Genetic and Phenotypic Variations of Inherited Retinal Diseases in Dogs: The Power of Within- and Across-Breed Studies

Considerable clinical and molecular variations have been known in retinal blinding diseases in man and also in dogs. Different forms of retinal diseases occur in specific breed(s) caused by mutations segregating within each isolated breeding population. While molecular studies to find genes and mutations underlying retinal diseases in dogs have benefited largely from the phenotypic and genetic uniformity within a breed, within- and across-breed variations have often played a key role in elucidating the molecular basis. The increasing knowledge of phenotypic, allelic, and genetic heterogeneities in canine retinal degeneration has shown that the overall picture is rather more complicated than initially thought. Over the past 20 years, various approaches have been developed and tested to search for genes and mutations underlying genetic traits in dogs, depending on the availability of genetic tools and sample resources. Candidate gene, linkage analysis, and genome-wide association studies have so far identified 24 mutations in 18 genes underlying retinal diseases in at least 58 dog breeds. Many of these genes have been associated with retinal diseases in humans, thus providing opportunities to study the role in pathogenesis and in normal vision. Application in therapeutic interventions such as gene therapy has proven successful initially in a naturally occurring dog model followed by trials in human patients. Other genes whose human homologs have not been associated with retinal diseases are potential candidates to explain equivalent human diseases and contribute to the understanding of their function in vision

Canine genome assembly correction facilitates identification of a MAP9 deletion as a potential age of onset modifier for RPGRIP1-associated canine retinal degeneration.

Retinal degeneration (RD) in the Miniature Long Haired Dachshund (MLHD) is a cone-rod dystrophy resulting in eventual blindness in affected individuals. In a previous study, a 44-nucleotide insertion (ins44) in exon 2 of RPGRIP1 was associated with RD. However, results on an extended population of MLHD revealed a variable RD onset age for ins44 homozygous dogs. Further investigations using a genome-wide association study comparing early onset and late onset RD cases identified an age of onset modifying locus for RD, approximately 30 Mb upstream of RPGRIP1 on chr15. In this investigation, target enriched sequencing identified a MAP9 deletion spanning approximately 22 kb associated with early RD onset. Identification of the deletion required correction to the CanFam3.1 genome build as canine MAP9 is part of a historic tandem duplication, resulting in incomplete assembly of this genome region. The deletion breakpoints were identified in MAP9 intron 10 and in a downstream partial MAP9 pseudogene. The fusion of these two genes, which we have called MAP9 EORD (microtubule-associated protein, early onset retinal degeneration), is in frame and is expressed at the RNA level, with the 3' region containing several predicted deleterious variants. We speculate that MAP9 associates with α-tubulin in the basal body of the cilium. RPGRIP1 is also known to locate to the cilium, where it is closely associated with RPGR. RPGRIP1 mutations also cause redistribution of α-tubulin away from the ciliary region in photoreceptors. Hence, a MAP9 partial deficit is a particularly attractive candidate to synergise with a partial RPGRIP1 deficit to cause a more serious disease.This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s00335-016-9627-

Recent advances in the understanding of cilia mechanisms and their applications as therapeutic targets

The primary cilium is a single immotile microtubule-based organelle that protrudes into the extracellular space. Malformations and dysfunctions of the cilia have been associated with various forms of syndromic and non-syndromic diseases, termed ciliopathies. The primary cilium is therefore gaining attention due to its potential as a therapeutic target. In this review, we examine ciliary receptors, ciliogenesis, and ciliary trafficking as possible therapeutic targets. We first discuss the mechanisms of selective distribution, signal transduction, and physiological roles of ciliary receptors. Next, pathways that regulate ciliogenesis, specifically the Aurora A kinase, mammalian target of rapamycin, and ubiquitin-proteasome pathways are examined as therapeutic targets to regulate ciliogenesis. Then, in the photoreceptors, the mechanism of ciliary trafficking which takes place at the transition zone involving the ciliary membrane proteins is reviewed. Finally, some of the current therapeutic advancements highlighting the role of large animal models of photoreceptor ciliopathy are discussed

Variabilities in Retinal Function and Structure in a Canine Model of Cone-Rod Dystrophy Associated With RPGRIP1 Support Multigenic Etiology

