A recessive CLN3 variant is responsible for delayed-onset retinal degeneration in Hereford cattle.

Thirteen American Hereford cattle were reported blind with presumed onset when ~12-mo-old. All blind cattle shared a common ancestor through both the maternal and paternal pedigrees, suggesting a recessive genetic origin. Given the pedigree relationships and novel phenotype, we characterized the ophthalmo-pathologic changes associated with blindness and identified the responsible gene variant. Ophthalmologic examinations of 5 blind cattle revealed retinal degeneration. Histologically, 2 blind cattle had loss of the retinal photoreceptor layer. Whole-genome sequencing (WGS) of 7 blind cattle and 9 unaffected relatives revealed a 1-bp frameshift deletion in ceroid lipofuscinosis neuronal 3 (CLN3; chr25 g.26043843del) for which the blind cattle were homozygous and their parents heterozygous. The identified variant in exon 16 of 17 is predicted to truncate the encoded protein (p. Pro369Argfs*8) battenin, which is involved in lysosomal function necessary for photoreceptor layer maintenance. Of 462 cattle genotyped, only blind cattle were homozygous for the deletion. A query of WGS data of > 5,800 animals further revealed that the variant was only observed in related Hereford cattle. Mutations in CLN3 are associated with human juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, which results in early-onset retinal degeneration and lesions similar to those observed in our cases. Our data support the frameshift variant of CLN3 as causative of blindness in these Hereford cattle, and provide additional evidence of the role of this gene in retinal lesions, possibly as a model for human non-syndromic JNCL.

Frameshift Variant in AMPD2 in Cirneco dell'Etna Dogs with Retinopathy and Tremors.

While the manifestations of many inherited retinal disorders are limited to loss of vision, others are part of a syndrome that affects multiple tissues, particularly the nervous system. Most syndromic retinal disorders are thought to be recessively inherited. Two dogs out of a litter of Cirneco dell' Etna dogs, both males, showed signs of retinal degeneration, along with tremors and signs described as either atypical seizures or paroxysmal dyskinesias, while the other two male littermates were normal. We named this oculo-neurological syndrome CONS (Cirneco oculo-neurological syndrome), and undertook homozygosity mapping and whole-genome sequencing to determine its potential genetic etiology. Notably, we detected a 1-bp deletion in chromosome 6 that was predicted to cause a frameshift and premature stop codon within the canine AMPD2 gene, which encodes adenosine monophosphate deaminase, an enzyme that converts adenosine 5'-monophosphate (AMP) to inosine 5'-monophosphate (IMP). Genotyping of the available Cirneco population suggested perfect segregation between cases and controls for the variant. Moreover, this variant was absent in canine genomic databases comprised of thousands of unaffected dogs. The AMPD2 genetic variant we identified in dogs presents with retinal manifestations, adding to the spectrum of neurological manifestations associated with AMPD2 variants in humans.

Olfaction evaluation in dogs with sudden acquired retinal degeneration syndrome.

PURPOSE: To evaluate olfaction in dogs with sudden acquired retinal degeneration syndrome (SARDS) compared with sighted dogs and blind dogs without SARDS as control groups. ANIMALS STUDIED: Forty client-owned dogs. PROCEDURE: Olfactory threshold testing was performed on three groups: SARDS, sighted, and blind/non-SARDS using eugenol as the test odorant. The olfactory threshold was determined when subjects indicated the detection of a specific eugenol concentration with behavioral responses. Olfactory threshold, age, body weight, and environmental room factors were evaluated. RESULTS: Sixteen dogs with SARDS, 12 sighted dogs, and 12 blind/non-SARDS dogs demonstrated mean olfactory threshold pen numbers of 2.8 (SD = 1.4), 13.8 (SD = 1.4), and 13.4 (SD = 1.1), respectively, which correspond to actual mean concentrations of 0.017 g/mL, 1.7 × 10-13 g/mL and 4.26 × 10-13 g/mL, respectively. Dogs with SARDS had significantly poorer olfactory threshold scores compared with the two control groups (p < .001), with no difference between the control groups (p = .5). Age, weight, and room environment did not differ between the three groups. CONCLUSIONS: Dogs with SARDS have severely decreased olfaction capabilities compared with sighted dogs and blind/non-SARDS dogs. This finding supports the suspicion that SARDS is a systemic disease causing blindness, endocrinopathy, and hyposmia. Since the molecular pathways are similar in photoreceptors, olfactory receptors, and steroidogenesis with all using G-protein coupled receptors in the cell membrane, the cause of SARDS may exist at the G-protein associated interactions with intracellular cyclic nucleotides. Further investigations into G-protein coupled receptors pathway and canine olfactory receptor genes in SARDS patients may be valuable in revealing the cause of SARDS.

