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The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Due to the limited availability of functional data for the equine genome, as well as the technical limitations of short-read RNA-seq, existing annotation of the equine genome contains limited information about important aspects of gene regulation, such as alternate isoforms and regulatory elements, which are either not transcribed or transcribed at a very low level. To solve above problems, the Functional Annotation of the Animal Genomes (FAANG) project proposed a systemic approach to tissue collection, phenotyping, and data generation, adopting the blueprint laid out by the Encyclopedia of DNA Elements (ENCODE) project. Here we detail the first comprehensive overview of gene expression and regulation in the horse, presenting 39,625 novel transcripts, 84,613 candidate cis-regulatory elements (CRE) and their target genes, 332,115 open chromatin regions genome wide across a diverse set of tissues. We showed substantial concordance between chromatin accessibility, chromatin states in different genic features and gene expression. This comprehensive and expanded set of genomics resources will provide the equine research community ample opportunities for studies of complex traits in the horse.

BackgroundWarmblood Fragile Foal Syndrome Type 1 (WFFS) is an autosomal recessive disorder reported previously only in warmbloods and thought to be caused by a variant in the gene procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1, c.2032G>A, p.Gly678Arg). Given the presentation of this Thoroughbred case, we hypothesised that a similar genetic mechanism caused this phenotype.ObjectivesTo describe the pathological and genetic findings on a foal presenting to a veterinary practice in the UK with skin lesions similar to other Ehlers-Danlos Syndromes, including those documented for warmbloods with WFFS.Study designA single case report describing a genetic investigation.MethodsA Thoroughbred foal presenting as dystocia was euthanised for multiple skin lesions and developmental abnormalities. DNA extracted from the foal was tested for the PLOD1 variant (c.2032G>A, p.Gly678Arg) using the commercially available assay. To confirm causality and further interrogate potential novel causes of Ehlers-Danlos Syndrome, 1799 functional candidate genes, including PLOD1, were analysed using whole genome sequencing data generated from DNA extracted from the foal's muscle. These data were compared to 34 control samples from at least 11 other breeds. Variants were prioritised for further evaluation based on predicted impact on protein function.ResultsPost-mortem evaluation concluded that this foal suffered from a condition of collagen dysplasia. The foal was homozygous for the c.2032G>A PLOD1 variant. Only two other missense variants identified from whole genome sequencing data were also computationally predicted to be deleterious to protein function, (NPHP3 c.1253T>C, p.Leu418Pro, EPDR1 c.154G>C, p.Glu52Gln). Neither of these genes have been linked to similar phenotypes, or Ehlers-Danlos Syndrome in humans or other species and thus further investigation of these variants as the cause of EDS was not warranted.Main limitationsThis study is a single case report in the Thoroughbred with no additional cases from this breed yet identified to replicate this finding.ConclusionsGiven the clinical presentation similar to WFFS, homozygosity for the PLOD1 variant, and absence of another more plausible causal variant from the WGS experiment, we conclude that PLOD1 c.2032G>A is the likely cause of this foal's condition. This is the first documented evidence of fragile foal syndrome caused by the PLOD1 variant in a breed outside of warmbloods, the Thoroughbred. We therefore recommend a change in the name of this disorder to fragile foal syndrome type 1 (FFS) and utilisation of genetic testing in Thoroughbreds to avoid producing affected foals.

Since domestication, horses have been selectively bred for various coat colors and white spotting patterns. To investigate breed distribution, allele frequencies, and potential lethal variants for recommendations on genetic testing, 29 variants within 14 genes were investigated in 11,281 horses from 28 breeds. The recessive chestnut ea allele in melanocortin 1 receptor (MC1R) (p.D84N) was identified in four breeds: Knabstrupper, Paint Horse, Percheron, and Quarter Horse. After filtering for relatedness, ea allele frequency in Knabstruppers was estimated at 0.035, thus illustrating the importance of testing for mate selection for base coat color. The Rocky Mountain Horse breed had the highest allele frequency for two of the dilution variants under investigation (Za.f. = 0.32 and Cha.f. = 0.026); marker-assisted selection in this breed could aid in the production of horses with desirable dilute coats with less severe ocular anomalies caused by the silver (Z) allele. With regard to white patterning, nine horses homozygous for the paired box 3 (PAX3) splashed white 2 (SW2) allele (p.C70Y) and six horses homozygous for the KIT proto-oncogene, receptor tyrosine kinase (KIT) sabino 1 (SB1) allele (ECA3g.79544206A>T) were identified, thus determining they are rare and confirming that homozygosity for SW2 is not embryonic lethal. The KIT dominant white 20 (W20) allele (p.R682H) was identified in all but three breeds: Arabian (n = 151), Icelandic Horse (n = 66), and Norwegian Fjord Horse (n = 90). The role of W20 in pigmentation across breeds is not well understood; given the different selection regimes of the breeds investigated, these data provide justification for further evaluating the functional role of this allele in pigmentation. Here, we present the largest dataset reported for coat color variants in horses to date, and these data highlight the importance of breed-specific studies to inform on the proper use of marker-assisted selection and to develop hypotheses related to pigmentation for further testing in horses.

