Epidemiological investigation of insulin dysregulation in Shetland and Welsh ponies in Australia.

BACKGROUND: Insulin dysregulation (ID) is central to equine metabolic syndrome. There are limited epidemiological studies investigating dynamic testing of ID in ponies. OBJECTIVES: To evaluate prevalence and risk factors for ID through dynamic testing of hyperinsulinaemia (DHI) and insulin resistance (IR). STUDY DESIGN: Cross-sectional. METHODS: Sex, age, breed, height, cresty neck score (CNS), body condition score (BCS), laminitis, HMGA2:c.83G>A genotype and pituitary pars intermedia dysfunction (PPID) status were documented. Dynamic hyperinsulinaemia was diagnosed with an oral sugar test (OST) and IR with an insulin tolerance test (ITT). Owners completed surveys reporting activity, laminitis history and perception of body condition using a (1-9) visual analogue scale (VASo). Ordinal scores were converted to binary outcomes for CNS (≤2/5 or ≥3/5), BCS and VASo (≤6/9 or ≥7/9). Variables associated with insulin concentrations, glucose reduction after the ITT and laminitis were evaluated with mixed effects regression models accounting for random effects of farms. RESULTS: Among 167 ponies tested, median (range) age was 9 (4-21) years and BCS was 6 (4-8). Prevalence (95% confidence interval [CI]) of ID was 61 (53-68)%. Factors associated with insulin concentrations (estimate [95% CI]; µIU/mL) 60 min post-OST were: age (1.07 [1.02-1.11]), CNS (≥3/5, 1.52 [1.04-2.23]) and VASo (≥7/9, 1.75 [1.09-2.79]); and 90 min post-OST were: age (1.08 [1.03-1.12]), CNS (≥3/5, 1.80 [1.22-2.64]), VASo (≥7/9, 2.49 [1.52-4.08]) and sex (male, 0.64 [0.45-0.91]). Factors associated with glucose reduction after the ITT (estimate [95% CI]; %) were: age (-1.34 [-2.01 to -0.67]), sex (female, -6.21 [-11.68 to -0.74]) and VASo (≥7/9, -1.74 [-18.89 to -4.78]). Factors associated with laminitis (odds ratio [95% CI]) were DHI (4.60 [1.68-12.58]), IR (3.66 [1.26-10.61]) and PPID (11.75 [1.54-89.40]). MAIN LIMITATIONS: Single time-point sampling, laminitis definition and diet analysis. CONCLUSIONS: Ageing, being female and owner-perceived obesity were associated with ID.

Evaluation of an HMGA2 variant contribution to height and basal insulin concentrations in ponies.

BACKGROUND: The HMGA2:c.83G>A variant was identified in Welsh ponies having pleiotropic effects on height and insulin concentration. OBJECTIVE: Determine whether the HMGA2:c.83G>A variant is associated with decreased height and higher basal insulin concentrations across pony breeds. ANIMALS: Two hundred thirty-six ponies across 6 breeds. METHODS: Cross-sectional study. Ponies were genotyped for the HMGA2:c.83G>A variant and phenotyped for height and basal insulin concentrations. Stepwise regression was performed for model analysis using a linear regression model for height and mixed linear model for insulin with farm as a random effect. Coefficient of determination, pairwise comparison of the estimated marginal means and partial correlation coefficients (parcor) were calculated to assess the relationship between HMGA2 genotype and height or insulin. RESULTS: Breed and genotype accounted for 90.5% of the variation in height across breeds, and genotype explained 21% to 44% of the variation within breeds. Breed, genotype, cresty neck score, sex, age, and farm accounted for 45.5% of the variation in insulin, with genotype accounting for 7.1%. The HMGA2 A allele frequency was 62% and correlated with both height (parcor = -0.39; P < .001) and insulin (parcor = 0.22; P = .02). Pairwise comparisons found A/A ponies were >10 cm shorter than other genotypes. Compared with G/G individuals, A/A and G/A individuals had 4.3 µIU/mL (95% confidence interval [CI]: 1.8-10.5) and 2.7 µIU/mL (95% CI: 1.4-5.3) higher basal insulin concentrations, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: These data demonstrate the pleiotropic effects of the HMGA2:c.83G>A variant and its role in identifying ponies at increased risk for insulin dysregulation.

