Steven G. Friedenberg

Canine hip dysplasia is predictable by genotyping

OBJECTIVE To establish a predictive method using whole genome genotyping for early intervention in canine hip dysplasia (CHD) risk management, for the prevention of the progression of secondary osteoarthritis (OA), and for selective breeding. DESIGN Two sets of dogs (six breeds) were genotyped with dense SNPs covering the entire canine genome. The first set contained 359 dogs upon which a predictive formula for genomic breeding value (GBV) was derived by using their estimated breeding value (EBV) of the Norberg angle (a measure of CHD) and their genotypes. To investigate how well the formula would work for an individual dog with genotype only (without using EBV), a cross validation was performed by masking the EBV of one dog at a time. The genomic data and the EBV of the remaining dogs were used to predict the GBV for the single dog that was left out. The second set of dogs included 38 new Labrador retriever dogs, which had no pedigree relationship to the dogs in the first set. RESULTS The cross validation showed a strong correlation (R>0.7) between the EBV and the GBV. The independent validation showed a moderate correlation (R=0.5) between GBV for the Norberg angle and the observed Norberg angle (no EBV was available for the new 38 dogs). Sensitivity, specificity, positive and negative predictive values of the genomic data were all above 70%. CONCLUSIONS Prediction of CHD from genomic data is feasible, and can be applied for risk management of CHD and early selection for genetic improvement to reduce the prevalence of CHD in breeding programs. The prediction can be implemented before maturity, at which age current radiographic screening programs are traditionally applied, and as soon as DNA is available.

Evaluation of a fibrillin 2 gene haplotype associated with hip dysplasia and incipient osteoarthritis in dogs

OBJECTIVE To determine whether a mutation in the fibrillin 2 gene (FBN2) is associated with canine hip dysplasia (CHD) and osteoarthritis in dogs. ANIMALS 1,551 dogs. Procedures-Hip conformation was measured radiographically. The FBN2 was sequenced from genomic DNA of 21 Labrador Retrievers and 2 Greyhounds, and a haplotype in intron 30 of FBN2 was sequenced in 90 additional Labrador Retrievers and 143 dogs of 6 other breeds. Steady-state values of FBN2 mRNA and control genes were measured in hip joint tissues of fourteen 8-month-old Labrador Retriever-Greyhound crossbreeds. RESULTS The Labrador Retrievers homozygous for a 10-bp deletion haplotype in intron 30 of FBN2 had significantly worse CHD as measured via higher distraction index and extended-hip joint radiograph score and a lower Norberg angle and dorsolateral subluxation score. Among 143 dogs of 6 other breeds, those homozygous for the same deletion haplotype also had significantly worse radiographic CHD. Among the 14 crossbred dogs, as the dorsolateral subluxation score decreased, the capsular FBN2 mRNA increased significantly. Those dogs with incipient hip joint osteoarthritis had significantly increased capsular FBN2 mRNA, compared with those dogs without osteoarthritis. Dogs homozygous for the FBN2 deletion haplotype had significantly less FBN2 mRNA in their femoral head articular cartilage. CONCLUSIONS AND CLINICAL RELEVANCE The FBN2 deletion haplotype was associated with CHD. Capsular gene expression of FBN2 was confounded by incipient secondary osteoarthritis in dysplastic hip joints. Genes influencing complex traits in dogs can be identified by genome-wide screening, fine mapping, and candidate gene screening.

Single nucleotide polymorphisms refine QTL intervals for hip joint laxity in dogs

Hip laxity is one characteristic of canine hip dysplasia (CHD), an inheritable disease that leads to hip osteoarthritis. Using a genome-wide screen with 250 microsatellites in a crossbreed pedigree of 159 dysplastic Labrador retrievers and unaffected greyhounds, we previously identified putative (P < 0.01) QTL on canine chromosomes 11 and 29 (CFA11 and CFA29). To refine these QTL locations, we have genotyped 257 dogs including 105 Labrador retrievers, seven greyhounds, four generations of their crossbreed offspring and three German shepherds for 111 and 171 SNPs on CFA11 and CFA29 respectively. The distraction index (DI, a measure of maximum hip laxity) was used as an intermediate phenotype that predicts whether a hip joint will or will not develop osteoarthritis. Using a multipoint linkage analysis, significant evidence (95% posterior probability) was found for QTL contributing to hip laxity in the 16.2-21 cM region on CFA11 that explained 15-18% of the total variance in DI. Evidence for an independent QTL on CFA29 was weaker than that on CFA11. Identification of the causative mutation(s) will lead to better understanding of biochemical pathways in both dogs and humans with hip laxity and dysplasia.

