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.

Investigation of the allele frequency of the G>A intron 10 ADAMTS17 mutation causing primary lens luxation in the Portuguese Podengo breed.

OBJECTIVE: To establish the allele frequency of the PLL-causing G>A intron 10 ADAMTS17 mutation in the Portuguese Podengo population in the UK and investigate a possible correlation between the mutation and short stature. METHODS: Two groups of dogs (Group 1 and Group 2) were recruited for the purpose of the study. Group 1 (n = 40) consisted of dogs which were genotyped only and Group 2 (n = 42) consisted of dogs which were genotyped, underwent a full ophthalmological examination and also had their height measured at the withers. RESULTS: In Group 1, genotyping for the ADAMTS17:c.1473+1G>A mutation confirmed 1/40 homozygous for the mutated allele (-/-), 7/40 heterozygous for the mutated allele (+/-), and 32/40 homozygous for the wild-type allele (+/+) dogs. In Group 2, genotyping of the dogs confirmed 6/42 heterozygous for the mutated allele (+/-) and homozygous for the wild-type allele (+/+) dogs. In total, 1/82 (1.2%) dogs were confirmed to be homozygous for the mutated allele, 13/82 (15.8%) heterozygous for the mutated allele and 68/82 (83%) homozygous for the wild-type allele. The frequency of the mutated allele across both groups was calculated as 0.09. A statistically significant correlation between the mutation and short stature could not be established (p = .590). CONCLUSIONS: The frequency of the mutation calculated in this study (0.09) is high. Genetic testing should be considered for each dog prior to breeding with a view of selective breeding.

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.

A LINE-1 insertion situated in the promoter of IMPG2 is associated with autosomal recessive progressive retinal atrophy in Lhasa Apso dogs.

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.

A variant in OLFML3 is associated with pectinate ligament abnormality and primary closed-angle glaucoma in Border Collies from the United Kingdom.

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.

Primary closed angle glaucoma in the Basset Hound: Genetic investigations using genome-wide association and RNA sequencing strategies.

Purpose: To investigate the genetic basis of primary closed angle glaucoma (PCAG) in European Basset Hounds using genome-wide association and RNA sequencing strategies. Methods: DNA samples from 119 European Basset Hounds were genotyped on the 170 K SNP CanineHD BeadChip array (Illumina) comprising 37 with normal iridocorneal angles (controls), 57 with pectinate ligament abnormality (PLA cases), and 25 with PCAG (PCAG cases). Genome-wide association studies (GWASs) of the PLA and PCAG cases were conducted. Whole transcriptome sequences of iridocorneal angle tissues from five Basset Hounds with PCAG were compared with those from four dogs with normal eyes to investigate differences in gene expression between the affected and unaffected eyes in GWAS-associated loci. A variant in NEB, previously reported to be associated with PCAG in American Basset Hounds, was genotyped in cohorts of European Basset Hounds and non-Basset Hounds. Results: The GWASs revealed 1.4 and 0.2 Mb regions, on chromosomes 24 and 37, respectively, that are statistically associated with PCAG. The former locus has previously been associated with glaucoma in humans. Whole transcriptome analysis revealed differential gene expression of eight genes within these two loci. The NEB variant was not associated with PLA or PCAG in this set of European Basset Hounds. Conclusions: We identified two novel loci for canine PCAG. Further investigation is required to elucidate candidate variants that underlie canine PCAG.

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.

Investigation of SLA4A3 as a candidate gene for human retinal disease.

SLC4A3 has been shown to cause retinal degeneration in a genetically engineered knockout mouse, and in a naturally occurring form of canine progressive retinal atrophy considered to be the equivalent of retinitis pigmentosa in humans (RP). This study was undertaken to investigate if SLC4A3 coding variants were implicated in human retinal degeneration. SLC4A3 exons were amplified and sequenced in 200 patients with autosomal recessive retinal degeneration who had no known molecular diagnosis for their condition, which included 197 unrelated individuals with suspected RP and three individuals with other forms of retinal disease. Three rare variants were identified that were predicted to be potentially pathogenic, however each variant was heterozygous in a single patient and therefore not considered disease-causing in isolation. Of these three variants, SNP-3 was the rarest, with an allele frequency of 7.06 x 10(-5) (>46,000 exomes from the ExAC database). In conclusion, no compound heterozygous or homozygous potentially pathogenic variants were identified that would account for recessive RP or retinal degeneration in this cohort, however the possibility remains that the rare variants identified could be acting with as yet undiscovered mutations in introns or regulatory regions. SLC4A3 remains an excellent candidate gene for human retinal degeneration, and with the advent of whole exome and whole genome sequencing of cohorts of molecularly unsolved patients with syndromic and non-syndromic forms of retinal degeneration, SLC4A3 may yet be implicated in human disease.

