Whole genome sequencing identified a 16 kilobase deletion on ECA13 associated with distichiasis in Friesian horses.

BACKGROUND: Distichiasis, an ocular disorder in which aberrant cilia (eyelashes) grow from the opening of the Meibomian glands of the eyelid, has been reported in Friesian horses. These misplaced cilia can cause discomfort, chronic keratitis, and corneal ulceration, potentially impacting vision due to corneal fibrosis, or, if secondary infection occurs, may lead to loss of the eye. Friesian horses represent the vast majority of reported cases of equine distichiasis, and as the breed is known to be affected with inherited monogenic disorders, this condition was hypothesized to be a simply inherited Mendelian trait. RESULTS: A genome wide association study (GWAS) was performed using the Axiom 670 k Equine Genotyping array (MNEc670k) utilizing 14 cases and 38 controls phenotyped for distichiasis. An additive single locus mixed linear model (EMMAX) approach identified a 1.83 Mb locus on ECA5 and a 1.34 Mb locus on ECA13 that reached genome-wide significance (pcorrected = 0.016 and 0.032, respectively). Only the locus on ECA13 withstood replication testing (p = 1.6 × 10- 5, cases: n = 5 and controls: n = 37). A 371 kb run of homozygosity (ROH) on ECA13 was found in 13 of the 14 cases, providing evidence for a recessive mode of inheritance. Haplotype analysis (hapQTL) narrowed the region of association on ECA13 to 163 kb. Whole-genome sequencing data from 3 cases and 2 controls identified a 16 kb deletion within the ECA13 associated haplotype (ECA13:g.178714_195130del). Functional annotation data supports a tissue-specific regulatory role of this locus. This deletion was associated with distichiasis, as 18 of the 19 cases were homozygous (p = 4.8 × 10- 13). Genotyping the deletion in 955 horses from 54 different breeds identified the deletion in only 11 non-Friesians, all of which were carriers, suggesting that this could be causal for this Friesian disorder. CONCLUSIONS: This study identified a 16 kb deletion on ECA13 in an intergenic region that was associated with distichiasis in Friesian horses. Further functional analysis in relevant tissues from cases and controls will help to clarify the precise role of this deletion in normal and abnormal eyelash development and investigate the hypothesis of incomplete penetrance.

SNP-based heritability and genetic architecture of cranial cruciate ligament rupture in Labrador Retrievers.

Cranial cruciate ligament rupture (CCLR) is one of the leading causes of pelvic limb lameness in dogs. About 6% of Labrador Retrievers suffer from this orthopedic problem. The aim of this study was to determine the heritability of CCLR in this breed using SNP array genotyping data. DNA samples were collected from CCLR-affected dogs (n = 190) and unaffected dogs over the age of 8 years (n = 143). All 333 dogs were genotyped directly or imputed up to approximately 710k SNPs on the Affymetrix Axiom CanineHD SNP array. Heritability of CCLR was calculated using multiple methodologies, including linear mixed models, Bayesian models and a model that incorporates LD. The covariates of sex and sterilization status were added to each analysis to assess their impact. Across the algorithms of these models, heritability ranged from 0.550 to 0.886, depending on covariate inclusion. The relatively high heritability for this disease indicates that a substantial genetic component contributes to CCLR in the Labrador Retriever.

Assessment of the FAM174A 11G allele as a risk allele for equine metabolic syndrome.

