GWAS using low-pass whole genome sequence reveals a novel locus in canine congenital idiopathic megaesophagus.

Congenital idiopathic megaesophagus (CIM) is a gastrointestinal disorder of dogs wherein the esophagus is dilated and swallowing activity is reduced, causing regurgitation of ingesta. Affected individuals experience weight loss and malnourishment and are at risk for aspiration pneumonia, intussusception, and euthanasia. Great Danes have among the highest incidences of CIM across dog breeds, suggesting a genetic predisposition. We generated low-pass sequencing data for 83 Great Danes and used variant calls to impute missing whole genome single-nucleotide variants (SNVs) for each individual based on haplotypes phased from 624 high-coverage dog genomes, including 21 Great Danes. We validated the utility of our imputed data set for genome-wide association studies (GWASs) by mapping loci known to underlie coat phenotypes with simple and complex inheritance patterns. We conducted a GWAS for CIM with 2,010,300 SNVs, identifying a novel locus on canine chromosome 1 (P-val = 2.76 × 10-10). Associated SNVs are intergenic or intronic and are found in two clusters across a 1.7-Mb region. Inspection of coding regions in high-coverage genomes from affected Great Danes did not reveal candidate causal variants, suggesting that regulatory variants underlie CIM. Further studies are necessary to assess the role of these non-coding variants.

Use of whole genome analysis to identify shared genomic variants across breeds in canine mitral valve disease.

Familial mitral valve prolapse in human beings has been associated with several genetic variants; however, in most cases, a known variant has not been identified. Dogs also have a naturally occurring form of familial mitral valve disease (MMVD) with similarities to the human disease. A shared genetic background and clinical phenotype of this disease in some dog breeds has indicated that the disease may share a common genetic cause. We evaluated DNA from 50 affected dogs from five different dog breeds in a whole genome sequencing approach to identify shared variants across and within breeds that could be associated with MMVD. No single causative genetic mutation was found from the 50 dogs with MMVD. Ten variants were identified in 37/50 dogs around and within the MED13L gene. These variants were no longer associated with MMVD when evaluated with a larger cohort including both affected and unaffected dogs. No high/moderate impact variants were identified in 10/10 miniature poodles, one was identified in 10/10 Yorkshire Terriers and 10/10 dachshunds, respectively, 14 were identified in 10/10 Miniature schnauzers, and 19 in 10/10 CKCS. Only one of these could be associated with the cardiac valve (Chr12:36801705, COL12A1; CKCS) but when evaluated in an additional 100 affected CKCS the variant was only identified in 84/100 affected dogs, perhaps indicating genetic heterogeneity in this disease. Our findings indicate that development of MMVD in the dog may be related to a combination of genetic and environmental factors that impact specific molecular pathways rather than a single shared genetic variant across or within breeds.

A defect in the NOG gene increases susceptibility to spontaneous superficial chronic corneal epithelial defects (SCCED) in boxer dogs.

BACKGROUND: Superficial chronic corneal epithelial defects (SCCEDs) are spontaneous corneal defects in dogs that share many clinical and pathologic characteristics to recurrent corneal erosions (RCE) in humans. Boxer dogs are predisposed to SCCEDs, therefore a search for a genetic defect was performed to explain this susceptibility. DNA was extracted from blood collected from Boxer dogs with and without SCCEDs followed by whole genome sequencing (WGS). RNA sequencing of corneal tissue and immunostaining of corneal sections from affected SCCED Boxer dogs with a deletion in the NOG gene and affected non-Boxer dogs without the deletion were performed. RESULTS: A 30 base pair deletion at a splice site in Noggin (NOG) (Chr 9:31453999) was identified by WGS and was significantly associated (P < 0.0001) with Boxer SCCEDs compared to unaffected non-Boxer dogs. NOG, BMP4, MMP13, and NCAM1 all had significant fold reductions in expression and SHH was significantly increased in Boxers with the NOG deletion as identified by RNA-Seq. Corneal IHC from NOG deletion dogs with SCCEDs had lower NOG and significantly higher scores of BMP2. CONCLUSIONS: Many Boxer dogs with SCCED have a genetic defect in NOG. NOG is a constitutive protein in the cornea which is a potent inhibitor of BMP, which likely regulate limbal epithelial progenitor cells (LEPC). Dysregulation of LEPC may play a role in the pathogenesis of RCE.

Canine junctional epidermolysis bullosa due to a novel mutation in LAMA3 with severe upper respiratory involvement.

