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The domestic cat (Felis silvestris catus) is a valued companion animal throughout the world. Over 60 different cat breeds are accepted for competition by the cat fancy registries in different countries. Genetic markers, including short tandem repeats and SNPs, are available to evaluate and manage levels of inbreeding and genetic diversity, population and breed structure relationships, and individual identification for forensic and registration purposes. The International Society of Animal Genetics (ISAG) hosts the Applied Genetics in Companion Animals Workshop, which supports the standardization of genetic marker panels and genotyping for the identification of cats via comparison testing. SNP panels have been in development for many species, including the domestic cat. An ISAG approved core panel of SNPs for use in cat identification and parentage analyses is presented. SNPs (n = 121) were evaluated by different university-based and commercial laboratories using 20 DNA samples as part of the ISAG comparison testing procedures. Different SNP genotyping technologies were examined, including DNA arrays, genotyping-by-sequencing and mass spectroscopy, to select a robust and efficient panel of 101 SNPs as the ISAG core panel for cats. The SNPs are distributed across all chromosomes including two on the X chromosome and an XY pseudo-autosomal sexing marker (zinc-finger XY; ZFXY). A population study demonstrated that the markers have an average polymorphic information content of 0.354 and a power of exclusion greater than 0.9999. The SNP panel should keep testing affordable while also allowing for the development of additional panels to monitor health, phenotypic traits, hybrid cats and highly inbred cats.

BackgroundDistichiasis, 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.ResultsA 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.ConclusionsThis 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.

BackgroundCatastrophic fractures are among the most common cause of fatalities in racehorses. Several factors, including genetics, likely contribute to increased risk for fatal injuries. A variant in the procollagen-lysine, 2-oxoglutarate 5-dioxygenase1 gene (PLOD1 c.2032G>A) was shown to cause Warmblood fragile foal syndrome type 1 (WFFS), a fatal recessive defect of the connective tissue. Screening of multiple horse breeds identified the presence of the WFFS allele in the Thoroughbred. PLOD1 is involved in cross-linking of collagen fibrils and thus could potentially increase the risk of catastrophic breakdown.ObjectivesEstimate the frequency of the WFFS allele (PLOD1 c.2032G>A) and determine if it is a risk factor for catastrophic breakdown in the Thoroughbred.Study designCase-control genetic study.MethodsGenomic DNA from hair and/or tissue samples was genotyped for the WFFS allele. Fisher's Exact tests were performed to compare allele and carrier frequencies between the case cohort (catastrophic breakdown, n = 22) and several cohorts with no record of injury (n = 138 raced/trained at same track and season and n = 185 older than 7 years and raced during same season), nonracers (n = 92), and a random sample without consideration for racing history (n = 279).ResultsThe frequency of the PLOD1 c.2032G>A variant in the Thoroughbred breed is low (1.2%). Seventeen of 716 Thoroughbreds tested were carriers (2.4%) and no WFFS homozygotes were detected. Only one catastrophic breakdown case carried the WFFS allele. No statistically significant difference in allele or carrier frequency was identified between case and control cohorts (P>0.05 in all comparisons performed).Main limitationsThis study evaluated cases from one single track.ConclusionsThis study demonstrated that the PLOD1 c.2032G>A associated with WFFS is present at very low frequency in Thoroughbreds and is not a genetic risk factor for catastrophic breakdown.

The horse reference genome from the Thoroughbred mare Twilight has been available for a decade and, together with advances in genomics technologies, has led to unparalleled developments in equine genomics. At the core of this progress is the continuing improvement of the quality, contiguity and completeness of the reference genome, and its functional annotation. Recent achievements include the release of the next version of the reference genome (EquCab3.0) and generation of a reference sequence for the Y chromosome. Horse satellite-free centromeres provide unique models for mammalian centromere research. Despite extremely low genetic diversity of the Y chromosome, it has been possible to trace patrilines of breeds and pedigrees and show that Y variation was lost in the past approximately 2300 years owing to selective breeding. The high-quality reference genome has led to the development of three different SNP arrays and WGSs of almost 2000 modern individual horses. The collection of WGS of hundreds of ancient horses is unique and not available for any other domestic species. These tools and resources have led to global population studies dissecting the natural history of the species and genetic makeup and ancestry of modern breeds. Most importantly, the available tools and resources, together with the discovery of functional elements, are dissecting molecular causes of a growing number of Mendelian and complex traits. The improved understanding of molecular underpinnings of various traits continues to benefit the health and performance of the horse whereas also serving as a model for complex disease across species.

