Two Independent Mutations in ADAMTS17 Are Associated with Primary Open Angle Glaucoma in the Basset Hound and Basset Fauve de Bretagne Breeds of Dog

PurposeMutations in ADAMTS10 (CFA20) have previously been associated with primary open angle glaucoma (POAG) in the Beagle and Norwegian Elkhound. The closely related gene, ADAMTS17, has also been associated with several different ocular phenotypes in multiple breeds of dog, including primary lens luxation and POAG. We investigated ADAMTS17 as a candidate gene for POAG in the Basset Hound and Basset Fauve de Bretagne dog breeds.MethodsWe performed ADAMTS17 exon resequencing in three Basset Hounds and three Basset Fauve de Bretagne dogs with POAG. Identified variants were genotyped in additional sample cohorts of both breeds and dogs of other breeds to confirm their association with disease.ResultsAll affected Basset Hounds were homozygous for a 19 bp deletion in exon 2 that alters the reading frame and is predicted to lead to a truncated protein. Fifty clinically unaffected Basset Hounds were genotyped for this mutation and all were either heterozygous or homozygous for the wild type allele. Genotyping of 223 Basset Hounds recruited for a different study revealed a mutation frequency of 0.081 and predicted frequency of affected dogs in the population to be 0.007. Based on the entire genotyping dataset the association statistic for the POAG-associated deletion was p = 1.26 x 10⁻¹⁰. All affected Basset Fauve de Bretagne dogs were homozygous for a missense mutation in exon 11 causing a glycine to serine amino acid substitution (G519S) in the disintegrin-like domain of ADAMTS17 which is predicted to alter protein function. Unaffected Basset Fauve de Bretagne dogs were either heterozygous for the mutation (5/24) or homozygous for the wild type allele (19/24). Based on the entire genotyping dataset the association statistic for the POAG-associated deletion was p = 2.80 x 10⁻⁷. Genotyping of 85 dogs of unrelated breeds and 90 dogs of related breeds for this variant was negative.ConclusionThis report documents strong associations between two independent ADAMTS17 mutations and POAG in two different dog breeds.     

Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.     

Cloning of canine rom-1 and its investigation as a candidate gene for generalized progressive retinal atrophies in dogs

Generalized progressive retinal atrophy (gPRA) represents a genetically heterogenous group of retinal degenerations affecting pedigree dogs. Currently, we are using a candidate gene approach in an attempt to identify mutations causing gPRA in dogs. Here we report the cloning, sequencing and analysis of canine rom-1 , a structural gene of the rod photoreceptor. Single-stranded conformation polymorphism (SSCP) analysis was used to look for polymorphisms segregating with gPRA in the English cocker spaniel, Labrador retriever, miniature poodle, miniature long-haired dachshund, Tibetan terrier, miniature schnauzer, Cardigan Welsh corgi and Irish wolfhound. Further investigation involved DNA sequencing and restriction fragment length polymorphism (RFLP) analysis. Our studies revealed the presence of three polymorphisms, none of which segregated with disease phenotype. Haplotype analysis identified four rom-1 alleles. Our results indicate that rom-1 is unlikely to be a cause of gPRA in the breeds of dog examined.     

A mixed-methods approach to understanding domestic dog health and disease transmission risk in an indigenous reserve in Guyana,South America

