A naturally occurring canine model of syndromic congenital microphthalmia.

In humans, the prevalence of congenital microphthalmia is estimated to be 0.2-3.0 for every 10,000 individuals, with nonocular involvement reported in ∼80% of cases. Inherited eye diseases have been widely and descriptively characterized in dogs, and canine models of ocular diseases have played an essential role in unraveling the pathophysiology and development of new therapies. A naturally occurring canine model of a syndromic disorder characterized by microphthalmia was discovered in the Portuguese water dog. As nonocular findings included tooth enamel malformations, stunted growth, anemia, and thrombocytopenia, we hence termed this disorder Canine Congenital Microphthalmos with Hematopoietic Defects. Genome-wide association study and homozygosity mapping detected a 2 Mb candidate region on canine chromosome 4. Whole-genome sequencing and mapping against the Canfam4 reference revealed a Short interspersed element insertion in exon 2 of the DNAJC1 gene (g.74,274,883ins[T70]TGCTGCTTGGATT). Subsequent real-time PCR-based mass genotyping of a larger Portuguese water dog population found that the homozygous mutant genotype was perfectly associated with the Canine Congenital Microphthalmos with Hematopoietic Defects phenotype. Biallelic variants in DNAJC21 are mostly found to be associated with bone marrow failure syndrome type 3, with a phenotype that has a certain degree of overlap with Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and reports of individuals showing thrombocytopenia, microdontia, and microphthalmia. We, therefore, propose Canine Congenital Microphthalmos with Hematopoietic Defects as a naturally occurring model for DNAJC21-associated syndromes.

Evaluation of tendon and ligament microstructure and mechanical properties in a canine model of mucopolysaccharidosis I.

Mucopolysaccharidosis (MPS) I is a lysosomal storage disorder characterized by deficient alpha-l-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans (GAGs) in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a strong clinical need for improved treatment approaches that specifically target joint tissues; however, their development is hampered by poor understanding of underlying disease pathophysiology, including how pathological changes to component tissues contribute to overall joint dysfunction. Ligaments and tendons, in particular, have received very little attention, despite the critical roles of these tissues in joint stability and biomechanical function. The goal of this study was to leverage the naturally canine model to undertake functional and structural assessments of the anterior (cranial) cruciate ligament (CCL) and Achilles tendon in MPS I. Tissues were obtained postmortem from 12-month-old MPS I and control dogs and tested to failure in uniaxial tension. Both CCLs and Achilles tendons from MPS I animals exhibited significantly lower stiffness and failure properties compared to those from healthy controls. Histological examination revealed multiple pathological abnormalities, including collagen fiber disorganization, increased cellularity and vascularity, and elevated GAG content in both tissues. Clinically, animals exhibited mobility deficits, including abnormal gait, which was associated with hyperextensibility of the stifle and hock joints. These findings demonstrate that pathological changes to both ligaments and tendons contribute to abnormal joint function in MPS I, and suggest that effective clinical management of joint disease in patients should incorporate treatments targeting these tissues.

German Shorthaired Pointer dogs with exfoliative cutaneous lupus erythematosus develop immune-complex membranous glomerulonephropathy.

German Shorthaired Pointer (GSHP) dogs with a UNC93B1 gene mutation develop exfoliative cutaneous lupus erythematosus (ECLE) and kidney disease resembling lupus nephritis in humans. The objective of this study was to characterize the kidney disease by light microscopy, immunofluorescence, and electron microscopy in a population of GSHP dogs with ECLE. Medical records were reviewed, and light microscopy of kidneys from 7 GSHP dogs with a previous histologic diagnosis of ECLE was performed. Immunofluorescence of fresh-frozen kidney from 1 dog and transmission electron microscopy of kidney from that dog and 2 additional dogs were performed. Five of 7 dogs had proteinuria diagnosed by urinalysis or urine protein-to-creatinine ratio. Two of 7 dogs were intermittently hypoalbuminemic, and none were azotemic. Histologic findings included early (2 dogs) to late (5 dogs) membranous glomerulonephropathy characterized by mild-to-severe glomerular capillary loop thickening and tubular proteinosis. In all 7 cases, trichrome staining revealed red granular immune deposits on the subepithelial surface of the glomerular basement membrane. Immunofluorescence revealed strong granular labeling for immunoglobulins and complement protein C3. Electron microscopy demonstrated subepithelial electron-dense immune deposits encircled by the remodeled glomerular basement membrane. These findings are diagnostic of immune-complex membranous glomerulonephropathy and are similar to class V lupus in humans. This cohort of GSHP dogs with ECLE developed immune-complex membranous glomerulonephropathy, which we hypothesize is a manifestation of systemic lupus erythematosus. GSHP dogs with ECLE should undergo clinical evaluation of renal function for early identification and treatment.

