Genetic Variants Affecting Skeletal Morphology in Domestic Dogs.

Purebred dog breeds provide a powerful resource for the discovery of genetic variants affecting skeletal morphology. Domesticated and subsequently purebred dogs have undergone strong artificial selection for a broad range of skeletal variation, which include both the size and shapes of their bones. While the phenotypic variation between breeds is high, within-breed morphological variation is typically low. Approaches for defining genetic variants associated with canine morphology include quantitative within-breed analyses, as well as across-breed analyses, using breed standards as proxies for individual measurements. The ability to identify variants across the genomes of individual dogs can now be paired with precise measures of morphological variation to define the genetic interactions and the phenotypic effect of variants on skeletal morphology.

Whole genome variant association across 100 dogs identifies a frame shift mutation in DISHEVELLED 2 which contributes to Robinow-like syndrome in Bulldogs and related screw tail dog breeds.

Domestic dog breeds exhibit remarkable morphological variations that result from centuries of artificial selection and breeding. Identifying the genetic changes that contribute to these variations could provide critical insights into the molecular basis of tissue and organismal morphogenesis. Bulldogs, French Bulldogs and Boston Terriers share many morphological and disease-predisposition traits, including brachycephalic skull morphology, widely set eyes and short stature. Unlike other brachycephalic dogs, these breeds also exhibit vertebral malformations that result in a truncated, kinked tail (screw tail). Whole genome sequencing of 100 dogs from 21 breeds identified 12.4 million bi-allelic variants that met inclusion criteria. Whole Genome Association of these variants with the breed defining phenotype of screw tail was performed using 10 cases and 84 controls and identified a frameshift mutation in the WNT pathway gene DISHEVELLED 2 (DVL2) (Chr5: 32195043_32195044del, p = 4.37 X 10-37) as the most strongly associated variant in the canine genome. This DVL2 variant was fixed in Bulldogs and French Bulldogs and had a high allele frequency (0.94) in Boston Terriers. The DVL2 variant segregated with thoracic and caudal vertebral column malformations in a recessive manner with incomplete and variable penetrance for thoracic vertebral malformations between different breeds. Importantly, analogous frameshift mutations in the human DVL1 and DVL3 genes cause Robinow syndrome, a congenital disorder characterized by similar craniofacial, limb and vertebral malformations. Analysis of the canine DVL2 variant protein showed that its ability to undergo WNT-induced phosphorylation is reduced, suggesting that altered WNT signaling may contribute to the Robinow-like syndrome in the screwtail breeds.

FGF4 retrogene on CFA12 is responsible for chondrodystrophy and intervertebral disc disease in dogs.

Chondrodystrophy in dogs is defined by dysplastic, shortened long bones and premature degeneration and calcification of intervertebral discs. Independent genome-wide association analyses for skeletal dysplasia (short limbs) within a single breed (PBonferroni = 0.01) and intervertebral disc disease (IVDD) across breeds (PBonferroni = 4.0 × 10-10) both identified a significant association to the same region on CFA12. Whole genome sequencing identified a highly expressed FGF4 retrogene within this shared region. The FGF4 retrogene segregated with limb length and had an odds ratio of 51.23 (95% CI = 46.69, 56.20) for IVDD. Long bone length in dogs is a unique example of multiple disease-causing retrocopies of the same parental gene in a mammalian species. FGF signaling abnormalities have been associated with skeletal dysplasia in humans, and our findings present opportunities for both selective elimination of a medically and financially devastating disease in dogs and further understanding of the ever-growing complexity of retrogene biology.

Evaluation of the major histocompatibility complex (MHC) class II as a candidate for sudden acquired retinal degeneration syndrome (SARDS) in Dachshunds.

OBJECTIVE: Sudden acquired retinal degeneration syndrome (SARDS) is one of the leading causes of acute blindness in dogs, with an unknown etiology and no effective treatment. Certain breeds such as Dachshunds are overrepresented among SARDS patients, and therefore, the syndrome is suspected to have a genetic component. The objective of this study was to determine if a genetic locus associated with SARDS in Dachshunds could be identified using a genome-wide association study (GWAS). PROCEDURES: Genome-wide association mapping was performed in 15 SARDS-affected and 16 unaffected Dachshunds. Genotyping of three classical DLA class II genes (DLA-DRB1, DLA-DQA1, and DLA-DQB1) was performed in 34 SARDS-affected and 66 unaffected Dachshunds to evaluate for an association in this region. RESULTS: Although no single nucleotide polymorphisms (SNPs) were of genome-wide statistical significance (PBonferroni  < 0.05), 5 of the top 9 SNPs were in the major histocompatibility complex (MHC). Using DLA typing, the allele DLA-DRB1*09401 was identified as a risk factor for the development of SARDS (P = 0.0032, OR = 4.0). The alleles DLA-DQB1*00101 (P = 0.0050, OR = 0.31), DLA-DQA1*00901 (P = 0.0087, OR = 0.33), and a previously identified DLA-DRB1allele described as "DRB1-T" (P = 0.0284, OR = 0.37) were identified as protective factors. CONCLUSIONS: Although far from definitive, association of SARDS with alleles of immunologic importance further supports the hypothesis that autoimmunity may play a role in the pathogenesis of SARDS.