BACKGROUND: Junctional epidermolysis bullosa (JEB) is a group of congenital blistering skin diseases characterized by clefting through the lamina lucida of the basement membrane zone. OBJECTIVES: To characterize the clinical and morphological features of a congenital mechanobullous disease in a litter of puppies with severe upper respiratory involvement, and to identify an associated genetic variant. ANIMALS: Five of eight puppies in an Australian cattle dog cross-bred litter showed signs of skin fragility. Three were stillborn and one died at one month of age. The two surviving puppies were presented with blistering skin disease and severe respiratory distress. Additionally, one unaffected sibling was examined and blood was obtained for genetic testing. METHODS AND MATERIALS: Post-mortem examination, histopathological evaluation and electron microscopy were performed. Whole genome sequencing (WGS) of one affected puppy was compared to a database of 522 dogs of 55 different breeds for variant analysis. Sanger sequencing of one additional affected and one unaffected sibling confirmed the variant. RESULTS: Clinically, severe mucocutaneous ulcers occurred in frictional areas with claw sloughing. Histopathological results revealed subepidermal clefts and electron microscopy confirmed the split in the lamina lucida. Post-mortem examination documented extensive pharyngeal and laryngeal lesions with granulation tissue and fibrinous exudate obscuring the airway. Moderate tracheal hypoplasia contributed. The WGS revealed a novel missense variant in the laminin α3-chain XP_537297.2p(Asp2867Val), with an autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL RELEVANCE: A novel variant in LAMA3 caused a generalized and severe phenotype of JEB with an unique clinical presentation of upper airway obstruction.

A missense variant in the titin gene in Doberman pinscher dogs with familial dilated cardiomyopathy and sudden cardiac death.

The dog provides a large animal model of familial dilated cardiomyopathy for the study of important aspects of this common familial cardiovascular disease. We have previously demonstrated a form of canine dilated cardiomyopathy in the Doberman pinscher breed that is inherited as an autosomal dominant trait and is associated with a splice site variant in the pyruvate dehydrogenase kinase 4 (PDK4) gene, however, genetic heterogeneity exists in this species as well and not all affected dogs have the PDK4 variant. Whole genome sequencing of a family of Doberman pinchers with dilated cardiomyopathy and sudden cardiac death without the PDK4 variant was performed. A pathologic missense variant in the titin gene located in an immunoglobulin-like domain in the I-band spanning region of the molecule was identified and was highly associated with the disease (p < 0.0001). We demonstrate here the identification of a variant in the titin gene highly associated with the disease in this spontaneous canine model of dilated cardiomyopathy. This large animal model of familial dilated cardiomyopathy shares many similarities with the human disease including mode of inheritance, clinical presentation, genetic heterogeneity and a pathologic variant in the titin gene. The dog is an excellent model to improve our understanding of the genotypic phenotypic relationships, penetrance, expression and the pathophysiology of variants in the titin gene.

DLA class II haplotypes show sex-specific associations with primary hypoadrenocorticism in Standard Poodle dogs.

Addison's disease (AD) is a life-threatening endocrine disorder that occurs spontaneously in both humans and dogs. Associations between MHC class II genes and AD have been shown in several human studies. Our goal was to identify MHC class II associations with AD in a large population of Standard Poodles, a breed highly predisposed to AD. We sequenced exon 2 of the class II genes DLA-DRB1, DLA-DQA1, and DLA-DQB1 in 110 affected and 101 unaffected Standard Poodles and tested for association with AD. After correcting for population structure, two haplotypes were found to confer risk of developing AD in a sex-specific manner: DLA-DRB1*015:01-DQA1*006:01-DQB1*023:01 in males (x2p = 0.03, OR 2.1) and DLA-DRB1*009:01-DQA1*001:01-DQB1*008:01:1 in females (x2p = 0.02, OR 8.43). Sex-specific associations have been previously described in human populations, but this is the first report of this kind in dogs. Consistent with findings in other studies, we found the DLA-DQA1*006:01 allele (x2p = 0.04) to be associated with AD in males independent of haplotype. In females, the haplotype DLA-DRB1*009:01-DQA1*001:01-DQB1*008:01:1 confers a very high risk for developing AD, although its frequency was rare (9 of 124 females) in our study population. Further studies are warranted to validate the findings of this exploratory dataset and to assess the usefulness of this haplotype as a risk marker for AD in female Standard Poodles. Our results highlight the importance of evaluating MHC class II disease associations in large populations, and accounting for both biological sex and population structure.