Proteomics identifies novel biomarkers of synovial joint disease in a canine model of mucopolysaccharidosis I.

Mucopolysaccharidosis I is a lysosomal storage disorder characterized by deficient alpha-L-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a critical need for improved understanding of joint disease pathophysiology in MPS I, including specific biomarkers to predict and monitor joint disease progression, and response to treatment. The objective of this study was to leverage the naturally-occurring MPS I canine model and undertake an unbiased proteomic screen to identify systemic biomarkers predictive of local joint disease in MPS I. Synovial fluid and serum samples were collected from MPS I and healthy dogs at 12 months-of-age, and protein abundance characterized using liquid chromatography tandem mass spectrometry. Stifle joints were evaluated postmortem using magnetic resonance imaging (MRI) and histology. Proteomics identified 40 proteins for which abundance was significantly correlated between serum and synovial fluid, including markers of inflammatory joint disease and lysosomal dysfunction. Elevated expression of three biomarker candidates, matrix metalloproteinase 19, inter-alpha-trypsin inhibitor heavy-chain 3 and alpha-1-microglobulin, was confirmed in MPS I cartilage, and serum abundance of these molecules was found to correlate with MRI and histological degenerative grades. The candidate biomarkers identified have the potential to improve patient care by facilitating minimally-invasive, specific assessment of joint disease progression and response to therapeutic intervention.

Dose-dependent effects of enzyme replacement therapy on skeletal disease progression in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme ß-glucuronidase. Skeletal abnormalities are common in patients and result in diminished quality of life. Enzyme replacement therapy (ERT) for MPS VII using recombinant human ß-glucuronidase (vestronidase alfa) was recently approved for use in patients; however, to date there have been no studies evaluating therapeutic efficacy in a large animal model of MPS VII. The objective of this study was to establish the effects of intravenous ERT, administered at either the standard clinical dose (4 mg/kg) or a high dose (20 mg/kg), on skeletal disease progression in MPS VII using the naturally occurring canine model. Untreated MPS VII animals exhibited progressive synovial joint and vertebral bone disease and were no longer ambulatory by age 6 months. Standard-dose ERT-treated animals exhibited modest attenuation of joint disease, but by age 6 months were no longer ambulatory. High-dose ERT-treated animals exhibited marked attenuation of joint disease, and all were still ambulatory by age 6 months. Vertebral bone disease was recalcitrant to ERT irrespective of dose. Overall, our findings indicate that ERT administered at higher doses results in significantly improved skeletal disease outcomes in MPS VII dogs.

KRT5 missense variant in a Cardigan Welsh Corgi with epidermolysis bullosa simplex.

Epidermolysis bullosa (EB) is a group of blistering disorders that includes several subtypes, classified according to their level of cleavage. Typical clinical signs are blisters and erosions resulting from minimal trauma. The disease has been described in many mammalian species and pathogenic variants in at least 18 different genes have been identified. In the present study, we investigated a Cardigan Welsh Corgi with congenital clinical signs consistent with epidermolysis bullosa. The puppy had blisters and erosions on the paw pads, and the oral mucosa. Histologic examination demonstrated the typical clefting between the dermis and epidermis and confirmed the clinical suspicion. We obtained whole genome sequencing data from the affected puppy and searched for variants in candidate genes known to cause EB. This revealed a heterozygous missense variant, KRT5:p.(E476K), affecting the highly conserved KLLEGE motif of keratin 5. The mutant allele in the affected puppy arose owing to a de novo mutation event as it was absent from both unaffected parents. Knowledge of the functional impact of KRT5 variants in other species together with the demonstration of the de novo mutation event establishes KRT5:p.(E476K) as causative variant for the observed EBS.