A Carbohydrate Sulfotransferase-6 (CHST6) gene mutation is associated with Macular Corneal Dystrophy in Labrador Retrievers.

PURPOSE: To locate and identify variants associated with macular corneal dystrophy (MCD) in Labrador Retriever (LR) dogs, in the candidate gene carbohydrate sulfotransferase-6 (CHST6). METHODS: The single coding exon of canine CHST6 was sequenced in one affected LR with MCD and one control LR clinically clear of ocular disease. A further 71 control LR with unknown clinical status were sequenced for the putative causal variant in CHST6. A TaqMan SNP genotyping assay was developed and used to screen an additional 84 dogs (five affected LR and 79 clinically clear LR). Finally, the variant was screened in a third cohort of 89 unrelated LR with unknown clinical status to estimate its allele frequency in the population of LR in the United Kingdom. RESULTS: A single nucleotide polymorphism (SNP) was identified within the coding exon of CHST6, resulting in a missense mutation (c.814C>A, p.R272S). All six LR affected with MCD were homozygous for the mutant allele, while 140/151 control LR were homozygous for the wild-type allele and 11/151 were heterozygous for the mutation, indicating an association with MCD (P < 10-5 ). The mutant allele was present in the unrelated LR cohort at a frequency of 0.017, suggesting carrier and affection rates of 3.3% and 0.028%, respectively. CONCLUSIONS: A missense mutation in the CHST6 gene is strongly associated with autosomal recessive MCD in the LR.

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.

Parallel mapping and simultaneous sequencing reveals deletions in BCAN and FAM83H associated with discrete inherited disorders in a domestic dog breed.

The domestic dog (Canis familiaris) segregates more naturally-occurring diseases and phenotypic variation than any other species and has become established as an unparalled model with which to study the genetics of inherited traits. We used a genome-wide association study (GWAS) and targeted resequencing of DNA from just five dogs to simultaneously map and identify mutations for two distinct inherited disorders that both affect a single breed, the Cavalier King Charles Spaniel. We investigated episodic falling (EF), a paroxysmal exertion-induced dyskinesia, alongside the phenotypically distinct condition congenital keratoconjunctivitis sicca and ichthyosiform dermatosis (CKCSID), commonly known as dry eye curly coat syndrome. EF is characterised by episodes of exercise-induced muscular hypertonicity and abnormal posturing, usually occurring after exercise or periods of excitement. CKCSID is a congenital disorder that manifests as a rough coat present at birth, with keratoconjunctivitis sicca apparent on eyelid opening at 10-14 days, followed by hyperkeratinisation of footpads and distortion of nails that develops over the next few months. We undertook a GWAS with 31 EF cases, 23 CKCSID cases, and a common set of 38 controls and identified statistically associated signals for EF and CKCSID on chromosome 7 (P(raw) 1.9×10(-14); P(genome) = 1.0×10(-5)) and chromosome 13 (P(raw) 1.2×10(-17); P(genome) = 1.0×10(-5)), respectively. We resequenced both the EF and CKCSID disease-associated regions in just five dogs and identified a 15,724 bp deletion spanning three exons of BCAN associated with EF and a single base-pair exonic deletion in FAM83H associated with CKCSID. Neither BCAN or FAM83H have been associated with equivalent disease phenotypes in any other species, thus demonstrating the ability to use the domestic dog to study the genetic basis of more than one disease simultaneously in a single breed and to identify multiple novel candidate genes in parallel.

Genetic screening for PRA-associated mutations in multiple dog breeds shows that PRA is heterogeneous within and between breeds.

