Identification of Genetic Variation and Haplotype Structure of the Canine \u3cem\u3eABCA4\u3c/em\u3e Gene for Retinal Disease Association Studies

Over 200 mutations in the retina specific member of the ATP-binding cassette transporter superfamily (ABCA4) have been associated with a diverse group of human retinal diseases. The disease mechanisms, and genotype–phenotype associations, nonetheless, remain elusive in many cases. As orthologous genes are commonly mutated in canine models of human blinding disorders, canine ABCA4 appears to be an ideal candidate gene to identify and study sequence changes in dogs affected by various forms of inherited retinal degeneration. However, the size of the gene and lack of haplotype assignment significantly limit targeted association and/or linkage approaches. This study assessed the naturally observed sequence diversity of ABCA4 in the dog, identifying 80% of novel variations. While none of the observed polymorphisms have been associated with blinding disorders to date, breed and potentially disease specific haplotypes have been identified. Moreover, a tag SNP map of 17 (15) markers has been established that accurately predicts common ABCA4 haplotypes (frequency \u3e 5%) explaining \u3e85% (\u3e80%) of the observed genetic diversity and will considerably advance future studies. Our sequence analysis of the complete canine ABCA4 coding region will clearly provide a baseline and tools for future association studies and comparative genomics to further delineate the role of ABCA4 in canine blinding disorders

Photoreceptor Dysplasia: An Inherited Progressive Retinal Atrophy of Miniature Schnauzer Dogs

A progressive retinal atrophy (PRA) affecting Miniature Schnauzer dogs is reported. Of the 287 individuals (148 female, 139 male) comprising the study population, 66 (23 percent) were affected (33 female, 33 male) and 221 animals (115 female, 106 male) were phenotypically normal. There was no sex predilection for the disease. Results of histologic and electroretinographic studies indicate that the disease is a new and different type of PRA, characterized by unique morphologic and functional deficits during rod and cone development. Accordingly, the disease has been termed photoreceptor dysplasia. Clinically, and particularly ophthalmoscopically, diagnosis is only practicable in very late stages of the disease. Electroretinography, however, can provide evidence of the disease in dogs at least as young as 8 weeks of age. Pedigree analysis and test-mating studies conclusively establish that inheritance is autosomal recessive. The gene symbol pd (for photoreceptor dysplasia) is assigned

Analysis of six candidate genes as potential modifiers of disease expression in canine XLPRA1, a model for human X-linked retinitis pigmentosa 3

Purpose: Canine X-linked progressive retinal atrophy (XLPRA) is caused by mutations in RPGR exon ORF15, which is also a mutation hotspot in human X-linked retinitis pigmentosa 3 (RP3). The XLPRA1 form of disease has shown extensive phenotypic variability in a colony of dogs that all inherited the same mutant X-chromosome. This variability in onset and severity makes XLPRA1 a valuable model to use to identify genes influencing photoreceptors degeneration in dog and to elucidate molecular mechanisms underlying RP in its human homolog. In this study, RPGRIP1, RANBP2, NPM1, PDE6D, NPHP5, and ABCA4 genes were selected on the basis of interaction with RPGR or RPGRIP1 or their implication in related retinal diseases, and were investigated as candidate genetic modifiers of XLPRA1. Methods: A pedigree derived from an affected male dog outcrossed to unrelated normal mix bred or purebred females was used. Morphologic examination revealed phenotypic variability in the affected dogs characterized as mild, moderate, or severe. Single nucleotide polymorphisms (SNPs) and indel-containing markers spanning the entire genes were designed, based on the canine sequence and the Broad Institute SNP library, and genotyped on the pedigree. For each candidate gene, haplotypes were identified and their frequencies in severely and moderately affected dogs were compared to detect a putative correlation between a gene-specific haplotype(s), and severity level of the disease. Primers were derived from expressed sequence tags (ESTs) and predicted transcripts to assess the relative retinal expression of the six genes of interest in normal and affected retinas of different ages. Results: Four to seven haplotypes per gene were identified. None of the haplotypes of RPGRIP1, NPM1, PDE6D, NPHP5, RANBP2, and ABCA4 were found to co-segregate with the moderate or severe phenotype. No significant difference in the retinal expression levels of the candidate genes was observed between normal and affected dogs. Conclusions: The haplotype distribution of RPGRIP1, NPM1, PDE6D, NPHP5, RANBP2, and ABCA4 suggests these genes are not modifiers of the disease phenotype observed in the XLPRA1 pedigree. The RPGRORF15 stop mutation does not affect the retinal expression of these genes at the mRNA level in the pre-degenerate stage of disease, but no conclusions can be made at this time about changes that may occur at the protein level

Blindness Due to Polymicrogyria and Asymmetrical Dilation of the Lateral Ventricles in Standard Poodles