Feline Fertility: Consequences of inbreeding and implications for reproductive fitness.

PRACTICAL RELEVANCE: When compared with the number of individuals that make up a dog breed, the population within a given cat breed is very small. Therefore, to maintain a breed standard, a certain degree of inbreeding is necessary. However, when inbreeding reaches a certain threshold, it can lead to decreased fertility, which manifests as failure to conceive, smaller litter size, increased neonatal illness and neonatal mortality. Breeders should be encouraged to keep comprehensive records on breeding outcomes, including number of kittens born, neonatal vitality, daily kitten weights and kitten health at weaning. Commercially available DNA panels are available to inform and facilitate excellent breeding choices and can estimate the coefficient of inbreeding. Clinicians should include a review of the degree of inbreeding in the work-up for any cattery or cat colony experiencing decreased fertility. AIM: The objective of this article is to provide clinicians, especially those working with cat breeders, with an easy-to-understand guide to genetics and to demonstrate how inbreeding influences fertility and neonatal survival. EQUIPMENT AND TECHNICAL SKILLS: Medical records and the pedigree of the cats in question are required to investigate cases of infertility that may be related to inbreeding. A DNA analysis kit that measures genetic diversity and health parameters can also be helpful; those that have been developed by geneticists and veterinarians at universities are preferable, as they include access to highly skilled genetic counselors and researchers who are open to working up newly discovered genetic diseases. EVIDENCE BASE: The material provided is based on current literature and the author's own studies examining outcomes in a closed cattery.

Effects of lithium administration on vertebral bone disease in mucopolysaccharidosis I dogs.

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease characterized by deficient activity of the enzyme alpha-L-iduronidase, leading to abnormal accumulation of heparan and dermatan sulfate glycosaminoglycans in cells and tissues. Patients commonly exhibit progressive skeletal abnormalities, in part due to failures of endochondral ossification during postnatal growth. Previously, using the naturally-occurring canine model, we showed that bone and cartilage cells in MPS I exhibit elevated lysosomal storage from an early age and that animals subsequently exhibit significantly diminished vertebral trabecular bone formation. Wnts are critical regulators of endochondral ossification that depend on glycosaminoglycans for signaling. The objective of this study was to examine whether lithium, a glycogen synthase kinase-3 inhibitor and stimulator of Wnt/beta-catenin signaling, administered during postnatal growth could attenuate progression of vertebral trabecular bone disease in MPS I. MPS I dogs were treated orally with therapeutic levels of lithium carbonate from 14 days to 6 months-of-age. Untreated heterozygous and MPS I dogs served as controls. Serum was collected at 3 and 6 months for assessment of bone turnover markers. At the study end point, thoracic vertebrae were excised and assessed using microcomputed tomography and histology. Lithium-treated animals exhibited significantly improved trabecular spacing, number and connectivity density, and serum bone-specific alkaline phosphatase levels compared to untreated animals. Growth plates from lithium-treated animals exhibited increased numbers of hypertrophic chondrocytes relative to both untreated MPS I and heterozygous animals. These findings suggest that bone and cartilage cells in MPS I are still capable of responding to exogenous osteogenic signals even in the presence of significant lysosomal storage, and that targeted osteogenic therapies may represent a promising approach for attenuating bone disease progression in MPS I.

ABHD5 frameshift deletion in Golden Retrievers with ichthyosis.