OBJECTIVE: To assess the extent of progressive retinal atrophy (PRA) genetic heterogeneity within and between domestic dog breeds. METHODS: DNA from 231 dogs with PRA, representing 36 breeds, was screened for 17 mutations previously associated with PRA in at least one breed of dog. Screening methods included amplified fragment size discrimination using gel electrophoresis or detection of fluorescence, (TaqMan(®) ; Life Technologies, Carlsbad, CA, USA) allelic discrimination, and Sanger sequencing. RESULTS: Of the 231 dogs screened, 129 were homozygous for a PRA-associated mutation, 29 dogs were carriers, and 73 were homozygous for the wild-type allele at all loci tested. In two of the 129 dogs, homozygous mutations were identified that had not previously been observed in the respective breeds: one Chinese Crested dog was homozygous for the RCD3-associated mutation usually found in the Cardigan Welsh Corgi, and one Standard Poodle was homozygous for the RCD4-associated mutation previously reported to segregate in Gordon and Irish Setters. In the majority of the breeds (15/21) in which a PRA-associated mutation is known to segregate, cases were identified that did not carry any of the known PRA-associated mutations. CONCLUSION: Progressive retinal atrophy in the dog displays significant genetic heterogeneity within as well as between breeds. There are also several instances where PRA-associated mutations segregate among breeds with no known close ancestry.

MHC class II association study in eight breeds of dog with hypoadrenocorticism.

Canine hypoadrenocorticism is an endocrine disorder characterised by inadequate secretion of steroid hormones from the adrenal glands. Pathology results from immune-mediated destruction of the adrenal cortex, which is similar to that seen in the human Addison's disease. Both the canine and human diseases have similar clinical presentation, with the diagnosis based on performing a dynamic adrenocorticotropic hormone stimulation test. MHC class II has previously been associated with the human and canine diseases. In the current study, we conducted an MHC class II association study in eight breeds of dog with diagnoses of hypoadrenocorticism. We demonstrated significant differences in dog leukocyte antigen (DLA) haplotype frequencies in six of these breeds: Cocker spaniel, Springer spaniel, Labrador, West Highland white terrier (WHWT), Bearded collie, and Standard poodle. In the Springer spaniel, the DLA-DRB1*015:01--DQA1*006:01--DQB1*023:01 haplotype was significantly associated with disease risk (p = 0.014, odds ratio (OR) = 5.14) and showed a similar trend in the Cocker spaniel. This haplotype is related to one associated with hypoadrenocorticism in the Nova Scotia duck tolling retriever. Similar haplotypes shared between breeds were demonstrated, with DLA-DRB1*001:01--DQA1*001:01--DQB1*002:01 more prevalent in both affected Labrador (p = 0.0002, OR = 3.06) and WHWT (p = 0.01, OR = 2.11). Other haplotypes that have not previously been associated with the disease were identified. The inter-breed differences in DLA haplotypes associated with susceptibility to canine hypoadrenocorticism could represent divergent aetiologies. This could have implications for clinical diagnosis and future comparative studies. Alternatively, it may suggest that the gene of interest is closely linked to the MHC.

Multiocular defect in the Old English Sheepdog: A canine form of Stickler syndrome type II associated with a missense variant in the collagen-type gene COL11A1.

Multiocular defect has been described in different canine breeds, including the Old English Sheepdog. Affected dogs typically present with multiple and various ocular abnormalities. We carried out whole genome sequencing on an Old English Sheepdog that had been diagnosed with hereditary cataracts at the age of five and then referred to a board-certified veterinary ophthalmologist due to owner-reported visual deterioration. An ophthalmic assessment revealed that there was bilateral vitreal degeneration, macrophthalmos, and spherophakia in addition to cataracts. Follow-up consultations revealed cataract progression, retinal detachment, uveitis and secondary glaucoma. Whole genome sequence filtered variants private to the case, shared with another Old English Sheepdog genome and predicted to be deleterious were genotyped in an initial cohort of six Old English Sheepdogs (three affected by multiocular defect and three control dogs without evidence of inherited eye disease). Only one of the twenty-two variants segregated correctly with multiocular defect. The variant is a single nucleotide substitution, located in the collagen-type gene COL11A1, c.1775T>C, that causes an amino acid change, p.Phe1592Ser. Genotyping of an additional 14 Old English Sheepdogs affected by multiocular defect revealed a dominant mode of inheritance with four cases heterozygous for the variant. Further genotyping of hereditary cataract-affected Old English Sheepdogs revealed segregation of the variant in eight out of nine dogs. In humans, variants in the COL11A1 gene are associated with Stickler syndrome type II, also dominantly inherited.

