Noa Safra

Mutations in the SLC2A9 Gene Cause Hyperuricosuria and Hyperuricemia in the Dog

Allantoin is the end product of purine catabolism in all mammals except humans, great apes, and one breed of dog, the Dalmatian. Humans and Dalmatian dogs produce uric acid during purine degradation, which leads to elevated levels of uric acid in blood and urine and can result in significant diseases in both species. The defect in Dalmatians results from inefficient transport of uric acid in both the liver and renal proximal tubules. Hyperuricosuria and hyperuricemia (huu) is a simple autosomal recessive trait for which all Dalmatian dogs are homozygous. Therefore, in order to map the locus, an interbreed backcross was used. Linkage mapping localized the huu trait to CFA03, which excluded the obvious urate transporter 1 gene, SLC22A12. Positional cloning placed the locus in a minimal interval of 2.5 Mb with a LOD score of 17.45. A critical interval of 333 kb containing only four genes was homozygous in all Dalmatians. Sequence and expression analyses of the SLC2A9 gene indicated three possible mutations, a missense mutation (G616T;C188F) and two promoter mutations that together appear to reduce the expression levels of one of the isoforms. The missense mutation is associated with hyperuricosuria in the Dalmatian, while the promoter SNPs occur in other unaffected breeds of dog. Verification of the causative nature of these changes was obtained when hyperuricosuric dogs from several other breeds were found to possess the same combination of mutations as found in the Dalmatian. The Dalmatian dog model of hyperuricosuria and hyperuricemia underscores the importance of SLC2A9 for uric acid transport in mammals.

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.

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; praw = 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.

Expanded dog leukocyte antigen (DLA) single nucleotide polymorphism (SNP) genotyping reveals spurious class II associations.

The dog leukocyte antigen (DLA) system contains many of the functional genes of the immune system, thereby making it a candidate region for involvement in immune-mediated disorders. A number of studies have identified associations between specific DLA class II haplotypes and canine immune hemolytic anemia, thyroiditis, immune polyarthritis, type I diabetes mellitus, hypoadrenocorticism, systemic lupus erythematosus-related disease complex, necrotizing meningoencephalitis (NME) and anal furunculosis. These studies have relied on sequencing approximately 300 bases of exon 2 of each of the DLA class II genes: DLA-DRB1, DLA-DQA1 and DLA-DQB1. In the present study, an association (odds ratio=4.29) was identified by this method between Weimaraner dogs with hypertrophic osteodystrophy (HOD) and DLA-DRB1∗01501. To fine map the association with HOD, a genotyping assay of 126 coding single nucleotide polymorphisms (SNPs) from across the entire DLA, spanning a region of 2.5 Mb (3,320,000-5,830,000) on CFA12, was developed and tested on Weimaraners with HOD, as well as two additional breeds with diseases associated with DLA class II: Nova Scotia duck tolling retrievers with hypoadrenocorticism and Pug dogs with NME. No significant associations were found between Weimaraners with HOD or Nova Scotia duck tolling retrievers with hypoadrenocorticism and SNPs spanning the DLA region. In contrast, significant associations were found with NME in Pug dogs, although the associated region extended beyond the class II genes. By including a larger number of genes from a larger genomic region, a SNP genotyping assay was generated that provides coverage of the extended DLA region and may be useful in identifying and fine mapping DLA associations in dogs.

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.

Clinical manifestations, response to treatment, and clinical outcome for Weimaraners with hypertrophic osteodystrophy: 53 cases (2009–2011)

OBJECTIVE To evaluate clinical manifestations, response to treatment, and outcome for Weimaraners with hypertrophic osteodystrophy (HOD). DESIGN Retrospective case series. ANIMALS 53 dogs. PROCEDURES Medical records were reviewed for signalment, vaccination history, clinical signs, laboratory test results, response to treatment, and relapses. Radiographs were reviewed. RESULTS Clinical signs included pyrexia, lethargy, and ostealgia; signs involving the gastrointestinal, ocular, or cutaneous systems were detected. Of the 53 dogs, 28 (52.8%) had HOD-affected littermates. Dogs with HOD-affected littermates were more likely to relapse, compared with the likelihood of relapse for dogs with no HOD-affected littermates. All 53 dogs had been vaccinated 1 to 30 days before HOD onset; no difference was found between the number of dogs with a history of vaccination with a recombinant vaccine (n … 21) versus a nonrecombinant vaccine (32). Fifty (94.3%) dogs had radiographic lesions compatible with HOD at disease onset, and the other 3 (5.7%) had HOD lesions 48 to 72 hours after the onset of clinical signs. Twelve of 22 (54.5%) dogs treated with NSAIDs did not achieve remission by 7 days after initiation of treatment. All dogs treated initially with corticosteroids achieved remission within 8 to 48 hours. Of the 33 dogs that reached adulthood, 28 (84.8%) were healthy and 5 (15.2%) had episodes of pyrexia and malaise. CONCLUSIONS AND CLINICAL RELEVANCE Treatment with corticosteroids was superior to treatment with NSAIDs in Weimaraners with HOD. It may be necessary to evaluate repeated radiographs to establish a diagnosis of HOD. Most HOD-affected Weimaraners had resolution of the condition with physeal closure.