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.

Evaluation of a DLA-79 allele associated with multiple immune-mediated diseases in dogs.

Immune-mediated diseases are common and life-threatening disorders in dogs. Many canine immune-mediated diseases have strong breed predispositions and are believed to be inherited. However, the genetic mutations that cause these diseases are mostly unknown. As many immune-mediated diseases in humans share polymorphisms among a common set of genes, we conducted a candidate gene study of 15 of these genes across four immune-mediated diseases (immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, immune-mediated polyarthritis (IMPA), and atopic dermatitis) in 195 affected and 206 unaffected dogs to assess whether causative or predictive polymorphisms might exist in similar genes in dogs. We demonstrate a strong association (Fisher's exact p = 0.0004 for allelic association, p = 0.0035 for genotypic association) between two polymorphic positions (10 bp apart) in exon 2 of one allele in DLA-79, DLA-79*001:02, and multiple immune-mediated diseases. The frequency of this allele was significantly higher in dogs with immune-mediated disease than in control dogs (0.21 vs. 0.12) and ranged from 0.28 in dogs with IMPA to 0.15 in dogs with atopic dermatitis. This allele has two non-synonymous substitutions (compared with the reference allele, DLA-79*001:01), resulting in F33L and N37D amino acid changes. These mutations occur in the peptide-binding pocket of the protein, and based upon our computational modeling studies, are likely to affect critical interactions with the peptide N-terminus. Further studies are warranted to confirm these findings more broadly and to determine the specific mechanism by which the identified variants alter canine immune system function.

Evaluation of artificial selection in Standard Poodles using whole-genome sequencing.

Identifying regions of artificial selection within dog breeds may provide insights into genetic variation that underlies breed-specific traits or diseases-particularly if these traits or disease predispositions are fixed within a breed. In this study, we searched for runs of homozygosity (ROH) and calculated the d i statistic (which is based upon F ST) to identify regions of artificial selection in Standard Poodles using high-coverage, whole-genome sequencing data of 15 Standard Poodles and 49 dogs across seven other breeds. We identified consensus ROH regions ≥1 Mb in length and common to at least ten Standard Poodles covering 0.6 % of the genome, and d i regions that most distinguish Standard Poodles from other breeds covering 3.7 % of the genome. Within these regions, we identified enriched gene pathways related to olfaction, digestion, and taste, as well as pathways related to adrenal hormone biosynthesis, T cell function, and protein ubiquitination that could contribute to the pathogenesis of some Poodle-prevalent autoimmune diseases. We also validated variants related to hair coat and skull morphology that have previously been identified as being under selective pressure in Poodles, and flagged additional polymorphisms in genes such as ITGA2B, CBX4, and TNXB that may represent strong candidates for other common Poodle disorders.

Variants in CLCN1 and PDE4C Associated with Muscle Hypertrophy, Dysphagia, and Gait Abnormalities in Young French Bulldogs.

(1) Background: Muscle hypertrophy, swallowing disorders, and gait abnormalities are clinical signs common to many muscle diseases, including muscular dystrophies, non-dystrophic myotonias, genetic myopathies associated with deficiency of myostatin, and acquired inflammatory myopathies. Here, we investigated underlying causes of this triad of clinical signs in four young French bulldogs via muscle histopathology coupled with whole genome and Sanger sequencing. (2) Methods: Dogs were evaluated by veterinary clinical internists and neurologists, and biopsies were obtained for histopathological diagnosis. DNA was submitted for whole genome sequencing, followed by bioinformatics evaluation and confirmation of variants via Sanger sequencing in two cases. (3) Results: Two novel variants were identified. The first, found in two related French bulldogs, was a homozygous variant in the chloride channel gene CLCN1 known to cause non-dystrophic congenital myotonia, and the second, found in an unrelated French bulldog, was a heterozygous variant in the cAMP phosphodiesterase gene PDE4C, which is the major phosphodiesterase expressed in skeletal muscle and may play a role in decreasing muscle atrophy. An underlying molecular basis in one other case has not yet been identified. (4) Conclusions: Here, we identified two novel variants, one in the CLCN1 and one in the PDE4C gene, associated with clinical signs of muscle hypertrophy, dysphagia, and gait abnormalities, and we suggested other bases of these phenotypes in French bulldogs that are yet to be discovered. Identification of genes and deleterious variants associated with these clinical signs may assist breeders in improving the overall health of this very popular breed and may lead to the identification of new therapies to reverse muscle atrophy in people and animals with neuromuscular diseases.