Identification of a Candidate Mutation in the COL1A2 Gene of a Chow Chow With Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a genetic disease that occurs in humans and animals. Individuals with OI exhibit signs of extreme bone fragility and osteopenia with frequent fractures and perinatal lethality in severe cases. In this study, we report the clinical diagnosis of OI in a dog and the use of targeted next-generation sequencing to identify a candidate autosomal dominant mutation in the COL1A2 gene. A 5-month-old male Chow Chow was examined with a fractured left humerus and resolving, bilateral femoral fractures. Radiographs revealed generalized osteopenia and bilateral humeral, radial, and femoral fractures. Targeted next-generation sequencing of genes associated with OI in humans (COL1A1, COL1A2, LEPRE1, SERPINH1, and CRTAP) revealed a G>A heterozygous mutation in the splice donor site of exon 18 of the COL1A2 gene (c.936 + 1G>A). The splice donor mutation was not detected among 91 control dogs representing 21 breeds. A comparative analysis of exon 18 and the exon-intron junction further showed that the mutated splice donor site is conserved among vertebrates. Altogether, these findings reveal a candidate autosomal splice donor site mutation causing OI in an individual Chow Chow.

SERPINB11 frameshift variant associated with novel hoof specific phenotype in Connemara ponies.

Horses belong to the order Perissodactyla and bear the majority of their weight on their third toe; therefore, tremendous force is applied to each hoof. An inherited disease characterized by a phenotype restricted to the dorsal hoof wall was identified in the Connemara pony. Hoof wall separation disease (HWSD) manifests clinically as separation of the dorsal hoof wall along the weight-bearing surface of the hoof during the first year of life. Parents of affected ponies appeared clinically normal, suggesting an autosomal recessive mode of inheritance. A case-control allelic genome wide association analysis was performed (ncases = 15, ncontrols = 24). Population stratification (λ = 1.48) was successfully improved by removing outliers (ncontrols = 7) identified on a multidimensional scaling plot. A genome-wide significant association was detected on chromosome 8 (praw = 1.37x10-10, pgenome = 1.92x10-5). A homozygous region identified in affected ponies spanned from 79,936,024-81,676,900 bp and contained a family of 13 annotated SERPINB genes. Whole genome next-generation sequencing at 6x coverage of two cases and two controls revealed 9,758 SNVs and 1,230 indels within the ~1.7-Mb haplotype, of which 17 and 5, respectively, segregated with the disease and were located within or adjacent to genes. Additional genotyping of these 22 putative functional variants in 369 Connemara ponies (ncases = 23, ncontrols = 346) and 169 horses of other breeds revealed segregation of three putative variants adjacent or within four SERPIN genes. Two of the variants were non-coding and one was an insertion within SERPINB11 that introduced a frameshift resulting in a premature stop codon. Evaluation of mRNA levels at the proximal hoof capsule (ncases = 4, ncontrols = 4) revealed that SERPINB11 expression was significantly reduced in affected ponies (p<0.001). Carrier frequency was estimated at 14.8%. This study describes the first genetic variant associated with a hoof wall specific phenotype and suggests a role of SERPINB11 in maintaining hoof wall structure.

Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas.

Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo.

Genome-wide association studies in dogs and humans identify ADAMTS20 as a risk variant for cleft lip and palate.