Adverse health events and recommended health research priorities in agility dogs as reported by dog owners.

Objective: To understand relative frequency of adverse health events, defined as injuries or infectious diseases, in dogs participating in agility and to determine health research priorities of agility dog owners. Procedures: An internet-based questionnaire distributed to agility dog owners included items related to experiences with infectious diseases and injuries in agility dogs, reasons for retirement of dogs from competition, and ranking of health research priorities. Frequencies of infectious diseases in US geographic regions were compared with Chi-square tests. Research priority rankings were determined as median and interquartile range (IQR) for each topic. Rank-based tests (Kruskal Wallis and Mann-Whitney) compared rankings between participants in different agility organizations, between veterinarian and non-veterinarian competitors, and between respondents who had competed in national championship events and other respondents. Results: There were 1,322 respondents who had competed in canine agility in the previous 6 months, with those respondents reporting a median time competing in the sport of 13 years (IQR = 8-20 years); 50% of respondents had competed in at least one national championship agility event in the preceding 5 years. Overall, 1,015 respondents (77%) indicated that one or more of their dogs had been injured and approximately one-third (n = 477, 36%) indicated that one or more dogs had likely acquired one or more infectious diseases as a result of agility activities. Specific types of infectious diseases acquired varied by geographic region in the US. Research priority rankings were similar regardless of preferred agility organization or respondent experience. The highest-ranking research topics were identification of risk factors for specific types of injuries, improvements in equipment and understanding of safe course design, and physical conditioning programs to prevent injury. Conclusions and clinical relevance: Agility competitors prioritize research in areas that advance understanding of injury prevention in their dogs. Research priorities are nearly uniform among competitors regardless of their preferred agility organization or level of experience, providing a strong rationale for agility organizations to collaborate in research initiatives that improve safety and well-being for dogs competing in the sport. There has been little published research focusing on the high-priority research areas identified by competitors.

Heritability of insidious uveitis in Appaloosa horses.

Equine recurrent uveitis (ERU) is a blinding ocular disorder among horses, and the Appaloosa horse breed is disproportionally affected by a chronic form of this intraocular inflammatory disease known as insidious uveitis. Strong breed predisposition and previous investigations suggest that there is a genetic component to the pathology of insidious uveitis among Appaloosa horses; however, no estimates of the heritability of the disease have previously been determined. This study aimed to characterize the genetic underpinning of the disease by estimating the heritability for insidious uveitis among Appaloosas. After combining two genotyping array datasets from the Illumina Equine SNP70 BeadChip and the Axiom Equine 670 K Genotyping Array, heritability was estimated for 59 affected and 83 unaffected horses using both restricted maximum likelihood (REML) and phenotype correlation - genotype correlation solvers from the linkage disequilibrium adjusted kinship software. Based on previous research, age and sex were used as covariates, and the locus responsible for the characteristic Appaloosa coat pattern (LP), previously associated with ERU risk, was included as a fixed effect ('top predictor'). Using prevalence values from 0.05 to 0.42, the heritability estimate for insidious uveitis ranged from 0.95 (SE = 0.14) to 1.74 (SE = 0.25) with LP contributing 0.16-0.33 to the estimate. This study suggests that insidious uveitis is highly heritable (REML 95% CI, h2  = 0.68-1.0) and additional loci outside of LP are contributing to the genetic risk for insidious uveitis for Appaloosas. Once identified, these other genetic factors may lead to new disease mitigation efforts in veterinary care and breeding practices.

Heritability and Genomic Architecture of Episodic Exercise-Induced Collapse in Border Collies.

