Improving the resolution of canine genome-wide association studies using genotype imputation: A study of two breeds.

Genotype imputation using a reference panel that combines high-density array data and publicly available whole genome sequence consortium variant data is potentially a cost-effective method to increase the density of extant lower-density array datasets. In this study, three datasets (two Border Collie; one Italian Spinone) generated using a legacy array (Illumina CanineHD, 173 662 SNPs) were utilised to assess the feasibility and accuracy of this approach and to gather additional evidence for the efficacy of canine genotype imputation. The cosmopolitan reference panels used to impute genotypes comprised dogs of 158 breeds, mixed breed dogs, wolves and Chinese indigenous dogs, as well as breed-specific individuals genotyped using the Axiom Canine HD array. The two Border Collie reference panels comprised 808 individuals including 79 Border Collies and 426 326 or 426 332 SNPs; and the Italian Spinone reference panel comprised 807 individuals including 38 Italian Spinoni and 476 313 SNPs. A high accuracy for imputation was observed, with the lowest accuracy observed for one of the Border Collie datasets (mean R2  = 0.94) and the highest for the Italian Spinone dataset (mean R2  = 0.97). This study's findings demonstrate that imputation of a legacy array study set using a reference panel comprising both breed-specific array data and multi-breed variant data derived from whole genomes is effective and accurate. The process of canine genotype imputation, using the valuable growing resource of publicly available canine genome variant datasets alongside breed-specific data, is described in detail to facilitate and encourage use of this technique in canine genetics.

Characterisation of canine KCNIP4: A novel gene for cerebellar ataxia identified by whole-genome sequencing two affected Norwegian Buhund dogs.

A form of hereditary cerebellar ataxia has recently been described in the Norwegian Buhund dog breed. This study aimed to identify the genetic cause of the disease. Whole-genome sequencing of two Norwegian Buhund siblings diagnosed with progressive cerebellar ataxia was carried out, and sequences compared with 405 whole genome sequences of dogs of other breeds to filter benign common variants. Nine variants predicted to be deleterious segregated among the genomes in concordance with an autosomal recessive mode of inheritance, only one of which segregated within the breed when genotyped in additional Norwegian Buhunds. In total this variant was assessed in 802 whole genome sequences, and genotyped in an additional 505 unaffected dogs (including 146 Buhunds), and only four affected Norwegian Buhunds were homozygous for the variant. The variant identified, a T to C single nucleotide polymorphism (SNP) (NC_006585.3:g.88890674T>C), is predicted to cause a tryptophan to arginine substitution in a highly conserved region of the potassium voltage-gated channel interacting protein KCNIP4. This gene has not been implicated previously in hereditary ataxia in any species. Evaluation of KCNIP4 protein expression through western blot and immunohistochemical analysis using cerebellum tissue of affected and control dogs demonstrated that the mutation causes a dramatic reduction of KCNIP4 protein expression. The expression of alternative KCNIP4 transcripts within the canine cerebellum, and regional differences in KCNIP4 protein expression, were characterised through RT-PCR and immunohistochemistry respectively. The voltage-gated potassium channel protein KCND3 has previously been implicated in spinocerebellar ataxia, and our findings suggest that the Kv4 channel complex KCNIP accessory subunits also have an essential role in voltage-gated potassium channel function in the cerebellum and should be investigated as potential candidate genes for cerebellar ataxia in future studies in other species.

Phenotypic Effects of FGF4 Retrogenes on Intervertebral Disc Disease in Dogs.

Two FGF4 retrogenes on chromosomes 12 (12-FGF4RG) and 18 (18-FGF4RG) contribute to short-limbed phenotypes in dogs. 12-FGF4RG has also been associated with intervertebral disc disease (IVDD). Both of these retrogenes were found to be widespread among dog breeds with allele frequencies ranging from 0.02 to 1; however, their additive contribution to disease is unknown. Surgical cases of IVDD (n = 569) were evaluated for age of onset, disc calcification, and genotypes for the FGF4 retrogenes. Multivariable linear regression analysis identified the presence of one or two copies of 12-FGF4RG associated with significantly younger age at first surgery in a dominant manner. 18-FGF4RG had only a minor effect in dogs with one copy. Multivariable logistic regression showed that 12-FGF4RG had an additive effect on radiographic disc calcification, while 18-FGF4RG had no effect. Multivariable logistic regression using mixed breed cases and controls identified only 12-FGF4RG as highly associated with disc herniation in a dominant manner (Odds Ratio, OR, 18.42, 95% Confidence Interval (CI) 7.44 to 50.26; P < 0.001). The relative risk for disc surgery associated with 12-FGF4RG varied from 5.5 to 15.1 within segregating breeds and mixed breeds. The FGF4 retrogene on CFA12 acts in a dominant manner to decrease the age of onset and increase the overall risk of disc disease in dogs. Other modifiers of risk may be present within certain breeds, including the FGF4 retrogene on CFA18.

Hereditary ataxia in four related Norwegian Buhunds.

CASE DESCRIPTION Two 12-week-old Norwegian Buhunds from a litter of 5 were evaluated because of slowly progressive cerebellar ataxia and fine head tremors. Two other females from the same pedigree had been previously evaluated for similar signs. CLINICAL FINDINGS Findings of general physical examination, CBC, and serum biochemical analysis were unremarkable for all affected puppies. Brain MRI and CSF analysis, including PCR assays for detection of Toxoplasma gondii, Neospora caninum, and canine distemper virus, were performed for 3 dogs, yielding unremarkable results. Urinary organic acid screening, enzyme analysis of fibroblasts cultured from skin biopsy specimens, and brainstem auditory-evoked response testing were performed for 2 puppies, and results were also unremarkable. TREATMENT AND OUTCOME The affected puppies were euthanized at the breeder's request, and their brains and spinal cords were submitted for histologic examination. Histopathologic findings included a markedly reduced expression of calbindin D28K and inositol triphosphate receptor 1 by Purkinje cells, with only mild signs of neuronal degeneration. Results of pedigree analysis suggested an autosomal recessive mode of inheritance. Candidate-gene analysis via mRNA sequencing for 2 of the affected puppies revealed no genetic variants that could be causally associated with the observed abnormalities. CLINICAL RELEVANCE Findings for the dogs of this report suggested the existence of a hereditary form of ataxia in Norwegian Buhunds with histologic characteristics suggestive of Purkinje cell dysfunction. The presence of hereditary ataxia in this breed must be considered both in clinical settings and for breeding strategies.

Ageing-associated DNA methylation dynamics are a molecular readout of lifespan variation among mammalian species.

BACKGROUND: Mammalian species exhibit a wide range of lifespans. To date, a robust and dynamic molecular readout of these lifespan differences has not yet been identified. Recent studies have established the existence of ageing-associated differentially methylated positions (aDMPs) in human and mouse. These are CpG sites at which DNA methylation dynamics show significant correlations with age. We hypothesise that aDMPs are pan-mammalian and are a dynamic molecular readout of lifespan variation among different mammalian species. RESULTS: A large-scale integrated analysis of aDMPs in six different mammals reveals a strong negative relationship between rate of change of methylation levels at aDMPs and lifespan. This relationship also holds when comparing two different dog breeds with known differences in lifespans. In an ageing cohort of aneuploid mice carrying a complete copy of human chromosome 21, aDMPs accumulate far more rapidly than is seen in human tissues, revealing that DNA methylation at aDMP sites is largely shaped by the nuclear trans-environment and represents a robust molecular readout of the ageing cellular milieu. CONCLUSIONS: Overall, we define the first dynamic molecular readout of lifespan differences among mammalian species and propose that aDMPs will be an invaluable molecular tool for future evolutionary and mechanistic studies aimed at understanding the biological factors that determine lifespan in mammals.

Evaluation of ADAMTS17 in Chinese Shar-Pei with primary open-angle glaucoma, primary lens luxation, or both.

OBJECTIVE To evaluate the coding regions of ADAMTS17 for potential mutations in Chinese Shar-Pei with a diagnosis of primary open-angle glaucoma (POAG), primary lens luxation (PLL), or both. ANIMALS 63 Shar-Pei and 96 dogs of other breeds. PROCEDURES ADAMTS17 exon resequencing was performed on buccal mucosal DNA from 10 Shar-Pei with a diagnosis of POAG, PLL, or both (affected dogs). A candidate causal variant sequence was identified, and additional dogs (53 Shar-Pei [11 affected and 42 unaffected] and 95 dogs of other breeds) were genotyped for the variant sequence by amplified fragment length polymorphism analysis. Total RNA was extracted from ocular tissues of 1 affected Shar-Pei and 1 ophthalmologically normal Golden Retriever; ADAMTS17 cDNA was reverse transcribed and sequenced, and ADAMTS17 expression was evaluated by quantitative reverse-transcription PCR assay. RESULTS All affected Shar-Pei were homozygous for a 6-bp deletion in exon 22 of ADAMTS17 predicted to affect the resultant protein. All unaffected Shar-Pei were heterozygous or homozygous for the wild-type allele. The variant sequence was significantly associated with affected status (diagnosis of POAG, PLL, or both). All dogs of other breeds were homozygous for the wild-type allele. The cDNA sequencing confirmed presence of the expected variant mRNA sequence in ocular tissue from the affected dog only. Gene expression analysis revealed a 4.24-fold decrease in the expression of ADAMTS17 in ocular tissue from the affected dog. CONCLUSIONS AND CLINICAL RELEVANCE Results supported that the phenotype (diagnosis of POAG, PLL, or both) is an autosomal recessive trait in Shar-Pei significantly associated with the identified mutation in ADAMTS17.

An Inversion Disrupting FAM134B Is Associated with Sensory Neuropathy in the Border Collie Dog Breed.

Sensory neuropathy in the Border Collie is a severe neurological disorder caused by the degeneration of sensory and, to a lesser extent, motor nerve cells with clinical signs starting between 2 and 7 months of age. Using a genome-wide association study approach with three cases and 170 breed matched controls, a suggestive locus for sensory neuropathy was identified that was followed up using a genome sequencing approach. An inversion disrupting the candidate gene FAM134B was identified. Genotyping of additional cases and controls and RNAseq analysis provided strong evidence that the inversion is causal. Evidence of cryptic splicing resulting in novel exon transcription for FAM134B was identified by RNAseq experiments. This investigation demonstrates the identification of a novel sensory neuropathy associated mutation, by mapping using a minimal set of cases and subsequent genome sequencing. Through mutation screening, it should be possible to reduce the frequency of or completely eliminate this debilitating condition from the Border Collie breed population.

Genome sequencing reveals a splice donor site mutation in the SNX14 gene associated with a novel cerebellar cortical degeneration in the Hungarian Vizsla dog breed.

BACKGROUND: Cerebellar cortical degeneration (CCD) is an increasingly recognised neurodegenerative disease process affecting many dog breeds. Typical presentation consists of a progressive cerebellar ataxia, with a variable age at onset and rate of progression between different breeds. Cerebellar histopathological findings typically consist of primary Purkinje neuronal degeneration and loss, with variable secondary depletion of the granular and molecular cell layers. Causative genes have been identified associated with CCD in several breeds, allowing screening for selective breeding to reduce the prevalence of these conditions. There have been no previous reports of CCD in Hungarian Vizslas. RESULTS: Two full-sibling Hungarian Vizsla puppies from a litter of nine presented with a history of progressive ataxia, starting around three months of age. Clinical signs included marked hypermetric and dysmetric ataxia, truncal sway, intention tremors and absent menace responses, with positional horizontal nystagmus in one dog. Routine diagnostic investigations were unremarkable, and magnetic resonance imaging performed in one dog revealed mild craniodorsal cerebellar sulci widening, supportive of cerebellar atrophy. Owners of both dogs elected for euthanasia shortly after the onset of signs. Histopathological examination revealed primary Purkinje neuron loss consistent with CCD. Whole genome sequencing was used to successfully identify a disease-associated splice donor site variant in the sorting nexin 14 gene (SNX14) as a strong causative candidate. An altered SNX14 splicing pattern for a CCD case was demonstrated by RNA analysis, and no SNX14 protein could be detected in CCD case cerebellum by western blotting. SNX14 is involved in maintaining normal neuronal excitability and synaptic transmission, and a mutation has recently been found to cause autosomal recessive cerebellar ataxia and intellectual disability syndrome in humans. Genetic screening of 133 unaffected Hungarian Vizslas revealed the presence of three heterozygotes, supporting the presence of carriers in the wider population. CONCLUSIONS: This is the first report of CCD in Hungarian Vizsla dogs and identifies a highly associated splice donor site mutation in SNX14, with an autosomal recessive mode of inheritance suspected.