Polymicrogyria and asymmetric dilation of the lateral ventricles were seen in related Standard poodles that had cortical blindness. Three of the affected dogs also had gait and postural abnormalities, and one of these had seizures.Two of the affected dogs were littermates. Thorough ophthalmologic and neurologic examinations (including electroretinography, electromyography, cerebrospinal fluid analysis, plain radiographs, and computerized tomography scans) revealed no significant abnormalities outside of the brain that would account for the blindness. Computerized tomography scans in three dogs demonstrated bilateral dilation of the lateral ventricles which was more severe in the right. All dogs were necropsied between 5 and 9 months of age and had strikingly similar brain abnormalities. Numerous small irregular gyri with shallow sulci covered the middle and caudal dorsal and lateral cerebral cortex. The bony ridges of the inner calvaria in this area conformed to the underlying microgyral pattern. The lateral ventricles were asymmetrically dilated with the right more severely affected, particularly in the occipital area, and the cortical grey and white matter, including the corpus callosum, were thinned in these areas. The third and fourth ventricles and mesencephalic aqueduct were normal. Histologically, there was thinning and simplification of the cortical grey matter with an increased density of medium to large neurons. The corona radiata and subcortical white matter were also thinner than normal with no evidence of demyelination of astrocytic scarring. This congenital anomaly of the visual cortex causing blindness in the Standard Poodle appears to be inherited as an autosomal recessive trait

Cloning and Characterization of Canine \u3cem\u3ePAX6\u3c/em\u3e and Evaluation as a Candidate Gene in a Canine Model of Aniridia

Purpose: Mutations in PAX6 cause human aniridia. The small eye (sey) mouse represents an animal model for aniridia. However, no large animal model currently exists. We cloned and characterized canine PAX6, and evaluated PAX6 for causal associations with inherited aniridia in dogs. Methods: Canine PAX6 was cloned from a canine retinal cDNA library using primers designed from human and mouse PAX6 consensus sequences. An RH3000 radiation hybrid panel was used to localize PAX6 within the canine genome. Genomic DNA was extracted from whole blood of dogs with inherited aniridia, and association testing was performed using markers on CFA18. Fourteen PAX6 exons were sequenced and scanned for mutations, and a Southern blot was used to test for large deletions. Results: Like the human gene, canine PAX6 has 13 exons and 12 introns, plus an alternatively spliced exon (5a). PAX6 nucleotide and amino acid sequences were highly conserved between dog, human, and mouse. The canine PAX6 cDNA sequence determined in this study spans 2 large gaps present in the current canine genomic sequence. Radiation hybrid mapping placed canine PAX6 on CFA18 in a region with synteny to HSA11p13. Exon-scanning revealed single nucleotide polymorphisms, but no pathological mutations, and Southern blot analysis revealed no differences between normal and affected animals. Conclusions: Canine PAX6 was cloned and characterized, and results provide sequence information for gaps in the current canine genome sequence. Canine PAX6 nucleotide and amino acid sequences, as well as gene organization and map location, were highly homologous with that of the human gene. PAX6 was evaluated in dogs with an inherited form of aniridia, and sequence analysis indicated no pathological mutations in the coding regions or splice sites of aniridia-affected dogs, and Southern blot analysis showed no large deletions

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 ∼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

Targeting Gene Expression to Cones With Human Cone Opsin Promoters in Recombinant AAV

Specific cone-directed therapy is of high priority in the treatment of human hereditary retinal diseases. However, not much information exists about the specific targeting of photoreceptor subclasses. Three versions of the human red cone opsin promoter (PR0.5, 3LCR-PR0.5 and PR2.1), and the human blue cone opsin promoter HB569, were evaluated for their specificity and robustness in targeting green fluorescent protein (GFP) gene expression to subclasses of cones in the canine retina when used in recombinant adeno-associated viral vectors of serotype 5. The vectors were administered by subretinal injection. The promoter PR2.1 led to most effective and specific expression of GFP in the long- and medium-wavelength-absorbing cones (L/M cones) of normal and diseased retinas. The PR0.5 promoter was not effective. Adding three copies of the 35-bp LCR in front of PR0.5 lead to weak GFP expression in L/M cones. The HB569 promoter was not specific, and GFP was expressed in a few L/M cones, some rods and the retinal pigment epithelium. These results suggest that L/M cones, the predominant class of cone photoreceptors in the retinas of dogs and most mammalian species can be successfully targeted using the human red cone opsin promoter

Photoreceptor Cell Death, Proliferation and Formation of Hybrid Rod/S-Cone Photoreceptors in the Degenerating STK38L Mutant Retina

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene. © 2011 Berta et al

Photoreceptor Cell Death, Proliferation and Formation of Hybrid Rod/S-Cone Photoreceptors in the Degenerating \u3cem\u3eSTK38L\u3c/em\u3e Mutant Retina

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7–14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene