Purpose Three related male English Cocker Spaniels (ECS) were reported to be congenitally blind. Examination of one of these revealed complete retinal detachment. A presumptive diagnosis of retinal dysplasia (RD) was provided and pedigree analysis was suggestive of an X-linked mode of inheritance. We sought to investigate the genetic basis of RD in this family of ECS. Methods Following whole genome sequencing (WGS) of the one remaining male RD-affected ECS, two distinct investigative approaches were employed: a candidate gene approach and a whole genome approach. In the candidate gene approach, COL9A2 , COL9A3 , NHEJ1 , RS1 and NDP genes were investigated based on their known associations with RD and retinal detachment in dogs and humans. In the whole genome approach, affected WGS was compared with 814 unaffected canids to identify candidate variants, which were filtered based on appropriate segregation and predicted pathogenic effects followed by subsequent investigation of gene function. Candidate variants were tested for appropriate segregation in the ECS family and association with disease was assessed using samples from a total of 180 ECS. Results The same variant in NDP (c.653_654insC, p.Met114Hisfs*16) that was predicted to result in 15 aberrant amino acids before a premature stop in norrin protein, was identified independently by both approaches and was shown to segregate appropriately within the ECS family. Association of this variant with X-linked RD was significant (P = 0.0056). Conclusions For the first time, we report a variant associated with canine X-linked RD. NDP variants are already known to cause X-linked RD, along with other abnormalities, in human Norrie disease. Thus, the dog may serve as a useful large animal model for research.

Canine progressive retinal atrophy (PRA) describes a group of hereditary diseases characterized by photoreceptor cell death in the retina, leading to visual impairment. Despite the identification of multiple PRA-causing variants, extensive heterogeneity of PRA is observed across and within dog breeds, with many still genetically unsolved. This study sought to elucidate the causal variant for a distinct form of PRA in the Shetland sheepdog, using a whole-genome sequencing approach. Filtering variants from a single PRA-affected Shetland sheepdog genome compared to 176 genomes of other breeds identified a single nucleotide variant in exon 11 of the Bardet–Biedl syndrome-2 gene (BBS2) (c.1222G>C; p.Ala408Pro). Genotyping 1386 canids of 155 dog breeds, 15 cross breeds and 8 wolves indicated the c.1222G>C variant was only segregated within Shetland sheepdogs. Out of 505 Shetland sheepdogs, seven were homozygous for the variant. Clinical history and photographs for three homozygotes indicated the presence of a novel phenotype. In addition to PRA, additional clinical features in homozygous dogs support the discovery of a novel syndromic PRA in the breed. The development and utilization of a diagnostic DNA test aim to prevent the mutation from becoming more prevalent in the breed.

Pigment-associated deafness is a common hereditary condition in a range of dog breeds. The aim of this study was to perform a genome-wide association analysis to investigate the genetic architecture of deafness in Australian Cattle Dogs. Genotypes for 104 757 polymorphisms in 216 dogs were available for analyses after quality control. A genomic relationship matrix was used in the mixed model analyses to account for polygenic effects, as we tested each polymorphism for its association with deafness, in a case/control experimental design. Three approaches were used to code the genotypes and test for additive, recessive and dominant SNP effects. The genome-wide association study analyses identified a clear association peak on CFA20, with the most significant SNPs on this chromosome (1.29 × 10-4 ) in the vicinity of MITF. Variants in MITF have been associated with white pigmentation in dogs and with deafness in humans and other species, supporting the premise that canine deafness is associated with variants in or near this gene. A recessive inheritance for the peak in CFA20 is possible given the significant results in the recessive model; however, the estimated heritability was low (4.54 × 10-5 ). Further validation, identification of variants and testing in other dog breeds are needed.