Cleft lip with or without cleft palate (CL/P) is the most commonly occurring craniofacial birth defect. We provide insight into the genetic etiology of this birth defect by performing genome-wide association studies in two species: dogs and humans. In the dog, a genome-wide association study of 7 CL/P cases and 112 controls from the Nova Scotia Duck Tolling Retriever (NSDTR) breed identified a significantly associated region on canine chromosome 27 (unadjusted p=1.1 x 10(-13); adjusted p= 2.2 x 10(-3)). Further analysis in NSDTR families and additional full sibling cases identified a 1.44 Mb homozygous haplotype (chromosome 27: 9.29 - 10.73 Mb) segregating with a more complex phenotype of cleft lip, cleft palate, and syndactyly (CLPS) in 13 cases. Whole-genome sequencing of 3 CLPS cases and 4 controls at 15X coverage led to the discovery of a frameshift mutation within ADAMTS20 (c.1360_1361delAA (p.Lys453Ilefs*3)), which segregated concordant with the phenotype. In a parallel study in humans, a family-based association analysis (DFAM) of 125 CL/P cases, 420 unaffected relatives, and 392 controls from a Guatemalan cohort, identified a suggestive association (rs10785430; p =2.67 x 10-6) with the same gene, ADAMTS20. Sequencing of cases from the Guatemalan cohort was unable to identify a causative mutation within the coding region of ADAMTS20, but four coding variants were found in additional cases of CL/P. In summary, this study provides genetic evidence for a role of ADAMTS20 in CL/P development in dogs and as a candidate gene for CL/P development in humans.

Genetic analysis of optic nerve head coloboma in the Nova Scotia Duck Tolling Retriever identifies discordance with the NHEJ1 intronic deletion (collie eye anomaly mutation).

Collie eye anomaly (CEA) encompasses a spectrum of different ophthalmic phenotypes from clinically inconsequential choroidal hypoplasia to blindness from coloboma of the optic nerve head (ONH). A previous study found a 7.8-kb deletion in intron 4 of the NHEJ1 gene to be associated with CEA. A genetic test based on this association is recommended for many breeds, including the Nova Scotia Duck Tolling Retriever (NSDTR). Collection of ONH coloboma-affected NSDTR showed lack of concordance of the NHEJ1 intronic deletion with ONH coloboma. Using genomewide single nucleotide polymorphism (SNP) genotyping in 7 ONH coloboma-affected NSDTR cases and 47 unaffected NSDTR controls with no ophthalmic signs, one SNP, located on chromosome 7, demonstrated genomewide significance. However, high genomic inflation may have confounded the results. Therefore, the genomewide association study was repeated using EMMAX to control for population structure in the cohort of 7 cases and 47 controls. However, no regions of the genome were significantly associated with ONH coloboma. These results failed to document significant association with the CEA locus. Due to the complex genetic etiology of ONH coloboma, the NHEJ1 intronic deletion test results should be carefully considered when making breeding decisions. If the goal is to select for visually competent dogs, our data suggest that eye examinations of puppies would be more effective as a guide in selection of breeding pairs than relying solely on currently available genetic tests.

Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds.

BACKGROUND: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. RESULTS: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. CONCLUSIONS: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

A LINE-1 insertion in DLX6 is responsible for cleft palate and mandibular abnormalities in a canine model of Pierre Robin sequence.

Cleft palate (CP) is one of the most commonly occurring craniofacial birth defects in humans. In order to study cleft palate in a naturally occurring model system, we utilized the Nova Scotia Duck Tolling Retriever (NSDTR) dog breed. Micro-computed tomography analysis of CP NSDTR craniofacial structures revealed that these dogs exhibit defects similar to those observed in a recognizable subgroup of humans with CP: Pierre Robin Sequence (PRS). We refer to this phenotype in NSDTRs as CP1. Individuals with PRS have a triad of birth defects: shortened mandible, posteriorly placed tongue, and cleft palate. A genome-wide association study in 14 CP NSDTRs and 72 unaffected NSDTRs identified a significantly associated region on canine chromosome 14 (24.2 Mb-29.3 Mb; p(raw )= 4.64 × 10(-15)). Sequencing of two regional candidate homeobox genes in NSDTRs, distal-less homeobox 5 (DLX5) and distal-less homeobox 6 (DLX6), identified a 2.1 kb LINE-1 insertion within DLX6 in CP1 NSDTRs. The LINE-1 insertion is predicted to insert a premature stop codon within the homeodomain of DLX6. This prompted the sequencing of DLX5 and DLX6 in a human cohort with CP, where a missense mutation within the highly conserved DLX5 homeobox of a patient with PRS was identified. This suggests the involvement of DLX5 in the development of PRS. These results demonstrate the power of the canine animal model as a genetically tractable approach to understanding naturally occurring craniofacial birth defects in humans.

Gonadectomy effects on the risk of immune disorders in the dog: a retrospective study.