Genome-wide association study in RPGRIP1(-/-) dogs identifies a modifier locus that determines the onset of retinal degeneration.

Cone-rod dystrophy (CRD) is a form of inherited retinal degeneration (RD) causing blindness in man as well as in several breeds of dog. Previously, a 44 bp insertion in RPGRIP1 (retinitis pigmentosa GTPase regulator interacting protein-1) was associated with a recessive early-onset CRD (cone-rod dystrophy 1, cord1) in a Miniature longhaired dachshund (MLHD) research colony. Yet in the MLHD pet population, extensive range of the onset age has been observed among RD cases, with some RPGRIP1(-/-) dogs lacking obvious clinical signs. Phenotypic variation has been known in human homologous diseases, including retinitis pigmentosa and Leber congenital amaurosis, indicating possible involvement of modifiers. To explore additional genetic loci associated with the phenotypic variation observed in MLHDs, a genome-wide association study was carried out using Canine SNP20 arrays in 83 RPGRIP1(-/-) MLHDs with variable ages of onset or no clinical abnormality. Using these samples, comparison of 31 early-onset RD cases against 49 controls (15 late-onset RD and 34 normal dogs combined) identified a strong association (P = 5.05 × 10(-13)) at a single locus on canine chromosome 15. At this locus, the majority of early-onset RD cases but few of the controls were homozygous for a 1.49 Mb interval containing ~11 genes. We conclude that homozygosity at both RPGRIP1 and the newly mapped second locus is necessary to develop early-onset RD, whereas RPGRIP1(-/-) alone leads to late-onset RD or no apparent clinical phenotype. This study establishes a unique model of canine RD requiring homozygous mutations at two distinct genetic loci for the manifestation of early-onset RD.

Genome-wide mRNA sequencing of a single canine cerebellar cortical degeneration case leads to the identification of a disease associated SPTBN2 mutation.

BACKGROUND: Neonatal cerebellar cortical degeneration is a neurodegenerative disease described in several canine breeds including the Beagle. Affected Beagles are unable to ambulate normally from the onset of walking and the main pathological findings include Purkinje cell loss with swollen dendritic processes. Previous reports suggest an autosomal recessive mode of inheritance. The development of massively parallel sequencing techniques has presented the opportunity to investigate individual clinical cases using genome-wide sequencing approaches. We used genome-wide mRNA sequencing (mRNA-seq) of cerebellum tissue from a single Beagle with neonatal cerebellar cortical degeneration as a method of candidate gene sequencing, with the aim of identifying the causal mutation. RESULTS: A four-week old Beagle dog presented with progressive signs of cerebellar ataxia and the owner elected euthanasia. Histopathology revealed findings consistent with cerebellar cortical degeneration. Genome-wide mRNA sequencing (mRNA-seq) of RNA from cerebellum tissue was used as a method of candidate gene sequencing. After analysis of the canine orthologues of human spinocerebellar ataxia associated genes, we identified a homozygous 8 bp deletion in the ß-III spectrin gene, SPTBN2, associated with spinocerebellar type 5 in humans. Genotype analysis of the sire, dam, ten clinically unaffected siblings, and an affected sibling from a previous litter, showed the mutation to fully segregate with the disorder. Previous studies have shown that ß-III spectrin is critical for Purkinje cell development, and the absence of this protein can lead to cell damage through excitotoxicity, consistent with the observed Purkinje cell loss, degeneration of dendritic processes and associated neurological dysfunction in this Beagle. CONCLUSIONS: An 8 bp deletion in the SPTBN2 gene encoding ß-III spectrin is associated with neonatal cerebellar cortical degeneration in Beagle dogs. This study shows that mRNA-seq is a feasible method of screening candidate genes for mutations associated with rare diseases when a suitable tissue resource is available.

Congenital keratoconjunctivitis sicca and ichthyosiform dermatosis in Cavalier King Charles spaniel dogs. Part II: candidate gene study.