Seizures following head trauma in dogs: 259 cases (1999–2009)

OBJECTIVE To determine whether dogs with head trauma have a greater incidence of seizures than the general canine patient population. DESIGN Retrospective case series. ANIMALS 259 client-owned dogs. PROCEDURES Medical records of dogs evaluated for head trauma at The Ohio State University Veterinary Medical Center from 1999 to 2009 were reviewed. Data were collected regarding the cause of the head trauma, physical examination and neurologic examination findings, comorbidities, and the development of seizures during hospitalization. A telephone survey was conducted to question owners regarding the development of seizures after discharge. Relationships between the nature of the head trauma and the development of seizures were then examined. RESULTS 3.5% of dogs with head trauma developed in-hospital seizures, and 6.8% of dogs with head trauma for which follow-up information was available developed seizures after hospital discharge, compared with an epilepsy rate of 1.4% in our hospital. Dogs that developed in-hospital seizures were significantly more likely to have been hit by a car or experienced acceleration-deceleration injury. Additionally, 10% of dogs with traumatic brain injury had in-hospital seizures. No visit or patient characteristics were significantly associated with the development of out-of-hospital seizures. CONCLUSIONS AND CLINICAL RELEVANCE Dogs with head trauma may develop seizures at a greater rate than dogs in the general canine patient population. Particularly in the immediate to early posttraumatic period, clinicians should remain vigilant for the development of posttraumatic seizures and treat patients accordingly.

Effect of disrupted mitochondria as a source of damage-associated molecular patterns on the production of tumor necrosis factor α by splenocytes from dogs

OBJECTIVE To evaluate the effects of damage-associated molecular patterns (DAMPs) derived from disrupted mitochondria on canine splenocytes and other immune cells. SAMPLES Liver, spleen, and bone marrow samples obtained from 8 cadavers of healthy research Beagles that had been euthanized for other purposes. PROCEDURES Mitochondria were obtained from canine hepatocytes, and mitochondrial DAMPs (containing approx 75% mitochondrial proteins) were prepared. Mitochondrial DAMPs and the nuclear cytokine high-mobility group box protein 1 were applied to splenocytes, bone marrow-differentiated dendritic cells, and a canine myelomonocytic cell (DH82) line for 6 or 24 hours. Cell culture supernatants from splenocytes, dendritic cells, and DH82 cells were assayed for tumor necrosis factor α with an ELISA. Expression of tumor necrosis factor α mRNA in splenocytes was evaluated with a quantitative real-time PCR assay. RESULTS In all cell populations evaluated, production of tumor necrosis factor α was consistently increased by mitochondrial DAMPs at 6 hours (as measured by an ELISA). In contrast, high-mobility group box protein 1 did not have any independent proinflammatory effects in this experimental system. CONCLUSIONS AND CLINICAL RELEVANCE The study revealed an in vitro inflammatory effect of mitochondrial DAMPs (containing approx 75% mitochondrial proteins) in canine cells and validated the use of an in vitro splenocyte model to assess DAMP-induced inflammation in dogs. This experimental system may aid in understanding the contribution of DAMPs to sepsis and the systemic inflammatory response syndrome in humans. Further studies in dogs are needed to validate the biological importance of these findings and to evaluate the in vivo role of mitochondrial DAMPs in triggering and perpetuating systemic inflammatory states.

Lymphocyte Subsets in the Adrenal Glands of Dogs With Primary Hypoadrenocorticism

Primary hypoadrenocorticism, or Addison’s disease, is an autoimmune condition common in certain dog breeds that leads to the destruction of the adrenal cortex and a clinical syndrome involving anorexia, gastrointestinal upset, and electrolyte imbalances. Previous studies have demonstrated that this destruction is strongly associated with lymphocytic-plasmacytic inflammation and that the lymphocytes are primarily T cells. In this study, we used both immunohistochemistry and in situ hybridization to characterize the T-cell subtypes involved. We collected postmortem specimens of 5 dogs with primary hypoadrenocorticism and 2 control dogs and, using the aforementioned techniques, showed that the lymphocytes are primarily CD4+ rather than CD8+. These findings have important implications for improving our understanding of the pathogenesis and in searching for the underlying causative genetic polymorphisms.

Evaluation of genes associated with human myxomatous mitral valve disease in dogs with familial myxomatous mitral valve degeneration

Myxomatous mitral valve disease (MMVD) is the most common heart disease in the dog. It is believed to be heritable in Cavalier King Charles spaniels (CKCS) and Dachshunds. Myxomatous mitral valve disease is a familial disease in human beings as well and genetic mutations have been associated with its development. We hypothesized that a genetic mutation associated with the development of the human form of MMVD was associated with the development of canine MMVD. DNA was isolated from blood samples from 10 CKCS and 10 Dachshunds diagnosed with MMVD, and whole genome sequences from each animal were obtained. Variant calling from whole genome sequencing data was performed using a standardized bioinformatics pipeline for all samples. After filtering, the canine genes orthologous to the human genes known to be associated with MMVD were identified and variants were assessed for likely pathogenic implications. No variant was found in any of the genes evaluated that was present in least eight of 10 affected CKCS or Dachshunds. Although mitral valve disease in the CKCS and Dachshund is a familial disease, we did not identify genetic cause in the genes responsible for the human disease in the dogs studied here.