Ichthyoses are hereditary skin disorders characterized by the formation of scales and defects in the outermost layer of the epidermis. In dogs, at least six different breed-specific ichthyoses including a relatively common PNPLA1-related autosomal recessive ichthyosis in Golden Retrievers are known. In this study, we investigated 14 Golden Retrievers with scales that were not homozygous for the mutant PNPLA1 allele suggesting a genetically distinct new form of ichthyosis. Histopathological examinations showed lamellar, orthokeratotic hyperkeratosis, and mildly hyperplastic epidermis that led to the diagnosis of a nonepidermolytic ichthyosis. Combined linkage and homozygosity mapping in 14 cases and 30 nonaffected family members delimited a critical interval of ∼12.7 Mb on chromosome 23. Whole-genome sequencing of an affected dog revealed a single protein-changing variant within this region that was not present in 795 control genomes. The identified variant is a 14 bp deletion in the ABHD5 gene (c.1006_1019del), leading to a frameshift and altering the last 14 codons p.(Asp336Serfs*6). The genotypes at this variant showed perfect cosegregation with the ichthyosis phenotype in a large family comprising 14 cases and 72 controls. ABHD5 encodes an acyltransferase required for lipid metabolism. In humans, variants in ABHD5 cause Chanarin-Dorfman syndrome, a neutral lipid storage disease with ichthyosis. Our data in dogs together with the knowledge on the effects of ABHD5 variants in humans strongly suggest ABHD5:c.1006_1019del as candidate causative genetic variant for a new canine form of ichthyosis, which we propose to designate as Golden Retriever ichthyosis type 2 (ICH2).

Development of an Informatics Algorithm to Link Seasonal Infectious Diseases to Birth-Dependent Diseases Across Species: A Case Study with Osteosarcoma.

Many diseases have been linked with birth seasonality, and these fall into four main categories: mental, cardiovascular, respiratory and women's reproductive health conditions. Informatics methods are needed to uncover seasonally varying infectious diseases that may be responsible for the increased birth month-dependent disease risk observed. We have developed a method to link seasonal infectious disease data from the USA to birth month dependent disease data from humans and canines. We also include seasonal air pollution and climate data to determine the seasonal factors most likely involved in the response. We test our method with osteosarcoma, a rare bone cancer. We found the Lyme disease incidence was the most strongly correlated significant factor in explaining the birth month-osteosarcoma disease pattern (R=0.418, p=2.80X10-23), and this was true across all populations observed: canines, pediatric, and adult populations.

Progression of vertebral bone disease in mucopolysaccharidosis VII dogs from birth to skeletal maturity.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient ß-glucuronidase activity, leading to accumulation of incompletely degraded heparan, dermatan and chondroitin sulfate glycosaminoglycans. Patients with MPS VII exhibit progressive spinal deformity, which decreases quality of life. Previously, we demonstrated that MPS VII dogs exhibit impaired initiation of secondary ossification in the vertebrae and long bones. The objective of this study was to build on these findings and comprehensively characterize how vertebral bone disease manifests progressively in MPS VII dogs throughout postnatal growth. Vertebrae were collected postmortem from MPS VII and healthy control dogs at seven ages ranging from 9 to 365 days. Microcomputed tomography and histology were used to characterize bone properties in primary and secondary ossification centers. Serum was analyzed for bone turnover biomarkers. Results demonstrated that not only was secondary ossification delayed in MPS VII vertebrae, but that it progressed aberrantly and was markedly diminished even at 365 days-of-age. Within primary ossification centers, bone volume fraction and bone mineral density were significantly lower in MPS VII at 180 and 365 days-of-age. MPS VII growth plates exhibited significantly lower proliferative and hypertrophic zone cellularity at 90 days-of-age, while serum bone-specific alkaline phosphatase (BAP) was significantly lower in MPS VII dogs at 180 days-of-age. Overall, these findings establish that vertebral bone formation is significantly diminished in MPS VII dogs in both primary and secondary ossification centers during postnatal growth.

Epiphyseal cartilage canal architecture and extracellular matrix remodeling in mucopolysaccharidosis VII dogs at the onset of postnatal growth.