Abstract Background Hereditary sensory and autonomic neuropathies (HSANs) are a group of genetic disorders affecting the peripheral nervous system. Two different associated variants have been identified in dogs: 1 in Border Collies and 1 in Spaniels and Pointers. Objectives Clinically and genetically characterize HSAN in a family of mixed breed dogs. Animals Five 7‐month‐old mixed breed dogs from 2 related litters were presented for evaluation of a 2‐month history of acral mutilation and progressive pelvic limb gait abnormalities. Methods Complete physical, neurological, electrodiagnostic, and histopathological evaluations were performed. Whole genome sequencing of 2 affected dogs (1 from each litter) was used to identify variants that were homozygous or heterozygous in both cases, but wild type in 217 control genomes of 100 breeds. Immunohistochemistry was used to assess protein expression. Results Complete physical, neurological, electrodiagnostic, and histopathological evaluations confirmed a disorder affecting sensory and autonomic nerves. Whole genome sequencing identified a missense variant in the RETREG1 (reticulophagy regulator 1) gene (c.656C > T, p.P219L). All affected dogs were homozygous for the variant, which was not detected in 1193 dogs from different breeds. Immunohistochemistry showed no expression of RETREG1 in the cerebellum of affected dogs. One of the affected dogs lived for 5 years and showed gradual progression of the clinical signs. Conclusions and Clinical Importance We confirmed the diagnosis of HSAN in a family of mixed breed dogs and identified a novel and possibly pathogenic RETREG1 variant. Affected dogs experienced gradual deterioration over several years.

Purpose Canine primary closed-angle glaucoma (PCAG) is a complex disease caused by multiple genetic factors. A c.590G>A variant in OLFML3 was recently reported to be a candidate for pectinate ligament abnormality (PLA) and PCAG in the Border Collie. We investigated the association of this variant with PLA and PCAG in Border Collies from the United Kingdom. Methods The OLFML3 variant was genotyped in 106 Border Collies comprising 90 with normal eyes (controls) and 16 with PLA (n = 11) and/or PCAG (n = 5) (cases). Genotyping was performed in an additional 103 Border Collies to estimate variant frequency within the population. To investigate the association of the variant with disease in other breeds, genotyping was performed in 337 non-Border Collies with PLA and/or PCAG. Results Of the 90 controls, 71 were homozygous for the wild-type allele, two were homozygous for the variant, and 17 were heterozygous. Of the 16 cases, three were homozygous for the wild-type allele, 11 were homozygous for the variant, and two were heterozygous. The association of the variant allele with disease was significant (P = 1.1 x 10(-9)). We estimated the frequency of this variant to be 4.4% within the United Kingdom Border Collie population, and it was not identified in clinically affected dogs of any other breed. Conclusions This study confirms the association of the OLFML3 variant with PLA and PCAG in Border Collies from the United Kingdom. DNA testing for the variant and selective breeding can reasonably be expected to result in a reduction of PLA and PCAG prevalence in the breed.

Abstract Background Canine progressive retinal atrophies are a group of hereditary retinal degenerations in dogs characterised by depletion of photoreceptor cells in the retina, which ultimately leads to blindness. PRA in the Lhasa Apso (LA) dog has not previously been clinically characterised or described in the literature, but owners in the UK are advised to have their dog examined through the British Veterinary Association/ Kennel Club/ International Sheep Dog Society (BVA/KC/ISDS) eye scheme annually, and similar schemes that are in operation in other countries. After the exclusion of 25 previously reported canine retinal mutations in LA PRA-affected dogs, we sought to identify the genetic cause of PRA in this breed. Results Analysis of whole-exome sequencing data of three PRA-affected LA and three LA without signs of PRA did not identify any exonic or splice site variants, suggesting the causal variant was non-exonic. We subsequently undertook a genome-wide association study (GWAS), which identified a 1.3 Mb disease-associated region on canine chromosome 33, followed by whole-genome sequencing analysis that revealed a long interspersed element-1 (LINE-1) insertion upstream of the IMPG2 gene. IMPG2 has previously been implicated in human retinal disease; however, until now no canine PRAs have been associated with this gene. The identification of this PRA-associated variant has enabled the development of a DNA test for this form of PRA in the breed, here termed PRA4 to distinguish it from other forms of PRA described in other breeds. This test has been used to determine the genotypes of over 900 LA dogs. A large cohort of genotyped dogs was used to estimate the allele frequency as between 0.07–0.1 in the UK LA population. Conclusions Through the use of GWAS and subsequent sequencing of a PRA case, we have identified a LINE-1 insertion in the retinal candidate gene IMPG2 that is associated with a form of PRA in the LA dog. Validation of this variant in 447 dogs of 123 breeds determined it was private to LA dogs. We envisage that, over time, the developed DNA test will offer breeders the opportunity to avoid producing dogs affected with this form of PRA.