An episodic nervous system disorder triggered by strenuous exercise, termed border collie collapse (BCC), exists in border collies and related breeds. The genetic basis of BCC is unknown but is believed to be a complex genetic disorder. Our goal was to estimate the heritability (h2SNP) of BCC, define its underlying genetic architecture, and identify associated genomic loci using dense whole-genome single-nucleotide polymorphism (SNP) genotyping data. Genotype data were obtained for ~440,000 SNPs from 343 border collies (168 BCC cases and 175 controls). h2SNP was calculated to be 49-61% depending on the estimated BCC prevalence. A total of 2407 SNPs across the genome accounted for nearly all the h2SNP of BCC, with an estimated 2003 SNPs of small effect, 349 SNPs of moderate effect, and 56 SNPs of large effect. Genome-wide association analyses identified significantly associated loci on chromosomes 1, 6, 11, 20, and 28, which accounted for ~5% of the total BCC h2SNP. We conclude that BCC is a moderately- to highly-heritable complex polygenetic disease resulting from contributions from hundreds to thousands of genetic variants with variable effect sizes. Understanding how much the BCC phenotype is determined by genetics and whether major gene mutations are likely to exist inform veterinarians and working/stock dog communities of the true nature of this condition.

Functional contexts of adipose and gluteal muscle tissue gene co-expression networks in the domestic horse.

A gene's response to an environment is tightly bound to the underlying genetic variation present in an individual's genome and varies greatly depending on the tissue it is being expressed in. Gene co-expression networks provide a mechanism to understand and interpret the collective transcriptional responses of genes. Here, we use the Camoco co-expression network framework to characterize the transcriptional landscape of adipose and gluteal muscle tissue in 83 domestic horses (Equus caballus) representing 5 different breeds. In each tissue, gene expression profiles, capturing transcriptional response due to variation across individuals, were used to build two separate, tissue-focused, genotypically-diverse gene co-expression networks. The aim of our study was to identify significantly co-expressed clusters of genes in each tissue, then compare the clusters across networks to quantify the extent that clusters were found in both networks as well as to identify clusters found in a single network. The known and unknown functions for each network were quantified using complementary, supervised and unsupervised approaches. First, supervised ontological enrichment was utilized to quantify biological functions represented by each network. Curated ontologies (GO and KEGG) were used to measure the known biological functions present in each tissue. Overall, a large percentage of terms (40.3% of GO and 41% of KEGG) were co-expressed in at least one tissue. Many terms were co-expressed in both tissues, however a small proportion of terms exhibited single tissue co-expression suggesting functional differentiation based on curated, functional annotation. To complement this, an unsupervised approach not relying on ontologies was employed. Strongly co-expressed sets of genes defined by Markov clustering identified sets of unannotated genes showing similar patterns of co-expression within a tissue. We compared gene sets across tissues and identified clusters of genes the either segregate in co-expression by tissue or exhibit high levels of co-expression in both tissues. Clusters were also integrated with GO and KEGG ontologies to identify gene sets containing previously curated annotations versus unannotated gene sets indicating potentially novel biological function. Coupling together these transcriptional datasets, we mapped the transcriptional landscape of muscle and adipose setting up a generalizable framework for interpreting gene function for additional tissues in the horse and other species.

Genetics of Equine Endocrine and Metabolic Disease.

A role for a genetic contribution to equine metabolic syndrome (EMS) and pars pituitary intermedia dysfunction (PPID) has been hypothesized. Heritability estimates of EMS biochemical measurements were consistent with moderately to highly heritable traits. Further, genome-wide association analyses have identified hundreds of regions of the genome contributing to EMS and candidate variants have been identified. The genetics of PPID has not yet been proven. Continued research for the specific genetic risk factors for both EMS and PPID is crucial for gaining a better understanding of the pathophysiology of both conditions and allowing development of genetic tests.

Equine Genotyping Arrays.

