Rod and cone photoreceptors are specialized retinal neurons that have a fundamental role in visual perception, capturing light and transducing it into a neuronal signal. Aberrant functioning of rod and/or cone photoreceptors can ultimately lead to progressive degeneration and eventually blindness. In man, many rod and rod-cone degenerative diseases are classified as forms of retinitis pigmentosa (RP). Dogs also have a comparable disease grouping termed progressive retinal atrophy (PRA). These diseases are generally due to single gene defects and follow Mendelian inheritance. We collected 51 DNA samples from Miniature Schnauzers affected by PRA (average age of diagnosis ∼3.9 ±1 years), as well as from 56 clinically normal controls of the same breed (average age ∼6.6 ±2.8 years). Pedigree analysis suggested monogenic autosomal recessive inheritance of PRA. GWAS and homozygosity mapping defined a critical interval in the first 4,796,806 bp of CFA15. Whole genome sequencing of two affected cases, a carrier and a control identified two candidate variants within the critical interval. One was an intronic SNV in HIVEP3, and the other was a complex structural variant consisting of the duplication of exon 5 of the PPT1 gene along with a conversion and insertion (named PPT1_dci). PPT1_dci was confirmed homozygous in a cohort of 22 cases, and 12 more cases were homozygous for the CFA15 haplotype. Additionally, the variant was found homozygous in 6 non-affected dogs of age higher than the average age of onset. The HIVEP3 variant was found heterozygous (n=4) and homozygous wild-type (n=1) in cases either homozygous for PPT1_dci or for the mapped CFA15 haplotype. We detected the wildtype and three aberrant PPT1 transcripts in isolated white blood cell mRNA extracted from a PRA case homozygous for PPT1_dci, and the aberrant transcripts involved inclusion of the duplicated exon 5 and novel exons following the activation of cryptic splice sites. No neurological signs were detected among the dogs homozygous for the PPT1_dci variant. Therefore, we propose PPT1_dci as causative for a non-syndromic form of PRA (PRA_PPT1) that shows incomplete penetrance in Miniature Schnauzers, potentially related to the presence of the wild-type transcript. To our knowledge, this is the first case of isolated retinal degeneration associated with a PPT1 variant.

A 9-month old male Jack Russell Terrier started showing paraparesis of the hindlimbs after a walk. Hospitalized, the dog went into cardiac arrest, and later died. Necroscopic examination revealed a severe thickness of the diaphragm, esophagus, and base of the tongue, leading to the diagnosis of muscular dystrophy. The histology confirmed the marked size variation, regeneration, and fibrosis replacement of the skeletal muscle fibers. Immunohistochemistry demonstrated the absence of dystrophin confirming the diagnosis. Transmission electron microscopy showed disarrangement of skeletal muscle fibers. Finally, whole-genome sequencing identified a ~368kb deletion spanning 19 exons of the canine dystrophin (DMD) gene. This pathogenic loss-of-function variant most likely explains the observed disease phenotype. The X-chromosomal variant was absent in seven controls of the same breed. Most likely, this partial deletion of the DMD gene was either transmitted on the maternal path within the family of the affected dog or arose de novo. This study revealed a spontaneous partial deletion in DMD gene in a Jack Russell Terrier showing a Duchenne-type muscular dystrophy due to non-functional dystrophin.

Congenital pseudomyotonia in cattle (PMT) is a rare skeletal muscle disorder, clinically characterized by stiffness and by delayed muscle relaxation after exercise. Muscle relaxation impairment is due to defective content of the Sarco(endo)plasmic Reticulum Ca²⁺ ATPase isoform 1 (SERCA1) protein, caused by missense mutations in the ATP2A1 gene. PMT represents the only mammalian model of human Brody myopathy. In the Romagnola breed, two missense variants occurring in the same allele were described, leading to Gly211Val and Gly286Val (G211V/G286V) substitutions. In this study, we analyzed the consequences of G211V and G286V mutations. Results support that the reduced amount of SERCA1 is a consequence of the G211V mutation, the G286V mutation almost being benign and the ubiquitin–proteasome system (UPS) being involved. After blocking the proteasome using a proteasome inhibitor, we found that the G211V mutant accumulates in cells at levels comparable to those of WT SERCA1. Our conclusion is that G211/286V mutations presumably originate in a folding-defective SERCA1 protein, recognized and diverted to degradation by UPS, although still catalytically functional, and that the main role is played by G211V mutation. Rescue of mutated SERCA1 to the sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca²⁺ concentration and prevent the appearance of pathological signs, paving the way for a possible therapeutic approach against Brody disease.