American Standardbreds were developed as a harness racing horse breed. The United States Trotting Association closed the studbook in 1973 and implemented a book size cap in 2009. This study aimed to investigate genetic diversity in the American Standardbred after the studbook cap was introduced using short tandem repeats (STRs) and single-nucleotide polymorphisms (SNPs). Sixteen STRs from horses foaled from 2010 to 2015 and their sires and dams (n = 50 621) were utilized to examine allelic richness (Ar), expected heterozygosity (HE), observed heterozygosity (HO), unbiased heterozygosity (HU), inbreeding coefficient (FIS), and fixation index (FST). These analyses found that trotting and pacing sires were less genetically diverse than dams (HEPBonferroni = 0.029 and 6.3 × 10-5, respectively) and their offspring (ArPBonferroni = 0.034 and 6.9 × 10-6, respectively), and pacing offspring were significantly less diverse than their dams (HEPBonferroni = 2 × 10-3). Inbreeding coefficients for trotters (FIS = -0.014) and pacers (FIS = -0.012) suggest that breeding practices have maintained diversity. Moderate levels of genetic differentiation (0.066 < FST < 0.11) were found between pacing and trotting groups. Additionally, 10 of the most prolific trotting sires and their male offspring (n = 84) were genotyped on the 670K Axiom Equine HD Array. HO values higher than HE (P < 0.001), low inbreeding coefficients (mean F = -0.064), and mean FROH = 21% indicate relatively high levels of diversity in this cohort, further supporting the STR data. However, in contrast, HO values were higher for trotting sires (0.41) than their offspring (0.36). This observation warrants further monitoring of diversity over time. These data provide an updated foundation of diversity indices for further, long-term analysis in the breed.

BackgroundThe prevalence of clinical signs and factors triggering muscle atrophy and rhabdomyolysis associated with an MYH1E321G mutation in Quarter Horses and related breeds (QH) remain poorly understood.Hypothesis/objectivesDetermine the prevalence and potential triggers of atrophy and stiffness in horses homozygous reference (N/N), heterozygous (My/N), and homozygous (My/My) for the MYH1E321G mutation.AnimalsTwo-hundred seventy-five N/N, 100 My/N, and 10 My/My QH.MethodsA retrospective case-control study using a closed-ended questionnaire completed by clients of the Veterinary Genetics Laboratory at the University of California, Davis. History of clinical signs, disease, vaccination and performance were analyzed by genotype using contingency testing.ResultsAtrophy occurred in proportionately more horses with MYH1E321G (My) than N/N QH and more frequently in My/My than My/N QH (P

The role of histone modifications in transcription remains incompletely understood. Here, we examine the relationship between histone modifications and transcription using experimental perturbations combined with sensitive machine-learning tools. Transcription predicted the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. Blocking transcription rapidly removed two punctate marks, H3K4me3 and H3K27ac, from chromatin indicating that transcription is required for active histone modifications. Transcription was also required for maintenance of H3K27me3, consistent with a role for RNA in recruiting PRC2. A subset of DNase-I-hypersensitive sites were refractory to prediction, precluding models where transcription initiates pervasively at any open chromatin. Our results, in combination with past literature, support a model in which active histone modifications serve a supportive, rather than an essential regulatory, role in transcription.

Cytosine methylation patterns have not yet been thoroughly studied in horses. Here, we profile n = 333 samples from 42 horse tissue types at loci that are highly conserved between mammalian species using a custom array (HorvathMammalMethylChip40). Using the blood and liver tissues from horses, we develop five epigenetic aging clocks: a multi-tissue clock, a blood clock, a liver clock and two dual-species clocks that apply to both horses and humans. In addition, using blood methylation data from three additional equid species (plains zebra, Grevy's zebras and Somali asses), we develop another clock that applies across all equid species. Castration does not significantly impact the epigenetic aging rate of blood or liver samples from horses. Methylation and RNA data from the same tissues define the relationship between methylation and RNA expression across horse tissues. We expect that the multi-tissue atlas will become a valuable resource.