Congenital deafness in the domestic dog is usually related to the presence of white pigmentation, which is controlled primarily by the piebald locus on chromosome 20 and also by merle on chromosome 10. Pigment-associated deafness is also seen in other species, including cats, mice, sheep, alpacas, horses, cows, pigs, and humans, but the genetic factors determining why some piebald or merle dogs develop deafness while others do not have yet to be determined. Here we perform a genome-wide association study (GWAS) to identify regions of the canine genome significantly associated with deafness in three dog breeds carrying piebald: Dalmatian, Australian cattle dog, and English setter. We include bilaterally deaf, unilaterally deaf, and matched control dogs from the same litter, phenotyped using the brainstem auditory evoked response (BAER) hearing test. Principal component analysis showed that we have different distributions of cases and controls in genetically distinct Dalmatian populations, therefore GWAS was performed separately for North American and UK samples. We identified one genome-wide significant association and 14 suggestive (chromosome-wide) associations using the GWAS design of bilaterally deaf vs. control Australian cattle dogs. However, these associations were not located on the same chromosome as the piebald locus, indicating the complexity of the genetics underlying this disease in the domestic dog. Because of this apparent complex genetic architecture, larger sample sizes may be needed to detect the genetic loci modulating risk in piebald dogs.

Canine progressive retinal atrophies (PRA) are genetically heterogeneous diseases characterized by retinal degeneration and subsequent blindness. PRAs are untreatable and affect multiple dog breeds, significantly impacting welfare. Three out of seven Giant Schnauzer (GS) littermates presented with PRA around four years of age. We sought to identify the causal variant to improve our understanding of the aetiology of this form of PRA and to enable development of a DNA test. Whole genome sequencing of two PRA-affected full-siblings and both unaffected parents was performed. Variants were filtered based on those segregating appropriately for an autosomal recessive disorder and predicted to be deleterious. Successive filtering against 568 canine genomes identified a single nucleotide variant in the gene encoding NECAP endocytosis associated 1 (NECAP1): c.544G>A (p.Gly182Arg). Five thousand one hundred and thirty canids of 175 breeds, 10 cross-breeds and 3 wolves were genotyped for c.544G>A. Only the three PRA-affected GS were homozygous (allele frequency in GS, excluding proband family = 0.015). In addition, we identified heterozygotes belonging to Spitz and Dachshund varieties, demonstrating c.544G>A segregates in other breeds of German origin. This study, in parallel with the known retinal expression and role of NECAP1 in clathrin mediated endocytosis (CME) in synapses, presents NECAP1 as a novel candidate gene for retinal degeneration in dogs and other species.

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Seven Northern Inuit Dogs (NID) were diagnosed by pedigree analysis with an autosomal recessive inherited oculoskeletal dysplasia (OSD). Short-limbed dwarfism, angular limb deformities and a variable combination of macroglobus, cataracts, lens coloboma, microphakia and vitreopathy were present in all seven dogs, while retinal detachment was diagnosed in five dogs. Autosomal recessive OSD caused by COL9A3 and COL9A2 mutations have previously been identified in the Labrador Retriever (dwarfism with retinal dysplasia 1—drd1) and Samoyed dog (dwarfism with retinal dysplasia 2—drd2) respectively; both of those mutations were excluded in all affected NID. Nine candidate genes were screened in whole genome sequence data; only one variant was identified that was homozygous in two affected NID but absent in controls. This variant was a nonsense single nucleotide polymorphism in COL9A3 predicted to result in a premature termination codon and a truncated protein product. This variant was genotyped in a total of 1,232 dogs. All seven affected NID were homozygous for the variant allele (T/T), while 31/116 OSD-unaffected NID were heterozygous for the variant (C/T) and 85/116 were homozygous for the wildtype allele (C/C); indicating a significant association with OSD (p = 1.41x10-11). A subset of 56 NID unrelated at the parent level were analysed to determine an allele frequency of 0.08, estimating carrier and affected rates to be 15% and 0.6% respectively in NID. All 1,109 non-NID were C/C, suggesting the variant is rare or absent in other breeds. Expression of retinal mRNA was similar between an OSD-affected NID and OSD-unaffected non-NID. In conclusion, a nonsense variant in COL9A3 is strongly associated with OSD in NID, and appears to be widespread in this breed.

Four full-sibling intact male Miniature Poodles were evaluated at 4-19 months of age. One was clinically normal and three were affected. All affected dogs were reluctant to exercise and had generalised muscle atrophy, a stiff gait and a markedly elevated serum creatine kinase activity. Two affected dogs also showed poor development, learning difficulties and episodes of abnormal behaviour. In these two dogs, investigations into forebrain structural and metabolic diseases were unremarkable; electromyography demonstrated fibrillation potentials and complex repetitive discharges in the infraspinatus, supraspinatus and epaxial muscles. Histopathological, immunohistochemical and immunoblotting analyses of muscle biopsies were consistent with dystrophin-deficient muscular dystrophy. DNA samples were obtained from all four full-sibling male Poodles, a healthy female littermate and the dam, which was clinically normal. Whole genome sequencing of one affected dog revealed a >5 Mb deletion on the X chromosome, encompassing the entire DMD gene. The exact deletion breakpoints could not be experimentally ascertained, but we confirmed that this region was deleted in all affected males, but not in the unaffected dogs. Quantitative polymerase chain reaction confirmed all three affected males were hemizygous for the mutant X chromosome, while the wildtype chromosome was observed in the unaffected male littermate. The female littermate and the dam were both heterozygous for the mutant chromosome. Forty-four Miniature Poodles from the general population were screened for the mutation and were homozygous for the wildtype chromosome. The finding represents a naturally-occurring mutation causing dystrophin-deficient muscular dystrophy in the dog.

About this supplement These abstracts have been published as Canine Genetics and Epidemiology Volume 5 Supplement 2, 2018: Selected canine abstracts from the Companion Animal Genetic Health conference 2018 (CAGH 2018): Canine Genetics and Epidemiology. A meeting report from the conference has been published as Canine Genetics and Epidemiology Volume 5 Supplement 1, 2018 and is available online at https://doi.org/10.1186/ s40575-018-0061-0. Selected feline abstracts from the conference have been published as Irish Veterinary Journal Volume 71 Supplement 1, 2018 and are available online at https://doi.org/10.1186/s13620-018-0126-0.