High-quality genomic tools have been integral in understanding genomic architecture and function in the modern-day horse. The equine genetics community has a long tradition of pooling resources to develop genomic tools. Since the equine genome was sequenced in 2006, several iterations of high throughput genotyping arrays have been developed and released, enabling rapid and cost-effective genotyping. This review highlights the design considerations of each iteration, focusing on data available during development and outlining considerations in selecting the genetic variants included on each array. Additionally, we outline recent applications of equine genotyping arrays as well as future prospects and applications.

Comparison between smartphone electrocardiography and standard three-lead base apex electrocardiography in healthy horses.

BACKGROUND: Cardiac arrhythmias are commonly auscultated during routine physical examinations in horses and determining the underlying electrical abnormality using an ECG is important. The most commonly used device is a three-lead base apex system (Televet), however few practitioners carry this for routine visits. With recognition of the utility of smartphone-based ECGs in humans, dogs and ruminants, the AliveCor single-lead bipolar smartphone-based ECG has gained popularity. The objective of this study was to determine if AliveCor and Televet ECG measurements were comparable in healthy horses using multiple observers. METHODS: ECGs were performed on 15 healthy horses simultaneously using the AliveCor and Televet. RESULTS: There was very good to perfect interdevice and interobserver agreement for heart rate and RR interval measurement, and moderate-to-good interdevice and interobserver agreement for detection of non-pathological arrhythmias. Interdevice agreement for measurement of P-wave and QRS duration, QT, PR and T-peak to T-end interval was poor to fair. Interestingly, interobserver agreement for P-wave and QRS duration, QT, PR, and T-peak to T-end interval measurements was fair to good. CONCLUSION: Overall, the AliveCor is comparable to the Televet for heart rate and RR measurement, and for the detection of non-pathogenic arrhythmias with acceptable agreement between observers.

Genome-Wide Association Analyses of Equine Metabolic Syndrome Phenotypes in Welsh Ponies and Morgan Horses.

Equine metabolic syndrome (EMS) is a complex trait for which few genetic studies have been published. Our study objectives were to perform within breed genome-wide association analyses (GWA) to identify associated loci in two high-risk breeds, coupled with meta-analysis to identify shared and unique loci between breeds. GWA for 12 EMS traits identified 303 and 142 associated genomic regions in 264 Welsh ponies and 286 Morgan horses, respectively. Meta-analysis demonstrated that 65 GWA regions were shared across breeds. Region boundaries were defined based on a fixed-size or the breakdown of linkage disequilibrium, and prioritized if they were: shared between breeds or across traits (high priority), identified in a single GWA cohort (medium priority), or shared across traits with no SNPs reaching genome-wide significance (low priority), resulting in 56 high, 26 medium, and seven low priority regions including 1853 candidate genes in the Welsh ponies; and 39 high, eight medium, and nine low priority regions including 1167 candidate genes in the Morgans. The prioritized regions contained protein-coding genes which were functionally enriched for pathways associated with inflammation, glucose metabolism, or lipid metabolism. These data demonstrate that EMS is a polygenic trait with breed-specific risk alleles as well as those shared across breeds.

Differential Gene Expression in Articular Cartilage and Subchondral Bone of Neonatal and Adult Horses.

Skeletogenesis is complex and incompletely understood. Derangement of this process likely underlies developmental skeletal pathologies. Examination of tissue-specific gene expression may help elucidate novel skeletal developmental pathways that could contribute to disease risk. Our aim was to identify and functionally annotate differentially expressed genes in equine neonatal and adult articular cartilage (AC) and subchondral bone (SCB). RNA was sequenced from healthy AC and SCB from the fetlock, hock, and stifle joints of 6 foals (≤4 weeks of age) and six adults (8-12 years of age). There was distinct clustering by age and tissue type. After differential expression analysis, functional annotation and pathway analysis were performed using PANTHER and Reactome. Approximately 1115 and 3574 genes were differentially expressed between age groups in AC and SCB, respectively, falling within dozens of overrepresented gene ontology terms and enriched pathways reflecting a state of growth, high metabolic activity, and tissue turnover in the foals. Enriched pathways were dominated by those related to extracellular matrix organization and turnover, and cell cycle and signal transduction. Additionally, we identified enriched pathways related to neural development and neurotransmission in AC and innate immunity in SCB. These represent novel potential mechanisms for disease that can be explored in future work.