Sequence Analysis of Six Candidate Genes in Miniature Schnauzers with Primary Hypertriglyceridemia.

Miniature Schnauzers are predisposed to primary hypertriglyceridemia (HTG). In this study, we performed whole genome sequencing (WGS) of eight Miniature Schnauzers with primary HTG and screened for risk variants in six HTG candidate genes: LPL, APOC2, APOA5, GPIHBP1, LMF1, and APOE. Variants were filtered to identify those present in ≥2 Miniature Schnauzers with primary HTG and uncommon (<10% allele frequency) in a WGS variant database including 613 dogs from 61 other breeds. Three variants passed filtering: an APOE TATA box deletion, an LMF1 intronic SNP, and a GPIHBP1 missense variant. The APOE and GPIHBP1 variants were genotyped in a cohort of 108 Miniature Schnauzers, including 68 with primary HTG and 40 controls. A multivariable regression model, including age and sex, did not identify an effect of APOE (estimate = 0.18, std. error = 0.14; p = 0.20) or GPIHBP1 genotypes (estimate = -0.26, std. error = 0.42; p = 0.54) on triglyceride concentration. In conclusion, we did not identify a monogenic cause for primary HTG in Miniature Schnauzers in the six genes evaluated. However, if HTG in Miniature Schnauzers is a complex disease resulting from the cumulative effects of multiple variants and environment, the identified variants cannot be ruled out as contributing factors.

A Novel CARMIL2 Immunodeficiency Identified in a Subset of Cavalier King Charles Spaniels with Pneumocystis and Bordetella Pneumonia.

Pet dogs are a valuable natural animal model for studying relationships between primary immunodeficiencies and susceptibility to Pneumocystis and other opportunistic respiratory pathogens. Certain breeds, such as the Cavalier King Charles Spaniel, are over-represented for Pneumocystis pneumonia (PCP), suggesting the presence of a primary immunodeficiency in the breed. Here, we report the discovery of a CARMIL2 nonsense variant in three Cavalier King Charles Spaniel dogs with either PCP (n = 2) or refractory Bordetella pneumonia (n = 1). CARMIL2 encodes a protein that plays critical roles in T-cell activation and other aspects of immune function. Deleterious CARMIL2 variants have recently been reported in human patients with PCP and other recurrent pneumonias. In addition to opportunistic respiratory infection, the affected dogs also exhibited other clinical manifestations of CARMIL2 deficiencies that have been reported in humans, including early-onset gastrointestinal disease, allergic skin disease, mucocutaneous lesions, abscesses, autoimmune disorders, and gastrointestinal parasitism. This discovery highlights the potential utility of a natural canine model in identifying and studying primary immunodeficiencies in patients affected by PCP.

Whole Animal Genome Sequencing: user-friendly, rapid, containerized pipelines for processing, variant discovery, and annotation of short-read whole genome sequencing data.

Advancements in massively parallel short-read sequencing technologies and the associated decreasing costs have led to large and diverse variant discovery efforts across species. However, processing high-throughput short-read sequencing data can be challenging with potential pitfalls and bioinformatics bottlenecks in generating reproducible results. Although a number of pipelines exist that address these challenges, these are often geared toward human or traditional model organism species and can be difficult to configure across institutions. Whole Animal Genome Sequencing (WAGS) is an open-source set of user-friendly, containerized pipelines designed to simplify the process of identifying germline short (SNP and indel) and structural variants (SVs) geared toward the veterinary community but adaptable to any species with a suitable reference genome. We present a description of the pipelines [adapted from the best practices of the Genome Analysis Toolkit (GATK)], along with benchmarking data from both the preprocessing and joint genotyping steps, consistent with a typical user workflow.

Current Classification of Canine Muscular Dystrophies and Identification of New Variants.

The spectrum of canine muscular dystrophies has rapidly grown with the recent identification of several more affected breeds and associated mutations. Defects include those in genes and protein products associated with the sarcolemma (dystrophin deficient X-linked muscular dystrophy and sarcoglycan-deficient limb-girdle muscular dystrophy) and with the extracellular matrix (collagen 6, laminin α2, and α-dystroglycan-deficient congenital muscular dystrophies). With the increasing application of whole genome sequencing and whole exome sequencing, the clinical and pathological spectra associated with specific neuromuscular genetic defects are constantly evolving. In this report, we provide a brief overview of the current status of gene defects reported in canine muscular dystrophies. We also report the causative mutations for novel forms of X-linked muscular dystrophy in Brittany spaniels and in a French bulldog.