Global Frequency Analyses of Canine Progressive Rod-Cone Degeneration-Progressive Retinal Atrophy and Collie Eye Anomaly Using Commercial Genetic Testing Data.

Hundreds of genetic variants associated with canine traits and disorders have been identified, with commercial tests offered. However, the geographic distributions and changes in allele and genotype frequencies over prolonged, continuous periods of time are lacking. This study utilized a large set of genotypes from dogs tested for the progressive rod-cone degeneration-progressive retinal atrophy (prcd-PRA) G>A missense PRCD variant (n = 86,667) and the collie eye anomaly (CEA)-associated NHEJ1 deletion (n = 33,834) provided by the commercial genetic testing company (Optigen/Wisdom Panel, Mars Petcare Science & Diagnostics). These data were analyzed using the chi-square goodness-of-fit test, time-trend graphical analysis, and regression modeling in order to evaluate how test results changed over time. The results span fifteen years, representing 82 countries and 67 breeds/breed mixes. Both diseases exhibited significant differences in genotype frequencies (p = 2.7 × 10-152 for prcd-PRA and 0.023 for CEA) with opposing graphical trends. Regression modeling showed time progression to significantly affect the odds of a dog being homozygous or heterozygous for either disease, as do variables including breed and breed popularity. This study shows that genetic testing informed breeding decisions to produce fewer affected dogs. However, the presence of dogs homozygous for the disease variant, especially for prcd-PRA, was still observed fourteen years after test availability, potentially due to crosses of unknown carriers. This suggests that genetic testing of dog populations should continue.

Hemostatic profiles in dogs with sudden acquired retinal degeneration syndrome.

BACKGROUND: Sudden acquired retinal degeneration syndrome (SARDS) is a common cause of irreversible blindness in dogs. It bears clinical resemblance to hypercortisolism, which can be associated with hypercoagulability. The role of hypercoagulability in dogs with SARDS is unknown. OBJECTIVE: Determine hemostatic profiles in dogs with SARDS. ANIMALS: Prospective pilot study: Dogs with a history of SARDS (n = 12). Prospective case-control study: Dogs with recent onset of SARDS (n = 7) and age-, breed-, and sex-matched controls (n = 7). METHODS: Prospective pilot study: We performed thromboelastography (TEG). Prospective case-control study: Dogs had CBC, serum biochemistry, urinalysis, TEG, fibrinogen concentration, antithrombin activity, D-dimers, thrombin-antithrombin complexes, and optical platelet aggregometry performed. RESULTS: Prospective pilot study: 9/12 dogs with a history of SARDS were hypercoagulable with increased TEG G value and 2/3 had hyperfibrinogenemia. Case-control study: All dogs with SARDS and 5/7 controls were hypercoagulable based on TEG G value. Dogs with SARDS had significantly higher G values (median, 12.7 kdynes/s; range, 11.2-25.4; P = .04) and plasma fibrinogen concentration (median, 463 mg/dL; range, 391-680; P < .001) compared to controls. CONCLUSIONS AND CLINICAL IMPORTANCE: Hypercoagulability was common in both dogs with SARDS and controls, but dogs with SARDS were significantly more hypercoagulable on TEG. The role of hypercoagulability in the pathogenesis of SARDS remains to be determined.