An 11G nucleotide repeat in the 3' UTR of FAM174A was recently postulated as a risk allele with a dominant mode of inheritance for equine metabolic syndrome (EMS) and laminitis status in Arabian horses. The objective of this project was to evaluate this hypothesis in a large and diverse across-breed population. A total of 301 ponies, 292 Morgans, 64 Arabians, 49 Tennessee Walking Horses and 59 Quarter Horses were genotyped for six observed G repeat alleles in the FAM174A 3' UTR. Phenotype data included laminitis status, baseline insulin, glucose, non-esterified fatty acids, triglycerides, adiponectin, leptin, ACTH, insulin and glucose post oral sugar test, and two proxies for insulin resistance. The 11G allele frequencies were 18.8, 6.9, 1.8, 0.2 and 0.0% in the Arabians, Tennessee Walkers, ponies, Morgans and Quarter Horses respectively. Association analyses between FAM174A genotype and EMS phenotypes, and between allele count and EMS phenotypes, identified no statistically significant associations. When a dominant effect for the 11G allele was evaluated, a statistically significant association with adiponectin levels was identified in the ponies, and pairwise comparisons revealed that the estimated marginal means were higher in ponies with the 11G allele vs. alternative alleles (i.e. the allele had a protective effect). In conclusion, our data do not support the FAM174A 11G allele as a risk allele for EMS in our studied breeds.

SNP-based heritability and genetic architecture of tarsal osteochondrosis in North American Standardbred horses.

Osteochondrosis is a common developmental orthopedic disease characterized by a failure of endochondral ossification. Standardbred horses are recognized as being predisposed to tarsal osteochondrosis. Prior heritability estimates for tarsal osteochondrosis in European Standardbreds and related trotting breeds have been based on pedigree data and range from 17-29%. Here, we report on genetic architecture and heritability based on high-density genotyping data in a cohort of North American Standardbreds (n = 479) stringently phenotyped for tarsal osteochondrosis. Whole-genome array genotyping data were imputed to ~2 million single nucleotide polymorphisms (SNPs). SNP-based heritability of osteochondrosis in this population was explained by 2326 SNPs. The majority of these SNPs (86.6%) had small effects, whereas fewer SNPs had moderate or large effects (10% and 2.9% respectively), which is consistent with a polygenic/complex disease. Heritability was estimated at 0.24 ± 0.16 using two methods of restricted maximum likelihood analysis, as implemented in gcta (with and without a weighted relatedness matrix) and ldak software. Estimates were validated using bootstrapping. Heritability estimates were within the range previously reported and suggest that osteochondrosis is moderately heritable but that a significant portion of disease risk is due to environmental factors and/or genotype × environment interactions. Future identification of the genes/variants that have the most impact on disease risk may allow early recognition of high-risk individuals.

A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase.

BACKGROUND: Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. METHODS: Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. RESULTS: PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower Km for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. CONCLUSIONS: Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. GENERAL SIGNIFICANCE: This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy.

Genetic risk factors for insidious equine recurrent uveitis in Appaloosa horses.

Appaloosa horses are predisposed to equine recurrent uveitis (ERU), an immune-mediated disease characterized by recurring inflammation of the uveal tract in the eye, which is the leading cause of blindness in horses. Nine genetic markers from the ECA1 region responsible for the spotted coat color of Appaloosa horses, and 13 microsatellites spanning the equine major histocompatibility complex (ELA) on ECA20, were evaluated for association with ERU in a group of 53 Appaloosa ERU cases and 43 healthy Appaloosa controls. Three markers were significantly associated (corrected P-value <0.05): a SNP within intron 11 of the TRPM1 gene on ECA1, an ELA class I microsatellite located near the boundary of the ELA class III and class II regions and an ELA class II microsatellite located in intron 1 of the DRA gene. Association between these three genetic markers and the ERU phenotype was confirmed in a second population of 24 insidious ERU Appaloosa cases and 16 Appaloosa controls. The relative odds of being an ERU case for each allele of these three markers were estimated by fitting a logistic mixed model with each of the associated markers independently and with all three markers simultaneously. The risk model using these markers classified ~80% of ERU cases and 75% of controls in the second population as moderate or high risk, and low risk respectively. Future studies to refine the associations at ECA1 and ELA loci and identify functional variants could uncover alleles conferring susceptibility to ERU in Appaloosa horses.

Predicted genetic burden and frequency of phenotype-associated variants in the horse.