and clinical homologies, with comparable subtypes 2 for which a BRAF-MBP gene fusion in one case using RNAseq and are currently for recurrence in the samples, as well as for the presence of this translocation in human glioma cases. Using affected cases and controls of the same breeds, to the identification of somatic alterations by transcriptome analyzes (RNAseq) and exome sequencing (WES) and to carry out genetic linkage and/or genetic association studies (GWAS) to identify genomic regions involved in predisposition. We will also search if and how the artificial selection that led to specific morphological characteristics, such as the shape of the dog ’ s skull (brachycephaly), would have also led to glioma predisposition. improvement in these traits, which is particularly important for HEART – heart conditions are believed to affect as many as 20% of Dobermans, and the symptoms of the disease often appear after a dog has already been used for breeding. Further, significant estimates obtained in the presented analyses indicate validity of the data, thus opening a new window of opportunity for genetic analyses of complex traits in numerically small breeds through the recruitment and collation of data by breed enthusiasts. each dog was measured at the withers. We used linear per allele re- gression to assess the association between ADAMTS17 mutations and height as a continuous variable, and linear regression and log- likelihood ratio tests to assess the shape of the association by comparing a general model with a linear per allele model. The mean heights of affected (n=21), carrier (n=84) and clear (n=122) Petit Basset Griffon Vendeen were 33.41 cm, 34.78 cm and 34.93 cm, respect- ively. The mean heights of affected (n=9), carrier (n=30) and clear (n=26) Shar Pei were 43.32 cm, 47.93 cm and 48.38 cm, respectively. Each breed-specific ADAMTS17 mutation showed a strong association with height in both breeds: Petit Basset Griffon Vendeen (P=7.9 x 10-3); Shar Pei (P=6.9 x 10-5). The shape of the associations ap-peared similar between the two breeds. In humans, ADAMTS17 af- fects skeletal development by modulating the extracellular matrix. A similar mechanism may be present in the dog. We speculate that se- lection for short stature might have inadvertently increased ADAMTS17 mutant allele frequencies and thus increased prevalence of primary open angle glaucoma in these breeds. and Epidemiology 2018, 5(Suppl 2): O5 In domestic dogs, the “ flat-faced ” brachycephalic head shape is a risk factor for developing the respiratory defect, Brachycephalic Obstructive Airway Syndrome (BOAS). As the popularity of breeds such as the French bulldog continues to increase in the UK, so too are the expected incidences of BOAS. For this reason, we be- came interested in the Norwich terrier, a non- brachycephalic breed which presents with Upper Airway Syndrome (UAS), a con- dition highly reminiscent of BOAS. Here, we have studied this single breed to identify genetic association(s) with UAS. Pathological assessments and grading from laryngoscopic examinations held at the Vetsuisse Faculty of the University of Bern, were used as phenotypes in conjunction with microarray genotypes to perform GWAS. In total, 233 Norwich terriers were examined. We identi- fied the same QTL on canine chromosome (CFA) 13 to be associated with the abnormal positioning of laryngeal cartilage and everted saccules in the dogs most severely affected by UAS. We phased genotypes at the CFA13 QTL to conduct haplotype map- ping, which led us to define a 413 kb critical interval which en-compasses a single positional candidate gene. The derived haplotype within this interval is overrepresented: it is found to be homozygous in 61 of 81 (74%) severely affected cases. In con- trast, this homozygous haplotype was identified among 7 of 86 (8.1%) mild/unaffected controls. We have resequenced four dogs representing phenotypic extremes to sixteen-fold depth to identify putatively causal variants. We will provide an update to this ongoing project, which is expected to guide Norwich terrier breeding and inspire additional exploration of the CFA13 locus to improve animal welfare. of multiple transcripts with alternative first exons. of multiple transcripts in the cerebellum through RNA sequencing, RT-PCR of samples from two Buhund cases and dogs of other RT-qPCR analysis and in-silico protein modelling have been used to further investigate the mutation ’ s effect on RNA ex- pression and protein stability. Chronic degenerative diseases (CDGs) are a major welfare concern in canine medicine myxomatous mitral valve disease (MMVD) an For an inher- polygenic and the mechanisms that requires examining molecular events in examination in both temporal and spatial terms changes in gene and pro- tein expression. examined the valvular gene expression at different stages of disease (temporal), different loca- tions (spatial) and in different cell culture models of MMVD. identified as the fundamen- tally important pathway in MMVD initiation and development, and progression to eventual end-stage valve pathology. stringent filtering and quality control, we tested 108,263 SNPs on 42 dogs, comprising 15 cases and 27 controls (call rate ≥ 97%; minor allele frequency ≥ 95%; genotype calls ≥ 90%). Analysis revealed a genome-wide significant association on canine chromosome 33 (-log praw = 2.2 x 10-16) which remained significant after correcting for multiple testing (pgenome = 0.9 x 10-5) and population substructure (p=raw1.6 x 10-17). A 1.3 megabase homozygous disease-associated region was defined, harbouring two candidate genes previously asso- ciated with human retinal degeneration. WGS was undertaken on a single PRA affected LA, and manual inter- rogation of the critical region in identified a long interspersed nu-clear element-1 (LINE-1) insertion, situated within the predicted promotor region of a retinal candidate gene. Due to the position of the LINE-1 insertion, it was not detected in the original WES data of the same case. The LINE-1 insertion was genotyped in 447 dogs across 122 breeds, including 63 LA dogs, and is private to the LA. Seventeen LA dogs (all clinically affected with PRA) were homozygous for the LINE-1 insertions, eight were heterozygous and thirty- eight were homozygous for the wildtype allele. As a result of this study a DNA test for this form of PRA, termed PRA4, has been developed at the Animal Health Trust. To date, 457 LA from 15 countries have been tested for PRA4 (354 UK dogs; car- rier frequency 17%; allele frequency 0.9054). This study highlights the power of utilising several genetic approaches to identify a PRA mutation and develop a diagnostic test to help dog breeders make informed breeding choices, minimising the risk of producing PRA- affected LA dogs. A point mutation in a lincRNA upstream of GDNF is associated with a canine insensitivity to pain: a spontaneous model for human sensory neuropathies. Plos Genetics 2016 Biomedical Variant Database Consortium and the Animal Health Trust Give a Dog a Genome bank) reduced an initial set of > 20 million variants down to a single candidate variant in a novel gene not previously associated with ret- inal degeneration in any species. The candidate variant was genotyped in a total of 1,444 dogs of 175 breeds, 10 cross breed dogs and 3 wolves, with our three PRA-affected Giant Schnauzers being the only homozygotes identified to date. Nine Giant Schnauzer heterozygotes were identified in addition to heterozygotes in three additional breeds of German origin, including the German Giant (Gross) and Medium (Mittel) Spitz and Miniature Longhaired Dachs-hund (MLHD). The genotyping of German Spitz varieties, including 110 Giant Spitz, 21 Medium Spitz, 24 Miniature (Klein) Spitz, 17 Pomeranian (Zwerg) Spitz, and an additional 27 Giant Schnauzers was carried out by collaborators at the University of Bern. We screened a total of 163 MLHD for the candidate variant. Seven German Giant Spitz, one German Medium Spitz and six MLHD heterozygotes were identified, suggesting this may be an ancestral, but rare, mutation. This study highlights the power of using WGS to identify novel genes associated with disease using a very small number of cases. This novel candidate gene, harbouring a variant that is predicted to be the causal mutation of PRA in the Giant Schnauzer, could provide in- sights into gene discoveries in human retinal degenerations. Further functional study options are being explored to confirm the candidate gene ’ s role in retinal function and maintenance. identify the genetic causes or predisposition to canine and human homologous genetic conditions. More recently, the idea emerged that dogs can be of help for therapeutic trials, to screen and validate new drugs in homologous human cancer types. To this aim, we created the canine Cani-DNA biobank in 2003 to col-lect blood and tissue samples from dogs affected with genetic dis- eases and healthy dogs. Cani-DNA contains the native samples and extracted nucleic acids as well as clinical and genealogical data, to distribute samples with high quality controlled procedures. This resource is implemented by a French veterinarian network based on vet practic- es, specialized vet centres and histopathology laborator-ies. In 2012, a contract signed between Cani-DNA and the 4 French vet schools and the Antagene Company allowed a national organization and international visibility. Cani-DNA joined the French consortium of domesticated animals, CRB-Anim (funded by French government 2012-2020), aiming to combine genetic and reproduct- ive resources. To date, Cani-DNA, with its primary site at CNRS Rennes and secondary sites at the fourth Vet Schools and Antagene contains almost 20 000 DNA extracted from blood and 3000 nucleic acids (DNA, RNA) extracted from tissue samples (tumoral and con- trols), representing 300 breeds and over 100 genetic diseases. Samples can be sent to implement Can