Erratum: Author Correction: Improved reference genome for the domestic horse increases assembly contiguity and composition.

[This corrects the article DOI: 10.1038/s42003-018-0199-z.].

Exploration of fine-scale recombination rate variation in the domestic horse.

Total genetic map length and local recombination landscapes typically vary within and across populations. As a first step to understanding the recombination landscape in the domestic horse, we calculated population recombination rates and identified likely recombination hotspots using approximately 1.8 million SNP genotypes for 485 horses from 32 distinct breeds. The resulting breed-averaged recombination map spans 2.36 Gb and accounts for 2939.07 cM. Recombination hotspots occur once per 23.8 Mb on average and account for ∼9% of the physical map length. Regions with elevated recombination rates in the entire cohort were enriched for genes in pathways involving interaction with the environment: immune system processes (specifically, MHC class I and class II genes), responses to stimuli, and serotonin receptor pathways. We found significant correlations between differences in local recombination rates and population differentiation quantified by F ST Analysis of breed-specific maps revealed thousands of hotspot regions unique to particular breeds, as well as unique "coldspots," regions where a particular breed showed below-average recombination, whereas all other breeds had evidence of a hotspot. Finally, we identified relative enrichment (P = 5.88 × 10-27) for the in silico-predicted recognition motif for equine PR/SET domain 9 (PRDM9) in recombination hotspots. These results indicate that selective pressures and PRDM9 function contribute to variation in recombination rates across the domestic horse genome.

Estimation of Actual and Ideal Bodyweight Using Morphometric Measurements of Miniature, Saddle-Type, and Thoroughbred Horses.

Adding breed type, height, and neck circumference to body length and girth circumference improves bodyweight (BW) estimation in different breeds of horses; however, equations have not been developed for all breed types. The objectives were to develop BW estimation equations for Miniature, saddle-type, and Thoroughbred horses using morphometric measurements. Measurements were collected on adult (≥3 years, nonpregnant) saddle-type (n = 209), adult (n = 249) and juvenile (<3 years, n = 61) Miniatures, and adult Thoroughbreds (n = 100). Personnel determined body condition score (BCS), measured withers height and girth circumference at the third thoracic vertebra, body length from the point of the shoulder to the point of the buttock and to a line perpendicular to the point of the buttock, and neck circumference at the midway point between the poll and withers. Each horse was weighed using a livestock scale. Bodyweight estimations equations were developed using linear regression modeling and log transformation. Mean (±standard deviation) BCS was 6.1 (±0.8), 5.4 (±0.6), 6.0 (±1.0), and 5.0 (±0.6) for adult and juvenile Miniatures, saddle-type, and Thoroughbreds, respectively. Bodyweight estimation equations developed through the current research were within 4% of the scale BW and offered improvements over previous BW estimation equations and weight tapes, which were off by 5%-25%. Owner-estimated BW was within 8%-15% of scale BW. Morphometric measurements were successfully used to develop BW equations for Miniature, saddle-type, and Thoroughbred horses. The equations will assist owners and professionals with managing horse BW and will be added to the Healthy Horse application.

Identification and validation of genetic variants predictive of gait in standardbred horses.

