Focal Bullous Retinal Detachments in PRA-affected Dogs

• The study reports the development of focal bullous retinal detachments (bullae) in dogs with progressive retinal atrophy (PRA).
• The study examined dogs with three distinct forms of PRA: PRA-affected Whippets, German Spitzes, and CNGB1-mutant Papillon crosses.
• Indirect ophthalmoscopy and spectral domain optical coherence tomography (SD-OCT) were used.
• 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.
• Multiple focal bullous retinal detachments (bullae) were identified in PRA-affected dogs of all three types.
• Bullae developed in 4 of 5 PRA-affected Whippets, 3 of 8 PRA-affected German 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 the formation of bullae.
• Retinal bullae were identified in dogs with three distinct forms of progressive retinal atrophy.
• The lesions develop prior to retinal thinning.
• Clinical change should be monitored in dogs with PRA.

Introduction

• Progressive retinal atrophy (PRA) is a common category of inherited retinal degeneration in dogs.
• PRA consists of a group of inherited degenerative disorders of the retina.
• PRA results in vision impairment and blindness.
• PRA has been identified in over 100 dog breeds.
• It is commonly a rod-led degeneration (rod-cone dystrophy).
• Slower loss of cones is secondary, leads to complete blindness.
• The molecular genetic basis has been investigated.
• Molecular genetic basis demonstrates the genetic heterogeneity of the condition.
• In some forms of PRA cones can be involved prior to, or at the same time as rods and are classified as cone-rod dystrophies.
• Funduscopic changes indicating a bilateral progressive retinal thinning are similar to those of the rod-cone dystrophies.
• Typical clinical signs include night vision loss and indications of progressive retinal thinning.
• Indications of tapetal hyperreflectivity with retinal vascular attenuation occur on funduscopic examination.
• Tapetal hyporeflectivity may be an early change in some PRA forms.
• Prior to photoreceptor death and retinal thinning, alterations may occur that result in more light absorption.
• Some PRA forms have specific changes, such as dominant PRA due to a rhodopsin mutation which renders the retina sensitive to light damage.
• Environmental light exposure can lead to patchy degeneration with rhodopsin mutation.
• A form of PRA in Whippets has been associated with the formation of multiple retinal bullae.
• Retinal Bullae are a change that had not been previously reported in dogs with PRA.
• Transient retinal separation has been occasionally observed in dogs with X-linked PRA type 2.
• Transient retinal separation is due to a mutation in the RPGR gene.
• The study states bullae formation in PRA-affected Whippets and reports that retinal bullae are a feature of two other PRA forms.
• Preliminary data suggests that gene therapy prevent bullae formation.

Materials and Methods

• Procedures were conducted according to the ARVO statement for Use of Animals in Ophthalmic and Vision Research.
• The study was approved by the Institutional Animal Care and Use Committee at Michigan State University.
• Studies in Brazil were approved by the Animal Use Ethics Committee of the Federal University of Paraná.
• Three groups of dogs with three different forms of autosomal recessive PRA were included and examined at early disease stages.
• Heterozygous littermates were examined for two groups maintained in a colony.
• Group 1: 5 Whippets (4 males, 1 female) PRA-affected, 3 males heterozygous for PRA.
• Whippets were from a colony at Michigan State University derived from previously reported PRA-affected Whippets.
• Dogs were monitored from age 3 months to at least 18 months.
• Group 2: 8 privately-owned German Spitzes (6 males, 2 females) PRA-affected, aged 3-12 months, diagnosed and examined at the Veterinary Teaching Hospital of the Federal University of Paraná (Brazil).
• Group 3: 20 Papillon beagle crosses (11 males, 9 females) homozygous for a mutation in rod cyclic nucleotide gated channel subunit beta 1 (CNGB1).
• Dogs were followed from 3 to at least 18 months of age.
• One dog underwent retinal gene augmentation therapy previously reported.
• Six dogs heterozygous for the CNGB1 mutation (1 male, 5 females) were examined.
• The Whippet and Papillon crosses were housed and fed with a 12/12 h light cycle.

Eye examination and fundus photographs

• Pupils were dilated with tropicamide for examination and spectral domain optical coherence tomography.
• Ophthalmic examination included slit-lamp biomicroscopy, indirect ophthalmoscopy, and fundus color images.

Spectral domain optical coherence tomography (SD-OCT)

• Retinal imaging was performed under general anesthesia after being pre-medicated with acepromazine, if needed buprenorphine was also used and anesthesia induced with intravenous propofol.
• Eyes were positioned in primary gaze using conjunctival stay sutures.
• Imaging was performed using combined confocal scanning laser ophthalmoscopy (cSLO) and spectral domain optical coherence tomography (SD-OCT).
• Infrared (IR) and autofluorescence (AF) cSLO images of the fundus were acquired using a 55° lens.
• Horizontal and vertical SD-OCT single line scans and raster volume scans from areas with bullae and from the rest of the retina were obtained using a 30° lens.