Domestic dogs (Canis lupus familiaris) can transmit a variety of pathogens due to their ubiquitousness in urban, rural and natural environments, and their close interactions with wildlife and humans. In this study, we used a mixed-methods approach to assess the role of domestic dogs as potential intermediaries of disease transmission from wildlife to humans among indigenous Waiwai in the Konashen Community Owned Conservation Area, Guyana. To address these objectives we 1) performed physical examinations and collected biological samples to assess Waiwai domestic dog health, and 2) administered questionnaires to characterize the role of dogs in the community and identify potential transmission pathways between wildlife, dogs, and humans. We observed ectoparasites on all dogs (n = 20), including: fleas (100%), ticks (15%), botflies (30%), and jigger flea lesions (Tunga penetrans) (80%). Ten percent of dogs were seropositive for Ehrlichia canis/ewingii, 10% were positive for Dirofilaria immitis, and one dog was seropositive for Leishmania infantum. All dogs (n = 20) were seronegative for: canine distemper virus, Brucella canis, Leptospira serovars, Trypanosoma cruzi, Anaplasma phagocytophilum/platys and Borrelia burgdorferi. Our questionnaire data revealed that the Waiwai remove ectoparasites from their dogs, clean up dog feces, and administer traditional and/or Western medicine to their dogs. White blood cell, strongyle-type ova, and eosinophil counts were lower in dogs that were not frequently used for hunting, dogs that did receive traditional and/or western medicine, and dogs that were frequently kept in elevated dog houses, although differences were not statistically significant. While our results suggest that the Waiwai have developed cultural practices that may promote dog health and/or prevent zoonotic disease transmission, more research is necessary to determine the efficacy of these practices. Our study provides important data on the health of dogs and the potential for disease transmission to humans in a zoonotic hotspot.     

Investigation of the role of opsin gene polymorphism in generalized progressive retinal atrophies in dogs

The generalized progressive retinal atrophies (gPRAs) form a group of retinal degenerations of pedigree dogs and cats, which have a variety of genetic origins (mostly unknown). We have examined the opsin gene for polymorphisms in several breeds of pedigree dog suffering from distinct forms of gPRA, by methods including single-strand conformation polymorphism analysis, microsatellite analysis and direct sequencing. The breeds examined included the Tibetan terrier, the miniature schnauzer, the Irish setter, the miniature poodle, the Labrador retriever and the English cocker spaniel, as well as individuals from breeds in which PRA has not been described and of mixed breed. Individuals from each of the named breeds suffering from PRA were compared with clinically normal dogs. Two polymorphisms were found. One, segregating within the Tibetan terrier population, but not seen in other breeds, was a synonymous transition at nucleotide position 780 in exon 3. Inheritance of this polymorphism suggests that opsin is unlikely to contain mutations causative of gPRA in this breed. The other polymorphism occurred between all miniature schnauzers examined and dogs of other breeds. It consisted of a single base insertion in intron 2. No polymorphisms in the opsin sequence were detected in any other breed. DNA sequencing allowed rigorous exclusion of mutations in opsin as a cause of gPRA in miniature poodles, English cocker spaniels or Labrador retrievers.     

Acrofacial Dysostosis,Cincinnati Type,a Mandibulofacial Dysostosis Syndrome with Limb Anomalies,Is Caused by POLR1A Dysfunction

We report three individuals with a cranioskeletal malformation syndrome that we define as acrofacial dysostosis, Cincinnati type. Each individual has a heterozygous mutation in POLR1A, which encodes a core component of RNA polymerase 1. All three individuals exhibit varying degrees of mandibulofacial dysostosis, and two additionally have limb anomalies. Consistent with this observation, we discovered that polr1a mutant zebrafish exhibited cranioskeletal anomalies mimicking the human phenotype. polr1a loss of function led to perturbed ribosome biogenesis and p53-dependent cell death, resulting in a deficiency of neural-crest-derived skeletal precursor cells and consequently craniofacial anomalies. Our findings expand the genotypic and phenotypic heterogeneity of congenital acrofacial disorders caused by disruption of ribosome biogenesis.     