Disease-causing variants have been identified for less than 20% of suspected equine genetic diseases. Whole genome sequencing (WGS) allows rapid identification of rare disease causal variants. However, interpreting the clinical variant consequence is confounded by the number of predicted deleterious variants that healthy individuals carry (predicted genetic burden). Estimation of the predicted genetic burden and baseline frequencies of known deleterious or phenotype associated variants within and across the major horse breeds have not been performed. We used WGS of 605 horses across 48 breeds to identify 32,818,945 variants, demonstrate a high predicted genetic burden (median 730 variants/horse, interquartile range: 613-829), show breed differences in predicted genetic burden across 12 target breeds, and estimate the high frequencies of some previously reported disease variants. This large-scale variant catalog for a major and highly athletic domestic animal species will enhance its ability to serve as a model for human phenotypes and improves our ability to discover the bases for important equine phenotypes.

Equine developmental orthopaedic diseases--a genome-wide association study of first phalanx plantar osteochondral fragments in Standardbred trotters.

Palmar/plantar osteochondral fragments (POF) in fetlock joints commonly affect and influence the athletic performance of horses. In this study, we used the Equine SNP50 BeadChip® to perform a genome-wide association study of metatarsophalangeal POF in 176 Norwegian Standardbred trotter yearlings. Putative quantitative trait loci (QTL) for medial and/or lateral POF, and medial POF only were identified on ECA1, 2, 7, 9 and 31, whereas for lateral POF, only on ECA7, 11, 27 and X. The moderate number of QTL evidences a complex inheritance and suggests various genes controlling POF development in medial and lateral locations.

Genetic mapping of recurrent exertional rhabdomyolysis in a population of North American Thoroughbreds.

Recurrent exertional rhabdomyolysis is a heritable disorder that results in painful skeletal muscle cramping with exercise in up to 10% of all Thoroughbred racehorses. Here, we report a genome-wide association study with 48 282 SNPs analyzed among 48 case and 37 control Thoroughbreds. The most significant SNPs spanned approximately 13 Mb on ECA16, and the P-value of the most significant SNP after correcting for population structure was 8.0 × 10(-6) . This region on ECA16 was further evaluated by genotyping 247 SNPs in both the initial population and a second population of 34 case and 98 control Thoroughbreds. Several SNPs across the 13-Mb region on ECA16 showed significance when each population was analyzed separately; however, the exact positions of the most significant SNPs within this region on ECA16 varied between populations. This variability in location may be attributed to lack of power owing to insufficient sample sizes within each population individually, or to the relative distribution of long, conserved haplotypes, characteristic of the Thoroughbred breed. Future genome-wide association studies with additional horses would likely improve the power to resolve casual loci located on ECA16 and increase the likelihood of detecting any additional loci on other chromosomes contributing to disease susceptibility.

Genetic Variation and the Distribution of Variant Types in the Horse.

Genetic variation is a key contributor to health and disease. Understanding the link between an individual's genotype and the corresponding phenotype is a major goal of medical genetics. Whole genome sequencing (WGS) within and across populations enables highly efficient variant discovery and elucidation of the molecular nature of virtually all genetic variation. Here, we report the largest catalog of genetic variation for the horse, a species of importance as a model for human athletic and performance related traits, using WGS of 534 horses. We show the extent of agreement between two commonly used variant callers. In data from ten target breeds that represent major breed clusters in the domestic horse, we demonstrate the distribution of variants, their allele frequencies across breeds, and identify variants that are unique to a single breed. We investigate variants with no homozygotes that may be potential embryonic lethal variants, as well as variants present in all individuals that likely represent regions of the genome with errors, poor annotation or where the reference genome carries a variant. Finally, we show regions of the genome that have higher or lower levels of genetic variation compared to the genome average. This catalog can be used for variant prioritization for important equine diseases and traits, and to provide key information about regions of the genome where the assembly and/or annotation need to be improved.

Genome-wide association analysis of osteochondrosis of the tibiotarsal joint in Norwegian Standardbred trotters.

