Genome-wide methylation patterns from canine nanopore assemblies.

Recent advances in long-read sequencing have enabled the creation of reference-quality genome assemblies for multiple individuals within a species. In particular, 8 long-read genome assemblies have recently been published for the canine model (dogs and wolves). These assemblies were created using a range of sequencing and computational approaches, with only limited comparisons described among subsets of the assemblies. Here we present 3 high-quality de novo reference assemblies based upon Oxford Nanopore long-read sequencing: 2 Bernese Mountain Dogs (BD & OD) and a Cairn terrier (CA611). These breeds are of particular interest due to the enrichment of unresolved genetic disorders. Leveraging advancement in software technologies, we utilized published data of Labrador Retriever (Yella) to generate a new assembly, resulting in a ∼280-fold increase in continuity (N50 size of 91 kbp vs 25.75 Mbp). In conjunction with these 4 new assemblies, we uniformly assessed 8 existing assemblies for generalized quality metrics, sequence divergence, and a detailed BUSCO assessment. We identified a set of ∼400 conserved genes during the BUSCO analysis missing in all assemblies. Genome-wide methylation profiles were generated from the nanopore sequencing, resulting in broad concordance with existing whole-genome and reduced-representation bisulfite sequencing, while highlighting superior overage of mobile elements. These analyses demonstrate the ability of Nanopore sequencing to resolve the sequence and epigenetic profile of canine genomes.

Development of a translatable gene augmentation therapy for CNGB1-retinitis pigmentosa.

In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development.

Preliminary characterization of a novel form of progressive retinal atrophy in the German Spitz dog associated with a frameshift mutation in GUCY2D.

OBJECTIVE: To describe the clinical, preliminary electroretinographic and optical coherence tomography features of a newly identified form of progressive retinal atrophy (PRA) in German Spitzes, and identify the causal gene mutation. ANIMALS: Thirty-three client-owned German Spitz dogs were included. PROCEDURES: All animals underwent a full ophthalmic examination, including vision testing. In addition, fundus photography, ERG, and OCT were performed. A DNA-marker-based association analysis was performed to screen potential candidate genes and the whole genomes of four animals were sequenced. RESULTS: Initial fundus changes were pale papilla and mild vascular attenuation. Oscillatory nystagmus was noted in 14 of 16 clinically affected puppies. Vision was impaired under both scotopic and photopic conditions. Rod-mediated ERGs were unrecordable in all affected dogs tested, reduced cone-mediated responses were present in one animal at 3 months of age and unrecordable in the other affected animals tested. Multiple small retinal bullae were observed in three clinically affected animals (two with confirmed genetic diagnosis). OCT showed that despite loss of function, retinal structure was initially well-preserved, although a slight retinal thinning developed in older animals with the ventral retina being more severely affected. Pedigree analysis supported an autosomal recessive inheritance. A mutation was identified in GUCY2D, which segregated with the disease (NM_001003207.1:c.1598_1599insT; p.(Ser534GlufsTer20)). Human subjects with GUCY2D mutations typically show an initial disconnect between loss of function and loss of structure, a feature recapitulated in the affected dogs in this study. CONCLUSION: We identified early-onset PRA in the German Spitz associated with a frameshift mutation in GUCY2D.

Elevated retinal cGMP is not associated with elevated circulating cGMP levels in a canine model of retinitis pigmentosa.

PURPOSE: To investigate whether raised levels of retinal cyclic guanosine monophosphate (cGMP) was reflected in plasma levels in PDE6A-/- dogs. MATERIALS AND METHODS: Retina was collected from 2-month-old wildtype dogs (PDE6A+/+, N = 6), heterozygous dogs (PDE6A+/-, N = 4) and affected dogs (PDE6A-/-, N = 3) and plasma was collected from 2-month-old wildtype dogs (PDE6A+/+, N = 5), heterozygous dogs (PDE6A+/-, N = 5) and affected dogs (PDE6A-/-, N = 5). Retina and plasma samples were measured by ELISA. RESULTS: cGMP levels in retinal samples of PDE6A-/- dogs at 2 months of age were significantly elevated. There was no significant difference in plasma cGMP levels between wildtype and PDE6A-/- or PDE6A+/- puppies. However, the plasma cGMP levels of the PDE6A-/- puppies were significantly lower than that of PDE6A+/- puppies. CONCLUSION: cGMP levels in the plasma from PDE6A-/- was not elevated when compared to control dogs. At the 2-month timepoint, cGMP plasma levels would not be a useful biomarker for disease.

