Retinal degenerative diseases, often genetic, lead to blindness. In Portuguese Water Dogs (PWDs), a *PRCD* variant causes prcd-PRA (progressive rod-cone degeneration), a late-onset (3-6 years) autosomal recessive progressive retinal atrophy (PRA). This study investigates a new early-onset PRA (EOPRA) in PWDs. Photoreceptor function is crucial for vision; these cells are highly polarized neurons with distinct compartments (outer segment, inner segment, cell body, synaptic body). Their maintenance relies on the retinal pigment epithelium. Defects in photoreceptor morphogenesis or development lead to progressive retinal degeneration (RD) and blindness. Recent research highlights the connecting cilium's critical role in photoreceptor integrity. Connecting cilia, analogous to primary sensory cilia, are implicated in ciliopathies, a group of inherited conditions where retinal dystrophy can be a sole or syndromic manifestation. Over 300 genes are linked to inherited retinal diseases in humans, and over 30 in dogs, mostly PRAs. Dogs are valuable animal models for studying RD and developing therapies translatable to humans. While the impact of genetic variants can sometimes be inferred, many domain of unknown function (DUF) families remain uncharacterized. Mapping techniques combined with sequencing help identify causative genes for autosomal recessive diseases and elucidate their roles in signaling pathways. The PWD breed, while once small, has gained popularity, and the limited genetic variation due to founder effects makes it advantageous for studying complex traits. Previous studies identified prcd-PRA in PWDs, but this study examines a new, early-onset form of RD.

Previous research on retinal degenerative diseases in dogs has focused on identifying genes associated with Progressive Retinal Atrophy (PRA). The *PRCD* gene has been identified as a major contributor to late-onset PRA in several breeds, including Portuguese Water Dogs. However, the early-onset form of PRA studied here had not been previously characterized. Other genes associated with PRA in various breeds have also been studied, demonstrating the genetic complexity of these diseases. Studies in mouse models have also highlighted the role of the connecting cilium in photoreceptor integrity, implicating ciliopathies in retinal dystrophy. The study highlights the importance of using a combined approach of SNP genotyping, whole-genome sequencing, and population screening to identify causative genes for inherited diseases.

Four full-sibling PWDs diagnosed with EOPRA (onset at 2-3 years) and their relatives were studied. Initial clinical signs included visual deficits worsening in dim light. Ophthalmoscopic findings included tapetal hyper-reflectivity, vascular attenuation, optic disc pallor, and depigmentation. One affected dog showed undetectable ERGs at age 6. Pedigree analysis suggested autosomal recessive inheritance. 173,661 SNPs were genotyped in four affected, two unaffected siblings, two parents, and 15 unrelated controls. GWAS showed suggestive peaks on CFA20 (p=0.00014) and CFA11 (p=0.00059). Linkage analysis in the closely related subset (parents and siblings) revealed positive LOD scores on CFA2, CFA12, and CFA20. Homozygosity mapping identified a shared homozygous region on CFA20 (21.9-48.5 Mb). Whole-genome sequencing (WGS) of one affected dog and its parents identified a 1 bp insertion (CFA20:g.33,717,704_33,717,705insT) in *CCDC66*, predicted to cause a frameshift and truncation (p.Val747SerfsTer8). Sanger sequencing confirmed the variant's presence in affected dogs and its absence in unaffected controls. Western blot analysis in COS-1 cells transfected with mutant *CCDC66* showed a truncated protein. Immunocytochemistry demonstrated nuclear mislocalization of the mutant protein compared to cytoplasmic localization of the wild-type protein. Further screening of PWDs confirmed co-segregation. Retinal RNA-seq data from normal dogs helped model the impact of the variant on transcripts and protein isoforms. Analysis of different canine CCDC66 transcripts was undertaken to better define potential impact of the mutation on CCDC66 protein.

A novel early-onset form of PRA (EOPRA) was identified in Portuguese Water Dogs. The disease showed autosomal recessive inheritance. GWAS, linkage analysis, and homozygosity mapping consistently pointed to a 26.55 Mb region on canine chromosome 20. Whole-genome sequencing revealed a 1 bp insertion in exon 15 of the *CCDC66* gene, leading to a frameshift mutation and a truncated protein (p.Val747SerfsTer8). The mutation was perfectly associated with EOPRA in the family studied. Western blot analysis confirmed the expression of a truncated CCDC66 protein in vitro. Importantly, immunocytochemistry experiments demonstrated that the mutant CCDC66 protein was mislocalized to the nucleus, unlike the wild-type protein, which showed predominantly cytoplasmic localization. This suggests altered functionality of the mutated protein. Analysis of retinal RNA-seq data suggested multiple isoforms, with the main isoform being 951 amino acids long and truncated to 755 amino acids by the mutation. Screening of additional PWDs showed the CCDC66 variant allele frequency of 1.1% in the population screened, with no homozygotes. Additional analysis was conducted on 4 other cases, with 3 being homozygous for the *PRCD* variant. The fourth case indicated a different genetic cause.

This study identifies a novel frameshift variant in *CCDC66* as the causative mutation for EOPRA in PWDs. This finding expands our understanding of genetic causes of PRA and demonstrates locus heterogeneity within a single breed. The observed nuclear mislocalization of the truncated CCDC66 protein strongly suggests a loss of function, contributing to the disease phenotype. The early onset of EOPRA, unlike the late-onset prcd-PRA, likely reflects the severity of the *CCDC66* mutation. Previous studies have linked CCDC66 variants to retinal degeneration in other canine breeds and mouse models, further supporting its role in retinal function. However, some conflicting evidence concerning CCDC66's involvement in human RDs does exist. The study highlights the importance of breed-specific genetic backgrounds in modifying disease expression. The variation in the age of onset between different breeds with CCDC66 mutations could be due to these background differences. Future research should focus on elucidating the precise functional role of the CCDC66 C-terminus and exploring potential therapeutic targets.

This study successfully identifies a novel frameshift mutation in the *CCDC66* gene as the causative factor for early-onset PRA in Portuguese Water Dogs. The study demonstrates the utility of a combined approach of GWAS, linkage analysis, WGS, and population screening in identifying causative genes for complex inherited diseases. The results expand our knowledge of the genetic basis of canine retinal dystrophies and provide insight into the potential role of CCDC66 in retinal function. Further studies are necessary to fully elucidate the functional implications of the CCDC66 mutation and to investigate potential therapeutic strategies.

The study's sample size, particularly the number of affected dogs, is relatively small. The lack of access to ocular tissues from affected dogs limited the ability to directly study the effect of the variant on retinal transcripts and protein levels in vivo. The impact of the RNF123 variant cannot be conclusively excluded due to the inability to identify homozygous individuals. The analysis focused on a specific breed, so the findings' generalizability to other breeds is not fully established. Further studies are needed to investigate the broader implications of the findings for human retinal diseases.