The Functional Annotation of Animal Genomes (FAANG) project aims to identify genomic regulatory elements in both sexes across multiple stages of development in domesticated animals. This study represents the first stage of the FAANG project for the horse, Equus caballus. A biobank of 80 tissue samples, two cell lines and six body fluids was created from two adult Thoroughbred mares. Ante-mortem assessments included full physical examinations, lameness, ophthalmologic and neurologic evaluations. Complete blood counts and serum biochemistries were also performed. At necropsy, in addition to tissue samples, aliquots of serum, ethylenediaminetetraacetic acid (EDTA) plasma, heparinized plasma, cerebrospinal fluid, synovial fluid, urine and microbiome samples from all regions of the gastrointestinal and urogenital tracts were collected. Epidermal keratinocytes and dermal fibroblasts were cultured from skin samples. All tissues were grossly and histologically evaluated by a board-certified veterinary pathologist. The results of the clinical and pathological evaluations identified subclinical eosinophilic and lymphocytic infiltration throughout the length of the gastrointestinal tract as well as a mild clinical lameness in both animals. Each sample was cryo-preserved in multiple ways, and nuclei were extracted from selected tissues. These samples represent the first published systemically healthy equine-specific biobank with extensive clinical phenotyping ante- and post-mortem. The tissues in the biobank are intended for community-wide use in the functional annotation of the equine genome. The use of the biobank will improve the quality of the reference annotation and allow all equine researchers to elucidate unknown genomic and epigenomic causes of disease.

BackgroundTranscriptome interpretation relies on a good-quality reference transcriptome for accurate quantification of gene expression as well as functional analysis of genetic variants. The current annotation of the horse genome lacks the specificity and sensitivity necessary to assess gene expression especially at the isoform level, and suffers from insufficient annotation of untranslated regions (UTR) usage. We built an annotation pipeline for horse and used it to integrate 1.9 billion reads from multiple RNA-seq data sets into a new refined transcriptome.ResultsThis equine transcriptome integrates eight different tissues from 59 individuals and improves gene structure and isoform resolution, while providing considerable tissue-specific information. We utilized four levels of transcript filtration in our pipeline, aimed at producing several transcriptome versions that are suitable for different downstream analyses. Our most refined transcriptome includes 36,876 genes and 76,125 isoforms, with 6474 candidate transcriptional loci novel to the equine transcriptome.ConclusionsWe have employed a variety of descriptive statistics and figures that demonstrate the quality and content of the transcriptome. The equine transcriptomes that are provided by this pipeline show the best tissue-specific resolution of any equine transcriptome to date and are flexible for several downstream analyses. We encourage the integration of further equine transcriptomes with our annotation pipeline to continue and improve the equine transcriptome.

BackgroundEfforts to resolve the transcribed sequences in the equine genome have focused on protein-coding RNA. The transcription of the intergenic regions, although detected via total RNA sequencing (RNA-seq), has yet to be characterized in the horse. The most recent equine transcriptome based on RNA-seq from several tissues was a prime opportunity to obtain a concurrent long non-coding RNA (lncRNA) database.ResultsThis lncRNA database has a breadth of eight tissues and a depth of over 20 million reads for select tissues, providing the deepest and most expansive equine lncRNA database. Utilizing the intergenic reads and three categories of novel genes from a previously published equine transcriptome pipeline, we better describe these groups by annotating the lncRNA candidates. These lncRNA candidates were filtered using an approach adapted from human lncRNA annotation, which removes transcripts based on size, expression, protein-coding capability and distance to the start or stop of annotated protein-coding transcripts.ConclusionOur equine lncRNA database has 20,800 transcripts that demonstrate characteristics unique to lncRNA including low expression, low exon diversity and low levels of sequence conservation. These candidate lncRNA will serve as a baseline lncRNA annotation and begin to describe the RNA-seq reads assigned to the intergenic space in the horse.