Purpose: Mucopolysaccharidosis (MPS) VII is a genetic, lysosomal storage disease characterized by abnormal accumulation of glycosaminoglycans in cells and tissues. MPS VII patients exhibit multiple failures of endochondral ossification during postnatal growth, including markedly delayed cartilage-to-bone conversion in the vertebrae and long bones. Cartilage canals provide the template for vascularization at the onset of secondary ossification. The objective of this study was to investigate whether abnormal cartilage canal architecture and enzyme-mediated extracellular matrix (ECM) remodeling contribute to delayed cartilage-to-bone conversion in MPS VII.Materials and Methods: The epiphyseal cartilage canal networks of 9-day-old healthy control and MPS VII-affected dog vertebrae were characterized using high-resolution, contrast-free quantitative susceptibility mapping magnetic resonance imaging. Relative expression levels of matrix metalloproteinases (MMPs) 9, 13 and 14 were examined using immunohistochemistry, while tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were examined using in situ enzyme staining.Results: Interestingly, the density, number, connectivity and thickness of cartilage canals was not significantly different between MPS VII and control vertebrae. Immunohistochemistry revealed diminished MMP-9, but normal MMP-13 and 14 expression by epiphyseal cartilage chondrocytes, while ALP and TRAP enzyme expression by chondrocytes and chondroclasts, respectively, were both diminished in MPS VII.Conclusions: Our findings suggest that while the epiphyseal cartilage canal network in MPS VII is normal at the onset of secondary ossification, expression of enzymes required for cartilage resorption and replacement with mineralized ECM, and initiation of angiogenesis, is impaired.

Ultrastructural analysis of different skeletal cell types in mucopolysaccharidosis dogs at the onset of postnatal growth.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Abnormal development of the vertebrae and long bones is a hallmark of skeletal disease in several MPS subtypes; however, the underlying cellular mechanisms remain poorly understood. The objective of this study was to conduct an ultrastructural examination of how lysosomal storage differentially affects major skeletal cell types in MPS I and VII using naturally occurring canine disease models. We showed that both bone and cartilage cells from MPS I and VII dog vertebrae exhibit significantly elevated storage from early in postnatal life, with storage generally greater in MPS VII than MPS I. Storage was most striking for vertebral osteocytes, occupying more than forty percent of cell area. Secondary to storage, dilation of the rough endoplasmic reticulum (ER), a marker of ER stress, was observed most markedly in MPS I epiphyseal chondrocytes. Significantly elevated immunostaining of light chain 3B (LC3B) in MPS VII epiphyseal chondrocytes suggested impaired autophagy, while significantly elevated apoptotic cell death in both MPS I and VII chondrocytes was also evident. The results of this study provide insights into how lysosomal storage differentially effects major skeletal cell types in MPS I and VII, and suggests a potential relationship between storage, ER stress, autophagy, and cell death in the pathogenesis of MPS skeletal defects.

Failures of Endochondral Ossification in the Mucopolysaccharidoses.

PURPOSE OF REVIEW: The mucopolysaccharidoses (MPS) are a group of inherited lysosomal storage disorders characterized by abnormal accumulation of glycosaminoglycans (GAGs) in cells and tissues. MPS patients frequently exhibit failures of endochondral ossification during postnatal growth leading to skeletal deformity and short stature. In this review, we outline the current understanding of the cellular and molecular mechanisms underlying failures of endochondral ossification in MPS and discuss associated treatment challenges and opportunities. RECENT FINDINGS: Studies in MPS patients and animal models have demonstrated that skeletal cells and tissues exhibit significantly elevated GAG storage from early in postnatal life and that this is associated with impaired cartilage-to-bone conversion in primary and secondary ossification centers, and growth plate dysfunction. Recent studies have begun to elucidate the underlying cellular and molecular mechanisms, including impaired chondrocyte proliferation and hypertrophy, diminished growth factor signaling, disrupted cell cycle progression, impaired autophagy, and increased cell stress and apoptosis. Current treatments such as hematopoietic stem cell transplantation and enzyme replacement therapy fail to normalize endochondral ossification in MPS. Emerging treatments including gene therapy and small molecule-based approaches hold significant promise in this regard. Failures of endochondral ossification contribute to skeletal deformity and short stature in MPS patients, increasing mortality and reducing quality of life. Early intervention is crucial for effective treatment, and there is a critical need for new approaches that normalize endochondral ossification by directly targeting affected cells and signaling pathways.