Screening canine BEST1 gene for variant

• Canine multifocal retinopathy (CMR) is associated with retinal bullae formation due to homozygous mutations in the BEST1 gene.
• The study screened for the unlikely possibility that a BEST1 mutation was segregating in the PRA-affected dog pedigrees the entire coding region
• Parts of the flanking introns were Sanger sequenced in two affected Whippets and one CNGB1-mutant dog and the sequence was obtained from whole genome sequencing of 2 PRA-affected dogs.
• Blood DNA was extracted using standard protocols and used to amplify coding region and intronic regions flanking exons of gene.
• Amplicons were submitted for Sanger sequencing.
• The resulting DNA sequence was aligned to the canine reference sequence and screened for polymorphisms.
• Aligned sequence files were viewed using Integrated Genomics Viewer and screened for variants from the Canfam3.1 genome in the coding exons and flanking intronic regions

Gene augmentation therapy

• An adeno-associated viral construct (serotype 5) packaged with CNGB1 cDNA was delivered by two subretinal injections of about 200 µl.

Fundus Imaging

• Multiple retinal bullae were detected in dogs with all three types of PRA; age at first identification varied between breeds, beginning as early as 3 months in Whippets and 6 months in German Spitz.
• The localization, number, and size of bullae differed.
• Subretinal fluid appeared clear on indirect ophthalmoscopy and color fundus imaging.
• Bullae were clearly visible in the tapetal fundus on cSLO IR imaging.
• Bullae within the non-tapetal fundus showed hyperfluorescence.
• SD-OCT imaging confirmed focal bullous retinal detachments and hyporeflective subretinal fluid.
• SD-OCT was a more sensitive method for detecting bullae, especially flatter detachments and those in the non-tapetal area.
• Retinal bullae localized to the central tapetal fundus developed in 4/5 PRA-affected Whippets (2 males, 2 females), but there were fewer bullae per eye.
• Bullae were detected from 3 months of age, up to 1210 µm in diameter.
• Bullae were first detectable by SD-OCT but most enlarged enough to be seen by indirect ophthalmoscopy and RetCam photography.
• In male PRA-affected Whippet serial SD-OCT examinations show that as the photoreceptor degeneration became apparent (ie, thinning on SD-OCT), bullae tended to flatten.
• The outer retina of the detached region degenerated more rapidly than the adjacent attached retina which can be clearly seen in the cross-sectional retinal images at 12 and 18 months ages.
• None of PRA carrier Whippets had bullae.
• Multiple Bullae (from 40-90 per eye) involving the whole tapetal area developed in of 3/8 PRA-affected German Spitz dogs with a tendency to be more concentrated near the dorsal blood vessels and smaller, from one fourth to one sixth of the optic disk diameter.
• Precise age at development is not known, but at 3 months of age bullae were not detected.
• By 6 months of age, they could be detected.
• Seven of the 8 PRA-affected German Spitz were examined by SD-OCT and indirect ophthalmoscopy, one was only examined by indirect ophthalmoscopy.
• No bullae were detected in this animal.
• Bullae were identified in 15 of the 20 CNGB1-mutant dogs, varying between size but numerous and first detected in peripheral tapetal and non-tapetal fundi, before developing in the more central tapetal regions
• The bullae appeared to become larger in the periphery with the largest measuring 3800 µm in diameter.
• These lesions were not present at the initial examinations of the German Spitz at 2–3 months of age, but appeared from about 4 months of age and became more obvious from 6-9 months of age and appeared to decrease 12-15 months of age.
• For 6 of the 15 subjects with bullae, these were detected by SD-OCT but were not apparent in the RetCam images or noted on indirect ophthalmoscopy.
• In the 5 CNGB1-mutant dogs in which bullae were not detected SD-OCT was not performed, they were only examined by indirect ophthalmoscopy and RetCam imaging.
• Bullae were not detected in any of the dogs heterozygous for the CNGB1 mutation.
• The CNGB1-mutant dog that had undergone gene augmentation therapy did not develop bullae in the treated retinal regions but developed multiple bullae in the surrounding untreated retina

Sequence of exons and adjacent portions of the introns of Best1

• Sequencing of the exons and flanking intronic regions of BEST1 in dogs of each breed revealed the presence of SNPs in the coding regions of exons 2, 4, 7 and 8.
• These SNPs were synonymous variants previously recorded in the canine SNP database.