Congenital stationary night blindness (CSNB) has been connected to the leopard complex spotting phenotype (LP) in various horse breeds. CSNB associated with LP is thought to be caused by a 1378bp insertion in TRPM1, with homozygotes being nightblind and having few to no spots of pigment in their white patterned area. This study aimed to assess the prevalence of CSNB alleles in tarant-colored horses in Poland through a three-primer system for an allele-specific Polymerase Chain Reaction (PCR). The TRPM1 gene insertion was genotyped in 221 horses belonging to Małopolska, Felin and Shetland Ponies. The chi-square (χ²) test indicates, that χ2 <5.991 suggesting that the population is in Hardy-Weinberg equilibrium. Of the horses carrying the LP allele, 7% of Małopolska horses, 4,8% of Felin ponies and 6.25 % of the Shetland ponies were homozygous for the TRMP1 insertion, indicating low-light vision issues, crucial for horses working in dim conditions. This study highlights the utility of genetic testing for accurate phenotype evaluation, and clinical and breeding management., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: K.R.M, M.S.S., and A.F., are affiliated with the National Research Institute of Animal Production, Department of Animal Molecular Biology. Department offers genetic testing but not for LP. R.R.B., is affiliated with the Veterinary Genetics Laboratory at UC Davis, a facility that offers genetic testing for LP and other veterinary genetics dialogistic testing., (Copyright © 2025. Published by Elsevier Inc.)

Equine spinal neurodegenerative conditions are frequently encountered in sport and racing horses and may be career-ending diagnoses. To further define the spatial transcriptomic landscape of equine dorsal root ganglia (DRG) in healthy adult horses, we investigated gene expression differences in distinct DRG regions using the GeoMx Digital Spatial Profiling from NanoString. Four human cell markers demonstrated high fidelity for equine cells; microtubule-associated protein 2 (MAP2), myelin basic protein (MBP), allograft inflammatory 104 factor 1/ionized calcium-binding adaptor molecule 1 (IBA1/AIF1), and Syto83 nuclear marker. Geometric regions of interest were then selected as MBP-rich, IBA1-high, and IBA1-low, and gene expression was compared. Experimental validation was achieved, with genes involved in myelination enriched in MBP-rich regions, and the identification of glia-specific genes enriched in IBA1-high regions. Thus, spatial transcriptomics with human cell markers was successful in equine DRG and can now be applied to determine cell-specific transcriptional changes during disease states., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: SR was employed by NanoString Technologies. Other authors have no competing interests to disclose.