Vitrification Using Soy Lecithin and Sucrose: A New Way to Store the Sperm for the Preservation of Canine Reproductive Function.

A challenge in freezing semen for short and long-term availability is avoiding damage to intact spermatozoa caused by the freezing process. Vitrification protocols provide better results through less manipulation of semen and shorter freezing time compared to slow freezing techniques. Our research was aimed at improving vitrification methods for canine semen. Semen quality was determined in 20 ejaculates after collection. Each ejaculate was divided into eight aliquots, each with a different extender. The control extender contained TRIS, citric acid, fructose, and antibiotics. Soy lecithin and sucrose were added to the control extender at different concentrations to make up the test extenders and final concentration of 50 × 106 spermatozoa/mL. From each group, a 33µL (1.65 × 106 spermatozoa) suspension of spermatozoa was dropped directly into liquid nitrogen and devitrified at least one week later and evaluated as before. Soy lecithin at 1% and 0.25 M sucrose added to the base vitrification media effectively preserved all sperm qualities. Our results demonstrate the effectiveness of our methods. Vitrification media containing sucrose and soy lecithin cause a minimal decline in quality of canine semen after devitrification. Furthermore, extenders used in our research did not contain egg yolk, which was replaced by soy lecithin, thus allowing for ease of shipping to other countries with strict requirements.

A DSG1 Frameshift Variant in a Rottweiler Dog with Footpad Hyperkeratosis.

A single male Rottweiler dog with severe footpad hyperkeratosis starting at an age of eight weeks was investigated. The hyperkeratosis was initially restricted to the footpads. The footpad lesions caused severe discomfort to the dog and had to be trimmed under anesthesia every 8-10 weeks. Histologically, the epidermis showed papillated villous projections of dense keratin in the stratum corneum. Starting at eight months of age, the patient additionally developed signs consistent with atopic dermatitis and recurrent bacterial skin and ear infections. Crusted hyperkeratotic plaques developed at sites of infection. We sequenced the genome of the affected dog and compared the data to 655 control genomes. A search for variants in 32 candidate genes associated with human palmoplantar keratoderma (PPK) revealed a single private protein-changing variant in the affected dog. This was located in the DSG1 gene encoding desmoglein 1. Heterozygous monoallelic DSG1 variants have been reported in human patients with striate palmoplantar keratoderma I (SPPK1), while biallelic DSG1 loss of function variants in humans lead to a more pronounced condition termed severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome. The identified canine variant, DSG1:c.2541_2545delGGGCT, leads to a frameshift and truncates about 20% of the coding sequence. The affected dog was homozygous for the mutant allele. The comparative data on desmoglein 1 function in humans suggest that the identified DSG1 variant may have caused the footpad hyperkeratosis and predisposition for allergies and skin infections in the affected dog.

A Missense Variant Affecting the C-Terminal Tail of UNC93B1 in Dogs with Exfoliative Cutaneous Lupus Erythematosus (ECLE).

Cutaneous lupus erythematosus (CLE) in humans encompasses multiple subtypes that exhibit a wide array of skin lesions and, in some cases, are associated with the development of systemic lupus erythematosus (SLE). We investigated dogs with exfoliative cutaneous lupus erythematosus (ECLE), a dog-specific form of chronic CLE that is inherited as a monogenic autosomal recessive trait. A genome-wide association study (GWAS) with 14 cases and 29 controls confirmed a previously published result that the causative variant maps to chromosome 18. Autozygosity mapping refined the ECLE locus to a 493 kb critical interval. Filtering of whole genome sequence data from two cases against 654 controls revealed a single private protein-changing variant in this critical interval, UNC93B1:c.1438C>A or p.Pro480Thr. The homozygous mutant genotype was exclusively observed in 23 ECLE affected German Shorthaired Pointers and an ECLE affected Vizsla, but absent from 845 controls. UNC93B1 is a transmembrane protein located in the endoplasmic reticulum and endolysosomes, which is required for correct trafficking of several Toll-like receptors (TLRs). The p.Pro480Thr variant is predicted to affect the C-terminal tail of the UNC93B1 that has recently been shown to restrict TLR7 mediated autoimmunity via an interaction with syndecan binding protein (SDCBP). The functional knowledge on UNC93B1 strongly suggests that p.Pro480Thr is causing ECLE in dogs. These dogs therefore represent an interesting spontaneous model for human lupus erythematosus. Our results warrant further investigations of whether genetic variants affecting the C-terminus of UNC93B1 might be involved in specific subsets of CLE or SLE cases in humans and other species.