Tabby patterns of fur coats are defining characteristics in wild and domestic felids. Historically, three autosomal alleles at one locus (Tabby): Abyssinian (Ta ; a.k.a. ticked), mackerel (Tm ; a.k.a. striped) and blotched (tb ; a.k.a. classic, blotched) were thought to control these patterns in domestic cats and their breeds. Currently, at least three loci influence cat tabby markings, two of which are designated Tabby and Ticked. The Tabby locus is laeverin (LVRN) and affects the mackerel and blotched patterns. The unidentified gene for the Ticked locus on cat chromosome B1 was suggested to control the presence or absence of the ticked pattern (Tabby - Abyssinian (Ta ; a.k.a. ticked). The cat reference genome (Cinnamon, the Abyssinian) has the ticked phenotype and the variant dataset and coat phenotypes from the 99 Lives Cat Genome Consortium (195 cats) were used to identify candidate genes and variants associated with the Ticked locus. Two strategies were used to find the Ticked allele(s), one considered Cinnamon with the reference allele or heterozygous (Strategy A) and the other considered Cinnamon as having the variant allele or heterozygous (Strategy B). For Strategy A, two variants in Dickkopf Wnt Signaling Pathway Inhibitor 4 (DKK4), a p.Cys63Tyr (B1:41621481, c.188G>A) and a less common p.Ala18Val (B1:42620835, c.53C>T) variant are suggested as two alleles influencing the Ticked phenotype. Bioinformatic and molecular modeling analysis suggests that these changes disrupt a key disulfide bond in the Dkk4 cysteine-rich domain 1 or Dkk4 signal peptide cleavage respectively. All coding variants were excluded as Ticked alleles using Strategy B.

Squamous cell carcinoma (SCC) occurs frequently in the human Xeroderma Pigmentosum (XP) syndrome and is characterized by deficient UV-damage repair. SCC is the most common equine ocular cancer and the only associated genetic risk factor is a UV-damage repair protein. Specifically, a missense mutation in horse DDB2 (T338M) was strongly associated with both limbal SCC and third eyelid SCC in three breeds of horses (Halflinger, Belgian, and Rocky Mountain Horses) and was hypothesized to impair binding to UV-damaged DNA. Here, we investigate DDB2-T338M mutant's capacity to recognize UV lesions in vitro and in vivo, together with human XP mutants DDB2-R273H and -K244E. We show that the recombinant DDB2-T338M assembles with DDB1, but fails to show any detectable binding to DNA substrates with or without UV lesions, due to a potential structural disruption of the rigid DNA recognition β-loop. Consistently, we demonstrate that the cellular DDB2-T338M is defective in its recruitment to focally radiated DNA damages, and in its access to chromatin. Thus, we provide direct functional evidence indicating the DDB2-T338M recapitulates molecular defects of human XP mutants, and is the causal loss-of-function allele that gives rise to equine ocular SCCs. Our findings shed new light on the mechanism of DNA recognition by UV-DDB and on the initiation of ocular malignancy.

Following the successful creation of a biobank from two adult Thoroughbred mares, this study aimed to recapitulate sample collection in two adult Thoroughbred stallions as part of the Functional Annotation of the Animal Genome (FAANG) initiative. Both stallions underwent thorough physical, lameness, neurologic, and ophthalmic (including electroretinography) examinations prior to humane euthanasia. Epididymal sperm was recovered from both stallions immediately postmortem and cryopreserved. Aseptically collected full thickness skin biopsies were used to isolate, culture and cryopreserve dermal fibroblasts. Serum, plasma, cerebrospinal fluid, urine, and gastrointestinal content from various locations were collected and cryopreserved. Under guidance of a board-certified veterinary anatomic pathologist, 102 representative tissue samples were collected from both horses. Whole tissue samples were flash-frozen and prioritized tissues had nuclei isolated and cryopreserved. Spatially contemporaneous samples of each tissue were submitted for histologic examination. Antemortem and gross pathologic examination revealed mild abnormalities in both stallions. One stallion (ECA_UCD_AH3) had unilateral thoracic limb lameness and bilateral chorioretinal scars. The second stallion (ECA_UCD_AH4) had subtle symmetrical pelvic limb ataxia, symmetrical prostatomegally, and moderate gastrointestinal nematodiasis. DNA from each was whole-genome sequenced and genotyped using the GGP Equine 70K SNP array. The genomic resources and banked biological samples from these animals augments the existing resource available to the equine genomics community. Importantly we may now improve the resolution of tissue-specific gene regulation as affected by sex, as well as add sex-specific tissues and gametes.

Coat color is a trait of economic significance in horses. Variants in seven genes have been documented to cause white patterning in horses. Of the 34 variants that have been identified in KIT proto-oncogene, receptor tyrosine kinase (KIT), 27 have only been reported in a single individual or family and thus not all are routinely offered for genetic testing. Therefore, to enable proper use of marker-assisted selection, determining breed specificity for these alleles is warranted. Screening 19 unregistered all-white Shetland ponies for 16 white patterning markers identified 14 individuals whose phenotype could not be explained by testing results. In evaluating other known dominant white variants, 14 horses were heterozygous for W13. W13 was previously only reported in two quarter horses and a family of Australian miniature horses. Genotyping known white spotting variants in 30 owner-reported white animals (25 Miniature Horses and five Shetland ponies) identified two additional W13/N American Miniature Horses. The estimated allele frequency of W13 in the American Miniature Horse was 0.0063 (79 N/N, 1 W13/N) and the allele was not detected in a random sample (n = 59) of Shetland ponies. No homozygous W13 individuals were identified and W13/N ponies had a similar all-white coat with pink skin phenotype, regardless of the other white spotting variants present, demonstrating that W13 results in a Mendelian inherited dominant white phenotype and homozygosity is likely lethal. These findings document the presence of W13 in the American Miniature Horse and Shetland pony populations at a low frequency and illustrate the importance of testing for this variant in additional breeds.

