgB like proteins belong to the family of PITP proteins. Using Northern and Western blotting, we demonstrated that the rdgB homologue is expressed in rat retina, olfactory bulb, and brain, but not in nonneuronal tissues. In the rat retina, immunoreactivity of the rdgB homologue was observed in photoreceptors and throughout the inner nuclear and plexiform layers; the strongest staining was in the inner plexiform layer. In the photoreceptor cells, the rdgB homologue was located primarily in the inner segment where sorting and traffic of membranes required for outer segment assembly take place. These data, together with recent findings showing PITPs as an important component of intracellular membrane traffic apparatus in mammalian cells, suggest that rdgB homologue may play a role in photoreceptor membrane renewal and in neurotransmitter release. Furthermore, using somatic hybrid cell hybridization and fluorescence in situ hybridization H-rdgB gene was mapped to human chromosome 11q13, a region known to contain several retinopathy loci, including Best disease and Bardet-Biedl syndrome I. Therefore, H-rdgB gene is an attractive candidate for several inherited retinal degenerative diseases. Dev. Genet. 20:235–245, 1997. © 1997 Wiley-Liss, Inc.     

Canine RPGRIP1 mutation establishes cone-rod dystrophy in miniature longhaired dachshunds as a homologue of human Leber congenital amaurosis

Cone-rod dystrophy 1 (cord1) is a recessive condition that occurs naturally in miniature longhaired dachshunds (MLHDs). We mapped the cord1 locus to a region of canine chromosome CFA15 that is syntenic with a region of human chromosome 14 (HSA14q11.2) containing the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) gene. Mutations in RPGRIP1 have been shown to cause Leber congenital amaurosis, a group of retinal dystrophies that represent the most common genetic causes of congenital visual impairment in infants and children. Using the newly available canine genome sequence we sequenced RPGRIP1 in affected and carrier MLHDs and identified a 44-nucleotide insertion in exon 2 that alters the reading frame and introduces a premature stop codon. All affected and carrier dogs within an extended inbred pedigree were homozygous and heterozygous, respectively, for the mutation. We conclude the mutation is responsible for cord1 and demonstrate that this canine disease is a valuable model for exploring disease mechanisms and potential therapies for human Leber congenital amaurosis.     

Chromosomal assignment of canine TSC2, PKD1 and CLN3 genes by radiation hybrid- and linkage analyses

The canine tuberous sclerosis 2 (TSC2) gene has been mapped to canine chromosome 6 using a canine whole genome radiation hybrid panel. There is close linkage between canine TSC2 and the polycystic kidney disease 1 gene (PKD1), as has been observed in humans and other mammalian species. The gene responsible for the human juvenile form of neuronal ceroid lipofuscinosis (CLN3), maps close to TSC2 and PKD1 in humans, and is also syntenic in the dog. We further demonstrate linkage to a group of polymorphic markers assigned to canine chromosome 6 (CFA6).     

Identification and characterization of C6orf37, a novel candidate human retinal disease gene on chromosome 6q14

We have identified a novel human gene, chromosome 6 open reading frame 37 (C6orf37), that is expressed in the retina and maps to human chromosome 6q14, a genomic region that harbors multiple retinal disease loci. The cDNA sequence contains an open reading frame of 1314 bp that encodes a 437-amino acid protein with a predicted molecular mass of 49.2 kDa. Northern blot analysis indicates that this gene is widely expressed, with preferential expression observed in the retina compared to other ocular tissues. The C6orf37 protein shares homology with putative proteins in R. norvegicus, M. musculus, D. melanogaster, and C. elegans, suggesting evolutionary conservation of function. Additional sequence analysis predicts that the C6orf37 gene product is a soluble, globular cytoplasmic protein containing several conserved phosphorylation sites. Furthermore, we have defined the genomic structure of this gene, which will enable its analysis as a candidate gene for chromosome 6q-associated inherited retinal disorders.     

Lysophosphatidylcholine acyltransferase 1 controls mitochondrial reactive oxygen species generation and survival of retinal photoreceptor cells

Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration–related retinal diseases.     