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.

Unilateral blindness presumed as sudden acquired retinal degeneration syndrome (SARDS) in one Dachshund and four Maltese dogs.

OBJECTIVE: To describe blindness, initially presenting as a unilateral condition and diagnosed as sudden acquired retinal degeneration syndrome (SARDS). ANIMALS STUDIED: One Dachshund and four Maltese dogs presented with unilateral blindness, for which the results of general ophthalmic examinations were insufficient to explain the blindness. All dogs were spayed females. RESULTS: Intraocular pressures were normal, and the optical media of the eyes were clear. Fundus appearances of the initially blind eyes were within normal variations, with slightly attenuated retinal blood vessels in some cases when compared with the sighted contralateral eyes. Electroretinography (ERG) amplitudes of the affected eyes were flat and reduced in the contralateral-sighted eyes in four dogs. One dog underwent ERG after the blindness progressed bilaterally 8 days after initial presentation (despite topical steroid medication). Two dogs had no recheck visits, but phone call follow-ups reported bilateral blindness 3 months later in one dog. One dog received no medication and retained vision in the contralateral eye until the last follow-up (94 days later). One dog received systemic cyclosporine and steroid medications and maintained vision in the contralateral eye; however, regular ERG rechecks showed a trend of declining amplitude (448 days). In this dog, optical coherence tomography (OCT) showed different stages of disorganized retinal layers as well as different retinal thickness between the eyes. CONCLUSIONS: Despite normal-looking fundi, ERG and OCT revealed different degrees of retinal changes between both eyes in this study. Eyes with vision might develop progressive blindness after a substantial amount of time in these presumed SARDS cases.

Comparison of the qualitative and quantitative optical coherence tomographic features between sudden acquired retinal degeneration syndrome and normal eyes in dogs.

OBJECTIVE: To quantitatively and qualitatively characterize the retinal optical coherence tomographic features of sudden acquired retinal degeneration syndrome (SARDS) and SARDS suspect dogs. ANIMALS STUDIED: Fourteen SARDS affected dogs, 11 age-, breed-, and sex-matched control dogs, and two SARDS suspect dogs. PROCEDURES: Spectral-domain optical coherence tomography (OCT) images were used to evaluate the quantitative features, including thickness, intereye asymmetry, and longitudinal changes in retinal layer thickness and the qualitative features, including retinal architecture and vitreous haze. RESULTS: Mean outer retinal layer thickness (ORT), outer nuclear layer thickness (ONL), and photoreceptor layer thickness (PRL) were significantly lower in the SARDS group, whereas mean inner retinal layer thickness was significantly higher in the SARDS group than in the control group. While thickness values of all retinal layers did not differ significantly between paired eyes in each group, the absolute intereye asymmetries in the ORT (p < .0001), ONL (p = .008), and PRL (p < .0001) were significantly higher in the SARDS group than in the control group. Some SARDS patients and SARDS suspects had a greater PRL than the control group, and serial OCT evaluation showed an increase in PRL in one SARDS suspect. Vitreous haze severity was greater in the SARDS group than in the control group (vitreous relative intensity, p = .030). CONCLUSIONS: We described the OCT features of SARDS patients and suspects. In particular, PRL thickening in the SARDS suspects might indicate an early change in SARDS. Although further studies are needed, this finding might provide new insights into the pathogenesis of SARDS.

An inherited night blindness in Wiltshire sheep.