Several horse breeds have been specifically selected for the ability to exhibit alternative patterns of locomotion, or gaits. A premature stop codon in the gene DMRT3 is permissive for "gaitedness" across breeds. However, this mutation is nearly fixed in both American Standardbred trotters and pacers, which perform a diagonal and lateral gait, respectively, during harness racing. This suggests that modifying alleles must influence the preferred gait at racing speeds in these populations. A genome-wide association analysis for the ability to pace was performed in 542 Standardbred horses (n = 176 pacers, n = 366 trotters) with genotype data imputed to ~74,000 single nucleotide polymorphisms (SNPs). Nineteen SNPs on nine chromosomes (ECA1, 2, 6, 9, 17, 19, 23, 25, 31) reached genome-wide significance (p < 1.44 x 10-6). Variant discovery in regions of interest was carried out via whole-genome sequencing. A set of 303 variants from 22 chromosomes with putative modifying effects on gait was genotyped in 659 Standardbreds (n = 231 pacers, n = 428 trotters) using a high-throughput assay. Random forest classification analysis resulted in an out-of-box error rate of 0.61%. A conditional inference tree algorithm containing seven SNPs predicted status as a pacer or trotter with 99.1% accuracy and subsequently performed with 99.4% accuracy in an independently sampled population of 166 Standardbreds (n = 83 pacers, n = 83 trotters). This highly accurate algorithm could be used by owners/trainers to identify Standardbred horses with the potential to race as pacers or as trotters, according to the genotype identified, prior to initiating training and would enable fine-tuning of breeding programs with designed matings. Additional work is needed to determine both the algorithm's utility in other gaited breeds and whether any of the predictive SNPs play a physiologically functional role in the tendency to pace or tag true functional alleles.

The horse Y chromosome as an informative marker for tracing sire lines.

Analysis of the Y chromosome is the best-established way to reconstruct paternal family history in humans. Here, we applied fine-scaled Y-chromosomal haplotyping in horses with biallelic markers and demonstrate the potential of our approach to address the ancestry of sire lines. We de novo assembled a draft reference of the male-specific region of the Y chromosome from Illumina short reads and then screened 5.8 million basepairs for variants in 130 specimens from intensively selected and rural breeds and nine Przewalski's horses. Among domestic horses we confirmed the predominance of a young'crown haplogroup' in Central European and North American breeds. Within the crown, we distinguished 58 haplotypes based on 211 variants, forming three major haplogroups. In addition to two previously characterised haplogroups, one observed in Arabian/Coldblooded and the other in Turkoman/Thoroughbred horses, we uncovered a third haplogroup containing Iberian lines and a North African Barb Horse. In a genealogical showcase, we distinguished the patrilines of the three English Thoroughbred founder stallions and resolved a historic controversy over the parentage of the horse 'Galopin', born in 1872. We observed two nearly instantaneous radiations in the history of Central and Northern European Y-chromosomal lineages that both occurred after domestication 5,500 years ago.

Evaluation of an HMGA2 variant for pleiotropic effects on height and metabolic traits in ponies.

BACKGROUND: Ponies are highly susceptible to metabolic derangements including hyperinsulinemia, insulin resistance, and adiposity. HYPOTHESIS/OBJECTIVES: Genetic loci affecting height in ponies have pleiotropic effects on metabolic pathways and increase the susceptibility to equine metabolic syndrome (EMS). ANIMALS: Two hundred ninety-four Welsh ponies and 529 horses. METHODS: Retrospective study of horses phenotyped for metabolic traits. Correlations between height and metabolic traits were assessed by Pearson's correlation coefficients. Complementary genome-wide analysis methods were used to identify a region of interest (ROI) for height and metabolic traits, determine the fraction of heritability contributed by the ROI, and identify candidate genes. RESULTS: There was an inverse relationship between height and baseline insulin (-0.26) in ponies. Genomic signature of selection and association analyses for both height and insulin identified the same ~1.3 megabase region on chromosome 6 that contained a shared ancestral haplotype between these traits. The ROI contributed ~40% of the heritability for height and ~20% of the heritability for insulin. High-mobility group AT-hook 2 was identified as a candidate gene, and Sanger sequencing detected a c.83G>A (p.G28E) variant associated with height in Shetland ponies. In our cohort of ponies, the A allele had a frequency of 0.76, was strongly correlated with height (-0.75), and was low to moderately correlated with metabolic traits including: insulin (0.32), insulin after an oral sugar test (0.25), non-esterified fatty acids (0.19), and triglyceride (0.22) concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: These data have important implications for identifying individuals at risk for EMS.