Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs.

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.

Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs.

Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog.

Differential targeting of feline photoreceptors by recombinant adeno-associated viral vectors: implications for preclinical gene therapy trials.

The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.

Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach.

Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset, and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface-exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected; four dogs received intravitreal injections and eight dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal) and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach.

RPE65 gene therapy slows cone loss in Rpe65-deficient dogs.

Recent clinical trials of retinal pigment epithelium gene (RPE65) supplementation therapy in Leber congenital amaurosis type 2 patients have demonstrated improvements in rod and cone function, but it may be some years before the effects of therapy on photoreceptor survival become apparent. The Rpe65-deficient dog is a very useful pre-clinical model in which to test efficacy of therapies, because the dog has a retina with a high degree of similarity to that of humans. In this study, we evaluated the effect of RPE65 gene therapy on photoreceptor survival in order to predict the potential benefit and limitations of therapy in patients. We examined the retinas of Rpe65-deficient dogs after RPE65 gene therapy to evaluate the preservation of rods and cone photoreceptor subtypes. We found that gene therapy preserves both rods and cones. While the moderate loss of rods in the Rpe65-deficient dog retina is slowed by gene therapy, S-cones are lost extensively and gene therapy can prevent that loss, although only within the treated area. Although LM-cones are not lost extensively, cone opsin mislocalization indicates that they are stressed, and this can be partially reversed by gene therapy. Our results suggest that gene therapy may be able to slow cone degeneration in patients if intervention is sufficiently early and also that it is probably important to treat the macula in order to preserve central function.

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function.

The purpose of this study was to evaluate whether immune responses interfered with gene therapy rescue using subretinally delivered recombinant adeno-associated viral vector serotype 2 carrying the RPE65 cDNA gene driven by the human RPE65 promoter (rAAV2.hRPE65p.hRPE65) in the second eye of RPE65-/- dogs that had previously been treated in a similar manner in the other eye. Bilateral subretinal injection was performed in nine dogs with the second eye treated 85-180 days after the first. Electroretinography (ERG) and vision testing showed rescue in 16 of 18 treated eyes, with no significant difference between first and second treated eyes. A serum neutralizing antibody (NAb) response to rAAV2 was detected in all treated animals, but this did not prevent or reduce the effectiveness of rescue in the second treated eye. We conclude that successful rescue using subretinal rAAV2.hRPE65p.hRPE65 gene therapy in the second eye is not precluded by prior gene therapy in the contralateral eye of the RPE65-/- dog. This finding has important implications for the treatment of human LCA type II patients.

The pattern of expression of guanine nucleotide-binding protein beta3 in the retina is conserved across vertebrate species.

Guanine nucleotide-binding protein beta3 (GNB3) is an isoform of the beta subunit of the heterotrimeric G protein second messenger complex that is commonly associated with transmembrane receptors. The presence of GNB3 in photoreceptors, and possibly bipolar cells, has been confirmed in murine, bovine and primate retinas [Lee RH, Lieberman BS, Yamane HK, Bok D, Fung BK (1992) J Biol Chem 267:24776-24781; Peng YW, Robishaw JD, Levine MA, Yau KW (1992) Proc Natl Acad Sci U S A 89:10882-10886; Huang L, Max M, Margolskee RF, Su H, Masland RH, Euler T (2003) J Comp Neurol 455:1-10]. Studies have indicated that a mutation in the GNB3 gene causes progressive retinopathy and globe enlargement (RGE) in chickens. The goals of this study were to (1) examine the expression pattern of GNB3 in wild-type and RGE mutant chickens, (2) characterize the types of bipolar cells that express GNB3 and (3) examine whether the expression of GNB3 in the retina is conserved across vertebrate species. We find that chickens homozygous for the RGE allele completely lack GNB3 protein. We find that the pattern of expression of GNB3 in the retina is highly conserved across vertebrate species, including teleost fish (Carassius auratus), frogs (Xenopus laevis), chickens (Gallus domesticus), mice (Mus musculata), guinea-pigs (Cavia porcellus), dogs (Canis familiaris) and non-human primates (Macaca fasicularis). Regardless of the species, we find that GNB3 is expressed by Islet1-positive cone ON-bipolar cells and by cone photoreceptors. In some vertebrates, GNB3-immunoreactivity was observed in both rod and cone photoreceptors. A protein-protein alignment of GNB3 across different vertebrates, from fish to humans, indicates a high degree (>92%) of sequence conservation. Given that analogous types of retinal neurons express GNB3 in different species, we propose that the functions and the mechanisms that regulate the expression of GNB3 are highly conserved.

