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.

Clinical, histopathological and genetic characterisation of oculoskeletal dysplasia in the Northern Inuit Dog.

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.

An intronic LINE-1 insertion in MERTK is strongly associated with retinopathy in Swedish Vallhund dogs.

The domestic dog segregates a significant number of inherited progressive retinal diseases, several of which mirror human retinal diseases and which are collectively termed progressive retinal atrophy (PRA). In 2014, a novel form of PRA was reported in the Swedish Vallhund breed, and the disease was mapped to canine chromosome 17. The causal mutation was not identified, but expression analyses of the retinas of affected Vallhunds demonstrated a 6-fold increased expression of the MERTK gene compared to unaffected dogs. Using 24 retinopathy cases and 97 controls with no clinical signs of retinopathy, we replicated the chromosome 17 association in Swedish Vallhunds from the UK and aimed to elucidate the causal variant underlying this association using whole genome sequencing (WGS) of an affected dog. This revealed a 6-8 kb insertion in intron 1 of MERTK that was not present in WGS of 49 dogs of other breeds. Sequencing and BLASTN analysis of the inserted segment was consistent with the insertion comprising a full-length intact LINE-1 retroelement. Testing of the LINE-1 insertion for association with retinopathy in the UK set of 24 cases and 97 controls revealed a strong statistical association (P-value 6.0 x 10-11) that was subsequently replicated in the original Finnish study set (49 cases and 89 controls (P-value 4.3 x 10-19). In a pooled analysis of both studies (73 cases and 186 controls), the LINE-1 insertion was associated with a ~20-fold increased risk of retinopathy (odds ratio 23.41, 95% confidence intervals 10.99-49.86, P-value 1.3 x 10-27). Our study adds further support for regulatory disruption of MERTK in Swedish Vallhund retinopathy; however, further work is required to establish a functional overexpression model. Future work to characterise the mechanism by which this intronic mutation disrupts gene regulation will further improve the understanding of MERTK biology and its role in retinal function.

Pectinate ligament dysplasia in the Border Collie, Hungarian Vizsla and Golden Retriever.

Pectinate ligament dysplasia (PLD) is significantly associated with primary closed angle glaucoma (PCAG) in several dog breeds. Gonioscopy screening for PLD is advised in breeds in which PLD and PCAG are particularly prevalent in order that affected dogs may be eliminated from the breeding population. The Border Collie (BC), Hungarian Vizsla (HV) and Golden Retriever (GR) breeds are currently under investigation for PLD by the British Veterinary Association/Kennel Club/International Sheep Dog Society (BVA/KC/ISDS) Eye Scheme. The authors aimed to determine the prevalence of PLD in UK populations of BC, HV and GR and to investigate possible associations between the degree of PLD and age and sex. Gonioscopy was performed in 102 BCs, 112 HVs and 230 GRs and the percentage of iridocorneal angle affected by PLD was estimated and classified as unaffected (0 per cent), mildly affected (<20 per cent), moderately affected (20-90 per cent) or severely affected (>90 per cent). Eleven of 102 (13.8 per cent) BCs, 16/112 (14.3 per cent) HVs and 60/230 (26.1 per cent) GRs were moderately or severely affected by PLD. The prevalence of PLD was significantly higher in GR than both BC and HV. There was a significant positive correlation between PLD and age in the HV and GR but not in the BC. There was no association between PLD and sex in any breed.

Prevalence of pectinate ligament dysplasia and associations with age, sex and intraocular pressure in the Basset hound, Flatcoated retriever and Dandie Dinmont terrier.

BACKGROUND: The aims of this study were to: determine the prevalence of pectinate ligament dysplasia (PLD) in populations of Basset hounds (BH), Flatcoated retrievers (FCR) and Dandie Dinmont terriers (DDT) resident in the UK; investigate possible associations between the degree of PLD and age, sex and intraocular pressure (IOP) and; investigate possible associations between IOP and age and sex. Gonioscopy was performed in both eyes of 198 BH, 170 FCR and 95 DDT and the percentage of iridocorneal angle affected by PLD was estimated and classified as unaffected (0 %), mildly affected (<20 %), moderately affected (20-90 %) or severely affected (>90 %). Rebound tonometry was performed bilaterally in the majority of enrolled dogs. RESULTS: Seventy-six of 198 (38.4 %) BH, 36/170 (21.2 %) FCR and 21/95 (22.1 %) DDT were moderately or severely affected by PLD. The prevalence of PLD was significantly higher in BH than both FCR and DDT. In all breeds there was a significant positive correlation between PLD and age. In the BH only there was a significant association between PLD and sex. In the DDT only there was a weak negative correlation between PLD and IOP and a moderately strong negative correlation between IOP and age. CONCLUSIONS: PLD is prevalent and significantly associated with age in all three breeds we investigated. The linear relationship between PLD and age can be explained by the progression of PLD over time which would contribute to the high prevalence of PLD despite widespread screening.