Residual rod function in CNGB1 mutant dogs.

PURPOSE: Mutations in the cyclic nucleotide-gated (CNG) channel beta subunit (CNGB1) are an important cause of recessive retinitis pigmentosa. We identified a large animal model with a truncating mutation of CNGB1. This study reports the persistence of small, desensitized rod ERG responses in this model. METHODS: Dark-, light-adapted and chromatic ERGs were recorded in CNGB1 mutant dogs and age and breed matched controls. Comparisons were made with a dog model known to completely lack rod function; young dogs with a mutation in the rod phosphodiesterase 6 alpha subunit (PDE6A-/-). Immunohistochemistry (IHC) to label the rod CNG alpha (CNGA1) and CNGB1 subunits was performed. RESULTS: The dark-adapted ERG of CNGB1 mutant dogs had a raised response threshold with lack of normal rod response and a remaining cone response. Increasing stimulus strength resulted in the appearance of a separate, slower positive waveform following the dark-adapted cone b-wave. With increasing stimulus strength this increased in amplitude and became faster to merge with the initial b-wave. Comparison of responses from PDE6A-/- (cone only dogs) with CNGB1 mutant dogs to red and blue flashes and between dark-adapted and light-adapted responses supported the hypothesis that the CNGB1 mutant dog had residual desensitized rod responses. CNGB1 mutant dogs had a small amount of CNGA1 detectable in the outer segments. CONCLUSIONS: CNGB1 mutant dogs have a residual ERG response from desensitized rods. This may be due to low levels of CNGA1 in outer segments.

An unusual inherited electroretinogram feature with an exaggerated negative component in dogs.

OBJECTIVES: To assess an inherited abnormal negative response electroretinogram (NRE) that originated in a family of Papillon dogs. ANIMALS STUDIED: Thirty-eight dogs (Papillons, or Papillon cross Beagles or Beagles). PROCEDURES: Dogs underwent routine ophthalmic examination and a detailed dark-adapted, light-adapted and On-Off electroretinographic study. Vision was assessed using a four-choice exit device. Spectral-domain optical coherence tomography (SD-OCT) was performed on a subset of dogs. Two affected males were outcrossed to investigate the mode of inheritance of the phenotype. RESULTS: The affected dogs had an increased underlying negative component to the ERG. This was most pronounced in the light-adapted ERG, resulting in a reduced b-wave and an exaggerated photopic negative response (PhNR). Changes were more pronounced with stronger flashes. Similarly, the On-response of the On-Off ERG had a reduced b-wave and a large post-b-wave negative component. The dark-adapted ERG had a significant increase in the scotopic threshold response (STR) and a significant reduction in the b:a-wave ratio. Significant changes could be detected at 2 months of age but became more pronounced with age. Vision testing using a four-choice device showed affected dogs had reduced visual performance under the brightest light condition. There was no evidence of a degenerative process in the affected dogs up to 8.5 years of age. Test breeding results suggested the NRE phenotype had an autosomal dominant mode of inheritance. CONCLUSIONS: We describe an inherited ERG phenotype in Papillon dogs characterized by an underlying negative component affecting both dark- and light-adapted ERG responses.

A large animal model of RDH5-associated retinopathy recapitulates important features of the human phenotype.

Pathogenic variants in retinol dehydrogenase 5 (RDH5) attenuate supply of 11-cis-retinal to photoreceptors leading to a range of clinical phenotypes including night blindness because of markedly slowed rod dark adaptation and in some patients, macular atrophy. Current animal models (such as Rdh5-/- mice) fail to recapitulate the functional or degenerative phenotype. Addressing this need for a relevant animal model we present a new domestic cat model with a loss-of-function missense mutation in RDH5 (c.542G > T; p.Gly181Val). As with patients, affected cats have a marked delay in recovery of dark adaptation. In addition, the cats develop a degeneration of the area centralis (equivalent to the human macula). This recapitulates the development of macular atrophy that is reported in a subset of patients with RDH5 mutations and is shown in this paper in seven patients with biallelic RDH5 mutations. There is notable variability in the age at onset of the area centralis changes in the cat, with most developing changes as juveniles but some not showing changes over the first few years of age. There is similar variability in development of macular atrophy in patients and while age is a risk factor, it is hypothesized that genetic modifying loci influence disease severity, and we suspect the same is true in the cat model. This novel cat model provides opportunities to improve molecular understanding of macular atrophy and test therapeutic interventions for RDH5-associated retinopathies.