Background: Allele-specific expression (ASE) analysis provides a nuanced view of cis-regulatory mechanisms affecting gene expression., Results: An equine ASE analysis was performed, using integrated Iso-seq and short-read RNA sequencing data from four healthy Thoroughbreds (2 mares and 2 stallions) across 9 tissues from the Functional Annotation of Animal Genomes (FAANG) project. Allele expression was quantified by haplotypes from long-read data, with 42,900 allele expression events compared. Within these events, 635 (1.48%) demonstrated ASE, with liver tissue containing the highest proportion. Genetic variants within ASE events were located in histone modified regions 64.2% of the time. Validation of allele-specific variants, using a set of 66 equine liver samples from multiple breeds, confirmed that 97% of variants demonstrated ASE., Conclusions: This valuable publicly accessible resource is poised to facilitate investigations into regulatory variation in equine tissues. Our results highlight the tissue-specific nature of allelic imbalance in the equine genome., Competing Interests: Declarations. Ethics approval and consent to participate: All protocols were approved by the University of California Davis Institutional Animal Care and Use Committee (Protocol #19037). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Determination of horse breeds predisposed to congenital and juvenile cataracts will enable investigations into potential genetic mechanisms for cataracts in horses., Objective: To investigate horse breed predispositions to congenital and juvenile cataracts in two academic referral populations., Study Design: Retrospective case series., Methods: Medical record identification of horses diagnosed with congenital or juvenile cataracts at the Cornell University Equine Hospital (2000-2022) and the University of California-Davis (UCD) Large Animal Clinic (1990-2021). Signalment, examination findings and treatments were recorded. Descriptive statistics were performed, and breed over-representations were determined using Chi-squared or Fisher's exact tests., Results: Thirty-one (Cornell) and 70 (UCD) horses with congenital or juvenile cataracts were identified, for a total of 101 affected horses. Seventy-eight horses were affected bilaterally and 23 were affected unilaterally, for a total of 179 affected eyes. Standardbreds were significantly over-represented at both institutions, comprising 32.5% of congenital/juvenile cataract cases and 10% of the equine hospital population at Cornell (p < 0.001) and 4.3% of cataract cases and 1.3% of the equine hospital population at UCD (p = 0.03). Thoroughbreds were under-represented for congenital and juvenile cataracts at both institutions (p = 0.03 Cornell, p = 0.01 UCD)., Main Limitations: Retrospective study, potential for selection bias., Conclusions: The over-representation of the Standardbred breed for congenital and juvenile cataracts at two institutions suggests an underlying genetic basis in the breed. Future genetic and genomic studies are warranted to investigate heritable cataracts in Standardbred horses., (© 2023 EVJ Ltd.)

The Greying with age phenotype in horses involves loss of hair pigmentation whereas skin pigmentation is not reduced, and a predisposition to melanoma. The causal mutation was initially reported as a duplication of a 4.6 kb intronic sequence in Syntaxin 17. The speed of greying varies considerably among Grey horses. Here we demonstrate the presence of two different Grey alleles, G2 carrying two tandem copies of the duplicated sequence and G3 carrying three. The latter is by far the most common allele, probably due to strong selection for the striking white phenotype. Our results reveal a remarkable dosage effect where the G3 allele is associated with fast greying and high incidence of melanoma whereas G2 is associated with slow greying and low incidence of melanoma. The copy number expansion transforms a weak enhancer to a strong melanocyte-specific enhancer that underlies hair greying (G2 and G3) and a drastically elevated risk of melanoma (G3 only). Our direct pedigree-based observation of the origin of a G2 allele from a G3 allele by copy number contraction demonstrates the dynamic evolution of this locus and provides the ultimate evidence for causality of the copy number variation of the 4.6 kb intronic sequence., (© 2024. The Author(s).)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: The pleiotropic effects of the melanocortin system show promise in overcoming limitations associated with large variations in opioid analgesic effectiveness observed in equine practice. Of particular interest is variation in the melanocortin-1-receptor (MC1R) gene, which dictates pigment type expression through its epistatic interaction with the agouti signalling protein (ASIP) gene. MC1R has previously been implicated in opioid efficacy in other species; however, this relationship is yet to be explored in horses. In this study, analgesic effectiveness was scored (1-3) based on noted response to dura penetration during the performance of cerebrospinal fluid centisis after sedation and tested for association with known genetic regions responsible for pigmentation variation in horses., Results: The chestnut phenotype was statistically significant (P < 0.05) in lowering analgesic effectiveness when compared to the bay base coat colour. The 11bp indel in ASIP known to cause the black base coat colour was not significant (P>0.05); however, six single nucleotide polymorphisms (SNPs) within the genomic region encoding the ASIP gene and one within MC1R were identified as being nominally significant (P<0.05) in association with opioid analgesic effectiveness. This included the location of the known e MC1R variant resulting in the chestnut coat colour., Conclusions: The current study provides promising evidence for important links between pigmentation genes and opioid effectiveness in horses. The application of an easily identifiable phenotype indicating variable sensitivity presents a promising opportunity for accessible precision medicine in the use of analgesics and warrants further investigation., (© 2024. Crown.)