Detailed four-way comparative mapping and gene order analysis of the canine ctvm locus reveals evolutionary chromosome rearrangements

Canine tricuspid valve malformation (CTVM) maps to canine chromosome 9 (CFA9), in a region syntenic with gene-dense human chromosome 17q. To define synteny blocks, we analyzed 148 markers on CFA9 using radiation hybrid mapping and established a four-way comparative map for human, mouse, rat, and dog. We identified a large number of rearrangements, allowing us to reconstruct the evolutionary history of individual synteny blocks and large chromosomal segments. A most parsimonious rearrangement scenario for all four species reveals that human chromosome 17q differs from CFA9 and the syntenic rodent chromosomes through two macroreversals of 9.2 and 23 Mb. Compared to a recovered ancestral gene order, CFA9 has undergone 11 reversals of <3 Mb and 2 reversals of >3 Mb. Interspecies reuse of breakpoints for micro- and macrorearrangements was observed. Gene order and content of the ctvm interval are best extrapolated from murine data, showing that multispecies genome rearrangement scenarios contribute to identifying gene content in canine mapping studies.     

Assignment of the beta-subunit of rod photoreceptor cGMP phosphodiesterase gene PDEB (homolog of the mouse rd gene) to human chromosome 4p16.

The gene encoding the beta-subunit of rod photoreceptor cGMP phosphodiesterase (gene symbol PDEB, homolog of the mouse rd gene) is mapped to human chromosome 4 using somatic cell hybrids and further localized to the chromosome band 4p16 using in situ hybridization. A mutation in the mouse gene underlies the recessive trait of retinal degeneration in the rd mouse. Thus, the human homolog is a candidate for lesions causing retinal degeneration.     

Localization of the gene for interphotoreceptor retinoid-binding protein to mouse chromosome 14 near Np-1

Interphotoreceptor retinoid-binding protein (IRBP) is a large glycoprotein known to bind retinoids and found primarily in the interphotoreceptor matrix of the retina between the retinal pigment epithelium and the photoreceptor cells. It is thought to transport retinoids between the retinal pigment epithelium and the photoreceptors, a critical role in the visual process. We have used a 900-bp bovine IRBP cDNA fragment to map the corresponding gene, Rbp-3, to mouse chromosome 14 with somatic cell hybrids and have positioned the gene near Np-1 (nucleoside phosphorylase-1) by analysis of the progeny of an intersubspecific backcross. In the human genome, NP maps to human chromosome 14 and RBP3 to human chromosome 10. Thus, these two genes span the putative site of a chromosomal translocation which contributed to divergent karyotype evolution of man and mouse.     

Characterization and chromosome assignment of the canine gamma-sarcoglycan gene (SGCG) to CFA 25q21-->q23

Mutations in the gene for gamma-sarcoglycan (SGCG) located on HSA 13q12 are responsible for limb girdle muscular dystrophy (LGMD2C) in human. Here we report the cloning of the canine SGCG gene together with its genomic structure and several intragenic polymorphisms. The coding part of the canine SGCG contains seven exons spanning at least 70 kb of genomic DNA. The chromosome assignment of the canine SGCG gene to CFA 25q21-->q23 confirms that the canine syntenic group 10 corresponds to CFA 25 and also supports the findings of human-canine reciprocal chromosome painting.     

PARP1 gene knock-out increases resistance to retinal degeneration without affecting retinal function

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP.     

Characterization and chromosomal localization of five canine ATOX1 pseudogenes

We have isolated six ATOX1 loci from the canine genome in BAC clones. Sequence analysis showed that five of these clones correspond to processed pseudogenes. Fluorescent in situ hybridization allowed us to map the genuine ATOX1 gene to CFA4q24-->q31 and the ATOX1 pseudogenes to CFA19q13.1, CFA4q24-->q31, CFA18q24-->q25, CFA9q22.1 -->q22.2 and CFA20q11-->q12.     

Apoptosis as the Mechanism of Neurodegeneration in Batten's Disease

Abstract: Batten's disease is a genetic neurodegenerative disease of childhood. Its hallmarks are retinitis pigmentosa and neuronal degeneration. As some types of photoreceptor death in mice are mediated by apoptosis, we investigated whether apoptosis is responsible for retinal and neuronal degeneration in the late infantile and juvenile forms of Batten's disease. Using the terminal dUDP nick end-labeling (TUNEL) staining method, we detected apoptotic neuronal cells in brain from patients and a canine model and in brain and retina from an ovine model for Batten's disease. We confirmed apoptosis by flow cytometry, electron microscopy, and DNA laddering. This is the first inherited neurodegenerative disease involving brain and retina in which apoptosis has been established as the mechanism of neuronal and photoreceptor cell death in both humans and animal models.     