Discussion

• Progressive retinal atrophy in dogs has been recognized since the 1900s.
• It is known that it is a genetically heterogeneous condition that varies in mode of inheritance, age of onset and rate of progression.
• The different forms of PRA share clinical features including a progressive photoreceptor loss leading to retinal thinning, retinal vascular attenuation and optic nerve head atrophy.
• Precise molecular mechanism underlying different genetic forms of PRA may differ and can include mechanisms such as failure of phototransduction leading to accumulation of cyclic GMP in photoreceptors
• Bullae formation occurred in a recent study in Whippets prior to retinal thinning which was also associated with an absence of the ERG b-wave suggesting impaired photoreceptor to bipolar cell synaptic transmission.
• 4 of the 5 PRA-affected Whippets examined were identified to have bullae formation.
• Retinal bullae formation occurred prior to retinal degeneration in two additional groups of dogs with two distinct forms of PRA: German Spitz with an early-onset autosomal recessive form of PRA and in dogs with PRA due to a mutation in CNGB1.
• In PRA-affected German Spitz 3 out of 8 of the examined dogs (prior to 12 months of age) and in CNGB1-mutant dogs 15 of 20 dogs examined were found to develop bullae.
• Bullae were not identified in any of the heterozygotes for the Whippet form of PRA nor heterozygotes for CNGB1-PRA.
• The presence of diseased photoreceptors prior to extensive cell death is necessary for the formation of bullae
• The bullae represent focal small bullous retinal detachments characterized by subretinal fluid accumulation.
• SD-OCT was a more sensitive method to detect small, flatter detachments, particularly those in the non-tapetal fundus.
• In dogs where the lesions were monitored over time, the detached retinal region tended to degenerate more rapidly than the adjacent attached region because of the additional deleterious effect of separation of the diseased photoreceptors from the supporting RPE.
• Whippets tended to have fewer bullae compared to PRA-affected German Spitz and the CNGB1-mutant dogs.
• Retinal gene therapy in one CNGB1 dog seemed to prevent bullae formation just in the treated retinal regions.
• The therapy restored normal retinal function and preserved structure suggesting that the presence of abnormal and degenerating photoreceptors was necessary for the formation of the bullae.
• The development of retinal bullae in dogs with 3 genetically distinct forms of PRA suggest that their formation is not specific to the PRA-causing gene mutation.
• Causal mutations in Whippets and German Spitz will be the subject of separate publications and not in genes related to CNGB1 which is mutated in the Papillon derived dogs.
• They are all photoreceptor specific genes and not expressed in RPE.
• There is an additional previous report of bullae formation in dogs with X-linked PRA type 2.
• Retinal bullae formation has also been reported by three independent groups in dogs with sudden acquired retinal degeneration syndrome (SARDS).
• The authors reported retinal bullae formation in dogs with SARDS found SD-OCT a more sensitive tool to detect the presence of bullae.
• Active photoreceptor degeneration is a common factor in several forms of PRA with different disease mechanisms and also in dogs with a non-inherited cause of photoreceptor degeneration.
• Several possible mechanisms can cause sub retinal fluid and lead to the formation of subretinal fluid.
• These can include disruption of the normal pump mechanism of the RPE or a loss of the integrity of the Blood Retinal Barrier (BRB).
• The RPE actively transports fluid out of the subretinal space, while keeping K+ and lactate levels tightly controlled.
• This function of the RPE maintains a negative hydrostatic pressure, essential for the adhesion between RPE and photoreceptors; failure of this transport system leads to retinal edema and retinal detachment.
• The BRB (inner/outer) plays an important role in the homeostatic regulation of the retinal microenvironment by controlling fluid and molecular movement between the ocular vascular meshwork and the retinal tissues and prevents leakage into the retina of macromolecules and other potentially noxious agents.
• The inner BRB (iBRB) is formed by the tight junctions between the capillary endothelial cells and Muller cells.
• The outer BRB (OBRB) is consisted by tight junctions between cells of RPE and separates the neural retina which is essential for transporting nutrients from the blood to the outer retina.
• Toxic products from the degenerating retina are suggested as a possible cause of breakdown of the BRB.
• Cystoid macular edema in humans may occur in up to 50% of RP patients.
• However, they accumulate fluid within retinal rather than in the subretinal space like Dogs.
• Genetic factors could include environmental influences or background genetics.
• Mutations in BEST1 cause canine multifocal retinopathy characterized by multiple retinal focal detachments.
• No variants that changed predicted coding were detected.
• Additional factors that lead to bullae formation required investigation.
• Although not reported in other PRA forms, the authors state that it is possible that lesions might develop transiently in the early stages of PRA in other dog breeds.
• Once retinal thinning is established the bullae seem to resolve and authors have seen dogs of other breeds with multiple bullae that have gone on to develop generalized retinal degeneration.
• Whether dogs with more advanced PRA that have retinal regions with more advanced retinal degeneration could have previously had bullae in those regions is speculated .

Conclusions

• Retinal bullae can develop in some forms of PRA and occur prior to retinal thinning and are no longer apparent once retinal degeneration is established.
• The retina in the affected region appears to degenerate more rapidly than in the adjacent regions not affected.
• SD-OCT is a sensitive tool for detecting these bullae.