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., (© 2023 The Authors. Veterinary Ophthalmology published by Wiley Periodicals LLC on behalf of American College of Veterinary Ophthalmologists.)

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., (© 2024. The Author(s).)

Background: Allele-specific expression (ASE) analysis provides a nuanced view of cis-regulatory mechanisms affecting gene expression., Results: In this work, we introduce and highlight the significance of an equine ASE analysis, containing integrated long- and short-read RNA sequencing data, along with insight from histone modification data, from four healthy Thoroughbreds (2 mares and 2 stallions) across 9 tissues., Conclusions: This valuable publicly accessible resource is poised to facilitate investigations into regulatory variation in equine tissues and foster a deeper understanding of the impact of allelic imbalance in equine health and disease at the molecular level.

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., (© 2023 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Splashed white in horses is characterized by extensive white patterning on the legs, face and abdomen and may be accompanied by deafness. To date, seven variants in microphthalmia-associated transcription factor (MITF) and two variants in Paired Box 3 (PAX3) have been identified to explain this phenotype. A splashed white Thoroughbred stallion, whose sire and dam were not patterned, was hypothesized to have a de novo variant leading to his white coat pattern. A whole-genome sequencing candidate gene approach identified two single nucleotide variants (SNVs) in SOX10, four SNVs in MITF and a 2.3 kb deletion in MITF with the alternative allele present in this stallion but absent in the other 18 horses analyzed. All six SNVs were annotated as modifiers and were not further considered. The deletion in MITF (NC&#95;009159.3:g.21555811&#95;21558139delinsAAAT) encompasses exon 9 encoding a part of the helix-loop-helix domain required for DNA binding. Sanger sequencing and parentage testing confirmed that this deletion was a de novo mutation of maternal origin. Consistent with the published nomenclature, we denote this likely causal variant as SW8. Genotyping three of this stallion's offspring identified SW8 only in the nearly all-white foal that was confirmed deaf by brainstem auditory evoked response testing. This foal was also a compound heterozygote for dominant white variants (W20/W22), but to date, W variants alone have not been connected to deafness. SW8 marks the fourth de novo MITF variant in horses reported to cause white patterning. The link between deafness and all MITF variants with and without other variants impacting melanocyte development and function needs to be further explored., (© 2023 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.)

Centromeres are epigenetically specified by the histone H3 variant CENP-A. Although mammalian centromeres are typically associated with satellite DNA, we previously demonstrated that the centromere of horse chromosome 11 (ECA11) is completely devoid of satellite DNA. We also showed that the localization of its CENP-A binding domain is not fixed but slides within an about 500 kb region in different individuals, giving rise to positional alleles. These epialleles are inherited as Mendelian traits but their position can move in one generation. It is still unknown whether centromere sliding occurs during meiosis or during development. Here, we first improve the sequence of the ECA11 centromeric region in the EquCab3.0 assembly. Then, to test whether centromere sliding may occur during development, we map the CENP-A binding domains of ECA11 using ChIP-seq in five tissues of different embryonic origin from the four horses of the equine FAANG (Functional Annotation of ANimal Genomes) consortium. Our results demonstrate that the centromere is localized in the same region in all tissues, suggesting that the position of the centromeric domain is maintained during development., (© 2023. Springer Nature Limited.)