Twelve cases of adult-onset blindness were identified in a flock of 130 polled Wiltshire sheep in New Zealand over a 3-year period. Affected sheep developed night blindness between 2 and 3 years of age, which progressed to complete blindness by 4 to 5 years of age. Fundic examination findings included progressive tapetal hyperreflectivity and attenuation of retinal blood vessels. Histologically, the retinas had a selective loss of rod photoreceptors with initial preservation of cone photoreceptors. Retinal degeneration was not accompanied by any other ocular or central nervous system abnormalities, and pedigree analysis suggested an inherited basis for the disease. Mating an affected Wiltshire ram to 2 affected Wiltshire ewes resulted in 6 progeny that all developed retinal degeneration by 2 years of age, while mating of the same affected ram to 6 unaffected ewes resulted in 8 unaffected progeny, consistent with autosomal recessive inheritance. Homozygosity mapping of 5 affected Wiltshire sheep and 1 unaffected Wiltshire sheep using an OvineSNP50 Genotyping BeadChip revealed an identical-by-descent region on chromosome 5, but none of the genes within this region were considered plausible candidate genes. Whole-genome sequencing of 2 affected sheep did not reveal any significant mutations in any of the genes associated with retinitis pigmentosa in humans or progressive retinal atrophy in dogs. Inherited progressive retinal degeneration affecting rod photoreceptors has not been previously reported in sheep, but this disease has several similarities to inherited retinal dystrophies in other species.

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.

A Missense Variant in the Bardet-Biedl Syndrome 2 Gene (BBS2) Leads to a Novel Syndromic Retinal Degeneration in the Shetland Sheepdog.

Canine progressive retinal atrophy (PRA) describes a group of hereditary diseases characterized by photoreceptor cell death in the retina, leading to visual impairment. Despite the identification of multiple PRA-causing variants, extensive heterogeneity of PRA is observed across and within dog breeds, with many still genetically unsolved. This study sought to elucidate the causal variant for a distinct form of PRA in the Shetland sheepdog, using a whole-genome sequencing approach. Filtering variants from a single PRA-affected Shetland sheepdog genome compared to 176 genomes of other breeds identified a single nucleotide variant in exon 11 of the Bardet-Biedl syndrome-2 gene (BBS2) (c.1222G>C; p.Ala408Pro). Genotyping 1386 canids of 155 dog breeds, 15 cross breeds and 8 wolves indicated the c.1222G>C variant was only segregated within Shetland sheepdogs. Out of 505 Shetland sheepdogs, seven were homozygous for the variant. Clinical history and photographs for three homozygotes indicated the presence of a novel phenotype. In addition to PRA, additional clinical features in homozygous dogs support the discovery of a novel syndromic PRA in the breed. The development and utilization of a diagnostic DNA test aim to prevent the mutation from becoming more prevalent in the breed.

Visual system pathology in a canine model of CLN5 neuronal ceroid lipofuscinosis.

CLN5 neuronal ceroid lipofuscinosis is a hereditary neurodegenerative disease characterized by progressive neurological decline, vision loss and seizures. Visual impairment in children with CLN5 disease is attributed to a progressive decline in retinal function accompanied by retinal degeneration as well as impaired central nervous system function associated with global brain atrophy. We studied visual system pathology in five Golden Retriever littermates homozygous for the CLN5 disease allele previously identified in the breed. The dogs exhibited signs of pronounced visual impairment by 21-22 months of age. Electroretinogram recordings showed a progressive decline in retinal function primarily affecting cone neural pathways. Altered visual evoked potential recordings indicated that disease progression affected visual signal processing in the brain. Aside from several small retinal detachment lesions, no gross retinal abnormalities were observed with in vivo ocular imaging and histologically the retinas did not exhibit apparent abnormalities by 23 months of age. However, there was extensive accumulation of autofluorescent membrane-bound lysosomal storage bodies in almost all retinal layers, as well as in the occipital cortex, by 20 months of age. In the retina, storage was particularly pronounced in retinal ganglion cells, the retinal pigment epithelium and in photoreceptor cells just interior to the outer limiting membrane. The visual system pathology of CLN5-affected Golden Retrievers is similar to that seen early in the human disease. It was not possible to follow the dogs to an advanced stage of disease progression due to the severity of behavioral and motor disease signs by 23 months of age. The findings reported here indicate that canine CLN5 disease will be a useful model of visual system disease in CLN5 neuronal ceroid lipofuscinosis. The baseline data obtained in this investigation will be useful in future therapeutic intervention studies. The findings indicate that there is a fairly broad time frame after disease onset within which treatments could be effective in preserving vision.