Osteochondrosis (OC), a disturbance in the process of endochondral ossification, is by far the most important equine developmental orthopaedic disease and is also common in other domestic animals and humans. The purpose of this study was to identify quantitative trait loci (QTL) associated with osteochondrosis dissecans (OCD) at the intermediate ridge of the distal tibia in Norwegian Standardbred (SB) using the Illumina Equine SNP50 BeadChip whole-genome single-nucleotide polymorphism (SNP) assay. Radiographic data and blood samples were obtained from 464 SB yearlings. Based on the radiographic examination, 162 horses were selected for genotyping; 80 of these were cases with an OCD at the intermediate ridge of the distal tibia, and 82 were controls without any developmental lesions in the joints examined. Genotyped horses descended from 22 sires, and the number of horses in each half-sib group ranged from 3 to 14. The population structure necessitated statistical correction for stratification. When conducting a case-control genome-wide association study (GWAS), mixed-model analyses displayed regions on chromosomes (Equus callabus chromosome - ECA) 5, 10, 27 and 28 that showed moderate evidence of association (P ≤ 5 × 10(-5); this P-value is uncorrected i.e. not adjusted for multiple comparisons) with OCD in the tibiotarsal joint. Two SNPs on ECA10 represent the most significant hits (uncorrected P=1.19 × 10(-5) in the mixed-model). In the basic association (chi-square) test, these SNPs achieved statistical significance with the Bonferroni correction (P=0.038) and were close in the permuted logistic regression test (P=0.054). Putative QTL on ECA 5, 10, 27 and 28 represent interesting areas for future research, validation studies and fine mapping of candidate regions. Results presented here represent the first GWAS of OC in horses using the recently released Illumina Equine SNP50 BeadChip.

Estimated prevalence of the Type 1 Polysaccharide Storage Myopathy mutation in selected North American and European breeds.

The GYS1 gene mutation that is causative of Type 1 Polysaccharide Storage Myopathy (PSSM) has been identified in more than 20 breeds of horses. However, the GYS1 mutation frequency or Type 1 PSSM prevalence within any given breed is unknown. The purpose of this study was to determine the frequency of the GYS1 mutation and prevalence of genetic susceptibility to Type 1 PSSM in selected breeds from Europe and North America. The GYS1 mutation was detected in 11 breeds, including, in order of increasing allele frequency, Shires, Morgans, Appaloosas, Quarter Horses, Paints, Exmoor Ponies, Saxon-Thuringian Coldbloods, South German Coldbloods, Belgians, Rhenish German Coldbloods and Percherons. The prevalence of genetic susceptibility to Type 1 PSSM in these breeds varied from 0.5% to 62.4%. The GYS1 mutation was not found in the sampled Thoroughbreds, Akhal-Tekes, Connemaras, Clydesdales, Norwegian Fjords, Welsh Ponies, Icelandics, Schleswig Coldbloods or Hanoverians, but failure to detect the mutation does not guarantee its absence. This knowledge will help breed associations determine whether they should screen for the GYS1 mutation and will alert veterinarians to a possible differential diagnosis for muscle pain, rhabdomyolysis or gait abnormalities.

Publisher Correction: A large intragenic deletion in the CLCN1 gene causes Hereditary Myotonia in pigs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Polysaccharide storage myopathy phenotype in quarter horse-related breeds is modified by the presence of an RYR1 mutation.

In this study we examined a family of Quarter Horses with Polysaccharide Storage Myopathy (PSSM) with a dominant mutation in the skeletal muscle glycogen synthase (GYS1) gene. A subset of horses within this family had a more severe and occasionally fatal PSSM phenotype. The purpose of this study was to identify a modifying gene(s) for the severe clinical phenotype. A genetic association analysis was used to identify RYR1 as a candidate modifying gene. A rare, known equine RYR1 mutation, associated with malignant hyperthermia (MH), was found to segregate in this GYS1 PSSM family. Retrospective analysis of patient records (n=179) demonstrated that horses with both the GYS1 and RYR1 mutations had a more severe clinical phenotype than horses with the GYS1 mutation alone. A treadmill trial (n=8) showed that serum creatine kinase activity was higher and exercise intolerance greater in horses with both mutations compared to the GYS1 mutation alone.