AAV retinal transduction in a large animal model species: comparison of a self-complementary AAV2/5 with a single-stranded AAV2/5 vector.

PURPOSE: To compare self-complementary (sc) and single-stranded (ss) adeno-associated viral 2/5 (AAV2/5) vectors for retinal cell transduction in the dog when delivered by subretinal injection. METHODS: ScAAV2/5 and ssAAV2/5 vectors encoding enhanced green fluorescent protein (GFP) under control of the chicken beta actin promoter were prepared to the same titer. Equal amounts of viral particles were delivered into the subretinal spaces of both eyes of two dogs. In each dog, one eye received the scAAV2/5 and the other the ssAAV2/5. In vivo expression of GFP was monitored ophthalmoscopically. The dogs were sacrificed, and their retinas were examined by fluorescent microscopy and immunohistochemistry to determine GFP expression patterns and to assay for glial reactivity. RESULTS: GFP expression in the scAAV2/5 injected eyes was detectable at a much earlier time point than in the ssAAV2/5 injected eyes. Expression of GFP was also at higher levels in the scAAV2/5-injected eyes. Expression levels remained stable for the seven month duration of the study. The types of cells transduced by both vectors were similar; there was strong reporter gene expression in the RPE and photoreceptors, although not all cones in the transduced area expressed GFP. Some horizontal and Müller cells were also transduced. CONCLUSIONS: When delivered by subretinal injection in the dog, scAAV2/5 induces faster and stronger transgene expression than ssAAV2/5. The spectrum of retinal neurons transduced is similar between the two vectors. These results confirm in a large animal model those previously reported in the mouse. ScAAV2/5 shows promise for use in the treatment of conditions where a rapid transgene expression is desirable. Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.

Antibiotic susceptibility of bacteria isolated from cats with ulcerative keratitis in Taiwan.

OBJECTIVES: To assess the antibiotic susceptibility of bacteria isolated from corneal ulcers in cats. METHODS: A total of 92 cats with infected corneal ulcers were swabbed for bacterial culture and the antibiotic sensitivity of the isolates analysed. RESULTS: Bacteria were isolated from 54 of 92 infected eyes with corneal ulcers and purulent discharge. A total of 59 bacterial isolates were obtained from the 54 ulcers. The ratio of Gram-positive to Gram-negative isolates was approximately 3:1. The most commonly isolated Gram-positive bacteria were Staphylococcus species (51 per cent of all isolates), while Pseudomonas aeruginosa (13.5 per cent of all isolates) was the most common Gram-negative bacteria isolated. The Gram-negative isolates demonstrated a greater incidence of antibiotic resistance than the Gram-positive ones. The most effective antibiotics against the isolates were ciprofloxacin, tobramycin and gentamicin, with erythromycin and lincomycin showing the greatest number of resistant isolates. CLINICAL SIGNIFICANCE: Staphylococcus and Pseudomonas species were the most common Gram-positive and Gram-negative bacteria, respectively, isolated from feline eyes with ulcerative keratitis. The second-generation fluoroquinolone, ciprofloxacin and the aminoglycoside gentamicin were found to be highly effective against the majority of isolates.

Antibiotic susceptibility of bacterial isolates from corneal ulcers of dogs in Taiwan.

