Neural tube defects in four Shetland sheepdog puppies: clinical characterisation and computed tomography investigation

Case report Here we report on the occurrence of neural tube defects in four related Shetland sheepdog puppies. Neural tube defects present as a range of congenital malformations affecting the spine, skull and associated structures. Despite the severity of these malformations and their relatively high prevalence in humans, the aetiology is not well understood. It is even less well characterised in veterinary medicine. Affected puppies were investigated using computed tomography and then post-mortem examination. Computed tomography identified a range of brain and spine abnormalities in the affected animals, including caudal anencephaly, encephalocele, spina bifida and malformed vertebrae. Other observed abnormalities in these puppies, including cranioschisis, atresia ani and hydrocephalus, may be secondary to, or associated with, the primary neural tube defects identified. Conclusion This case report describes multiple related cases of neural tube defects in an Australian cohort of dogs. This study also highlights the potential of advanced imaging techniques in identifying congenital anomalies in stillborn and neonatal puppies. Further research is required to investigate the aetiology of neural tube defects in this group of affected Shetland sheepdogs

Therapeutic Neonatal Hepatic Gene Therapy in Mucopolysaccharidosis VII Dogs

Dogs with mucopolysaccharidosis VII (MPS VII) were injected intravenously at 2–3 days of age with a retroviral vector (RV) expressing canine β-glucuronidase (cGUSB). Five animals received RV alone, and two dogs received hepatocyte growth factor (HGF) before RV in an attempt to increase transduction efficiency. Transduced hepatocytes expanded clonally during normal liver growth and secreted enzyme with mannose 6-phosphate. Serum GUSB activity was stable for up to 14 months at normal levels for the RV-treated dogs, and for 17 months at 67-fold normal for the HGF/RV-treated dog. GUSB activity in other organs was 1.5–60% of normal at 6 months for two RV-treated dogs, which was likely because of uptake of enzyme from blood by the mannose 6-phosphate receptor. The body weights of untreated MPS VII dogs are 50% of normal at 6 months. MPS VII dogs cannot walk or stand after 6 months, and progressively develop eye and heart disease. RV- and HGF/RV-treated MPS VII dogs achieved 87% and 84% of normal body weight, respectively. Treated animals could run at all times of evaluation for 6–17 months because of improvements in bone and joint abnormalities, and had little or no corneal clouding and no mitral valve thickening. Despite higher GUSB expression, the clinical improvements in the HGF/RV-treated dog were similar to those in the RV-treated animals. This is the first successful application of gene therapy in preventing the clinical manifestations of a lysosomal storage disease in a large animal

Breed risk, immunophenotypes and genomic studies of canine atopic dermatitis

BREED RISK, IMMUNOPHENOTYPES AND GENOMIC STUDIES OF CANINE ATOPIC DERMATITIS Canine atopic dermatitis (AD) is a complex genetically-linked immunological hypersensitivity which has similar clinical signs and pathological features to human AD, and involves immune dysregulation and skin barrier impairment. This thesis examines genetic factors underpinning canine AD and focuses on breed prevalence and evaluation of changes in the blood. Sixteen breeds with increased relative risk (≥1.5) were identified and gender predisposition in two dog breeds was revealed. One clade of dog breeds is highly represented amongst AD patients worldwide, and with increased RR in Australia. A 19 cytokine/chemokine multiplex bio-assay measured significantly elevated CXCL8, IL-7 and IL-15 concentrations, and reduced Stem-cell factor (SCF) in plasma of canine AD patients (n=27) compared to controls (n=11). Microarray gene expression data from leukocytes of atopic dogs (n=6) and controls (n=6) revealed 603 differentially expressed (DE) genes. Amongst these, candidate immune-related genes were highlighted, including the most under-represented gene, IL-7 receptor alpha subunit (IL7R). Quantitative real time PCR confirmed reduced gene expression for genes of the IL-7/IL7R pathway. A significant enrichment cluster consisting of immune-related pathways was identified, including T-cell receptor and B-cell receptor pathways. Expression analysis of miRNA from leukocytes of AD dogs identified15 with differential patterns of expression. The miRNA cfa-miR-31 had low levels in atopic dogs, suggesting a potential immune impairment dysregulation. Integrating the data gathered in the array studies reported in this thesis together with the genetic structure of dog breeds provides a powerful model to enable better characterisation of Australian dog breed susceptibility to canine AD and breed-related phenotypes. The IL-7/IL7R pathway may play a key role in the immune response in canine AD, may provide biomarkers to assist with diagnoses, and is a potential target for therapeutic agents

