Altered Basal Autophagy Affects Extracellular Vesicle Release in Cells of Lagotto Romagnolo Dogs With a Variant ATG4D

Lagotto Romagnolo breed dogs develop a progressive neurological disease with intracellular vacuolar storage when homozygous for a variant in the autophagy-related gene 4D (ATG4D). A lysosomal enzyme deficiency has not been proven in this disease, despite its overlapping morphology with lysosomal storage diseases. Instead, basal autophagy was altered in fibroblasts from affected dogs. The aim of this study was to clarify the origin of the limiting membrane of the accumulating vacuoles and determine whether altered basal autophagy affects the extracellular release of vesicles in cells from diseased dogs. When assessed by immunoelectron microscopy, the membrane of the cytoplasmic vacuoles in affected tissues contained ATG4D, markers for autolysosomes (microtubule-associated protein 1A/B light chain 3 and lysosome-associated membrane protein 2) and for recycling endosomes (transferrin receptor 2), indicating that the vacuoles are hybrid organelles between endocytic and autophagic pathways. Ultracentrifugation, nanoparticle tracking analysis, and mass spectrometry were used to analyze the vesicles released from cultured fibroblasts of affected and control dogs. The amount of extracellular vesicles (EVs) released from affected fibroblasts was significantly increased during basal conditions in comparison to controls. This difference disappeared during starvation. The basal EV proteome of affected cells was enriched with cytosolic, endoplasmic reticulum, and mitochondrial proteins. Heat shock proteins and chaperones, some of which are known substrates of basal autophagy, were identified among the proteins unique to EVs of affected cells. An increased release of extracellular vesicles may serve as a compensatory mechanism in disposal of intracellular proteins during dysfunctional basal autophagy in this spontaneous disease

Genetic Panel Screening of Nearly 100 Mutations Reveals New Insights into the Breed Distribution of Risk Variants for Canine Hereditary Disorders

Background The growing number of identified genetic disease risk variants across dog breeds challenges the current state-of-the-art of population screening, veterinary molecular diagnostics, and genetic counseling. Multiplex screening of such variants is now technologically feasible, but its practical potential as a supportive tool for canine breeding, disease diagnostics, pet care, and genetics research is still unexplored. Results To demonstrate the utility of comprehensive genetic panel screening, we tested nearly 7000 dogs representing around 230 breeds for 93 disease-associated variants using a custom-designed genotyping microarray (the MyDogDNA1panel test). In addition to known breed disease-associated mutations, we discovered 15 risk variants in a total of 34 breeds in which their presence was previously undocumented. We followed up on seven of these genetic findings to demonstrate their clinical relevance. We report additional breeds harboring variants causing factor VII deficiency, hyperuricosuria, lens luxation, von Willebrand's disease, multifocal retinopathy, multidrug resistance, and rod-cone dysplasia. Moreover, we provide plausible molecular explanations for chondrodysplasia in the Chinook, cerebellar ataxia in the Norrbottenspitz, and familiar nephropathy in the Welsh Springer Spaniel. Conclusions These practical examples illustrate how genetic panel screening represents a comprehensive, efficient and powerful diagnostic and research discovery tool with a range of applications in veterinary care, disease research, and breeding. We conclude that several known disease alleles are more widespread across different breeds than previously recognized. However, careful follow up studies of any unexpected discoveries are essential to establish genotype-phenotype correlations, as is readiness to provide genetic counseling on their implications for the dog and its breed.Peer reviewe

The canine era : the rise of a biomedical model

Since the annotation of its genome a decade ago, the dog has proven to be an excellent model for the study of inherited diseases. A large variety of spontaneous simple and complex phenotypes occur in dogs, providing physiologically relevant models to corresponding human conditions. In addition, gene discovery is facilitated in clinically less heterogeneous purebred dogs with closed population structures because smaller study cohorts and fewer markers are often sufficient to expose causal variants. Here, we review the development of genomic resources from microsatellites to whole-genome sequencing and give examples of successful findings that have followed the technological progress. The increasing amount of whole-genome sequence data warrants better functional annotation of the canine genome to more effectively utilise this unique model to understand genetic contributions in morphological, behavioural and other complex traits.Peer reviewe

A Missense Change in the ATG4D Gene Links Aberrant Autophagy to a Neurodegenerative Vacuolar Storage Disease

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10(-136)) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to themacroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.Peer reviewe

Risk of anal furunculosis in German Shepherd dogs is associated with the major histocompatibility complex

Anal furunculosis (AF) is a chronic, progressive inflammatory disease of the perianal tissues most frequently affecting middle-aged or older German Shepherd dogs (GSD). Because this breed accounts for over 80% of all reported cases, there is likely to be a genetic association with disease susceptibility. Although there are some similarities with perianal fistulation that occurs in human Crohn's disease, the aetiology and pathogenesis of AF are still poorly understood. Recent research has suggested an immune-mediated aetiology, and evidence for this has been further provided by clinical responses to the immunosuppressive drug cyclosporin. The aim of the current study was to investigate canine major histocompatibility complex immune response genes. Dog leucocyte antigen class II alleles and haplotypes were characterised by sequence-based typing of 107 GSD affected with AF and 196 breed-matched controls collected in the UK. A highly significant association of DLA-DRB1*00101 with the presence of AF was observed (OR = 5.01, CI = 2.7-9.3, P < 0.00000001). This association was confirmed in a second cohort of GSD collected in Finland. Homozygosity for this allele is associated with an earlier disease onset