Photoreceptor dysplasia (pd) in miniature schnauzer dogs: evaluation of candidate genes by molecular genetic analysis.

Photoreceptor dysplasia (pd) is one of a group of at least six distinct autosomal and one X-linked retinal disorders identified in dogs which are collectively known as progressive retinal atrophy (PRA). It is an early onset retinal disease identified in miniature schnauzer dogs, and pedigree analysis and breeding studies have established autosomal recessive inheritance of the disease. Using a gene-based approach, a number of retina-expressed genes, including some members of the phototransduction pathway, have been causally implicated in retinal diseases of humans and other animals. Here we examined seven such potential candidate genes (opsin, RDS/peripherin, ROM1, rod cGMP-gated cation channel alpha-subunit, and three subunits of transducin) for their causal association with the pd locus by testing segregation of intragenic markers with the disease locus, or, in the absence of informative polymorphisms, sequencing of the coding regions of the genes. Based on these results, we have conclusively excluded four photoreceptor-specific genes as candidates for pd by linkage analysis. For three other photoreceptor-specific genes, we did not find any mutation in the coding sequences of the genes and have excluded them provisionally. Formal exclusion would require investigation of the levels of expression of the candidate genes in pd-affected dogs relative to age-matched controls. At present we are building suitable informative pedigrees for the disease locus with a sufficient number of meiosis to be useful for genomewide screening. This should identify markers linked to the disease locus and eventually permit progress toward the identification of the photoreceptor dysplasia gene and the disease-causing mutation.

Characterization of canine photoreceptor phosducin cDNA and identification of a sequence variant in dogs with photoreceptor dysplasia.

Photoreceptor dysplasia (pd) is an autosomal recessive disease of miniature schnauzer dogs causing retinal degeneration. The disease is a homologue of retinitis pigmentosa, a group of genetically heterogeneous diseases, causing blindness in humans. A subtraction library was prepared from retinas of pd affected and age-matched normal control dogs to isolate de novo candidate genes for further examination. From the subtraction library, cDNA for phosducin (PDC), a member of the phototransduction pathway, was isolated as a transcript expressed at a higher level in the affected retina. First, the normal canine PDC cDNA was characterized to evaluate the PDC gene in the pd-affected retina. The characterized region of normal PDC cDNA spans 1258 nucleotides (nt) that include 738 nt of coding sequence predicted to encode a protein (Mr=28 209) of 245 amino acids (aa). Over the coding region, PDC shares 86-95% nt sequence identity and 90-95% identity in the deduced aa sequence with homologous mammalian sequences. A major transcript (1.9 kb) was observed only in retina by Northern analysis, but low levels of transcript were detected in brain, liver and kidney by reverse transcription and polymerase chain reaction. Retinal immunocytochemistry showed that PDC was detected only in rod photoreceptors, mainly in the inner segment and perinuclear region. By Northern blot analysis, increased PDC expression was observed in pre-degenerate affected retina relative to the age-matched normal. In pd- affected miniature schnauzer pedigree, a missense mutation was detected in codon 82 (CGA to GGA) that would create a non-conservative substitution (Arg to Gly) in close vicinity to the residue (Glu 85) which directly interacts with the betagamma-subunits of transducin. Only pd-affected dogs were found to be homozygous for the mutant allele, and none among 48 dogs tested from 20 other dog breeds had this allele, suggesting that the mutation could be causally associated with pd in miniature schnauzers. However, since some affected dogs are heterozygous for the mutant allele, and some are homozygous for the wild-type allele, this putative PDC missense mutation, if it is indeed a disease causing mutation, does not account entirely for the genetics of inherited retinal degeneration in the miniature schnauzer breed.

In vivo erythrocyte sickling in the Japanese sika deer (Cervus nippon): methodology.

The Japanese sika deep (Cervus nippon) proved to be a suitable animal model for the study of acute phases of in vivo erythrocyte sickling. Ophthalmologic studies can be conducted during or after 1 to 6 hours of effective in vivo sickling. Intravenous administration of 1.75 to 3.5% sodium bicarbonate solution at a rate of 500 to 1,000 ml per hour produced a transient state of alkalosis and in vivo erythrocyte sickling in the sika deer. The percentage of sickled erythrocytes increased as the blood pH increased. Concurrently, the packed cell volume decreased. Sickling was enhanced by 100% oxygen ventilation after endotracheal intubation and light anesthetization. After the induction of erythrocyte sickling, a sickling-reversal phenomenon occurred despite continued bicarbonate administration. During the course of this reversal, the percentage of sickled erythrocytes steadily decreased, the venous blood pH decreased, and the packed cell volume slowly increased. Because of the sickling-reversal phenomenon, chronic erythrocyte sickling was not achieved.

Ophthalmic manifestations of induced sickling of erythrocytes in Japanese sika deer (Cervus nippon).

Nine female Japanese sika deer (Cervus nippon) were used in a total of 25 experiments in which sickling was chemically induced. During these experiments, color fundic and color fluorescein photographs were taken. Fundic changes included retinal vascular attenuation, blood column pallor, and decreased tapetal reflectivity. These changes were most likely directly associated with a decreased hematocrit and a generalized shocklike condition. Three deer had a congested appearance in retinal blood vessels and tapetum lucidum. Two of the 3 deer developed serous detachment of the retina. These changes seemingly were associated with severe venous statis; all 3 deer died shortly after the experiment was terminated. These experiments yielded data only for the acutely affected deer. None of the ocular changes could be considered the result of chronic sickling because of the reversal of sickling that occurred despite continued intravenous administration of bicarbonate. None of the deer developed ocular changes characteristic of sickle cell retinopathy in human beings. The changes in human beings probably result from continued stress and prolongation of sickling, and especially from a multiplicity of repeated severe episodes of sickling occurring over many years.