OBJECTIVES: To analyse antibiotic susceptibility of bacteria associated with ulcerative keratitis in dogs. METHODS: Bacteria isolated from 190 eyes with ulcerative keratitis were identified, and the antibiotic susceptibility of isolates was studied. RESULTS: In total, 258 species of bacteria were isolated from the 190 eyes. Of the isolates, 78 per cent were Gram-positive and 28 per cent were Gram-negative bacteria. The most commonly isolated Gram-positive bacteria in dogs were Staphylococcus spp (49 per cent), Streptococcus spp (7 per cent) and Corynebacterium spp (7 per cent); while Pseudomonas aeruginosa (7.6 per cent) and Escherichia coli (5.8 per cent) were the commonest Gram-negative pathogens. Resistance to commonly used ophthalmic antibiotics was seen in Staphylococcus, Streptococcus, Corynebacterium, Pseudomonas and Escherichia species isolates. CLINICAL SIGNIFICANCE: Isolates from dogs with corneal ulcers in Taiwan may be resistant to several commonly used ophthalmic preparations. Ciprofloxacin showed good action against most isolates, with the notable exception of Streptococcus species. Chloramphenicol or cephalothin had the best in vitro action against the Streptococcus species isolates.

Advances in the molecular understanding of canine retinal diseases.

Retinal dystrophies are a common cause of blindness in purebred dogs. Progressive retinal atrophy, the canine equivalent of retinitis pigmentosa in humans, is the most common dystrophy. Molecular studies have led to the identification of the genetic defect underlying some forms of progressive retinal atrophy and the mapping of the chromosomal location of others. Additionally, the gene mutation that causes a severe retinal dystrophy in the briard, which is the equivalent of Leber congenital amaurosis in humans, has been identified. These advances have led to the development of DNA-based diagnostic tests for some retinal dystrophies, thus facilitating their eradication. The study of these dystrophies in dogs has also provided useful information about the equivalent diseases in humans. Recently, gene therapy has been used to restore vision to dogs with a retinal dystrophy due to a mutation in the RPE65 gene. Such studies are important in the quest to develop therapies for similar conditions in humans.

Spontaneous lacquer crack lesions in the retinopathy, globe enlarged (rge) chick.

Lacquer crack lesion (LCL), a complication of myopia in human patients, is characterized by loss of retinal pigment epithelium and breaks in Bruch's membrane. This report describes comparable lesions in the "retinopathy, globe enlarged" (rge) chick. Twenty-six birds, (nine rge/rge [affected], 12 rge/+ [carriers] and five +/+ [normal]), were examined ophthalmoscopically from hatching up to 336 days of age. Ophthalmoscopically detected fundus lesions were investigated by light and transmission electron microscopy. Pale, linear fundus lesions were detected in both eyes of seven of the nine rge/rge chicks, from as early as 45 days of age. Histological and ultrastructural examination of four of the affected rge/rge chicks revealed areas of ruptured Bruch's membrane, with focal absence of retinal pigment epithelium. Fibroblasts covered the interface of the abnormal Bruch's membrane and choriocapillaris. There was disorganization of overlying photoreceptor outer and inner segments, and thinning of inner and outer nuclear layers, while the rest of the inner retina appeared unaltered. The lesions present in rge chicks showed histological changes similar to those of LCLs described in human patients with pathological myopia. The formation of LCLs in rge chicks is probably due to stretching of Bruch's membrane secondary to abnormal globe enlargement, resulting in linear rupture of the membrane and associated changes. The rge chick may prove a useful model for human LCL formation.

Basic concepts in statistics for veterinary ophthalmologists.

Abstract This review article provides a comprehensive coverage of basic statistical procedures commonly used in biologic experiments and is intended to be a basic guide to the selection of appropriate tests and also to aid the reader in the evaluation of published studies. The focus is on the use, interpretation and presentation of the main statistical tests and their key concepts for the practicing veterinary ophthalmologist. Several examples derived from actual ophthalmic research are presented in the text. Lastly, the article discusses computer-based statistical analysis, provides a list of different software available and gives a practical example of a 'real life' statistical analysis.