The horse reference genome assemblies, EquCab2.0 and EquCab3.0, have enabled great advancements in the equine genomics field, from tools to novel discoveries. However, significant gaps of knowledge regarding genome function remain, hindering the study of complex traits in horses. In an effort to address these gaps and with inspiration from the Encyclopedia of DNA Elements (ENCODE) project, the equine Functional Annotation of Animal Genome (FAANG) initiative was proposed to bridge the gap between genome and gene expression, providing further insights into functional regulation within the horse genome. Three years after launching the initiative, the equine FAANG group has generated data from more than 400 experiments using over 50 tissues, targeting a variety of regulatory features of the equine genome. In this review, we examine how valuable lessons learned from the ENCODE project informed our decisions in the equine FAANG project. We report the current state of the equine FAANG project and discuss how FAANG can serve as a template for future expansion of functional annotation in the equine genome and be used as a reference for studies of complex traits in horse. A well-annotated reference functional atlas will also help advance equine genetics in the pan-genome and precision medicine era.

Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A+ selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, both polyadenylated and non-polyadenylated transcripts. The purpose of this study was to determine which library preparation method is optimal for lncRNA investigations in the horse. Using liver and cerebral parietal lobe tissues from two healthy Thoroughbred mares, RNA-seq libraries were prepared using standard poly-A+ selection and rRNA-depletion methods. Averaging the two biologic replicates, poly-A+ selection yielded 327 and 773 more unique lncRNA transcripts for liver and parietal lobe, respectively. More lncRNA were found to be unique to poly-A+ selected libraries, and rRNA-depletion identified small nucleolar RNA (snoRNA) to have a higher relative expression than in the poly-A+ selected libraries. Overall, poly-A+ selection provides a more thorough identification of total lncRNA in equine tissues while rRNA-depletion may allow for easier detection of snoRNAs.

Splashed white is a coat color pattern in horses characterized by extensive white patterning on the legs, belly, and face often accompanied by blue eyes and deafness. Three mutations in microphthalmia-associated transcription factor (MITF) and two mutations in Paired Box 3 (PAX3) have been identified that explain splashed white patterns (SW1-SW5). An American Paint Horse stallion with a splashed white phenotype and blue eyes, whose parents were not white patterned, was negative for the 5 known splashed white variants and other known white spotting alleles. This novel splashed white phenotype (SW6) was hypothesized to be caused by a de novo mutation in MITF or PAX3. Analysis of whole-genome sequencing using the EquCab3.0 reference genome for comparison identified an 8.7 kb deletion in MITF on ECA16 (NC_009159.3:g.21551060-21559770del). The deletion encompassed part of intron 7 through the 3' UTR of exon 9 of MITF, including the helix-loop-helix DNA-binding domain (ENSECAT00000006375.3). This variant is predicted to truncate protein and impair binding to DNA. Sanger sequencing confirmed the stallion was heterozygous for the MITF deletion. No single nucleotide polymorphisms (SNPs) or structural variants were identified in PAX3 or any of the other candidate genes that were unique to the stallion or predicted to affect protein function. Genotyping five of the stallion's splashed white offspring, including one all white foal, found that they were also heterozygous for the deletion. Given the role of MITF in producing white pattern phenotypes, and the predicted deleterious effect of this mutation, this 8.7 kb deletion is the likely causal variant for SW6.

One of the primary aims of the Functional Annotation of ANimal Genomes (FAANG) initiative is to characterize tissue-specific regulation within animal genomes. To this end, we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in eight prioritized tissues collected as part of the FAANG equine biobank from two thoroughbred mares. Data were generated according to optimized experimental parameters developed during quality control testing. To ensure that we obtained sufficient ChIP and successful peak-calling, data and peak-calls were assessed using six quality metrics, replicate comparisons, and site-specific evaluations. Tissue specificity was explored by identifying binding motifs within unique active regions, and motifs were further characterized by gene ontology (GO) and protein-protein interaction analyses. The histone marks identified in this study represent some of the first resources for tissue-specific regulation within the equine genome. As such, these publicly available annotation data can be used to advance equine studies investigating health, performance, reproduction, and other traits of economic interest in the horse.