Development of retinal bullae in dogs with progressive retinal atrophy.

OBJECTIVE: To report the development of focal bullous retinal detachments (bullae) in dogs with different forms of progressive retinal atrophy (PRA). PROCEDURES: Dogs with three distinct forms of PRA (PRA-affected Whippets, German Spitzes and CNGB1-mutant Papillon crosses) were examined by indirect ophthalmoscopy and spectral domain optical coherence tomography (SD-OCT). Retinal bullae were monitored over time. One CNGB1-mutant dog was treated with gene augmentation therapy. The canine BEST1 gene coding region and flanking intronic sequence was sequenced in at least one affected dog of each breed. RESULTS: Multiple focal bullous retinal detachments (bullae) were identified in PRA-affected dogs of all three types. They developed in 4 of 5 PRA-affected Whippets, 3 of 8 PRA-affected Germans Spitzes and 15 of 20 CNGB1-mutant dogs. The bullae appeared prior to marked retinal degeneration and became less apparent as retinal degeneration progressed. Bullae were not seen in any heterozygous animals of any of the types of PRA. Screening of the coding region and flanking intronic regions of the canine BEST1 gene failed to reveal any associated pathogenic variants. Retinal gene augmentation therapy in one of the CNGB1-mutant dogs appeared to prevent formation of bullae. CONCLUSIONS: Retinal bullae were identified in dogs with three distinct forms of progressive retinal atrophy. The lesions develop prior to retinal thinning. This clinical change should be monitored for in dogs with PRA.

Comparison of Developmental Dynamics in Human Fetal Retina and Human Pluripotent Stem Cell-Derived Retinal Tissue.

Progressive vision loss, caused by retinal degenerative (RD) diseases such as age-related macular degeneration, retinitis pigmentosa, and Leber congenital amaurosis, severely impacts quality of life and affects millions of people. Finding efficient treatment for blinding diseases is among the greatest unmet clinical needs. The evagination of optic vesicles from developing pluripotent stem cell-derived neuroepithelium and self-organization, lamination, and differentiation of retinal tissue in a dish generated considerable optimism for developing innovative approaches for treating RD diseases, which previously were not feasible. Retinal organoids may be a limitless source of multipotential retinal progenitors, photoreceptors (PRs), and the whole retinal tissue, which are productive approaches for developing RD disease therapies. In this study we compared the distribution and expression level of molecular markers (genetic and epigenetic) in human fetal retina (age 8-16 weeks) and human embryonic stem cell (hESC)-derived retinal tissue (organoids) by immunohistochemistry, RNA-seq, flow cytometry, and mass-spectrometry (to measure methylated and hydroxymethylated cytosine level), with a focus on PRs to evaluate the clinical application of hESC-retinal tissue for vision restoration. Our results revealed high correlation in gene expression profiles and histological profiles between human fetal retina (age 8-13 weeks) and hESC-derived retinal tissue (10-12 weeks). The transcriptome signature of hESC-derived retinal tissue from retinal organoids maintained for 24 weeks in culture resembled the transcriptome of human fetal retina of more advanced developmental stages. The histological profiles of 24 week-old hESC-derived retinal tissue displayed mature PR immunophenotypes and presence of developing inner and outer segments. Collectively, our work highlights the similarity of hESC-derived retinal tissue at early stages of development (10 weeks), and human fetal retina (age 8-13 weeks) and it supports the development of regenerative medicine therapies aimed at using tissue from hESC-derived retinal organoids (hESC-retinal implants) for mitigating vision loss.

A CNTNAP1 Missense Variant Is Associated with Canine Laryngeal Paralysis and Polyneuropathy.