The advent of whole genome sequencing (WGS) has promised to revolutionise genetic research, and the rapid fall in per-sample costs in recent years has made the revolution an affordable reality for geneticists. The technology is especially useful for the study of simple Mendelian conditions where disease-causing mutations have the potential to be identified from the WGS of a single case. However, when comparing a typical canine genome with the reference sequence (CanFam3.1) or a control genome, at least 2-3 million variants will typically be identified. Many of these variants are likely common polymorphisms which could be excluded by comparing with multiple control genomes. We devised the Give a Dog a Genome (GDG) project to build a resource of canine genetic variants across the genome using WGS; currently projected to contain 90 genomes from 78 breeds, and investigate genetic diseases in at least 69 breeds. We used a crowd-funding approach, with the costs of the project being shared between multiple stakeholders. To date (two years after GDG was launched), 74 samples from 69 breeds have been sequenced comprising 62 dogs affected with a suspected genetic condition (27 conditions in total) and 12 apparently healthy older dogs. The GDG variant bank has been used to validate several disease-associated mutations and DNA tests have been developed to improve the health and wellbeing of dogs. All of the WGS data generated through GDG will be shared with the Dog Biomedical Variant Database Consortium (DBVDC), and specific sequences will be shared with at least 20 scientists from Europe and the USA to contribute to their research.