Identification of a novel frameshift mutation in the DMD gene as the cause of muscular dystrophy in a Norfolk terrier dog.

BACKGROUND: A Norfolk terrier was referred to the Animal Health Trust neurology department with suspected dystrophin-deficient muscular dystrophy (DD-MD), which was confirmed by clinical workup and immunohistochemistry. FINDINGS: Exon resequencing of the canine Duchenne Muscular Dystrophy (DMD) gene was undertaken to screen for potential disease causing mutations. The sequence data generated from all coding DMD exons revealed a 1 bp deletion in exon 22, causing a frameshift and premature termination of the coding sequence. Gene expression analysis indicated reduced levels of dystrophin transcript in the DD-MD case and western blot confirmed the absence of full length protein. CONCLUSIONS: The finding represents a novel mutation causing DD-MD in the dog.

A novel locus on canine chromosome 13 is associated with cataract in the Australian Shepherd breed of domestic dog.

Hereditary cataract is a common ocular disorder in the purebred dog population and is a leading cause of visual impairment and blindness in dogs. Despite this, little is known to date about the genetics underlying this condition. We have used a genome-wide association study and targeted resequencing approach to identify a novel locus for cataracts in the Australian Shepherd breed of dog, using dogs that are clear of an HSF4 mutation, previously identified as the major susceptibility locus in this breed. Cataract cases were defined as dogs with bilateral posterior cataracts, or bilateral nuclear cataracts. Controls were at least 8 years of age with no evidence of cataracts or other ocular abnormality. Using 15 bilateral posterior polar cataract cases and 68 controls, we identified a genome-wide statistical association for cataracts in the Australian Shepherd on canine chromosome 13 at 46.4 Mb (P value: 1.5 × 10(-7)). We sequenced the 14.16 Mb associated region in ten Australian Shepherds to search for possible causal variants underlying the association signal and conducted additional fine-mapping of the region by genotyping 28 intronic variants that segregated correctly in our ten sequenced dogs. From this analysis, the strongest associated variants were located in intron 5 of the SCFD2 gene. Further study will require analysis of additional cases and controls and ocular tissue from dogs affected with bilateral cataracts that are free of the HSF4 mutation.

Comparison of canine mandibular bone regeneration by distraction osteogenesis versus acute resection and rigid external fixation.

The present study was performed (1) to explore the mechanism of skeletal healing following distraction osteogenesis of the mandible and to evaluate whether the same process is involved following acute mandibular resection and rigid external fixation, and (2) to examine the role of the periosteum in skeletal healing in both models. The study was performed using 16 mongrel dogs divided into two equal groups. In the first group, distraction of 20 mm was performed at a rate of 1 mm/day. In the second group, bone resection of 20 mm was performed, followed by rigid external fixation. The buccal periosteum was stripped in four dogs from each group, and the periosteum was left intact in the remaining four dogs. Dogs were euthanized after a survival period of either 2 or 3 months, and the new bone regenerate was evaluated. Analysis consisted of three-dimensional computed tomography scanning, histometric analysis, and immunostaining. Analysis of bone mineral content in the residual gap was conducted. Bone mineral content was increased in 3- versus 2-month survival for all groups (p < 0.05). The distracted groups had greater bone mineral content than their acutely resected counterparts, with the difference achieving statistical significance by 3-month survival (p < 0.05). Although periosteal preservation resulted in increased bone mineral content over time for all groups (p = 0.044), periosteal preservation had no significant effect on bone mineral content in the distracted groups. After periosteal stripping, however, bone mineral content was significantly increased in dogs that underwent distraction rather than acute resection and rigid external fixation (p = 0.022). Regarding histometric analysis, analysis of fibrous tissue content in the bone regenerate demonstrated that by 3 months the distracted groups had significantly less fibrous tissue in the new bone regenerate than did the acutely resected groups (p < 0.001). Regarding immunostaining, diffuse localization of transforming growth factor-beta1 was observed in all groups at 2 months, returning to nearly baseline levels by 3 months. These data demonstrate that significant bone formation in a segmental gap can be achieved after acute mandibular resection and rigid external fixation if the periosteum is preserved. However, after periosteal injury or stripping, significant bone formation can only be achieved by distraction osteogenesis. In both processes, bone formation is preceded by up-regulation of transforming growth factor-beta1.