Validation of a urine test and characterization of the putative genetic mutation for hyperuricosuria in Bulldogs and Black Russian Terriers

OBJECTIVE To determine whether hyperuricosuria was a predisposing factor for urate urolithiasis in Bulldogs and Black Russian Terriers (BRTs) and to estimate the allele frequency of the Cys181Phe genetic mutation in urate transporter SLC2A9 in these breeds. ANIMALS 192 Bulldogs, 101 BRTs, 10 Dalmatians, and 9 dogs of other breeds. PROCEDURES Uric acid (UA) and creatinine (Cr) concentrations were quantified in urine samples collected from all dogs via midstream catch during natural voiding. Buccal swab or blood samples were also obtained, and DNA was extracted and used to genotype SLC2A9 sequence variants by use of pyrosequencing assays. A urine test for hyperuricosuria was validated in adult dogs by comparing urinary UA:Cr ratios between known hyperuricosuric and nonhyperuricosuric dogs. RESULTS Significantly higher UA:Cr ratios were found in some Bulldogs and BRTs, compared with ratios in other dogs from these breeds. These dogs were also homozygous for the SLC2A9 Cys181Phe mutation. The allele frequency of the Cys181Phe mutation was 0.16 in Bulldogs and 0.51 in BRTs. On the basis of these allele frequencies, 3% of the Bulldog population and 27% of the BRT population were estimated to be hyperuricosuric. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested the genetic mutation associated with hyperuricosuria, first identified in Dalmatians, also appears to cause hyperuricosuria in Bulldogs and BRTs, indicating that similar management strategies for urate urolithiasis can be used in these breeds. The allele frequency of the mutation was high in both breeds, and DNA testing can be used to select against the mutation.

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. GLIA 2014;62:39–51

Serum levels of innate immunity cytokines are elevated in dogs with metaphyseal osteopathy (hypertrophic osteodytrophy) during active disease and remission

Metaphyseal osteopathy (MO) (hypertrophic osteodystrophy) is a developmental disorder of unexplained etiology affecting dogs during rapid growth. Affected dogs experience relapsing episodes of lytic/sclerotic metaphyseal lesions and systemic inflammation. MO is rare in the general dog population; however, some breeds (Weimaraner, Great Dane and Irish Setter) have a much higher incidence, supporting a hereditary etiology. Autoinflammatory childhood disorders of parallel presentation such as chronic recurrent multifocal osteomyelitis (CRMO), and deficiency of interleukin-1 receptor antagonist (DIRA), involve impaired innate immunity pathways and aberrant cytokine production. Given the similarities between these diseases, we hypothesize that MO is an autoinflammatory disease mediated by cytokines involved in innate immunity. To characterize immune dysregulation in MO dogs we measured serum levels of inflammatory markers in 26 MO and 102 control dogs. MO dogs had significantly higher levels (pg/ml) of serum Interleukin-1beta (IL-1β), IL-18, IL-6, Granulocyte-macrophage colony stimulating factor (GM-CSF), C-X-C motif chemokine 10 (CXCL10), tumor necrosis factor (TNF), and IL-10. Notably, recovered MO dogs were not different from dogs during active MO disease, providing a suggestive mechanism for disease predisposition. This is the first documentation of elevated immune markers in MO dogs, uncovering an immune profile similar to comparable autoinflammatory disorders in children.

Association of early onset myasthenia gravis in Newfoundland dogs with the canine major histocompatibility complex class I

Acquired Myasthenia Gravis (MG) is an autoimmune neuromuscular disorder whose development in humans has been associated with the Major Histocompatibility Complex (MHC) or Human Leukocyte Antigen (HLA). There is a form of early onset MG (EOMG) in Newfoundland dogs that mimics the clinical presentation in humans and appears to have familial inheritance. Genotyping of three classical Dog Leukocyte Antigen (DLA) class II genes, DLA-DRB1, DLA-DQA1 and DLA-DQB1, in 16 Newfoundlands with EOMG and 46 unaffected Newfoundlands, identified DLA-DQB1 *00301 (p-value = 0.0051 OR: 7.41) as a risk locus for the development of EOMG in this breed. In order to further investigate the extent of the association to the entire MHC region, 208 additional SNPs were genotyped in two phases. Both a risk locus for EOMG to the DLA class I (chr12: 458483-506460) and a protective locus for EOMG susceptibility that extends outside of the DLA class I (chr12: 89701-475348) were identified. Four additional dog breeds with an elevated risk for the development of MG were SNP genotyped, but no shared or significant associations were found. MHC involvement in canine MG disease manifestation overlaps with loci identified in human studies and highlights the value of dogs as a model for genetic studies of naturally occurring diseases.