Genome-Wide Association Analysis Identifies a Mutation in the Thiamine Transporter 2 (SLC19A3) Gene Associated with Alaskan Husky Encephalopathy

Alaskan Husky Encephalopathy (AHE) has been previously proposed as a mitochondrial encephalopathy based on neuropathological similarities with human Leigh Syndrome (LS). We studied 11 Alaskan Husky dogs with AHE, but found no abnormalities in respiratory chain enzyme activities in muscle and liver, or mutations in mitochondrial or nuclear genes that cause LS in people. A genome wide association study was performed using eight of the affected dogs and 20 related but unaffected control AHs using the Illumina canine HD array. SLC19A3 was identified as a positional candidate gene. This gene controls the uptake of thiamine in the CNS via expression of the thiamine transporter protein THTR2. Dogs have two copies of this gene located within the candidate interval (SLC19A3.2 – 43.36–43.38 Mb and SLC19A3.1 – 43.411–43.419 Mb) on chromosome 25. Expression analysis in a normal dog revealed that one of the paralogs, SLC19A3.1, was expressed in the brain and spinal cord while the other was not. Subsequent exon sequencing of SLC19A3.1 revealed a 4bp insertion and SNP in the second exon that is predicted to result in a functional protein truncation of 279 amino acids (c.624 insTTGC, c.625 C>A). All dogs with AHE were homozygous for this mutation, 15/41 healthy AH control dogs were heterozygous carriers while 26/41 normal healthy AH dogs were wild type. Furthermore, this mutation was not detected in another 187 dogs of different breeds. These results suggest that this mutation in SLC19A3.1, encoding a thiamine transporter protein, plays a critical role in the pathogenesis of AHE.     

Duration of Plague (Yersinia pestis) Outbreaks in Black-Tailed Prairie Dog (Cynomys ludovicianus) Colonies of Northern Colorado

Plague, caused by the bacterium Yersinia pestis, triggers die-offs in colonies of black-tailed prairie dogs (Cynomys ludovicianus), but the time-frame of plague activity is not well understood. We document plague activity in fleas from prairie dogs and their burrows on three prairie dog colonies that suffered die-offs. We demonstrate that Y. pestis transmission occurs over periods from several months to over a year in prairie dog populations before observed die-offs.     

Partial Deletion of the Sulfate Transporter SLC13A1 Is Associated with an Osteochondrodysplasia in the Miniature Poodle Breed

A crippling dwarfism was first described in the Miniature Poodle in Great Britain in 1956. Here, we resolve the genetic basis of this recessively inherited disorder. A case-control analysis (8∶8) of genotype data from 173 k SNPs revealed a single associated locus on CFA14 (P_raw <10^–8). All affected dogs were homozygous for an ancestral haplotype consistent with a founder effect and an identical-by-descent mutation. Systematic failure of nine, nearly contiguous SNPs, was observed solely in affected dogs, suggesting a deletion was the causal mutation. A 130-kb deletion was confirmed both by fluorescence in situ hybridization (FISH) analysis and by cloning the physical breakpoints. The mutation was perfectly associated in all cases and obligate heterozygotes. The deletion ablated all but the first exon of SLC13A1, a sodium/sulfate symporter responsible for regulating serum levels of inorganic sulfate. Our results corroborate earlier findings from an Slc13a1 mouse knockout, which resulted in hyposulfatemia and syndromic defects. Interestingly, the metabolic disorder in Miniature Poodles appears to share more clinical signs with a spectrum of human disorders caused by SLC26A2 than with the mouse Slc13a1 model. SLC26A2 is the primary sodium-independent sulfate transporter in cartilage and bone and is important for the sulfation of proteoglycans such as aggregan. We propose that disruption of SLC13A1 in the dog similarly causes undersulfation of proteoglycans in the extracellular matrix (ECM), which impacts the conversion of cartilage to bone. A co-dominant DNA test of the deletion was developed to enable breeders to avoid producing affected dogs and to selectively eliminate the mutation from the gene pool.     

Molecular analysis of congenital scoliosis: a candidate gene approach

The etiology of congenital scoliosis is largely unknown. The severe vertebral disorder, spondylocostal dysostosis type 1, is associated with a homozygous delta-like 3 (DLL3) mutation. Scoliosis has been observed in a heterozygous DLL3 carrier, raising the possibility of its involvement in congenital scoliosis. We present the first molecular study of congenital scoliosis by analysis of the candidate gene DLL3 and demonstrate one novel missense variant. However, no novel or previously described mutations are present in our cohort, indicating that DLL3 mutations may not be a major cause of congenital scoliosis. Additionally, we have evaluated patients with congenital scoliosis not diagnosed with a known syndrome and identified a significant number of associated renal and cardiac anomalies and familial incidence of idiopathic scoliosis in this group.     