Visualization of Genome Diversity in German Shepherd Dogs

A loss of genetic diversity may lead to increased disease risks in subpopulations of dogs. The canine breed structure has contributed to relatively small effective population size in many breeds and can limit the options for selective breeding strategies to maintain diversity. With the completion of the canine genome sequencing project, and the subsequent reduction in the cost of genotyping on a genomic scale, evaluating diversity in dogs has become much more accurate and accessible. This provides a potential tool for advising dog breeders and developing breeding programs within a breed. A challenge in doing this is to present complex relationship data in a form that can be readily utilized. Here, we demonstrate the use of a pipeline, known as NetView, to visualize the network of relationships in a subpopulation of German Shepherd Dogs

Roan, ticked and clear coat patterns in the canine are associated with three haplotypes near usherin on CFA38

White coat patterning is a feature of many dog breeds and is known to be coded primarily by the gene micropthalmia-associated transcription factor (MITF). This patterning in the coat can be modified by other factors to produce the attractive phenotypes termed ‘ticked’ and ‘roan’ that describe the presence of flecks of color that vary in distribution and intensity within otherwise ‘clear’ white markings. The appearance of the pigment in the white patterning caused by ticking and roaning intensifies in the weeks after birth. We applied genome-wide association to compare English Cocker Spaniels of roan phenotype (N = 34) with parti-color (non-roan) English Cocker Spaniels (N = 9) and identified an associated locus on CFA 38, CFA38:11 057 040 (Praw = 8.9 × 10−10, Pgenome = 2.7 × 10−5). A local case–control association in English Springer Spaniels comparing 11 ticked and six clear dogs identified indicative association with a different haplotype, CFA38:11 122 467G>T (Praw = 1.7 × 10−5) and CFA38:11 124 294A>C (Praw = 1.7 × 10−5). We characterize three haplotypes in Spaniels according to their putative functional variant profiles at CFA38:11 111 286C>T (missense), CFA38:11 131 841–11 143 239DUP.insTTAA (using strongly linked marker CFA38:11 143 243C>T) and CFA38:11 156 425T>C (splice site). In Spaniels, the haplotypes work as an allelic series including alleles (t, recessive clear; T, dominant ticked/parti-color; and TR, incomplete dominant roan) to control the appearance of pigmented spots or flecks in otherwise white areas of the canine coat. In Spaniels the associated haplotypes are t (CCT), T (TCC) and TR (TTT) for SNP markers on CFA38 at 11 111 286C>T, 11 143 243C>T and 11 156 425T>C respectively. It is likely that other alleles exist in this series and together the haplotypes result in a complex range of patterning that is only visible when dogs have white patterning resulting from the epistatic gene Micropthalmia-associated transcription factor (the S-locus)

Genome-Wide Association Analysis for Chronic Superficial Keratitis in the Australian Racing Greyhound

Chronic superficial keratitis (CSK) is a progressive inflammatory condition of the eye (cornea) that can cause discomfort and blindness. Differential disease risk across dog breeds strongly suggests that CSK has a genetic basis. In addition to genetic risk, the occurrence of CSK is exacerbated by exposure to ultraviolet light. Genome-wide association analysis considered 109 greyhounds, 70 with CSK and the remainder with normal phenotype at an age over four years. Three co-located variants on CFA18 near the 5′ region of the Epidermal Growth Factor Receptor (EGFR) gene were associated with genome-wide significance after multiple-test correction (BICF2P579527, CFA18: 6,068,508, praw = 1.77 × 10−7, pgenome = 0.017; BICF2P1310662, CFA18: 6,077,388, praw = 4.09 × 10−7, pgenome = 0.040; BICF2P160719, CFA18: 6,087,347, praw = 4.09 × 10−7, pgenome = 0.040) (canFam4)). Of the top 10 associated markers, eight were co-located with the significantly associated markers on CFA18. The associated haplotype on CFA18 is protective for the CSK condition. EGFR is known to play a role in corneal healing, where it initiates differentiation and proliferation of epithelial cells that in turn signal the involvement of stromal keratocytes to commence apoptosis. Further validation of the putative functional variants is required prior to their use in genetic testing for breeding programs