A case-control survey study of environmental risk factors for primary hypoadrenocorticism in dogs.

BACKGROUND: Primary hypoadrenocorticism in dogs is thought to be multifactorial with roles for both genetic and environmental factors. The contributions of environmental factors remain unexplored. OBJECTIVE: Identify environmental and lifestyle exposures associated with primary hypoadrenocorticism in 2 dog breeds with high risk of developing the disease. ANIMALS: Animals were not used in this study. Owners of Standard Poodles (STPDs) and Portuguese water dogs (POWDs) participated in a survey. METHODS: Retrospective case-control study. Dog owners were invited to participate in an online survey through convenience sampling. Questions regarded the demographics, health histories, and indoor/outdoor environments in which their dogs live and play. Responses for dogs with primary hypoadrenocorticism were compared to those without the disease using univariate and multivariate logistic regression models. RESULTS: Five thousand forty-seven responses (358 cases, 4689 controls) met initial inclusion criteria. Significant associations with modest effect size were found for community type, ingestion of canned food, and use of lawn fertilizer in some analysis models. Reproductive (spay/neuter) status exhibited the strongest association with high effect size across all models with adjusted odds ratio (OR) 2.5 (95% confidence interval [CI], 1.4-4.5; P = .003) for spayed females and 6.0 (95% CI, 2.6-13.9; P < .001) for neutered males. CONCLUSIONS AND CLINICAL IMPORTANCE: The large effect size for reproductive status reflects its high potential clinical relevance, whereas modest effect sizes for other environmental variables suggest lower potential clinical relevance. These findings are associations and do not necessarily imply causation. Before any actionable recommendations are warranted, additional evidence regarding biological mechanisms is needed.

Canine models of Charcot-Marie-Tooth: MTMR2, MPZ, and SH3TC2 variants in golden retrievers with congenital hypomyelinating polyneuropathy.

Congenital hypomyelinating polyneuropathy (HPN) restricted to the peripheral nervous system was reported in 1989 in two Golden Retriever (GR) littermates. Recently, four additional cases of congenital HPN in young, unrelated GRs were diagnosed via neurological examination, electrodiagnostic evaluation, and peripheral nerve pathology. Whole-genome sequencing was performed on all four GRs, and variants from each dog were compared to variants found across >1,000 other dogs, all presumably unaffected with HPN. Likely causative variants were identified for each HPN-affected GR. Two cases shared a homozygous splice donor site variant in MTMR2, with a stop codon introduced within six codons following the inclusion of the intron. One case had a heterozygous MPZ isoleucine to threonine substitution. The last case had a homozygous SH3TC2 nonsense variant predicted to truncate approximately one-half of the protein. Haplotype analysis using 524 GR established the novelty of the identified variants. Each variant occurs within genes that are associated with the human Charcot-Marie-Tooth (CMT) group of heterogeneous diseases, affecting the peripheral nervous system. Testing a large GR population (n = >200) did not identify any dogs with these variants. Although these variants are rare within the general GR population, breeders should be cautious to avoid propagating these alleles.

Novel COL6A3 frameshift variant in American Staffordshire Terrier dogs with Ullrich-like congenital muscular dystrophy.

Two (male and female) 10-month-old American Staffordshire Terrier littermates presented for progressive weakness, joint contracture, and distal limb joint hyperlaxity beginning around 6 months of age. Neurological examination, serum creatine kinase activity, infectious disease titers, cerebrospinal fluid analysis, and electrodiagnostic testing were performed. Muscle biopsies were collected for histopathology and immunofluorescence staining for localization of dystrophy associated proteins. Whole-genome sequencing (WGS) was performed on 1 affected dog. Variants were compared to a database of 671 unaffected dogs of multiple breeds. Histopathology confirmed a dystrophic phenotype and immunofluorescence staining of muscle cryosections revealed an absence of staining for collagen-6. WGS identified a homozygous 1 bp deletion in the COL6A3 gene, unique to the first affected dog. Sanger sequencing confirmed the homozygous presence of the frameshift variant in both affected dogs. This report describes the clinical features and most likely genetic basis of an Ullrich-like recessively inherited form of congenital muscular dystrophy in American Staffordshire Terriers.

The S-measurement in the diagnosis of canine hip dysplasia.