Squamous cell carcinoma (SCC) is the most common cancer affecting the equine eye. A missense variant within the gene damage-specific DNA binding protein 2 (DDB2 c.1013C>T, p.Thr338Met) was previously identified as a causal recessive genetic risk factor for the development of ocular SCC within Haflingers, Belgian Draft horses, and Rocky Mountain Horses, but not in the Appaloosa or Arabian breeds. This study aimed to evaluate three cases of ocular SCC in additional breeds and determine if DNA testing for the DDB2 variant in warmblood horses and Connemara ponies is warranted. Histopathology confirmed ocular SCC in all three cases and DNA testing confirmed each horse was homozygous for the DDB2 risk factor. The DDB2 risk allele frequency was estimated to be 0.0043 for Holsteiners (N = 115), 0.014 for Belgian Warmbloods (N = 71), and 0.22 for Connemara Ponies (N = 86). Taken together these data support using DNA testing for DDB2 in Connemara Ponies to assist in mate selection and clinical management. Given the low observed allele frequencies in both the Holsteiner and Belgian Warmblood breeds and that the case under investigation was a warmblood cross-bred, evaluating additional SCC affected warmbloods is warranted to fully determine the importance of DDB2 genotyping as a risk factor in warmblood breeds.

Warmblood fragile foal syndrome (WFFS) is an autosomal recessive disorder caused by a single nucleotide variant in the procollagen-lysine-2-oxoglutarate-5-dioxygenase 1 gene (PLOD1:c.2032G>A, p.Gly678Arg). Homozygosity for the PLOD1 variant causes an Ehler-Danlos-like syndrome, which has to date only been reported in warmblood breeds but the WFFS allele has been also detected in the Thoroughbred. To investigate the breed distribution of the WFFS allele, 4081 horses belonging to 38 different breeds were screened. In total, 4.9% of the horses representing 21 breeds carried the WFFS allele. The affected breeds were mainly warmbloods, with carrier frequency as high as 17% in the Hanoverian and Danish Warmblood. The WFFS allele was not detected in most non-warmblood breeds. Exceptions include WFFS carriers in the Thoroughbred (17/716), Haflinger (2/48), American Sport Pony (1/12), and Knabstrupper (3/46). The origin of the WFFS allele remains unknown. The Arabian breed and specifically the stallion Bairactar Or. Ar. (1813), whose offspring were reported to have a similar phenotype in the 19th century, were hypothesized as the origin. DNA from a museum sample of Bairactar Or. Ar. showed that he did not carry the mutated allele. This result, together with the genotypes of 302 Arabians, all homozygous for the reference allele, does not support an Arabian origin of the WFFS allele. Our extensive survey shows the WFFS allele to be of moderate frequency and concern in warmbloods and also in breeds where it may not be expected.

Equine recurrent uveitis (ERU) is an immune-mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T-cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T-cell phenotype. Adipose-derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4+ T-cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T-cells and B-cells in horses with ERU were similar to normal horses. However, CD4+ T-cells from horses with ERU expressed higher amounts of IFNγ indicating a pro-inflammatory Th1 phenotype. When co-incubated with MSCs, activated CD4+ T-cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell-cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4+ T-cell activation phenotype through a combination of cell-cell contact and prostaglandin signaling.

One of the primary aims of the Functional Annotation of ANimal Genomes (FAANG) initiative is to characterize tissue-specific regulation within animal genomes. To this end, we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in eight prioritized tissues collected as part of the FAANG equine biobank from two thoroughbred mares. Data were generated according to optimized experimental parameters developed during quality control testing. To ensure that we obtained sufficient ChIP and successful peak-calling, data and peak-calls were assessed using six quality metrics, replicate comparisons, and site-specific evaluations. Tissue specificity was explored by identifying binding motifs within unique active regions, and motifs were further characterized by gene ontology (GO) and protein-protein interaction analyses. The histone marks identified in this study represent some of the first resources for tissue-specific regulation within the equine genome. As such, these publicly available annotation data can be used to advance equine studies investigating health, performance, reproduction, and other traits of economic interest in the horse.

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

Mushroom is a unique coat color phenotype in Shetland Ponies characterized by the dilution of the chestnut coat color to a sepia tone and is hypothesized to be a recessive trait. A genome wide association study (GWAS), utilizing the Affymetrix 670K array (MNEc670k) and a single locus mixed linear model analysis (EMMAX), identified a locus on ECA7 for further investigation (Pcorrected = 2.08 × 10-10). This locus contained a 3 Mb run of homozygosity in the 12 mushroom ponies tested. Analysis of high throughput Illumina sequencing data from one mushroom Shetland pony compared to 87 genomes from horses of various breeds, uncovered a frameshift variant, p.Asp201fs, in the MFSD12 gene encoding the major facilitator superfamily domain containing 12 protein. This variant was perfectly concordant with phenotype in 96 Shetland Ponies (P = 1.15 × 10-22), was identified in the closely related Miniature Horse for which the mushroom phenotype is suspected to occur (fmu = 0.02), and was absent in 252 individuals from seven additional breeds not reported to have the mushroom phenotype. MFSD12 is highly expressed in melanocytes and variants in this gene in humans, mice, and dogs impact pigmentation. Given the role of MFSD12 in melanogenesis, we propose that p.Asp201fs is causal for the dilution observed in mushroom ponies.