BACKGROUND: Gonadectomy is one of the most common procedures performed on dogs in the United States. Neutering has been shown to reduce the risk for some diseases although recent reports suggest increased prevalence for structural disorders and some neoplasias. The relation between neuter status and autoimmune diseases has not been explored. This study evaluated the prevalence and risk of atopic dermatitis (ATOP), autoimmune hemolytic anemia (AIHA), canine myasthenia gravis (CMG), colitis (COL), hypoadrenocorticism (ADD), hypothyroidism (HYPO), immune-mediated polyarthritis (IMPA), immune-mediated thrombocytopenia (ITP), inflammatory bowel disease (IBD), lupus erythematosus (LUP), and pemphigus complex (PEMC), for intact females, intact males, neutered females, and neutered males. Pyometra (PYO) was evaluated as a control condition. RESULTS: Patient records (90,090) from the William R. Pritchard Veterinary Medical Teaching Hospital at the University of California, Davis from 1995 to 2010 were analyzed in order to determine the risk of immune-mediated disease relative to neuter status in dogs. Neutered dogs had a significantly greater risk of ATOP, AIHA, ADD, HYPO, ITP, and IBD than intact dogs with neutered females being at greater risk than neutered males for all but AIHA and ADD. Neutered females, but not males, had a significantly greater risk of LUP than intact females. Pyometra was a greater risk for intact females. CONCLUSIONS: The data underscore the importance of sex steroids on immune function emphasizing a role of these hormones on tissue self-recognition. Neutering is critically important for population control, reduction of reproductive disorders, and offers convenience for owners. Despite these advantages, the analyses of the present study suggest that neutering is associated with increased risk for certain autoimmune disorders and underscore the need for owners to consult with their veterinary practitioner prior to neutering to evaluate possible benefits and risks associated with such a procedure.

Genome-wide association mapping in dogs enables identification of the homeobox gene, NKX2-8, as a genetic component of neural tube defects in humans.

Neural tube defects (NTDs) is a general term for central nervous system malformations secondary to a failure of closure or development of the neural tube. The resulting pathologies may involve the brain, spinal cord and/or vertebral column, in addition to associated structures such as soft tissue or skin. The condition is reported among the more common birth defects in humans, leading to significant infant morbidity and mortality. The etiology remains poorly understood but genetic, nutritional, environmental factors, or a combination of these, are known to play a role in the development of NTDs. The variable conditions associated with NTDs occur naturally in dogs, and have been previously reported in the Weimaraner breed. Taking advantage of the strong linkage-disequilibrium within dog breeds we performed genome-wide association analysis and mapped a genomic region for spinal dysraphism, a presumed NTD, using 4 affected and 96 unaffected Weimaraners. The associated region on canine chromosome 8 (pgenome  =3.0 × 10(-5)), after 100,000 permutations, encodes 18 genes, including NKX2-8, a homeobox gene which is expressed in the developing neural tube. Sequencing NKX2-8 in affected Weimaraners revealed a G to AA frameshift mutation within exon 2 of the gene, resulting in a premature stop codon that is predicted to produce a truncated protein. The exons of NKX2-8 were sequenced in human patients with spina bifida and rare variants (rs61755040 and rs10135525) were found to be significantly over-represented (p=0.036). This is the first documentation of a potential role for NKX2-8 in the etiology of NTDs, made possible by investigating the molecular basis of naturally occurring mutations in dogs.

Single nucleotide polymorphism (SNP) variation of wolves (Canis lupus) in Southeast Alaska and comparison with wolves, dogs, and coyotes in North America.

There is considerable interest in the genetics of wolves (Canis lupus) because of their close relationship to domestic dogs (C. familiaris) and the need for informed conservation and management. This includes wolf populations in Southeast Alaska for which we determined genotypes of 305 wolves at 173662 single nucleotide polymorphism (SNP) loci. After removal of invariant and linked SNP, 123801 SNP were used to quantify genetic differentiation of wolves in Southeast Alaska and wolves, coyotes (C. latrans), and dogs from other areas in North America. There is differentiation of SNP allele frequencies between the species (wolves, coyotes, and dogs), although differentiation is relatively low between some wolf and coyote populations. There are varying levels of differentiation among populations of wolves, including low differentiation of wolves in interior Alaska, British Columbia, and the northern US Rocky Mountains. There is considerable differentiation of SNP allele frequencies of wolves in Southeast Alaska from wolves in other areas. However, wolves in Southeast Alaska are not a genetically homogeneous group and there are comparable levels of genetic differentiation among areas within Southeast Alaska and between Southeast Alaska and other geographic areas. SNP variation and other genetic data are discussed regarding taxonomy and management.