A GYS1 gene mutation is highly associated with polysaccharide storage myopathy in Cob Normand draught horses.

Glycogen storage diseases or glycogenoses are inherited diseases caused by abnormalities of enzymes that regulate the synthesis or degradation of glycogen. Deleterious mutations in many genes of the glyco(geno)lytic or the glycogenesis pathways can potentially cause a glycogenosis, and currently mutations in fourteen different genes are known to cause animal or human glycogenoses, resulting in myopathies and/or hepatic disorders. The genetic bases of two forms of glycogenosis are currently known in horses. A fatal neonatal polysystemic type IV glycogenosis, inherited recessively in affected Quarter Horse foals, is due to a mutation in the glycogen branching enzyme gene (GBE1). A second type of glycogenosis, termed polysaccharide storage myopathy (PSSM), is observed in adult Quarter Horses and other breeds. A severe form of PSSM also occurs in draught horses. A mutation in the skeletal muscle glycogen synthase gene (GYS1) was recently reported to be highly associated with PSSM in Quarter Horses and Belgian draught horses. This GYS1 point mutation appears to cause a gain-of-function of the enzyme and to result in the accumulation of a glycogen-like, less-branched polysaccharide in skeletal muscle. It is inherited as a dominant trait. The aim of this work was to test for possible associations between genetic polymorphisms in four candidate genes of the glycogen pathway or the GYS1 mutation in Cob Normand draught horses diagnosed with PSSM by muscle biopsy.

Comparative skeletal muscle histopathologic and ultrastructural features in two forms of polysaccharide storage myopathy in horses.

Polysaccharide storage myopathy (PSSM) has been found in more than 35 different horse breeds through identification of abnormal storage of polysaccharide in muscle biopsies. A dominant mutation in the glycogen synthase 1 gene (GYS1) accounts for a substantial proportion of PSSM cases in at least 17 breeds, including Quarter Horses, but some horses diagnosed with PSSM by muscle histopathologic analysis are negative for the mutation. We hypothesized that a second distinct form of glycogen storage disease exists in GYS1-negative horses with PSSM. The objectives of this study were to compare the histopathologic features, ultrastructure of polysaccharide, signalment, history, and presenting complaints of GYS1-negative Quarter Horses and related breeds with PSSM to those of GYS1-positive horses with PSSM. The total histopathologic score in frozen sections of skeletal muscle stained with hematoxylin and eosin, periodic acid Schiff (PAS) and amylase-PAS stains from 53 GYS1-negative horses did not differ from that of 52 GYS1-positive horses. Abnormal polysaccharide was fine granular or homogenous in appearance (49/53; 92%), often amylase-sensitive (28/53; 53%), more commonly located under the sarcolemma, and consisting of beta glycogen particles in GYS1-negative horses. However, in GYS1-positive horses, abnormal polysaccharide was usually coarse granular (50/52; 96%), amylase-resistant (51/52; 98%), more commonly cytoplasmic, and consisting of beta glycogen particles or, in some myofibers, filamentous material surrounded by beta glycogen particles. Retrospective analysis found that GYS1-negative horses (n = 43) were younger at presentation (4.9 +/- 0.6 years vs. 6.7 +/- 0.3 years for GYS1-positive horses) and were more likely to be intact males than GYS1-positive horses (n = 160). We concluded that 2 forms of PSSM exist and often have distinctive abnormal polysaccharide. However, because evaluation of the histologic appearance of polysaccharide can be subjective and affected by age, the gold standard for diagnosis of PSSM at present would appear to be testing for the GYS1 mutation followed by evaluating muscle biopsy for characteristic abnormal polysaccharide in those horses that are negative for the mutation.

A glycogen synthase 1 mutation associated with equine polysaccharide storage myopathy and exertional rhabdomyolysis occurs in a variety of UK breeds.