Squamous cell carcinoma (SCC) is the most common periocular cancer in horses and the second most common tumor of the horse overall. A missense mutation in damage-specific DNA-binding protein 2 (DDB2, c.1012 C>T, p.Thr338Met) was previously found to be strongly associated with ocular SCC in Haflinger and Belgian horses, explaining 76% of cases across both breeds. To determine if this same variant in DDB2 contributes to risk for ocular SCC in the Arabian, Appaloosa, and Percheron breeds and to determine if the variant contributes to risk for oral or urogenital SCC, histologically confirmed SCC cases were genotyped for the DDB2 variant and associations were investigated. Horses with urogenital SCC that were heterozygous for the DDB2 risk allele were identified in the Appaloosa breed, but a significant association between the DDB2 variant and SCC occurring at any location in this breed was not detected. The risk allele was not identified in Arabians, and no Percherons were homozygous for the risk allele. High-throughput sequencing data from six Haflingers were analyzed to ascertain if any other variant from the previously associated 483 kb locus on ECA12 was more concordant with the SCC phenotype than the DDB2 variant. Sixty polymorphisms were prioritized for evaluation, and no other variant from this locus explained the genetic risk better than the DDB2 allele (P = 3.39 × 10-17, n = 118). These data provide further support of the DDB2 variant contributing to risk for ocular SCC, specifically in the Haflinger and Belgian breeds.

Clinical studies provide strong evidence that obesity and associated adipose tissue (AT) inflammation are risk factors for breast cancer (BrCA); however, mechanistic knowledge of the interaction of obesity, BrCA, and menopausal status has proven to be not only lacking, but contradictory. Obesity-induced inflammation and elevated biosynthesis of estrogens, through aromatase-mediated metabolism of precursors, have been linked with hormone receptor positive (HP) postmenopausal BrCA but not previously associated with premenopausal BrCA risk. Thus, further delineation of the interaction of obesity, inflammation, and aromatase is required for the development of therapeutic treatment options. The purpose of this study was to examine the effect of high fat diet (HFD)-induced inflammation on tumorigenesis in a model of pre and postmenopausal HP BrCA. Female PyMT/MMTV ovary intact and ovariectomized mice were fed low and HFD diets to examine the role of obesity-induced inflammation and hormone production in the development of HP BrCA. Tumor statistics for number, volume, weight, histopathology scoring and gene expression of macrophage and inflammatory mediators were measured in the AT and mammary gland at sacrifice. HFD feedings of ovary intact mice resulted in increased adiposity and tumorigenesis, indicated by increased primary tumor volume, multiplicity, tumor burden, and increased tumor progression represented by histopathological scoring. HFD-induced obesity significantly upregulated aromatase and macrophage marker expression in the AT (F4/80 and CD11c) and mammary gland (Mertk) in a premenopausal model of BrCA. Conversely, HFD feedings had no significant effect on tumorigenesis in a postmenopausal model of BrCA despite large increases in adiposity in ovariectomized mice; however, limitations within the model may have precluded any significant findings. This data suggests that obesity-induced increases in inflammation and hormone production, via aromatase expression, is associated with increases in tumorigenesis in a model of premenopausal HP BrCA in the PyMT/MMTV strain.

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

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Chromosomal abnormalities are a common cause of human miscarriage but rarely reported in any other species. As a result, there are currently inadequate animal models available to study this condition. Horses present one potential model since mares receive intense gynecological care. This allowed us to investigate the prevalence of chromosomal copy number aberrations in 256 products of conception (POC) in a naturally occurring model of pregnancy loss (PL). Triploidy (three haploid sets of chromosomes) was the most common aberration, found in 42% of POCs following PL over the embryonic period. Over the same period, trisomies and monosomies were identified in 11.6% of POCs and subchromosomal aberrations in 4.2%. Whole and subchromosomal aberrations involved 17 autosomes, with chromosomes 3, 4, and 20 having the highest number of aberrations. Triploid fetuses had clear gross developmental anomalies of the brain. Collectively, data demonstrate that alterations in chromosome number contribute to PL similarly in women and mares, with triploidy the dominant ploidy type over the key period of organogenesis. These findings, along with highly conserved synteny between human and horse chromosomes, similar gestation lengths, and the shared single greatest risk for PL being advancing maternal age, provide strong evidence for the first animal model to truly recapitulate many key features of human miscarriage arising due to chromosomal aberrations, with shared benefits for humans and equids. [ABSTRACT FROM AUTHOR]

Recent advances in genomic sequencing technology and computational assembly methods have allowed scientists to improve reference genome assemblies in terms of contiguity and composition. EquCab2, a reference genome for the domestic horse, was released in 2007. Although of equal or better quality compared to other first-generation Sanger assemblies, it had many of the shortcomings common to them. In 2014, the equine genomics research community began a project to improve the reference sequence for the horse, building upon the solid foundation of EquCab2 and incorporating new short-read data, long-read data, and proximity ligation data. Here, we present EquCab3. The count of non-N bases in the incorporated chromosomes is improved from 2.33 Gb in EquCab2 to 2.41 Gb in EquCab3. Contiguity has also been improved nearly 40-fold with a contig N50 of 4.5 Mb and scaffold contiguity enhanced to where all but one of the 32 chromosomes is comprised of a single scaffold.