High incidence of Trypanosoma cruzi infections in dogs directly detected through longitudinal tracking at 10 multi-dog kennels,Texas, USA

Canine Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is increasingly recognized as a health concern for dogs in the USA, and infected dogs may signal geographic regions of risk for human disease. Dogs living in multi-dog kennel environments (kennels with more than one dog) where triatomine vectors are endemic may be at high risk for infection. We monitored a cohort of 64 T. cruzi-infected and uninfected dogs across 10 kennels in Texas, USA, to characterize changes in infection status over one year. We used robust diagnostic criteria in which reactivity on multiple independent platforms was required to be considered positive. Among the 30 dogs enrolled as serologically- and/or PCR-positive, all but one dog showed sustained positive T. cruzi diagnostic results over time. Among the 34 dogs enrolled as serologically- and PCR-negative, 10 new T. cruzi infections were recorded over a 12-month period. The resulting incidence rate for dogs initially enrolled as T. cruzi-negative was 30.7 T. cruzi infections per 100 dogs per year. This study highlights the risk of T. cruzi infection to dogs in kennel environments. To protect both dog and human health, there is an urgent need to develop more integrated vector control methods as well as prophylactic and curative antiparasitic treatment options for T. cruzi infection in dogs.     

Whole-Genome Sequencing Reveals Lactase Persistence Adaptation in European Dogs

Coexistence and cooperation between dogs and humans over thousands of years have supported convergent evolutionary processes in the two species. Previous studies found that Eurasian dogs evolved into a distinct geographic cluster. In this study, we used the genomes of 242 European dogs, 38 Southeast Asian indigenous (SEAI) dogs, and 41 gray wolves to identify adaptation of European dogs . We report 86 unique positively selected genes in European dogs, among which is LCT (lactase). LCT encodes lactase, which is fundamental for the digestion of lactose. We found that an A-to-G mutation (chr19:38,609,592) is almost fixed in Middle Eastern and European dogs. The results of two-dimensional site frequency spectrum (2D SFS) support that the mutation is under soft sweep . We inferred that the onset of positive selection of the mutation is shorter than 6,535 years and behind the well-developed dairy economy in central Europe. It increases the expression of LCT by reducing its binding with ZEB1, which would enhance dog’s ability to digest milk-based diets. Our study uncovers the genetic basis of convergent evolution between humans and dogs with respect to diet, emphasizing the import of the dog as a biomedical model for studying mechanisms of the digestive system.     

Müllerian inhibiting substance is present in testes of dogs with persistent müllerian duct syndrome

Breeding studies in a strain of miniature schnauzer dogs with Persistent Müllerian Duct Syndrome (PMDS) indicate this syndrome is inherited as an autosomal recessive trait, as it is in man. Testes of neonatal dogs affected with PMDS and normal male littermates were examined for Müllerian Inhibiting Substance (MIS) production by immunohistochemistry and bioassay. MIS immunoactivity was detected in Sertoli cells of normal and affected pups using an avidin-biotin complex-enhanced method. Rat embryonic Müllerian ducts regressed when cocultured with testis fragments of both normal and affected pups in a graded organ culture bioassay, demonstrating that the MIS produced was bioactive. These findings indicate that Müllerian duct persistence in affected dogs is not due to a mutation in the structural gene for MIS, but rather, by inference, to a failure of response to MIS at the receptor level.     