A high density SNP array for the domestic horse and extant Perissodactyla: utility for association mapping, genetic diversity, and phylogeny studies.

An equine SNP genotyping array was developed and evaluated on a panel of samples representing 14 domestic horse breeds and 18 evolutionarily related species. More than 54,000 polymorphic SNPs provided an average inter-SNP spacing of ∼43 kb. The mean minor allele frequency across domestic horse breeds was 0.23, and the number of polymorphic SNPs within breeds ranged from 43,287 to 52,085. Genome-wide linkage disequilibrium (LD) in most breeds declined rapidly over the first 50-100 kb and reached background levels within 1-2 Mb. The extent of LD and the level of inbreeding were highest in the Thoroughbred and lowest in the Mongolian and Quarter Horse. Multidimensional scaling (MDS) analyses demonstrated the tight grouping of individuals within most breeds, close proximity of related breeds, and less tight grouping in admixed breeds. The close relationship between the Przewalski's Horse and the domestic horse was demonstrated by pair-wise genetic distance and MDS. Genotyping of other Perissodactyla (zebras, asses, tapirs, and rhinoceros) was variably successful, with call rates and the number of polymorphic loci varying across taxa. Parsimony analysis placed the modern horse as sister taxa to Equus przewalski. The utility of the SNP array in genome-wide association was confirmed by mapping the known recessive chestnut coat color locus (MC1R) and defining a conserved haplotype of ∼750 kb across all breeds. These results demonstrate the high quality of this SNP genotyping resource, its usefulness in diverse genome analyses of the horse, and potential use in related species.

A mutation in the canine gene encoding folliculin-interacting protein 2 (FNIP2) associated with a unique disruption in spinal cord myelination.

Novel mutations in myelin and myelin-associated genes have provided important information on oligodendrocytes and myelin and the effects of their disruption on the normal developmental process of myelination of the central nervous system (CNS). We report here a mutation in the folliculin-interacting protein 2 (FNIP2) gene in the Weimaraner dog that results in hypomyelination of the brain and a tract-specific myelin defect in the spinal cord. This myelination disruption results in a notable tremor syndrome from which affected dogs recover with time. In the peripheral tracts of the lateral and ventral columns of the spinal cord, there is a lack of mature oligodendrocytes. A genome-wide association study of DNA from three groups of dogs mapped the gene to canine chromosome 15. Sequencing of all the genes in the candidate region identified a frameshift mutation in the FNIP2 gene that segregated with the phenotype. While the functional role of FNIP2 is not known, our data would suggest that production of truncated protein results in a delay or failure of maturation of a subpopulation of oligodendrocytes.

A variant in the 5'UTR of ERBB4 is associated with lifespan in Golden Retrievers.

Genome-wide association studies (GWAS) in long-lived human populations have led to identification of variants associated with Alzheimer's disease and cardiovascular disease, the latter being the most common cause of mortality in people worldwide. In contrast, naturally occurring cancer represents the leading cause of death in pet dogs, and specific breeds like the Golden Retriever (GR) carry up to a 65% cancer-related death rate. We hypothesized that GWAS of long-lived GRs might lead to the identification of genetic variants capable of modifying longevity within this cancer-predisposed breed. A GWAS was performed comparing GR dogs ≥ 14 years to dogs dying prior to age 12 which revealed a significant association to ERBB4, the only member of the epidermal growth factor receptor family capable of serving as both a tumor suppressor gene and an oncogene. No coding variants were identified, however, distinct haplotypes in the 5'UTR were associated with reduced lifespan in two separate populations of GR dogs. When all GR dogs were analyzed together (n = 304), the presence of haplotype 3 was associated with shorter survival (11.8 years vs. 12.8 years, p = 0.024). GRs homozygous for haplotype 3 had the shortest survival, and GRs homozygous for haplotype 1 had the longest survival (11.6 years vs. 13.5 years, p = 0.0008). Sub-analyses revealed that the difference in lifespan for GRs carrying at least 1 copy of haplotype 3 was specific to female dogs (p = 0.009), whereas survival remained significantly different in both male and female GRs homozygous for haplotype 1 or haplotype 3 (p = 0.026 and p = 0.009, respectively). Taken together, these findings implicate a potential role for ERBB4 in GR longevity and provide evidence that within-breed canine lifespan studies could serve as a mechanism to identify favorable or disease-modifying variants important to the axis of aging and cancer.