BackgroundCongenital stationary night-blindness (CSNB) is a recessive autosomal defect in low-light vision in Appaloosa and other horse breeds. This condition has been mapped by linkage analysis to a gene coding for the Transient Receptor Potential cation channel Member 1 (TRPM1). TRPM1 is normally expressed in the ON-bipolar cells of the inner nuclear layer of the retina. Down-regulation of TRPM1 expression in CSNB results from a transposon-like insertion in intron 1 of the TRPM1 gene. Stop transcription signals in this transposon significantly reduce TRPM1 primary transcript levels in CSNB horses. This study describes additional contributions by a second mutation of the TRPM1 gene, the ECA1 108,249,293 C > T SNP, to down-regulation of transcription of the TRPM1 gene in night-blind horses. This TRPM1 SNP introduces a consensus binding site for neuro-oncological ventral antigen 1 (Nova-1) protein in the primary transcript. Nova-1 binding disrupts normal splicing signals, producing unstable, non-functional mRNA transcripts.ResultsRetinal bipolar cells express both TRPM1 and Nova-1 proteins. In vitro addition of Nova-1 protein retards electrophoretic migration of TRPM1 RNA containing the ECA1 108,249,293 C > T SNP. Up-regulating Nova-1 expression in primary cultures of choroidal melanocytes carrying the intron 11 SNP caused an average log 2-fold reduction of ~6 (64-fold) of TRPM1 mRNA expression.ConclusionsThese finding suggest that the equine TRPM1 SNP can act independently to reduce survival of TRPM1 mRNA escaping the intron 1 transcriptional stop signals in CSNB horses. Coexistence and co-inheritance of two independent TRPM1 mutations across 1000 equine generations suggests a selective advantage for the apparently deleterious CSNB trait.

Congenital stationary night blindness (CSNB) is an ocular disorder characterized by nyctalopia. An autosomal recessive missense mutation in glutamate metabotropic receptor 6 (GRM6 c.533C>T, p.(Thr178Met)), called CSNB2, was previously identified in one Tennessee Walking Horse and predicted to reduce binding affinity of the neurotransmitter glutamate, impacting the retinal rod ON‐bipolar cell signaling pathway. Thus, the first aim was to identify the allele frequency (AF) of CSNB2 in breeds with reported cases of CSNB and breeds closely related to the Tennessee Walking Horse. The second aim was to perform ocular examinations in multiple breeds to confirm the link between genotype and CSNB phenotype. In evaluating 3518 horses from 14 breeds, the CSNB2 allele was identified in nine previously unreported breeds. The estimated AF was highest in pacing Standardbreds (0.17) and lowest in American Quarter Horses (0.0010). Complete ophthalmic examinations and electroretinograms (ERG) were performed on 19 horses from three breeds, including one CSNB2 homozygote from each breed. All three CSNB2/CSNB2 horses had an electronegative ERG waveform under scotopic light conditions consistent with CSNB. The remaining 16 horses (seven CSNB2/N and nine N/N) had normal scotopic ERG results. All horses had normal photopic ERGs. This study provides additional evidence that GRM6 c.533C>T homozygosity is likely causal to CSNB in Tennessee Walking Horses, Standardbreds, and Missouri Fox Trotting Horses. Genetic testing is recommended for breeds with the CSNB2 allele to limit the production of affected horses. This study represents the largest across‐breed identification of CSNB in the horse and suggests that this disorder is likely underdiagnosed. [ABSTRACT FROM AUTHOR]

The Functional Annotation of Animal Genomes (FAANG) Consortium recently held a Gathering On FAANG (GO-FAANG) Workshop in Washington, DC on October 7-8, 2015. This consortium is a grass-roots organization formed to advance the annotation of newly assembled genomes of domesticated and non-model organisms (www.faang.org). The workshop gathered together from around the world a group of 100+ genome scientists, administrators, representatives of funding agencies and commodity groups to discuss the latest advancements of the consortium, new perspectives, next steps and implementation plans. The workshop was streamed live and recorded, and all talks, along with speaker slide presentations, are available at www.faang.org. In this report, we describe the major activities and outcomes of this meeting. We also provide updates on ongoing efforts to implement discussions and decisions taken at GO-FAANG to guide future FAANG activities. In summary, reference datasets are being established under pilot projects; plans for tissue sets, morphological classification and methods of sample collection for different tissues were organized; and core assays and data and meta-data analysis standards were established.