A de novo mutation in the EXT2 gene associated with osteochondromatosis in a litter of American Staffordshire Terriers

Background We aimed to identify mutations associated with osteochondromatosis in a litter of American Staffordshire Terrier puppies. Hypothesis We hypothesized that the associated mutation would be located in a gene that causes osteochondromatosis in humans. Animals A litter of 9 American Staffordshire puppies, their sire and dam, 3 of 4 grandparents, 26 healthy unrelated American Staffordshire Terriers, and 154 dogs of 27 different breeds. Methods Whole genome sequencing was performed on the proband, and variants were compared against polymorphisms derived from 154 additional dogs across 27 breeds, as well as single nucleotide polymorphism database 146. One variant was selected for follow‐up sequencing. Parentage and genetic mosaicism were evaluated across the litter. Results We found 56,301 genetic variants unique to the proband. Eleven variants were located in or near the gene exostosin 2 (EXT2), which is strongly associated with osteochondromatosis in humans. One heterozygous variant (c.969C > A) is predicted to result in a stop codon in exon 5 of the gene. Sanger sequencing identified the identical mutation in all affected offspring. The mutation was absent in the unaffected offspring, both parents, all available grandparents, and 26 healthy unrelated American Staffordshire Terriers. Conclusions and Clinical Importance These findings represent the first reported mutation associated with osteochondromatosis in dogs. Because this mutation arose de novo, the identical mutation is unlikely to be the cause of osteochondromatosis in other dogs. However, de novo mutations in EXT2 are common in humans with osteochondromatosis, and by extension, it is possible that dogs with osteochondromatosis could be identified by sequencing the entire EXT2 gene.

Deafness and vestibular dysfunction in a Doberman Pinscher puppy associated with a mutation in the PTPRQ gene

Background A congenital syndrome of hearing loss and vestibular dysfunction affects Doberman Pinschers. Its inheritance pattern is suspected to be autosomal recessive and it potentially represents a spontaneous animal model of an autosomal recessive syndromic hearing loss. Hypothesis/Objectives The objectives of this study were to use whole genome sequencing (WGS) to identify deleterious genetic variants in candidate genes associated with the syndrome and to study the prevalence of candidate variants among a population of unaffected Doberman Pinschers. Animals One affected Doberman Pinscher and 202 unaffected Doberman Pinschers. Methods WGS of the affected dog with filtering of variants against a database of 154 unaffected dogs of diverse breeds was performed. Confirmation of candidate variants was achieved by Sanger sequencing followed by genotyping of the control population of unaffected Doberman Pinschers. Results WGS and variant filtering identified an alteration in a gene associated with both deafness and vestibular disease in humans: protein tyrosine phosphatase, receptor type Q (PTPRQ). There was a homozygous A insertion at CFA15: 22 989 894, causing a frameshift mutation in exon 39 of the gene. This insertion is predicted to cause a protein truncation with a premature stop codon occurring after position 2054 of the protein sequence that causes 279 C‐terminal amino acids to be eliminated. Prevalence of the variant was 1.5% in a cohort of 202 unaffected Doberman Pinschers; all unaffected Doberman Pinschers were heterozygous or heterozygous for the reference allele. Conclusion and Clinical Importance We report the identification of a genetic alteration on the PTPRQ gene that is associated with congenital hearing and vestibular disorder in a young Doberman Pinscher dog.

Evaluation of the genetic basis of primary hypoadrenocorticism in Standard Poodles using SNP array genotyping and whole-genome sequencing.

Primary hypoadrenocorticism, also known as Addison’s disease, is an autoimmune disorder leading to the destruction of the adrenal cortex and subsequent loss of glucocorticoid and mineralocorticoid hormones. The disease is prevalent in Standard Poodles and is believed to be highly heritable in the breed. Using genotypes derived from the Illumina Canine HD SNP array, we performed a genome-wide association study of 133 carefully phenotyped Standard Poodles (61 affected, 72 unaffected) and found no markers significantly associated with the disease. We also sequenced the entire genomes of 20 Standard Poodles (13 affected, 7 unaffected) and analyzed the data to identify common variants (including SNPs, indels, structural variants, and copy number variants) across affected dogs and variants segregating within a single pedigree of highly affected dogs. We identified several candidate genes that may be fixed in both Standard Poodles and a small population of dogs of related breeds. Further studies are required to confirm these findings more broadly, as well as additional gene-mapping efforts aimed at fully understanding the genetic basis of what is likely a complex inherited disorder.