Whole genome sequencing identifies a homozygous nonsense mutation in the JPH2 gene in Shih Tzu dogs with progressive retinal atrophy.

Progressive retinal atrophy (PRA), common autosomal recessive disorder affecting several dog breeds including Shih Tzu, is characterized by degeneration of photoreceptors leading to blindness. To identify PRA genetic variants, three affected and 15 unaffected Shih Tzu and 20 non-Shih Tzu were recruited. Dogs underwent ophthalmologic examination and electroretinography, revealing hallmark retina pathological changes and an abnormal electroretinography in all affected dogs but not in unaffected dogs. WGS was performed. Non-synonymous homozygous variants were searched in coding regions of genes involved in retinal diseases/development; the criterion was that variants should only be present in affected dogs and should be absent in both unaffected and 46 genomes of dogs (from an available evolutionary database). Only one out of the 109 identified variants is predicted to harbor a high-impact consequence, a nonsense c.452A>C (p.L151X) in the JPH2 gene. The genotype of JPH2 variant in all 38 dogs was determined with Sanger sequencing. All three affected dogs, but none of the 35 unaffected, were homozygous for the nonsense variant. JPH2 has been previously found to be expressed in several excitable cells/tissues including retina photoreceptors. Hence, JPH2 is a candidate gene for PRA in Shih Tzu.

Presumed neuroprotective therapies prescribed by veterinary ophthalmologists for canine degenerative retinal and optic nerve diseases.

OBJECTIVE: To investigate veterinary ophthalmologists' use of presumed neuroprotective therapies for degenerative retinal and optic nerve diseases in dogs. PROCEDURES: An online survey was sent to 663 board-certified veterinary ophthalmologists who were Diplomates of the American College of Veterinary Ophthalmologists (ACVO), Asian College of Veterinary Ophthalmologists (AiCVO), Latin American College of Veterinary Ophthalmologists (Colegio Latinoamericano de Oftalmólogos Veterinarios, CLOVE), or European College of Veterinary Ophthalmologists (ECVO). The survey was created using Qualtrics® software and focused on the prescription of presumed neuroprotective treatments for canine glaucoma, sudden acquired retinal degeneration syndrome (SARDS), progressive retinal atrophy (PRA), and retinal detachment (RD). RESULTS: A total of 165 completed surveys were received, representing an overall response rate of 25%, which was comparable across the four specialty colleges. Of all respondents, 140/165 (85%) prescribed some form of presumed neuroprotective therapies at least once in the last five years: 114/165 (69%) for glaucoma, 51/165 (31%) for SARDS, 116/165 (70%) for PRA, and 50/165 (30%) for RD. The three most recommended neuroprotective reagents were the commercial Ocu-GLO™ Vision Supplement for animals, amlodipine, and human eye supplements. CONCLUSIONS: Despite lack of published clinical efficacy data, the majority of surveyed board-certified veterinary ophthalmologists previously prescribed a presumed neuroprotective therapy at least once in the last five years in dogs with degenerative retinal and optic nerve diseases.

A missense variant in IFT122 associated with a canine model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a blinding eye disease affecting nearly two million people worldwide. Dogs are affected with a similar illness termed progressive retinal atrophy (PRA). Lapponian herders (LHs) are affected with several types of inherited retinal dystrophies, and variants in PRCD and BEST1 genes have been associated with generalized PRA and canine multifocal retinopathy 3 (cmr3), respectively. However, all retinal dystrophy cases in LHs are not explained by these variants, indicating additional genetic causes of disease in the breed. We collected DNA samples from 10 PRA affected LHs, with known PRCD and BEST1 variants excluded, and 34 unaffected LHs. A genome-wide association study identified a locus on CFA20 (praw = 2.4 × 10-7, pBonf = 0.035), and subsequent whole-genome sequencing of an affected LH revealed a missense variant, c.3176G>A, in the intraflagellar transport 122 (IFT122) gene. The variant was also found in Finnish Lapphunds, in which its clinical relevancy needs to be studied further. The variant interrupts a highly conserved residue, p.(R1059H), in IFT122 and likely impairs its function. Variants in IFT122 have not been associated with retinal degeneration in mammals, but the loss of ift122 in zebrafish larvae impaired opsin transport and resulted in progressive photoreceptor degeneration. Our study establishes a new spontaneous dog model to study the role of IFT122 in RP biology, while the affected breed will benefit from a genetic test for a recessive condition.

