Correction: Identification of a variant in NDP associated with X-linked retinal dysplasia in the English cocker spaniel dog.

[This corrects the article DOI: 10.1371/journal.pone.0251071.].

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.

Identification of a variant in NDP associated with X-linked retinal dysplasia in the English cocker spaniel dog.

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.

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.

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.

Whole Genome Sequencing of Giant Schnauzer Dogs with Progressive Retinal Atrophy Establishes NECAP1 as a Novel Candidate Gene for Retinal Degeneration.

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.

Clinical and genetic characterisation of dystrophin-deficient muscular dystrophy in a family of Miniature Poodle dogs.

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.