DNA Sequence Variants in the Five Prime Untranslated Region of the Cyclooxygenase-2 Gene Are Commonly Found in Healthy Dogs and Gray Wolves.

The aim of this study was to investigate the frequency of regional DNA variants upstream to the translation initiation site of the canine Cyclooxygenase-2 (Cox-2) gene in healthy dogs. Cox-2 plays a role in various disease conditions such as acute and chronic inflammation, osteoarthritis and malignancy. A role for Cox-2 DNA variants in genetic predisposition to canine renal dysplasia has been proposed and dog breeders have been encouraged to select against these DNA variants. We sequenced 272-422 bases in 152 dogs unaffected by renal dysplasia and found 19 different haplotypes including 11 genetic variants which had not been described previously. We genotyped 7 gray wolves to ascertain the wildtype variant and found that the wolves we analyzed had predominantly the second most common DNA variant found in dogs. Our results demonstrate an elevated level of regional polymorphism that appears to be a feature of healthy domesticated dogs.

Genome-wide association study identifies a novel canine glaucoma locus.

Glaucoma is an optic neuropathy and one of the leading causes of blindness. Its hereditary forms are classified into primary closed-angle (PCAG), primary open-angle (POAG) and primary congenital glaucoma (PCG). Although many loci have been mapped in human, only a few genes have been identified that are associated with the development of glaucoma and the genetic basis of the disease remains poorly understood. Glaucoma has also been described in many dog breeds, including Dandie Dinmont Terriers (DDT) in which it is a late-onset (>7 years) disease. We designed clinical and genetic studies to better define the clinical features of glaucoma in the DDT and to identify the genetic cause. Clinical diagnosis was based on ophthalmic examinations of the affected dogs and 18 additionally investigated unaffected DDTs. We collected DNA from over 400 DTTs and a genome wide association study was performed in a cohort of 23 affected and 23 controls, followed by a fine mapping, a replication study and candidate gene sequencing. The clinical study suggested that ocular abnormalities including abnormal iridocorneal angles and pectinate ligament dysplasia are common (50% and 72%, respectively) in the breed and the disease resembles human PCAG. The genetic study identified a novel 9.5 Mb locus on canine chromosome 8 including the 1.6 Mb best associated region (p = 1.63 × 10(-10), OR = 32 for homozygosity). Mutation screening in five candidate genes did not reveal any causative variants. This study indicates that although ocular abnormalities are common in DDTs, the genetic risk for glaucoma is conferred by a novel locus on CFA8. The canine locus shares synteny to a region in human chromosome 14q, which harbors several loci associated with POAG and PCG. Our study reveals a new locus for canine glaucoma and ongoing molecular studies will likely help to understand the genetic etiology of the disease.

Missense mutation in CAPN1 is associated with spinocerebellar ataxia in the Parson Russell Terrier dog breed.

Spinocerebellar ataxia (SCA) in the Parson Russell Terrier (PRT) dog breed is a disease of progressive incoordination of gait and loss of balance. Clinical signs usually become notable between 6 and 12 months of age with affected dogs presenting with symmetric spinocerebellar ataxia particularly evident in the pelvic limbs. The degree of truncal ataxia, pelvic limb hypermetria and impaired balance is progressive, particularly during the initial months of disease. A certain degree of stabilisation as well as intermittent worsening may occur. At the later stages of the disease ambulation often becomes difficult, with owners often electing to euthanise affected dogs on welfare grounds. Using a GWAS approach and target-enriched massively-parallel sequencing, a strongly associated non-synonymous SNP in the CAPN1 gene, encoding the calcium dependent cysteine protease calpain1 (mu-calpain), was identified. The SNP is a missense mutation causing a cysteine to tyrosine substitution at residue 115 of the CAPN1 protein. Cysteine 115 is a highly conserved residue and forms a key part of a catalytic triad of amino acids that are crucial to the enzymatic activity of cysteine proteases. The CAPN1 gene shows high levels of expression in the brain and nervous system and roles for the protein in both neuronal necrosis and maintenance have been suggested. Given the functional implications and high level of conservation observed across species, the CAPN1 variant represents a provocative candidate for the cause of SCA in the PRT and a novel potential cause of ataxia in humans.

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.