Laryngeal paralysis associated with a generalized polyneuropathy (LPPN) most commonly exists in geriatric dogs from a variety of large and giant breeds. The purpose of this study was to discover the underlying genetic and molecular mechanisms in a younger-onset form of this neurodegenerative disease seen in two closely related giant dog breeds, the Leonberger and Saint Bernard. Neuropathology of an affected dog from each breed showed variable nerve fiber loss and scattered inappropriately thin myelinated fibers. Using across-breed genome-wide association, haplotype analysis, and whole-genome sequencing, we identified a missense variant in the CNTNAP1 gene (c.2810G>A; p.Gly937Glu) in which homozygotes in both studied breeds are affected. CNTNAP1 encodes a contactin-associated protein important for organization of myelinated axons. The herein described likely pathogenic CNTNAP1 variant occurs in unrelated breeds at variable frequencies. Individual homozygous mutant LPPN-affected Labrador retrievers that were on average four years younger than dogs affected by geriatric onset laryngeal paralysis polyneuropathy could be explained by this variant. Pathologic changes in a Labrador retriever nerve biopsy from a homozygous mutant dog were similar to those of the Leonberger and Saint Bernard. The impact of this variant on health in English bulldogs and Irish terriers, two breeds with higher CNTNAP1 variant allele frequencies, remains unclear. Pathogenic variants in CNTNAP1 have previously been reported in human patients with lethal congenital contracture syndrome and hypomyelinating neuropathy, including vocal cord palsy and severe respiratory distress. This is the first report of contactin-associated LPPN in dogs characterized by a deleterious variant that most likely predates modern breed establishment.

A novel mutation in PDE6B in Spanish Water Dogs with early-onset progressive retinal atrophy.

OBJECTIVE: To identify the underlying mutation in a recently identified early-onset progressive retinal atrophy (PRA) in the Spanish Water Dog (SWD) breed. ANIMAL STUDIED: Eighteen SWDs were used in this study. Six SWDs diagnosed with PRA and 12 phenotypically normal SWDs. PROCEDURES: An exclusion analysis using an established microsatellite panel to screen PRA candidate genes was combined with whole genome sequencing of two affected SWD siblings and two phenotypically normal SWDs (a sibling and the dam). RESULTS: A 6-bp deletion was identified in exon 19 of PDE6B removing two highly conserved amino acids from the enzymatic domain of the PDE6B protein (c.2218-2223del; p.Phe740_Phe741del). This segregated with the disease status in the small study pedigree. CONCLUSIONS: Identification of this novel PDE6B mutation adds to the already described PDE6B mutations responsible for PRA in the Irish Setter, Sloughi, and American Staffordshire Terrier dog breeds. A DNA-based test was designed to allow breeders to genotype their animals and make informed breeding decisions in the effort to eradicate PRA from the SWD breed.

Large Animal Models of Inherited Retinal Degenerations: A Review.

Studies utilizing large animal models of inherited retinal degeneration (IRD) have proven important in not only the development of translational therapeutic approaches, but also in improving our understanding of disease mechanisms. The dog is the predominant species utilized because spontaneous IRD is common in the canine pet population. Cats are also a source of spontaneous IRDs. Other large animal models with spontaneous IRDs include sheep, horses and non-human primates (NHP). The pig has also proven valuable due to the ease in which transgenic animals can be generated and work is ongoing to produce engineered models of other large animal species including NHP. These large animal models offer important advantages over the widely used laboratory rodent models. The globe size and dimensions more closely parallel those of humans and, most importantly, they have a retinal region of high cone density and denser photoreceptor packing for high acuity vision. Laboratory rodents lack such a retinal region and, as macular disease is a critical cause for vision loss in humans, having a comparable retinal region in model species is particularly important. This review will discuss several large animal models which have been used to study disease mechanisms relevant for the equivalent human IRD.

Architecture of the TRPM2 channel and its activation mechanism by ADP-ribose and calcium.

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that has an essential role in diverse physiological processes such as core body temperature regulation, immune response and apoptosis1-4. TRPM2 is polymodal and can be activated by a wide range of stimuli1-7, including temperature, oxidative stress and NAD+-related metabolites such as ADP-ribose (ADPR). Its activation results in both Ca2+ entry across the plasma membrane and Ca2+ release from lysosomes8, and has been linked to diseases such as ischaemia-reperfusion injury, bipolar disorder and Alzheimer's disease9-11. Here we report the cryo-electron microscopy structures of the zebrafish TRPM2 in the apo resting (closed) state and in the ADPR/Ca2+-bound active (open) state, in which the characteristic NUDT9-H domains hang underneath the MHR1/2 domain. We identify an ADPR-binding site located in the bi-lobed structure of the MHR1/2 domain. Our results provide an insight into the mechanism of activation of the TRPM channel family and define a framework for the development of therapeutic agents to treat neurodegenerative diseases and temperature-related pathological conditions.