Background: Globoid cell leukodystrophy (GCL) is a fatal autosomal recessive disease caused by variants in the galactosylceramidase (GALC) gene. Two dog breed-specific variants are reported., Objectives: Characterize the putatively causative GALC variant for GCL in a family of dogs and determine population allele frequency., Animals: Four related mixed-breed puppies with signs of neurologic disease were evaluated. Subsequently, 33 related dogs were tested for genetic markers for parentage and the identified GALC variant. Additional GALC genotyping was performed on 278 banked samples from various breeds., Methods: The 4 affected puppies had neurological exams and necropsies. DNA was isolated from blood samples. Variants in GALC were identified via Sanger sequencing. Parentage testing was performed using short tandem repeat markers. Prevalence of the GALC variant of interest was investigated in other breeds., Results: GCL was confirmed histopathologically. A novel missense variant in GALC (NC&#95;006590.4:g.58893972G>A) was homozygous in all affected animals (n = 4). A recessive mode of inheritance was confirmed by parentage testing as was variant linkage with the phenotype (LOD = 3.36). Among the related dogs (n = 33), 3 dogs were homozygous and 7 heterozygous. The variant allele was not detected in screening 278 dogs from 5 breeds. The novel variant is either unique to this family or has an extremely low allele frequency in the general population., Conclusions and Clinical Importance: A novel GALC variant was identified that likely explains GCL in this cohort. The identification of multiple causal variants for GCL in dogs is consistent with findings in humans., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

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., (© 2022 EVJ Ltd.)

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., (© 2022 EVJ Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Peng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Equine recurrent uveitis (ERU) is an ocular inflammatory disease that can be difficult to manage clinically. As such, it is the leading cause of bilateral blindness for horses. ERU is suspected to have a complex autoimmune etiology with both environmental and genetic risk factors contributing to onset and disease progression in some or all cases. Work in recent years has aimed at unraveling the primary triggers, such as infectious agents and inherited breed-specific risk factors, for disease onset, persistence, and progression. This review has aimed at encompassing those factors that have been associated, implicated, or substantiated as contributors to ERU, as well as identifying areas for which additional knowledge is needed to better understand risk for disease onset and progression. A greater understanding of the risk factors for ERU will enable earlier detection and better prognosis through prevention and new therapeutics., (© 2022 American College of Veterinary Ophthalmologists.)

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, h 2  = 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., (© 2022 Stichting International Foundation for Animal Genetics.)

Deleterious genetic variants are an important cause of skeletal muscle disease. Immunohistochemical evaluation of muscle biopsies is standard for the diagnosis of muscle disorders. The prevalence of alleles causing hyperkalemic periodic paralysis (HYPP), malignant hyperthermia (MH), polysaccharide storage myopathy 1 (PSSM1), glycogen branching enzyme deficiency (GBED), myotonia congenita (MC), and myosin heavy chain myopathy (MYHM) in horses with muscle disease is unknown. Archived slides processed for immunohistochemical analysis from 296 horses with muscle disease were reviewed blinded and clinical information obtained. DNA isolated from stored muscle samples from these horses were genotyped for disease variants. Histological findings were classified as myopathic in 192, neurogenic in 41, and normal in 63 horses. A third of the population had alleles that explained disease which constituted 45% of the horses with confirmed histological myopathic process. Four of six muscle disease alleles were identified only in Quarter horse breeds. The allele causing PSSM1 was detected in other breeds, and MC was not detected in these samples. The My allele, associated with susceptibility for MYHM, was the most common (62%) with homozygotes (16/27) presenting a more severe phenotype compared to heterozygotes (6/33). All cases with the MH allele were fatal upon triggering by anesthesia, stress or concurrent myopathy. Both, muscle histological and genetic analyses are essential in the investigation of muscle disease, since 10% of the horses with muscle disease and normal histology had a muscle disease causing genetic variant, and 63% of histologically confirmed muscle with alterations had no known genetic variants., (Copyright © 2022. Published by Elsevier Inc.)

Background: Warmblood 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., Objectives: To 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 Design: A single case report describing a genetic investigation., Methods: A 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., Results: Post-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 Limitations: This study is a single case report in the Thoroughbred with no additional cases from this breed yet identified to replicate this finding., Conclusions: Given 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., (© 2021 EVJ Ltd.)

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., (© The American Genetic Association. 2021.)