Canine sudden acquired retinal degeneration syndrome: Owner perceptions on the time to vision loss, treatment outcomes, and prognosis for life.

BACKGROUND: Canine sudden acquired retinal degeneration syndrome (SARDS) causes blindness for which there are no proven effective treatments. We aimed to clarify the time to vision loss, treatment response/side effects, and prognosis for life in dogs with SARDS. METHODS: An online questionnaire was administered to owners of dogs with a historical diagnosis of SARDS. Mortality data were compared with a published purebred reference population. Select parameters were analyzed statistically using general linear model with least square means, two-sample t tests, and chi-squared or Fisher's exact tests. RESULTS: Responses from owners that stated that their dog visited an ophthalmologist and had electroretinography performed (n = 434) were analyzed. The majority of owners (65.4%) reported the time from vision disturbance to complete vision loss as <2 weeks; 19.4% reported >4 weeks. Onset of systemic clinical signs to complete vision loss was >4 weeks in 44.5% of responses. A higher proportion of owners reported some vision recovery with combination treatment (14.4%) compared with monotherapy (3.2%, P = .0004). Side effects of treatment were commonly reported. Dogs with SARDS did not have a shorter lifespan than the reference population but had higher incidence of kidney disease (P = .0001) and respiratory disease (P = .0004) at death. CONCLUSIONS: Dogs with SARDS have a rapid onset of vision loss. In the owner's opinion, treatment is unlikely to restore vision and is associated with systemic side effects. The potential for systemic pathologies that arise after SARDS diagnosis warrants further study.

Presumed cancer-associated retinopathy (CAR) mimicking Sudden Acquired Retinal Degeneration Syndrome (SARDS) in canines.

OBJECTIVE: To describe functional and structural features of presumed cancer-associated retinopathy (CAR) mimicking sudden acquired retinal degeneration syndrome (SARDS) in dogs and describe treatment outcomes. ANIMALS: Subjects were 17 dogs from 8 eight US states and Canada diagnosed with SARDS or immune-mediated retinitis (IMR) by 12 ophthalmologists. Nine eyes from seven deceased patients were used for microarray (MA), histology, or immunohistochemical (IHC) analysis. PROCEDURES: Dogs underwent complete ophthalmic examination, including retinal photography, optical coherence tomography (OCT), chromatic pupil light reflex testing (cPLR), and electroretinography (ERG), in addition to complete systemic examination. Histology, microarray, and IHC analysis were performed in CAR retinas to evaluate histological and molecular changes in retinal tissue. RESULTS: None of the patients evaluated satisfied previously established criteria for diagnosis of SARDS (flat ERG+ no red - good blue PLR), and all were diagnosed with IMR. All patients were diagnosed with a cancer: meningioma (24%), sarcoma (18%), pituitary tumor (12%), and squamous cell carcinoma (12%), other (34%). Median survival time was 6 months from diagnosis (range 1-36 months). Most frequent systemic abnormalities were as follows: proteinuria (78%); elevated liver enzymes (47%); and metabolic changes (PU/PD, polyphagia - 24%). Immunosuppressive therapy resulted in the reversal of blindness in 44% of treated patients, with 61% of all treated patients recovering and/or maintaining vision. Median time for preservation of vision was 5 months (range 1-35 months). CONCLUSIONS: Observed changes are highly suggestive of immune-mediated damage in IMR-CAR eyes. A relatively high percentage of patients with CAR responded positively to immunosuppressive therapy.