FGF4L2 retrogene copy number is associated with intervertebral disc calcification and vertebral geometry in Nova Scotia Duck Tolling Retrievers.

OBJECTIVES: To evaluate the effects of the chondrodystrophy-associated FGF4L2 retrogene on intervertebral disc (IVD) calcification and vertebral geometry. ANIMALS: 22 Nova Scotia Duck Tolling Retrievers (NSDTR) with no FGF4L2 retrogene (n = 7, wild-type dogs), 1 retrogene copy (8, heterozygous dogs), or 2 retrogene copies (7, homozygous dogs). PROCEDURES: Computed tomography (CT) scans of the vertebral column were analyzed using computer-aided design (CAD) software. IVD calcification, vertebral column length, and vertebral geometry of the third cervical (C3), 13th thoracic (T13), and first lumbar (L1) vertebrae were compared. RESULTS: IVD calcification was not found in wild-type dogs. IVD calcification was more frequent in homozygous dogs than heterozygous (P = .008) or wild-type dogs (P < .001) and in heterozygous dogs compared to wild-type dogs (P < .001). Four IVDs were subclinically herniated in 3 dogs (2 homozygous, 1 heterozygous). Calcified IVD had a greater volume and surface area in heterozygous dogs than homozygous dogs. C3 vertebral canal height-to-width ratio was greater in homozygous dogs than heterozygous dogs (P = .044) and wild-type dogs (P = .010). CLINICAL RELEVANCE: IVD calcification and vertebral geometry can be analyzed using CAD software. The presence of 1 or 2 FGF4L2 copies in the absence of the FGF4L1 retrogene has an additive effect on the number of calcified IVD and a minor effect on vertebral geometry in NSDTR dogs. Data support the use of FGF4L2 phenotyping to reduce clinical disease in segregating breeds and to monitor the introduction of wild-type alleles into fixed breed populations.

Naturally occurring canine laminopathy leading to a dilated and fibrosing cardiomyopathy in the Nova Scotia Duck Tolling Retriever.

Dilated cardiomyopathy (DCM) is characterized by decreased systolic function and dilation of one or both ventricles, often leading to heart failure or sudden death. Two 10-month-old sibling Nova Scotia Duck Tolling Retrievers (NSDTR) died acutely with evidence of dilated cardiomyopathy with myocardial fibrosis. Association analysis using two cases and 35 controls identified three candidate regions homozygous in the two cases. Whole genome sequencing identified a frameshift deletion in the LMNA gene (NC_049228.1:g.41688530del, NP_001274080:p.(Asp576ThrfsTer124)). Three retrospectively identified NSDTRs with sudden death before 2 years of age and severe myocardial fibrosis were also homozygous for the deletion. One 5 year old with sudden death and myocardial fibrosis was heterozygous for the deletion. This variant was not identified in 722 dogs of other breeds, nor was it identified to be homozygous in 784 NSDTR. LMNA codes for lamin A/C proteins, which are type V intermediate filaments that provide structural support to the nuclear membrane. In humans, LMNA variants can cause DCM with sudden death as well as diseases of striated muscles, lipodystrophy, neuropathies, and accelerated aging disorders. This frameshift deletion is predicted to affect processing of prelamin A into lamin A. Pedigree analysis in the NSDTR and functional evaluation of heterozygotes is consistent with a predominantly recessive mode of inheritance and possibly low penetrance in heterozygotes in contrast to people, where most pathogenic LMNA variants are dominantly inherited.

Electrophysiological studies in American Quarter horses with neuroaxonal dystrophy.

OBJECTIVE: Neuroaxonal dystrophy (NAD) is a disease characterized by the sudden onset of neurologic signs in horses ranging from 4 to 36 months of age. Equine degenerative myeloencephalopathy (EDM), a disease that has been associated with low vitamin E concentrations, is considered a more advanced form of NAD. The objective of this report is to describe the electrophysiological features of NAD/EDM in American Quarter horses (QHs). HORSES: Six NAD/EDM-affected QHs and six unaffected QHs were evaluated by ophthalmic examination and electroretinography. Five of the NAD/EDM-affected QH and five unaffected QHs were also evaluated by electroencephalography (EEG). RESULTS: Ophthalmic examination, ERGs, and EEGs were unremarkable in NAD/EDM cases. CONCLUSIONS: Neuroaxonal dystrophy/EDM does not appear to cause clinical signs of ocular disease or functional ERG/EEG deficits in QHs.