Applied Veterinary Informatics: Development of a Semantic and Domain-Specific Method to Construct a Canine Data Repository.

Animals are used to study the pathogenesis of various human diseases, but typically as animal models with induced disease. However, companion animals develop disease spontaneously in a way that mirrors disease development in humans. The purpose of this study is to develop a semantic and domain-specific method to enable construction of a data repository from a veterinary hospital that would be useful for future studies. We developed a two-phase method that combines semantic and domain-specific approaches to construct a canine data repository of clinical data collected during routine care at the Matthew J Ryan Veterinary Hospital of the University of Pennsylvania (PennVet). Our framework consists of two phases: (1) a semantic data-cleaning phase and (2) a domain-specific data-cleaning phase. We validated our data repository using a gold standard of known breed predispositions for certain diseases (i.e., mitral valve disease, atrial fibrillation and osteosarcoma). Our two-phase method allowed us to maximize data retention (99.8% of data retained), while ensuring the quality of our result. Our final population contained 84,405 dogs treated between 2000 and 2017 from 194 distinct dog breeds. We observed the expected breed associations with mitral valve disease, atrial fibrillation, and osteosarcoma (P < 0.05) after adjusting for multiple comparisons. Precision ranged from 60.0 to 83.3 for the three diseases (avg. 74.2) and recall ranged from 31.6 to 83.3 (avg. 53.3). Our study describes a two-phase method to construct a clinical data repository using canine data obtained during routine clinical care at a veterinary hospital.

Imputation of canine genotype array data using 365 whole-genome sequences improves power of genome-wide association studies.

Genomic resources for the domestic dog have improved with the widespread adoption of a 173k SNP array platform and updated reference genome. SNP arrays of this density are sufficient for detecting genetic associations within breeds but are underpowered for finding associations across multiple breeds or in mixed-breed dogs, where linkage disequilibrium rapidly decays between markers, even though such studies would hold particular promise for mapping complex diseases and traits. Here we introduce an imputation reference panel, consisting of 365 diverse, whole-genome sequenced dogs and wolves, which increases the number of markers that can be queried in genome-wide association studies approximately 130-fold. Using previously genotyped dogs, we show the utility of this reference panel in identifying potentially novel associations, including a locus on CFA20 significantly associated with cranial cruciate ligament disease, and fine-mapping for canine body size and blood phenotypes, even when causal loci are not in strong linkage disequilibrium with any single array marker. This reference panel resource will improve future genome-wide association studies for canine complex diseases and other phenotypes.

Molecular profiling of failed endochondral ossification in mucopolysaccharidosis VII.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of ß-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan, and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification. Epiphyseal tissue was isolated postmortem from the vertebrae of control and MPS VII-affected dogs at 9 and 14 days-of-age (n = 5 for each group). Differences in global gene expression across this developmental window for both cohorts were measured using whole-transcriptome sequencing (RNA-Seq). Principal Component Analysis revealed clustering of samples within each group, indicating clear effects of both age and disease state. At 9 days-of-age, 1375 genes were significantly differentially expressed between MPS VII and control, and by 14 days-of-age, this increased to 4719 genes. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification was performed, and a subset of gene expression differences were validated using qPCR. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. In control samples, temporal changes in gene expression from 9 to 14 days-of-age were consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/ß-catenin and BMP signaling were not upregulated during this same developmental window suggesting that important bone formation pathways are not activated. In conclusion, this study represents an important step towards identifying therapeutic targets and biomarkers for bone disease in MPS VII patients during postnatal growth.