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ(2)=1022.00, p<

Background: Genetic testing is required for the registration of foals of most equine breeds. Objectives: To describe two clinical cases of marked delayed embryonic development or delayed fertilisation in pregnancies generated by embryo transfer. Study design: Case report. Methods: Donor mares were inseminated with semen from one stallion during one oestrous cycle and semen from a different stallion on the subsequent oestrous cycle. Embryo(s) were collected 8 days after ovulation during the second oestrous cycle and transferred into synchronised recipient mares. Genetic testing was performed to determine parentage of the two foals. Results: For both foals, DNA parentage testing excluded the second stallion as the genetic sire and confirmed that the first stallion, whose semen was inseminated on the previous oestrous cycle, was the actual genetic sire. Main limitations: Rare event in horses; two clinical cases are described. Conclusions: It is hypothesised that either marked delayed embryonic development or extended sperm survival occurred in the donor mares. Without genetic testing, parentage assignment based solely on breeding records would have been incorrect. [ABSTRACT FROM AUTHOR]

Background: Equine recurrent uveitis (ERU) is the leading cause of blindness for horses; previous research implicated the leopard complex spotting allele (LP) as a genetic risk factor for insidious uveitis in the Appaloosa. There is limited information about risk in the Knabstrupper. Objective: To evaluate clinical manifestations, disease frequency and potential risk factors for ERU in Knabstrupper horses. Study design: Cross‐sectional study. Methods: Ocular examinations were performed on 116 horses, and based on identified anomalies, horses were classified as suspect, ERU‐affected or having no clinical signs. Microagglutination testing (MAT) of serum assessed exposure to Leptospira spp. Clinical signs, age, sex, base colour, coat pattern, LP and PATN1 genotypes, percent white at birth, progressive roaning and Leptospira were assessed as risk factors using multivariable exact logistic regression, accounting for clustering at the barn level. Additionally, a pedigree analysis was performed (n = 20 cases and 21 controls), and coefficients of coancestry (CC) and inbreeding were calculated. Results: Prevalence of insidious uveitis in this sample of Knabstruppers was 20.7%. Similar to findings for Appaloosas, LP homozygotes had higher odds of uveitis compared with true solid (N/N) horses (LP/LP OR = 7.64, 95% CI [0.8 to +INF], p = 0.04) and age was also identified as a risk factor. After accounting for LP, the 16–20 age group had higher odds compared with the youngest group (OR = 13.36, 95% CI [1.4–213.4], p = 0.009). The distributions of average CC were significantly different between cases and controls (p = 0.01). Main limitations: A relatively small sample size decreased the power for detecting additional associations. The progressive nature of insidious uveitis may have prevented identification of younger affected horses. Conclusions: Our data support genotyping for LP to assess risk of ERU in Knabstruppers. Additional studies are necessary to develop more robust risk models across LP breeds for earlier detection and improved clinical management. [ABSTRACT FROM AUTHOR]

Background: Equine familial isolated hypoparathyroidism (EFIH) and fragile foal syndrome (FFS) are both fatal recessive conditions reported in Thoroughbred foals. The causal variants for EFIH (RAPGEF5 c.2624C>A; EquCab3.0. chr4: g.54108297G>T) and FFS (PLOD1 c.2032G>A; EquCab3.0, chr2: g.39927817) were recently reported. Prevalence assessment for these variants in a large cohort of samples is needed to provide evidence‐based recommendations for genetic testing. Objectives: To estimate the frequency of the EFIH and FFS variant alleles in the United States Thoroughbred population between 1988 and 2019, and determine whether these are recent mutations or are increasing in frequency due to current breeding practices. Study design: Population allele frequency study. Methods: Genomic DNA from hair and serum samples were genotyped for the EFIH and FFS. Allele frequencies between cohorts, based on year of birth (1988–2000, n = 728) and (2001–2019, n = 1059), as well as across the seven geographical regions of the United States were compared by Fisher's Exact tests. Results: EFIH and FFS allele frequencies were not significantly different between the two time points studied (0.008 and 0.004, respectively, in the older cohorts and 0.008 and 0.009 in most recent years). No EFIH or FFS homozygotes were detected. A sample from 1992 was identified as a carrier for EFIH and one from 1993 a carrier for FFS. Non‐significant changes in geographical distribution of carriers for both traits were observed. Main limitations: The earliest samples available for study were from foals born in 1988. Conclusions: The EFIH and FFS variants are present at low frequency in the United States Thoroughbred population but are not recent mutations. There is no evidence to support changes in allele frequency over time. However, given the closed studbook and breeding practices, continued monitoring of breed allele frequencies and genetic testing is recommended to avoid the mating of carriers and production of affected foals. [ABSTRACT FROM AUTHOR]