Equine recurrent uveitis (ERU) is an autoimmune disease defined by inflammation of the uveal tract of the eye. The cause of ERU is thought to be complex, involving both genetic and environmental factors. The purpose of this study was to investigate potential genetic risk factors for ERU in the Icelandic horse. Fifty-six Icelandic horses (11 affected with ERU and 45 controls) living in Denmark and the USA, eight years or older, were included in the study. A case-control GWAS was performed using the GGP Equine 80K array on the Illumina Infinium HD Beadchip using 40 horses. A mixed linear model analysis identified a single SNP on ECA 11 (BIEC2&#95;141650; NC&#95;009154.3:g.3817009A>G) that reached genome-wide significance (p = 1.79 × 10 -7 ). This variant was within an intron of tissue inhibitor of metalloproteinase 2 (TIMP2), a gene previously implicated in ERU. Sanger sequencing identified a single coding variant in this gene; however it was a synonymous mutation (NC&#95;009154.3:g.3858193C>T) and was not perfectly concordant with ERU phenotype (p = 0.68). Further investigation of TIMP2 is warranted. Additional horses and markers are needed to identify other potential loci worthy of further investigation as contributors to ERU risk in Icelandic horses., (© 2022 Stichting International Foundation for Animal Genetics.)

Background: The prevalence of clinical signs and factors triggering muscle atrophy and rhabdomyolysis associated with an MYH1 E321G mutation in Quarter Horses and related breeds (QH) remain poorly understood., Hypothesis/objectives: Determine the prevalence and potential triggers of atrophy and stiffness in horses homozygous reference (N/N), heterozygous (My/N), and homozygous (My/My) for the MYH1 E321G mutation., Animals: Two-hundred seventy-five N/N, 100 My/N, and 10 My/My QH., Methods: A 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., Results: Atrophy occurred in proportionately more horses with MYH1 E321G (My) than N/N QH and more frequently in My/My than My/N QH (P < .001; My/My 8/10 [80%], My/N 17/100 [17%], N/N 29/275 [11%]). More My/My horses had rapid atrophy (P < .001), with recurrence in 50%. Fewer My/My horses recovered versus My/N QH (P < .001). Stiffness was common across genotypes (P = .100; My/My 4/10 [40%], My/N 18/100 [18%], N/N 48/275 [17%]). Three months before the observed atrophy and stiffness, 47% of MYH1 E321G QH were vaccinated or had respiratory or gastrointestinal disease. Horses achieving 100% expected performance did not differ across genotypes (50% My/My, 71% My/N, 55% N/N), but, only 4/10 My/My QH were competing. My/N horses achieved national or world championships or both., Conclusion and Clinical Importance: Approximately 20% of My/N QH develop rapid atrophy. Atrophy is more common (80%) in homozygous My/My QH and less likely to resolve. Inciting causes such as vaccination and infection are inapparent in over half of cases., (© 2022 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (© 2022. The Author(s).)

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.

The domestic cat (Felis silvestris catus) is a valued companion animal throughout the world. Over 60 different cat breeds are accepted for competition by the cat fancy registries in different countries. Genetic markers, including short tandem repeats and SNPs, are available to evaluate and manage levels of inbreeding and genetic diversity, population and breed structure relationships, and individual identification for forensic and registration purposes. The International Society of Animal Genetics (ISAG) hosts the Applied Genetics in Companion Animals Workshop, which supports the standardization of genetic marker panels and genotyping for the identification of cats via comparison testing. SNP panels have been in development for many species, including the domestic cat. An ISAG approved core panel of SNPs for use in cat identification and parentage analyses is presented. SNPs (n = 121) were evaluated by different university-based and commercial laboratories using 20 DNA samples as part of the ISAG comparison testing procedures. Different SNP genotyping technologies were examined, including DNA arrays, genotyping-by-sequencing and mass spectroscopy, to select a robust and efficient panel of 101 SNPs as the ISAG core panel for cats. The SNPs are distributed across all chromosomes including two on the X chromosome and an XY pseudo-autosomal sexing marker (zinc-finger XY; ZFXY). A population study demonstrated that the markers have an average polymorphic information content of 0.354 and a power of exclusion greater than 0.9999. The SNP panel should keep testing affordable while also allowing for the development of additional panels to monitor health, phenotypic traits, hybrid cats and highly inbred cats., (© 2021 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.)