PURPOSE: To identify causative mutation(s) for congenital keratoconjunctivitis sicca and ichthyosiform dermatosis (CKCSID) in Cavalier King Charles spaniel (CKCS) dogs using a candidate gene approach. METHODS: DNA samples from 21 cases/parents were collected. Canine candidate genes (CCGs) for similar inherited human diseases were chosen. Twenty-eight candidate genes were identified by searching the Pubmed OMIM database (http://www.ncbi.nlm.nih.gov/omim). Canine orthologues of human candidate genes were identified using the Ensembl orthologue prediction facility (http://www.ensembl.org/index.html). Two microsatellites flanking each candidate gene were selected, and primers to amplify each microsatellite were designed using the Whitehead Institute primer design website (http://frodo.wi.mit.edu/primer3/). The microsatellites associated with all 28 CCGs were genotyped on a panel of 21 DNA samples from CKCS dogs (13 affected and eight carriers). Genotyping data was analyzed to identify markers homozygous in affected dogs and heterozygous in carriers (homozygosity mapping). RESULTS: None of the microsatellites associated with 25 of the CCGs displayed an association with CKCSID in the 21 DNA samples tested. Three CCGs associated microsatellites were monomorphic across all samples tested. CONCLUSIONS: Twenty-five CCGs were excluded as cause of CKCSID. Three CCGs could not be excluded from involvement in the inheritance of CKCSID.

Ophthalmic and cone derived electrodiagnostic findings in outbred Miniature Long-haired Dachshunds homozygous for a RPGRIP1 mutation.

OBJECTIVE: To investigate ophthalmic and cone-derived electrodiagnostic findings in outbred Miniature Long-haired Dachshunds (MLHD) homozygous for a mutation in the RPGRIP1 gene previously associated with cone-rod dystrophy 1 (cord1). ANIMALS: A total of 36 MLHD homozygous for the RPGRIP1 mutation and 23 dogs clear of the mutation (control group). PROCEDURES: The dogs underwent ophthalmic examination and photopic electroretinogram (ERG) recordings. RESULTS: None of the control dogs presented with clinical or ophthalmic signs consistent with cord1. Amongst the dogs homozygous for the mutation one presented with bilateral symmetrical total retinal atrophy. None of the other dogs in this group showed signs consistent with cord1. Photopic ERG recordings were available in 23 control dogs and 34 dogs homozygous for the mutation. Photopic a- and b-waves following four light stimuli (3 cdS/m(2) ) at a rate of 5.1 Hz were not significantly different between groups. The amplitudes of the 30 Hz flicker (128 flashes, 3 cdS/m(2) ) response were significantly reduced in the dogs homozygous for the PRGRIP1 mutation. The difference in age between the two groups did not significantly affect the difference. CONCLUSION: Homozygosity of the RPGRIP1 mutation does not invariably result in early onset cord1. However, cone derived ERG recordings show evidence of a reduced cone or inner retinal function in homozygous but clinically normal MLHD. Modifying genes that have yet to be identified may influence an individual dog's risk of developing the blinding cord1 and also the age of onset and rate of progression.

A Missense Variant in the Bardet-Biedl Syndrome 2 Gene (BBS2) Leads to a Novel Syndromic Retinal Degeneration in the Shetland Sheepdog.

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.

A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs.

Double muscling is a trait previously described in several mammalian species including cattle and sheep and is caused by mutations in the myostatin (MSTN) gene (previously referred to as GDF8). Here we describe a new mutation in MSTN found in the whippet dog breed that results in a double-muscled phenotype known as the "bully" whippet. Individuals with this phenotype carry two copies of a two-base-pair deletion in the third exon of MSTN leading to a premature stop codon at amino acid 313. Individuals carrying only one copy of the mutation are, on average, more muscular than wild-type individuals (p = 7.43 x 10(-6); Kruskal-Wallis Test) and are significantly faster than individuals carrying the wild-type genotype in competitive racing events (Kendall's nonparametric measure, tau = 0.3619; p approximately 0.00028). These results highlight the utility of performance-enhancing polymorphisms, marking the first time a mutation in MSTN has been quantitatively linked to increased athletic performance.