Canine inherited retinal degenerations: update on molecular genetic research and its clinical application.

Inherited retinal degenerations in the dog include generalised progressive retinal atrophy, retinal pigment epithelial dystrophy, congenital stationary night blindness and day blindness (hemeralopia). The clinical phenotype and pathology of these diseases closely resemble some types of human inherited retinal degeneration, in particular retinitis pigmentosa, one of the most common inherited causes of blindness in man. Molecular genetic investigations aim to identify the genetic mutations underlying the canine inherited retinal degenerations. Two major research strategies, candidate gene analysis and linkage analysis, have been used. To date, candidate gene analysis has definitively identified the genetic mutations underlying nine inherited retinal degenerations, each in a different breed of dog, and linkage studies have identified genetic markers for a further retinal degeneration which is found in at least six different breeds. This review outlines the research strategy behind candidate gene and linkage studies and summarises recent results in the search for genetic causes of canine inherited retinal degenerations. The aim is to increase awareness of this rapidly changing field and to show how the research can be used to develop genetic tests for these diseases and thereby reduce the incidence of inherited eye disease in dogs.

Development and use of a polymerase chain reaction-based diagnostic test for the causal mutation of progressive retinal atrophy in Cardigan Welsh Corgis.

OBJECTIVE: To develop an allele-specific polymerase chain reaction (ASPCR)-based diagnostic test for the mutation in the cyclic guanosine monophosphate phosphodiesterase alpha subunit gene (PDE6A) that causes the rcd3 form of progressive retinal atrophy (PRA) in Cardigan Welsh Corgis. ANIMALS: 1 affected homozygote, 1 unaffected carrier, 1 genotypically normal dog, and 500 unknown-PRA status Cardigan Welsh Corgis. PROCEDURE: Control blood samples were collected from Cardigan Welsh Corgis of known PRA status (ie, affected homozygote, unaffected carrier, and a genotypically normal dog) for test development. Test blood samples were collected from 500 Cardigan Welsh Corgis of unknown PRA status. Genomic DNA was used as a template in ASPCR. One pair of primers was designed to specifically amplify only the mutant allele, and another set to amplify only the wildtype allele. The PCR conditions were adjusted to ensure each reaction was 100% specific. RESULTS: The PCR conditions were identified so that each ASPCR only amplified the allele it was designed to amplify. Of the 500 Cardigan Welsh Corgis tested using the newly developed ASPCR, 457 were homozygous for the normal allele (genotypically normal), 43 were heterozygous (phenotypically normal carriers), and none were homozygous for the mutant allele. CONCLUSION AND CLINICAL RELEVANCE: A rapid, ASPCR diagnostic test able to detect the PDE6A gene mutation responsible for the rcd3 form of PRA in Cardigan Welsh Corgis was developed. The test provides a useful service for Cardigan Welsh Corgi breeders and will enable them to prevent the birth of homozygote mutant dogs.

cGMP phosphodiesterase-alpha mutation causes progressive retinal atrophy in the Cardigan Welsh corgi dog.