Patients and animal models of CNGß1-deficient retinitis pigmentosa support gene augmentation approach.

Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel ß 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGß1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials.

Electron cryo-microscopy structure of a human TRPM4 channel.

Ca2+-activated, non-selective (CAN) ion channels sense increases of the intracellular Ca2+ concentration, producing a flux of Na+ and/or K+ ions that depolarizes the cell, thus modulating cellular Ca2+ entry. CAN channels are involved in cellular responses such as neuronal bursting activity and cardiac rhythm. Here we report the electron cryo-microscopy structure of the most widespread CAN channel, human TRPM4, bound to the agonist Ca2+ and the modulator decavanadate. Four cytosolic C-terminal domains form an umbrella-like structure with a coiled-coil domain for the 'pole' and four helical 'ribs' spanning the N-terminal TRPM homology regions (MHRs), thus holding four subunits in a crown-like architecture. We observed two decavanadate-binding sites, one in the C-terminal domain and another in the intersubunit MHR interface. A glutamine in the selectivity filter may be an important determinant of monovalent selectivity. Our structure provides new insights into the function and pharmacology of both the CAN and the TRPM families.

A tool set to allow rapid screening of dog families with PRA for association with candidate genes.

OBJECTIVE: To develop a method to rapidly screen candidate genes for association with recessively inherited progressive retinal atrophy (PRA) in pedigrees of dog in which a causative mutation has not been identified. ANIMAL STUDIED: Thirteen PRA-affected dogs were used in this study. PROCEDURES: Two microsatellite markers (MS) were designed flanking 45 candidate genes. MS markers were analyzed for heterozygosity and allelic richness. Two dog breeds, in which the causative mutation has been identified (Entlebucher Sennenhunds [ES] and PDE6A-mutant dogs [PDE6A]), were used to validate the MS marker panel. One breed in which the causative mutation is currently unknown (Old English Sheepdog [OES]) was investigated in this study utilizing the MS panel. RESULTS: Marker heterozygosity excluded 38 of 45 and 41 of 45 candidate genes (ES and PDE6A, respectively) with each true culprit gene remaining on the list of nonexcluded candidate genes. Additionally, 41 of 45 genes were excluded for OES. CONCLUSIONS: This tool set was used quickly and efficiently to narrow down 45 candidate genes for recessively inherited PRA in two types of dogs with known mutations and one type of dog with an unknown mutation.

Progressive retinal atrophy in the Polski Owczarek Nizinny dog: a clinical and genetic study.

OBJECTIVE: To describe ophthalmic, functional, structural, and genetical characteristics of progressive retinal atrophy (PRA) in the polski owczarek nizinny (PON) breed of dog. ANIMALS STUDIED CLINICALLY: Client-owned PON dogs (n = 82) from Sweden. PROCEDURES: Routine examination for presumed inherited eye disease was performed in all dogs. Bilateral full-field electroretinography (ERG) was performed in 11 affected and 4 control dogs. Eyes from one affected dog were studied with light microscopy. DNA samples from 34 Swedish and 30 PON dogs collected by Michigan State University (MSU) were tested for the mutations causing the rcd4 and prcd forms of PRA. RESULTS: Sixteen of the eighty-two Swedish dogs were diagnosed with PRA. Slight vascular attenuation, first seen at 4.5 years of age, preceded changes in tapetal reflectivity. The initial ERG changes in affected dogs showed markedly diminished rod responses, while cone responses were barely affected. Eventually, cone responses were also reduced. Retinal morphology showed approximately a 50% reduction of photoreceptor nuclei in the outer nuclear layer. Fourteen of fifteen PRA-affected Swedish dogs and eighteen of twenty of the MSU PRA-affected dogs tested genetically were positive for the rcd4 mutation. All tested dogs were negative for the mutation causing prcd-PRA. CONCLUSIONS: PRA of PON dogs is a late-onset degenerative disease with slow progression. There is early loss of rod function, while the cone system deteriorates later. The rcd4 mutation in the C2ORF71 gene was associated with the majority of the PRA cases tested. The possibility of additional forms of PRA in the breed cannot be excluded.

Isolation and cultivation of canine uveal melanocytes.