Candidate Genetic Modifiers for RPGR Retinal Degeneration.

Purpose: To define genetic variants associated with variable severity of X-linked progressive retinal atrophy 1 (XLPRA1) caused by a five-nucleotide deletion in canine RPGR exon ORF15. Methods: A genome-wide association study (GWAS) was performed in XLPRA1 phenotype informative pedigree. Whole genome sequencing (WGS) was used for mutational analysis of genes within the candidate genomic region. Retinas of normal and mutant dogs were used for gene expression, gene structure, and RNA duplex analyses. Results: GWAS followed by haplotype phasing identified an approximately 4.6 Mb candidate genomic interval on CFA31 containing seven protein-coding genes expressed in retina (ROBO1, ROBO2, RBM11, NRIP1, HSPA13, SAMSN1, and USP25). Furthermore, we identified and characterized two novel lncRNAs, ROBO1-AS and ROBO2-AS, that display overlapping gene organization with axon guidance pathway genes ROBO1 and ROBO2, respectively, producing sense-antisense gene pairs. Notably, ROBO1-AS and ROBO2-AS act in cis to form lncRNA/mRNA duplexes with ROBO1 and ROBO2, respectively, suggesting important roles for these lncRNAs in the ROBO regulatory network. A subsequent WGS identified candidate genes within the genomic region on CFA31 that might be implicated in modifying severity of XLPRA1. This approach led to discovery of genetic variants in ROBO1, ROBO1-AS, ROBO2-AS, and USP25 that are strongly associated with the XLPRA1 moderate phenotype. Conclusions: The study provides new insights into the genetic basis of phenotypic variation in severity of RPGRorf15-associated retinal degeneration. Our findings suggest an important role for ROBO pathways in disease progression further expanding on our previously reported changes of ROBO1 expression in XLPRA1 retinas.

A LINE-1 insertion situated in the promoter of IMPG2 is associated with autosomal recessive progressive retinal atrophy in Lhasa Apso dogs.

BACKGROUND: Canine progressive retinal atrophies are a group of hereditary retinal degenerations in dogs characterised by depletion of photoreceptor cells in the retina, which ultimately leads to blindness. PRA in the Lhasa Apso (LA) dog has not previously been clinically characterised or described in the literature, but owners in the UK are advised to have their dog examined through the British Veterinary Association/ Kennel Club/ International Sheep Dog Society (BVA/KC/ISDS) eye scheme annually, and similar schemes that are in operation in other countries. After the exclusion of 25 previously reported canine retinal mutations in LA PRA-affected dogs, we sought to identify the genetic cause of PRA in this breed. RESULTS: Analysis of whole-exome sequencing data of three PRA-affected LA and three LA without signs of PRA did not identify any exonic or splice site variants, suggesting the causal variant was non-exonic. We subsequently undertook a genome-wide association study (GWAS), which identified a 1.3 Mb disease-associated region on canine chromosome 33, followed by whole-genome sequencing analysis that revealed a long interspersed element-1 (LINE-1) insertion upstream of the IMPG2 gene. IMPG2 has previously been implicated in human retinal disease; however, until now no canine PRAs have been associated with this gene. The identification of this PRA-associated variant has enabled the development of a DNA test for this form of PRA in the breed, here termed PRA4 to distinguish it from other forms of PRA described in other breeds. This test has been used to determine the genotypes of over 900 LA dogs. A large cohort of genotyped dogs was used to estimate the allele frequency as between 0.07-0.1 in the UK LA population. CONCLUSIONS: Through the use of GWAS and subsequent sequencing of a PRA case, we have identified a LINE-1 insertion in the retinal candidate gene IMPG2 that is associated with a form of PRA in the LA dog. Validation of this variant in 447 dogs of 123 breeds determined it was private to LA dogs. We envisage that, over time, the developed DNA test will offer breeders the opportunity to avoid producing dogs affected with this form of PRA.