REASONS FOR PERFORMING STUDY: A glycogen synthase (GYS1) mutation has been described in horses with histopathological evidence of polysaccharide storage myopathy (PSSM) in the USA. It is unknown whether the same mutation is present in horses from the UK. OBJECTIVES: To determine whether the GYS1 mutation occurs in UK horses with histopathological evidence of PSSM and exertional rhabdomyolysis. HYPOTHESIS: The R309H GYS1 mutation is present in a variety of UK horse breeds and that the mutation is commonly associated with exertional rhabdomyolysis. METHODS: DNA was extracted from 47 muscle or blood samples from UK horses with histories of exertional rhabdomyolysis in which muscle biopsy diagnosis had been pursued. The proportions of GYS1 mutation positive cases were compared among histopathologically defined groups. In addition, breeds that carried the GYS1 mutation were identified from a total of 37 grade 2 (amylase-resistant) PSSM cases. RESULTS: Of 47 horses with exertional rhabdomyolysis in which a muscle biopsy diagnosis was pursued, 10 (21%) carried the GYS1 mutation. The mutation was only found in horses with grade 2 PSSM (i.e. not in horses with normal, idiopathic myopathy or grade 1 PSSM biopsy samples). In total, the GYS1 mutation was found in 24/37 (65%) of grade 2 PSSM cases. A variety of breeds, including Quarter Horse, Appaloosa, Warmblood, Connemara-cross, Cob, Polo Pony and Thoroughbred cross carried the mutation. CONCLUSIONS: The GYS1 mutation is an important cause of exertional rhabdomyolysis of UK horse breeds but does not account for all forms of PSSM. POTENTIAL RELEVANCE: Genotyping is recommended in cases of exertional rhabdomyolysis, prior to or in combination with, muscle biopsy. However a significant proportion of horses with histopathological evidence of PSSM and/or exertional rhabdomyolysis have different diseases.

Heritability of metabolic traits associated with equine metabolic syndrome in Welsh ponies and Morgan horses.

BACKGROUND: Equine metabolic syndrome (EMS) is a complex clinical disorder with both environmental and genetic factors contributing to EMS phenotypes. Estimates of heritability determine the proportion of variation in a trait that is attributable to genetics. OBJECTIVES: To provide heritability estimates for nine metabolic traits associated with EMS in two high-risk breeds. STUDY DESIGN: Retrospective cohort study. METHODS: High-density single-nucleotide polymorphism (SNP) genotype data was used to estimate the heritability (h2 SNP ) of nine metabolic traits relevant to EMS in a cohort of 264 Welsh ponies and 286 Morgan horses. Traits included measurements of insulin, glucose, non-esterified fatty acids (NEFA), triglycerides, leptin, adiponectin, ACTH, and glucose (GLU-OST) and insulin (INS-OST) following an oral sugar challenge. RESULTS: In Welsh ponies, seven of the nine traits had statistically significant h2 SNP estimates that were considered moderately to highly heritable (h2 SNP >0.20) including: triglycerides (0.313; s.e. = 0.146), glucose (0.408; s.e. = 0.135), NEFA (0.434; s.e. = 0.136), INS-OST (0.440; s.e. = 0.148), adiponectin (0.488; s.e. = 0.143), leptin (0.554; s.e. = 0.132) and insulin (0.808; s.e. = 0.108). In Morgans, six of the nine traits had statistically significant h2 SNP estimates that were also determined to be moderately to highly heritable including: INS-OST (0.359; s.e. = 0.185), leptin (0.486; s.e. = 0.177), GLU-OST (0.566 s.e. = 0.175), insulin (0.592; s.e. = 0.195), NEFA (0.684; s.e. = 0.164), and adiponectin (0.913; s.e. = 0.181). MAIN LIMITATIONS: Insufficient population size may have limited power to obtain statistically significant h2 SNP estimates for ACTH (both breeds), glucose and triglycerides in Morgans and GLU-OST in Welsh ponies. CONCLUSIONS: This study provides the first concrete evidence of a genetic contribution to key phenotypes associated with EMS. Eight of these nine traits had moderate to high h2 SNP estimates in this cohort. These data demonstrate that continued research for identification of the genetic risk factors for EMS phenotypes within and across breeds is warranted.