OBJECTIVES: To establish a method for isolation and culture of canine uveal melanocytes. ANIMALS STUDIED: Uveal explants from five mixed-breed dogs. PROCEDURES: Donor globes were dissected, and the anterior uvea removed. The uveal explants were placed in trypsin solution for enzymatic digestion. Extracted cells were cultured in modified F12 media. Immunocytochemistry was performed to confirm the identity of the extracted cells. RESULTS: Melanocytes were successfully isolated from uveal explants. Contaminating cell types were not observed. Repeated passaging of the melanocytes resulted in a gradual decrease in intracellular pigment. Melanocyte cell lines could be cryopreserved, thawed, and cultures successfully reestablished. CONCLUSIONS: This extraction technique allows for generation of large populations of canine uveal melanocytes in a relatively short period of time. This technique could be a useful tool for future studies investigating both normal cellular characteristics and alterations found in melanocytes from dogs with ocular melanocytic disorders.

A partial gene deletion of SLC45A2 causes oculocutaneous albinism in Doberman pinscher dogs.

The first white Doberman pinscher (WDP) dog was registered by the American Kennel Club in 1976. The novelty of the white coat color resulted in extensive line breeding of this dog and her offspring. The WDP phenotype closely resembles human oculocutaneous albinism (OCA) and clinicians noticed a seemingly high prevalence of pigmented masses on these dogs. This study had three specific aims: (1) produce a detailed description of the ocular phenotype of WDPs, (2) objectively determine if an increased prevalence of ocular and cutaneous melanocytic tumors was present in WDPs, and (3) determine if a genetic mutation in any of the genes known to cause human OCA is causal for the WDP phenotype. WDPs have a consistent ocular phenotype of photophobia, hypopigmented adnexal structures, blue irides with a tan periphery and hypopigmented retinal pigment epithelium and choroid. WDPs have a higher prevalence of cutaneous melanocytic neoplasms compared with control standard color Doberman pinschers (SDPs); cutaneous tumors were noted in 12/20 WDP (<5 years of age: 4/12; >5 years of age: 8/8) and 1/20 SDPs (p<0.00001). Using exclusion analysis, four OCA causative genes were investigated for their association with WDP phenotype; TYR, OCA2, TYRP1 and SLC45A2. SLC45A2 was found to be linked to the phenotype and gene sequencing revealed a 4,081 base pair deletion resulting in loss of the terminus of exon seven of SLC45A2 (chr4∶77,062,968-77,067,051). This mutation is highly likely to be the cause of the WDP phenotype and is supported by a lack of detectable SLC45A2 transcript levels by reverse transcriptase PCR. The WDP provides a valuable model for studying OCA4 visual disturbances and melanocytic neoplasms in a large animal model.

A large animal model for CNGB1 autosomal recessive retinitis pigmentosa.

Retinal dystrophies in dogs are invaluable models of human disease. Progressive retinal atrophy (PRA) is the canine equivalent of retinitis pigmentosa (RP). Similar to RP, PRA is a genetically heterogenous condition. We investigated PRA in the Papillon breed of dog using homozygosity mapping and haplotype construction of single nucleotide polymorphisms within a small family group to identify potential positional candidate genes. Based on the phenotypic similarities between the PRA-affected Papillons, mouse models and human patients, CNGB1 was selected as the most promising positional candidate gene. CNGB1 was sequenced and a complex mutation consisting of the combination of a one basepair deletion and a 6 basepair insertion was identified in exon 26 (c.2387delA;2389_2390insAGCTAC) leading to a frameshift and premature stop codon. Immunohistochemistry (IHC) of pre-degenerate retinal sections from a young affected dog showed absence of labeling using a C-terminal CNGB1 antibody. Whereas an antibody directed against the N-terminus of the protein, which also recognizes the glutamic acid rich proteins arising from alternative splicing of the CNGB1 transcript (upstream of the premature stop codon), labeled rod outer segments. CNGB1 combines with CNGA1 to form the rod cyclic nucleotide gated channel and previous studies have shown the requirement of CNGB1 for normal targeting of CNGA1 to the rod outer segment. In keeping with these previous observations, IHC showed a lack of detectable CNGA1 protein in the rod outer segments of the affected dog. A population study did not identify the CNGB1 mutation in PRA-affected dogs in other breeds and documented that the CNGB1 mutation accounts for ~70% of cases of Papillon PRA in our PRA-affected canine DNA bank. CNGB1 mutations are one cause of autosomal recessive RP making the CNGB1 mutant dog a valuable large animal model of the condition.