Localization of the gene for interphotoreceptor retinoid-binding protein to mouse chromosome 14 near Np-1

Interphotoreceptor retinoid-binding protein (IRBP) is a large glycoprotein known to bind retinoids and found primarily in the interphotoreceptor matrix of the retina between the retinal pigment epithelium and the photoreceptor cells. It is thought to transport retinoids between the retinal pigment epithelium and the photoreceptors, a critical role in the visual process. We have used a 900-bp bovine IRBP cDNA fragment to map the corresponding gene, Rbp-3, to mouse chromosome 14 with somatic cell hybrids and have positioned the gene near Np-1 (nucleoside phosphorylase-1) by analysis of the progeny of an intersubspecific backcross. In the human genome, NP maps to human chromosome 14 and RBP3 to human chromosome 10. Thus, these two genes span the putative site of a chromosomal translocation which contributed to divergent karyotype evolution of man and mouse.     

Structure,chromosomal location,and analysis of the canine Cu/Zn superoxide dismutase (SOD1) gene

Mutations in Cu/Zn superoxide dismutase (SOD1), a major cytosolic antioxidant enzyme in eukaryotic cells, have been reported in approximately 20% of familial amyotrophic lateral sclerosis (FALS) patients. Hereditary canine spinal muscular atrophy (HCSMA), a fatal inherited motor neuron disease in Brittany spaniels, shares many clinical and pathological features with human motor neuron disease, including FALS. The SOD1 coding region has been sequenced and cloned from several animal species, but not from the dog. We have mapped the chromosomal location, sequenced, and characterized the canine SOD1 gene. Extending this analysis, we have evaluated SOD1 as a candidate for HCSMA. The 462 bp SOD1 coding region in the dog encodes 153 amino acid residues and exhibits more than 83% and 79% sequence identity to other mammalian homologues at both the nucleotide and amino acid levels, respectively. The canine SOD1 gene maps to CFA31 close to syntenic group 13 on the radiation hybrid (RH) map in the vicinity of sodium myo/inositol transporter (SMIT) gene. The human orthologous SOD1 and SMIT genes have been localized on HSA 21q22.1 and HSA 21q21, respectively, confirming the conservation of synteny between dog syntenic group 13 and HSA 21. Direct sequencing of SOD1 cDNA from six dogs with HCSMA revealed no mutations. Northern analysis indicated no differences in steady-state levels of SOD1 mRNA.     

A Novel Form of Progressive Retinal Atrophy in Swedish Vallhund Dogs

Inherited retinal degenerations, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), represent leading causes of incurable blindness in humans. This is also true in dogs, where the term progressive retinal atrophy (PRA) is used to describe inherited photoreceptor degeneration resulting in progressive vision loss. Because of the similarities in ocular anatomy, including the presence of a cone photoreceptor-rich central retinal region, and the close genotype-phenotype correlation, canine models contribute significantly to the understanding of retinal disease mechanisms and the development of new therapies. The screening of the pure-bred dog population for new forms of PRA represents an important strategy to establish new large animal models. By examining 324 dogs of the Swedish vallhund breed in seven countries and across three continents, we were able to describe a new and unique form of PRA characterized by the multifocal appearance of red and brown discoloration of the tapetal fundus followed over time by thinning of the retina. We propose three stages of the disease based on the appearance of the ocular fundus and associated visual deficits. Electroretinography revealed a gradual loss of both rod and cone photoreceptor-mediated function in Stages 2 and 3 of the disease. In the few dogs that suffered from pronounced vision loss, night-blindness occurred first in late Stage 2, followed by decreased day-vision in Stage 3. Histologic examinations confirmed the loss of photoreceptor cells at Stage 3, which was associated with the accumulation of autofluorescent material in the adjacent retinal pigment epithelium. Pedigree analysis was suggestive of an autosomal-recessive mode of inheritance. Mutations in six known canine retinal degeneration genes as well as hypovitaminosis E were excluded as causes of the disease. The observed variability in the age of disease onset and rate of progression suggest the presence of genetic and/or environmental disease modifiers.