OBJECTIVE: To propose a direct measure of subluxation of the femoral head (S) in the assessment of hip joint laxity and evaluate it for clinical use. STUDY DESIGN: Method comparison study. ANIMALS: Dogs (n = 51). METHODS: Dogs were sedated or anesthetized for a dorsolateral subluxation (DLS) examination. Two sets of radiographs were acquired, 1 each by a different technologist. A calibrated measuring bar was included on the image at the height of the hip to assess magnification. The DLS was calculated for each hip and different persons unaware of these details measured the "S"-value. One person measured the S-value 3 times over 3 days. Box plots were used to determine a cut-off for the empiric (8 mm) and corrected (4 mm) S-value. RESULTS: Of 51 dogs, 33 were dysplastic based on a DLS score <55%. Magnification and body weight were strongly correlated (r = 0.4922, P = .0006). Both empiric and corrected S measurements showed good agreement with the DLS score (κ = 0.688 and κ = 0.681, respectively). The corrected S measurement produced more false negatives. Bland-Altman analysis showed interobserver and technician variance acceptable for clinical use (limits of agreement < ±3 mm). Intraobserver repeatability was acceptable for the right hip (95% of differences were ≤1.3 mm and 100% ≤ 1.9) but not for the left hip. CONCLUSION: Using a cut-off value of 5 mm, the empirical S measurement can be used to exclude hip dysplasia in young dogs of various body proportions.

A scoping review of autoantibodies as biomarkers for canine autoimmune disease.

BACKGROUND: Autoantibody biomarkers are valuable tools used to diagnose and manage autoimmune diseases in dogs. However, prior publications have raised concerns over a lack of standardization and sufficient validation for the use of biomarkers in veterinary medicine. OBJECTIVES: Systematically compile primary research on autoantibody biomarkers for autoimmune disease in dogs, summarize their methodological features, and evaluate their quality; synthesize data supporting their use into a resource for veterinarians and researchers. ANIMALS: Not used. METHODS: Five indices were searched to identify studies for evaluation: PubMed, CAB Abstracts, Web of Science, Agricola, and SCOPUS. Two independent reviewers (AET and ELC) screened titles and abstracts for exclusion criteria followed by full-text review of remaining articles. Relevant studies were classified based on study objectives (biomarker, epitope, technique). Data on study characteristics and outcomes were synthesized in independent data tables for each classification. RESULTS: Ninety-two studies qualified for final analysis (n = 49 biomarker, n = 9 epitope, and n = 34 technique studies). A high degree of heterogeneity in study characteristics and outcomes reporting was observed. Opportunities to strengthen future studies could include: (1) routine use of negative controls, (2) power analyses to inform sample sizes, (3) statistical analyses when appropriate, and (4) multiple detection techniques to confirm results. CONCLUSIONS: These findings provide a resource that will allow veterinary clinicians to efficiently evaluate the evidence supporting the use of autoantibody biomarkers, along with the varied methodological approaches used in their development.

Red cell distribution width is a predictor of all-cause mortality in hospitalized dogs.

OBJECTIVE: To determine whether RBC distribution width (RDW) is associated with an increased odds of mortality in hospitalized dogs and cats. DESIGN: Retrospective, single-center study; data collected from 2007 to 2017. SETTING: University teaching hospital. ANIMALS: Six thousand six hundred and sixty-one animals (5183 dogs and 1478 cats). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Medical records were identified from animals presented to the emergency service and admitted to the ICU with a CBC and serum biochemistry performed on admission. Patients were stratified into quintiles based upon presenting RDW, and logistic regression modeling was performed to evaluate the relationship between RDW and in-hospital mortality. Canine patients with a presenting RDW in the upper fourth and fifth quintiles had an increased odds of all-cause in-hospital mortality (p < 0.0001). Specifically, dogs in the upper fifth quintile had a 2.1-fold greater odds of death compared to dogs in the first quintile, and dogs in the upper fourth quintile had a 1.9-fold greater odds of death compared to dogs in the first quintile. This relationship remained significant after adjusting for age, sex, key laboratory values excluding HCT, medical versus surgical disease, and diagnosis category. This relationship was no longer significant with the inclusion of HCT. No significant association was identified between presenting RDW and in-hospital mortality in cats. CONCLUSIONS: Hospitalized dogs with higher RDW on presentation to the emergency service have greater odds of all-cause in-hospital mortality compared to dogs with a lower RDW. A similar association between RDW and mortality was not found in cats. Further studies are warranted to assess the usefulness of this biomarker for specific diseases in dogs and to better understand the mechanisms driving the association between increased RDW and mortality in canine patients.