Purpose: In the premolecular era, mammalian samples were embedded in epoxy resin blocks, such as Epon or Poly/Bed, for future evaluation by electron microscopy. However, use of these archival specimens for more modern mutation characterization studies can be challenging. The aim of this study was to determine if genomic DNA could be extracted from osmicated archival epoxy-embedded tissues to a quality suitable for short-amplicon PCR amplification. Methods: We selected nine archived Epon, Araldite, or Poly/Bed embedded blocks of mammalian retinal and corneal tissue that were ~10 mm in length, embedded in the 1970s to 1990s, and had an extensive phenotypic description. Tissues were fixed in several combinations of glutaraldehyde and osmium before embedding. The blocks were shaved of excess resin, fragmented, and digested using an epoxy resin removal solution. The softened plastic was cut with a scalpel, washed, drained, and incubated at 56 °C overnight in a tissue lysis solution containing Proteinase K. Trizol was added to the samples, which were further mechanically homogenized. Chloroform was added, and the samples were centrifuged at 4 °C and 12,000 g. Upon phase separation, the upper clear phase was removed, 95% EtOH was added, the mix was filtered through a mini-genomic DNA extraction column and washed twice, and DNA was eluted with 10 mM Tris-HCL. Following final removal of phenol contamination using water-saturated ether, the purified DNA was quantified and used for PCR amplification. Results: The extraction success was tested by targeted PCR amplification using primers that produced amplicons 90 to 260 bp in length and targeted genes relevant for inherited eye studies (progressive rod-cone degeneration-PRCD; rhodopsin-RHO; glucuronidase beta-GUSB1), plus an additional control gene receptor accessory protein 1 (REEP1). All but one of the epoxy-embedded eye samples were successfully amplified. Sanger sequencing confirmed the gene identity of amplified products. Conclusions: By identifying methods to extract DNA from osmicated epoxy-embedded mammalian eye tissues, our results provide a valuable resource for determining the genetic basis of inherited diseases and for retroactively confirming molecular diagnoses based on microscopic analysis.

Background: Progressive retinal atrophy (PRA) was diagnosed in four related male English Cocker Spaniels (ECS) between the ages of 3 and 4 years. All affected dogs were born to clinically unaffected parents that had tested negative for known PRA-causing variants through commercial vendors. Pedigree analysis revealed that the mothers of the affected dogs were paternal half-siblings, and their father had been reported as visually impaired, suggesting an X-linked recessive mode of inheritance. Results: To identify the underlying genetic cause, we performed homozygosity mapping, identifying six candidate regions, and whole-genome sequencing. A single high-impact coding variant—a 1-bp deletion in exon 36 of the CACNA1F gene on X chromosome (UU_Cfam_GSD_1.0 NC_049260.1 g.42,516,353del) —was identified. This variant is predicted to cause a frameshift and premature stop codon (XM_038587436.1, c.4,481del, XP_038443364.1:Phe1482LeufsTer8) and was found in a hemizygous state in all affected males and heterozygous in their mothers, while being absent in fathers as well as unaffected control dogs. A specifically designed PCR test was developed and applied to a broader cohort of 92 related and unrelated ECS dogs, confirming the segregation of this variant. Conclusions: This study identified a novel segregating CACNA1F variant, likely to be responsible for an X-linked recessive form of PRA in the ECS. Given that CACNA1F is already associated with retinal disorders in other mammals, including humans, this finding adds to its relevance across species. This study highlights the importance for breeders of regular health examinations for late-onset conditions and prompt reporting of hereditary disorders of unknown cause to enable rapid identification of their genetic basis. Development and implementation of specific genetic screening tests are recommended to assess the frequency of this variant in ECS and other populations, inform breeding strategies, and prevent further dissemination of this deleterious variant.