Defects in the cilia gene RPGRIP1 cause Leber congenital amaurosis and cone-rod dystrophy in humans. A form of canine cone-rod dystrophy (cord1) was originally associated with a homozygous insertion in RPGRIP1 (RPGRIP1ins/ins) as the primary disease locus while a homozygous deletion in MAP9(MAP9del/del) was later identified as a modifier associated with the early onset form. However, we find further variability in cone electroretinograms (ERGs) ranging from normal to absent in an extended RPGRIP1ins/ins canine colony, irrespective of the MAP9 genotype. Ophthalmoscopically, cone ERGabsentRPGRIP1ins/ins eyes show discolouration of the tapetal fundus with varying onset and disease progression, while sd-OCT reveals atrophic changes. Despite marked changes in cone ERG and retinal morphology, photopic vision-guided behaviour is comparable between normal and cone ERGabsentRPGRIP1ins/ins littermates. Cone morphology of the dogs lacking cone ERG are truncated with shortened outer and inner segments. Immunohistochemically, cone ERGabsentRPGRIP1ins/ins retinas have extensive L/M-opsin mislocalization, lack CNGB3 labelling in the L/M-cones, and lack GC1 in all cones. Our results indicate that cord1 is a multigenic disease in which mutations in neither RPGRIP1 nor MAP9 alone lead to visual deficits, and additional gene(s) contribute to cone-specific functional and morphologic defects

Molecular characterization of MAP9 in the photoreceptor sensory cilia as a modifier in canine RPGRIP1-associated cone-rod dystrophy

Photoreceptors possess a highly specialized primary cilium containing expanded ciliary membrane discs called the outer segment. The photoreceptor cilium is essential for the maintenance of the outer segment, and pathogenic variants in more than 50 cilia-related genes have been identified as causing non-syndromic inherited retinal diseases in patients. The retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) is a structural protein localized to the photoreceptor cilium and biallelic RPGRIP1 variants have been associated with non-syndromic human inherited retinal diseases. In a canine cone-rod dystrophy model, a naturally occurring 44-bp exonic insertion in RPGRIP1 (RPGRIP1ins44/ins44) is the primary disease locus while an additional homozygous variant in MAP9 (microtubule associated protein 9) (MAP9aff/aff) acts as a modifier associated with early disease onset. MAP9 was originally identified as a microtubule-binding protein stabilizing microtubule structure during both mitosis and interphase in human cell lines. However, the roles of MAP9 in primary cilia, including photoreceptor neurosensory cilia, have not been well understood. Hence, we characterized the pathogenic phenotypes associated with homozygous MAP9 variant, and investigated the molecular function of MAP9 in primary cilia using the RPGRIP1-associated oligogenic canine cone-rod dystrophy model as well as cultured cells. Both functionally and structurally, the RPGRIP1ins44/ins44MAP9aff/aff retina exhibited progressive cone photoreceptor degeneration starting earlier than the retina affected by RPGRIP1ins44/ins44 alone. Based on immunostaining of canine retinal sections and cultured cells, we found that MAP9 is prominently localized in the basal body of primary cilia and played an important role in maintaining the structure of ciliary microtubule axoneme. These findings suggest that the affected MAP9, together with mutant RPGRIP1, is deprived of critical roles in cilia organization and maintenance resulting in altered cilia structure and function giving rise to early onset and accelerated disease progression in the RPGRIP1ins44/ins44MAP9aff/aff double homozygote cone-rod dystrophy canine model

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB

Canine CNGA3 Gene Mutations Provide Novel Insights Into Human Achromatopsia-Associated Channelopathies and Treatment

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivoand channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivochannels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients

CCDC66 frameshift variant associated with a new form of early-onset progressive retinal atrophy in Portuguese Water Dogs.

Aberrant photoreceptor function or morphogenesis leads to blinding retinal degenerative diseases, the majority of which have a genetic aetiology. A variant in PRCD previously identified in Portuguese Water Dogs (PWDs) underlies prcd (progressive rod-cone degeneration), an autosomal recessive progressive retinal atrophy (PRA) with a late onset at 3-6 years of age or older. Herein, we have identified a new form of early-onset PRA (EOPRA) in the same breed. Pedigree analysis suggested an autosomal recessive inheritance. Four PWD full-siblings affected with EOPRA diagnosed at 2-3 years of age were genotyped (173,661 SNPs) along with 2 unaffected siblings, 2 unaffected parents, and 15 unrelated control PWDs. GWAS, linkage analysis and homozygosity mapping defined a 26-Mb candidate region in canine chromosome 20. Whole-genome sequencing in one affected dog and its obligatory carrier parents identified a 1 bp insertion (CFA20:g.33,717,704_33,717,705insT (CanFam3.1); c.2262_c.2263insA) in CCDC66 predicted to cause a frameshift and truncation (p.Val747SerfsTer8). Screening of an extended PWD population confirmed perfect co-segregation of this genetic variant with the disease. Western blot analysis of COS-1 cells transfected with recombinant mutant CCDC66 expression constructs showed the mutant transcript translated into a truncated protein. Furthermore, in vitro studies suggest that the mutant CCDC66 is mislocalized to the nucleus relative to wild type CCDC66. CCDC66 variants have been associated with inherited retinal degenerations (RDs) including canine and murine ciliopathies. As genetic variants affecting the primary cilium can cause ciliopathies in which RD may be either the sole clinical manifestation or part of a syndrome, our findings further support a role for CCDC66 in retinal function and viability, potentially through its ciliary function