Auditory loss has been reported in camelids using brainstem auditory evoked responses (BAER). Differentiation between conductive versus sensorineural dysfunction has not been investigated. Therefore, the objective of the study was to investigate auditory function using BAER and bone conduction (BC). Twenty-four alpacas: 15 females, 9 intact males (2-16 years of age) were included in a randomized clinical trial. BAER and BC were recorded using two derivations (vertex to mastoid and vertex to cranial aspect of second cervical vertebra). All alpacas underwent complete physical examinations and were sedated with xylazine hydrochloride at 0.6 mg/kg IM. Peaks, when present, were identified and latencies, amplitudes, and amplitude ratios were determined. Eleven alpacas had normal responses and 13 had auditory loss based on BAER. The latter consisted of complete absence of peaks bilaterally (n = 3), absence of peaks unilaterally (n = 1), delayed latencies bilaterally (n = 4), and delayed latencies unilaterally (n = 5). Distinct peaks on BC supported conductive auditory loss in 6 alpacas, difficult to interpret due to stimulus artifact and additional undefined peaks in 4, and absent peaks in 3 alpacas. The cause of auditory loss was presumed to be due to otitis in 6, aging in 4 (10-16 years old), and congenital sensorineural (absent peaks on BAER and BC) in 3 alpacas with unpigmented fiber and irises. BAER and BC are useful and non-invasive to perform techniques for the investigation of auditory loss in alpacas, and further characterization as conductive or sensorineural., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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&#95;UCD&#95;AH3) had unilateral thoracic limb lameness and bilateral chorioretinal scars. The second stallion (ECA&#95;UCD&#95;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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Donnelly, Bellone, Hales, Nguyen, Katzman, Dujovne, Knickelbein, Avila, Kalbfleisch, Giulotto, Kingsley, Tanaka, Esdaile, Peng, Dahlgren, Fuller, Mienaltowski, Raudsepp, Affolter, Petersen and Finno.)

Background: The only known genetic cause of congenital stationary night blindness (CSNB) in horses is a 1378 bp insertion in TRPM1. However, an affected Tennessee Walking Horse was found to have no copies of this variant., Objectives: To identify the genetic cause for CSNB in an affected Tennessee Walking Horse., Study Design: Case report detailing a whole-genome sequencing (WGS) approach to identify a causal variant., Methods: A complete ophthalmic exam, including an electroretinogram (ERG), was performed on suspected CSNB-affected horse. WGS data were generated from the case and compared with data from seven other breeds (n = 29). One hundred candidate genes were evaluated for coding variants homozygous in the case and absent in all other horses. Protein modelling was used to assess the functional effects of the identified variant. A random cohort of 90 unrelated Tennessee Walking Horses and 273 horses from additional breeds were screened to estimate allele frequency of the GRM6 variant., Results: ERG results were consistent with CSNB. WGS analysis identified a missense mutation in metabotropic glutamate receptor 6 (GRM6) (c.533C>T p.Thr178Met). This single nucleotide polymorphism (SNP) is predicted to be deleterious and protein modelling supports impaired binding of the neurotransmitter glutamate. This variant was not detected in 273 horses from three additional breeds. The estimated allele frequency in Tennessee Walking Horses is 10%., Main Limitations: Limited phenotype information for controls and no additional cases with which to replicate this finding., Conclusions: We identified a likely causal recessive missense variant in GRM6. Based on protein modelling, this variant alters GRM6 binding, and thus signalling from the retinal rod cell to the ON-bipolar cell, impairing vision in low light conditions. Given the 10% population allele frequency, it is likely that additional affected horses exist in this breed and further work is needed to identify and examine these animals., (© 2020 EVJ Ltd.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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.1013 C>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.