Molecular detection of Giardia duodenalis and Cryptosporidium spp. from stray dogs residing in monasteries in Bangkok,Thailand

Both Cryptosporidium spp. and Giardia duodenalis are enteric protozoan parasites that infect a wide variety of domestic animals as well as humans worldwide, causing diarrheal diseases. Giardia duodenalis assemblages C and D are specific to canine hosts and zoonotic assemblages A and B are also found in dogs as a reservoir host. In dogs, Cryptosporidium canis is the host-specific species while humans are infected by C. hominis and C. parvum and at least another 16 zoonotic Cryptosporidium species have been reported causing human infections, with C. meleagridis, C. viatorum, and C. ubiquitum being the most frequent. The objective of this study was to determine the prevalence of Cryptosporidium spp. and G. duodenalis from stray dogs in areas of Bangkok and to identify the species and assemblages. Fecal samples (540) were collected from dogs residing in 95 monasteries in 48 districts in the Bangkok metropolitan area. Nested Polymerase Chain Reaction (PCR) was performed using the ssu-rRNA gene for both parasites. In total, 3.0% (16/540) samples were positive for G. duodenalis, with most being G. duodenalis assemblage D (7/16) followed by assemblage C (7/16) and zoonotic assemblage A (2/16). The prevalence of Cryptosporidium spp. was 0.7% (4/540) based on the PCR results and all were the dog genotype C. canis. These results indicated that dogs residing in Bangkok monasteries poses a limited role as source of human giardiosis and cryptosporidiosis.     

De novo GLI3 mutation in esophageal atresia: Reproducing the phenotypic spectrum of Gli3 defects in murine models

Esophageal atresia is a common and life-threatening birth defect with a poorly understood etiology. In this study, we analyzed the sequence variants of coding regions for a set of esophageal atresia-related genes including MYCN, SOX2, CHD7, GLI3, FGFR2 and PTEN for mutations using PCR-based target enrichment and next-generation sequencing in 27 patients with esophageal atresia. Genomic copy number variation analysis was performed using Affymetrix SNP 6.0. We found a de novo heterozygous mutation in the N-terminal region of the GLI3 gene (c.332 T > C, p.M111T) in a patient with esophageal atresia and hemivertebrae. The N-terminal region (amino acids 1–397) of GLI3 contains the repressor domain, which interacts with SKI family proteins. Using the co-immunoprecipitation assay, we found that interaction of GLI3 with the SKI family protein SKIL was significantly compromised by the p.M111T mutation of GLI3. Thus far, all the identified mutations mapped within the repressor domain of GLI3 were nonsense and frame-shift mutations. In this study, a missense mutation was initially detected in this region. Our finding is the first to link this GLI3 gene mutation with esophageal atresia in humans, which was previously suggested in an animal model.     

A Frameshift Mutation in the Cubilin Gene (CUBN) in Border Collies with Imerslund-Gr?sbeck Syndrome (Selective Cobalamin Malabsorption)

Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies.     

Analysis of structural diversity in wolf-like canids reveals post-domestication variants

Background

Although a variety of genetic changes have been implicated in causing phenotypic differences among dogs, the role of copy number variants (CNVs) and their impact on phenotypic variation is still poorly understood. Further, very limited knowledge exists on structural variation in the gray wolf, the ancestor of the dog, or other closely related wild canids. Documenting CNVs variation in wild canids is essential to identify ancestral states and variation that may have appeared after domestication.

Results

In this work, we genotyped 1,611 dog CNVs in 23 wolf-like canids (4 purebred dogs, one dingo, 15 gray wolves, one red wolf, one coyote and one golden jackal) to identify CNVs that may have arisen after domestication. We have found an increase in GC-rich regions close to the breakpoints and around 1 kb away from them suggesting that some common motifs might be associated with the formation of CNVs. Among the CNV regions that showed the largest differentiation between dogs and wild canids we found 12 genes, nine of which are related to two known functions associated with dog domestication; growth (PDE4D, CRTC3 and NEB) and neurological function (PDE4D, EML5, ZNF500, SLC6A11, ELAVL2, RGS7 and CTSB).

Conclusions

Our results provide insight into the evolution of structural variation in canines, where recombination is not regulated by PRDM9 due to the inactivation of this gene. We also identified genes within the most differentiated CNV regions between dogs and wolves, which could reflect selection during the domestication process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-465) contains supplementary material, which is available to authorized users.