PURPOSE: To screen the alpha-subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6A) as a potential candidate gene for progressive retinal atrophy (PRA) in the Cardigan Welsh corgi dog. METHODS: Single-strand conformation polymorphism (SSCP) analysis was used to screen short introns of the canine PDE6A gene for informative polymorphisms in members of an extended pedigree of PRA-affected Cardigan Welsh corgis. After initial demonstration of linkage of a polymorphism in the PDE6A gene with the disease locus, the complete coding region of the PDE6A gene of a PRA-affected Cardigan Welsh corgi was cloned in overlapping fragments and sequenced. SSCP-based and direct DNA sequencing tests were developed to detect the presence of a PDE6A gene mutation that segregated with disease status in the extended pedigree of PRA-affected Cardigan Welsh corgis. Genomic DNA sequencing was developed as a diagnostic test to establish the genotype of Cardigan Welsh corgis in the pet population. RESULTS: A polymorphism within intron 18 of the canine PDE6A gene was invariably present in the homozygous state in PRA-affected Cardigan Welsh corgis. The entire PDE6A gene was cloned from one PRA-affected dog and the gene structure and intron sizes established and compared with those of an unaffected animal. Intron sizes were identical in affected and normal dogs. Sequencing of exons and splice junctions in the affected animal revealed a 1-bp deletion in codon 616. Analysis of PRA-affected anti obligate carrier Cardigan Welsh corgis showed that this mutation cosegregated with disease status. CONCLUSIONS: A single base deletion at codon 616 in the PDE6A gene cosegregated with PRA status with zero discordance in Cardigan Welsh corgis with PRA. A lod score of 4.816 with a recombination fraction (theta) of zero strongly suggests that this mutation is responsible for PRA in the breed. The mutation is predicted to lead to a frame shift resulting in a string of 28 altered codons followed by a premature stop codon. The authors suggest that this type of PRA be given the name rod-cone dysplasia 3 (rcd3).

Corneal thickness, intraocular pressure, and optical corneal diameter in Rocky Mountain Horses with cornea globosa or clinically normal corneas.

OBJECTIVE: To compare corneal thickness, intraocular pressure, and optical corneal diameter in Rocky Mountain Horses with cornea globosa and those with clinically normal corneas. ANIMALS: 129 Rocky Mountain Horses. PROCEDURE: Ultrasonic pachymetry was used to measure corneal thickness. Applanation tonometry was used to measure intraocular pressure. A Jameson caliper was used to measure optical corneal diameter. RESULTS: The central and temporal peripheral portions of the cornea were significantly thicker in horses with cornea globosa than in horses with clinically normal corneas, but corneal thicknesses in the dorsal, ventral, and medial peripheral portions of the cornea were not significantly different between groups. There were no differences in corneal thickness between male and female horses or between right and left eyes. However, there was a positive correlation between age and corneal thickness. Intraocular pressure was not significantly different between horses with cornea globosa and those with clinically normal corneas, or between right and left eyes, or male and female horses. Optical corneal diameter for horses with cornea globosa was not significantly different from diameter for horses with clinically normal corneas, but optical corneal diameter was positively correlated with age. CONCLUSIONS AND CLINICAL RELEVANCE: Cornea globosa in Rocky Mountain Horses is not associated with increased intraocular pressure. Corneal thickness and optical corneal diameter increase with age in Rocky Mountain Horses.

Isolation and investigation of canine phosducin as a candidate for canine generalized progressive retinal atrophies.

A subtractive cDNA cloning strategy was used to isolate canine retina-specific genes. Canine phosducin cDNA was cloned from a canine subtracted retinal cDNA library and was analysed as a candidate for canine generalized progressive retinal atrophies (gPRA). Canine phosducin cDNA is 1230 bp in length encoding 245 amino acids. The nucleotide and amino acid sequences of canine phosducin are highly conserved when compared with those of five other mammalian species, namely human, cat, cow, rat, and mouse. Northern blot analysis demonstrated that the mRNA transcript for phosducin was approximately 1.3 kb in size and was present in canine retina, but showed no visible signals in 13 other canine tissues. The phosducin gene was examined for polymorphisms in a total of 101 pedigree dogs of eight breeds, including normal, obligate gPRA carriers, and gPRA-affected dogs, by single-stranded conformation polymorphisms (SSCP) analysis. Polymorphisms in the phosducin gene were detected only in the 3' untranslated region of the gene in two breeds of dogs: allelic heterozygous polymorphisms in miniature poodles suffering from one form of gPRA (progressive rod-cone degeneration, prcd), and a different polymorphism in a single normal Irish wolfhound. The polymorphisms of phosducin in prcd-affected miniature poodles did not segregate with the autosomal recessive form of gPRA. Heterozygous inheritance of the polymorphisms suggests that phosducin is very unlikely to carry the mutation causing prcd, so phosducin was probably excluded as a candidate for prcd-affected miniature poodles in this study.