Horses with equine recurrent uveitis have an activated CD4+ T-cell phenotype that can be modulated by mesenchymal stem cells in vitro.

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

Successful ATAC-Seq From Snap-Frozen Equine Tissues

An assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) has become an increasingly popular method to assess genome-wide chromatin accessibility in isolated nuclei from fresh tissues. However, many biobanks contain only snap-frozen tissue samples. While ATAC-seq has been applied to frozen brain tissues in human, its applicability in a wide variety of tissues in horse remains unclear. The Functional Annotation of Animal Genome (FAANG) project is an international collaboration aimed to provide high quality functional annotation of animal genomes. The equine FAANG initiative has generated a biobank of over 80 tissues from two reference female animals and experiments to begin to characterize tissue specificity of genome function for prioritized tissues have been performed. Due to the logistics of tissue collection and storage, extracting nuclei from a large number of tissues for ATAC-seq at the time of collection is not always practical. To assess the feasibility of using stored frozen tissues for ATAC-seq and to provide a guideline for the equine FAANG project, we compared ATAC-seq results from nuclei isolated from frozen tissue to cryopreserved nuclei (CN) isolated at the time of tissue harvest in liver, a highly cellular homogenous tissue, and lamina, a relatively acellular tissue unique to the horse. We identified 20,000– 33,000 accessible chromatin regions in lamina and 22–61,000 in liver, with consistently more peaks identified using CN isolated at time of tissue collection. Our results suggest that frozen tissues are an acceptable substitute when CN are not available. For more challenging tissues such as lamina, nuclei extraction at the time of tissue collection is still preferred for optimal results. Therefore, tissue type and accessibility to intact nuclei should be considered when designing ATAC-seq experiments

Functionally Annotating Regulatory Elements in the Equine Genome Using Histone Mark ChIP-Seq.

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

Decoding the Equine Genome: Lessons from ENCODE

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

Frameshift Variant in MFSD12 Explains the Mushroom Coat Color Dilution in Shetland Ponies

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

"Adopt-a-Tissue" Initiative Advances Efforts to Identify Tissue-Specific Histone Marks in the Mare

International audienc

An intronic copy number variation in Syntaxin 17 determines speed of greying and melanoma incidence in Grey horses.

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

Accumulating mutations in series of haplotypes at the KIT and MITF loci are major determinants of white markings in Franches-Montagnes horses.

Coat color and pattern variations in domestic animals are frequently inherited as simple monogenic traits, but a number are known to have a complex genetic basis. While the analysis of complex trait data remains a challenge in all species, we can use the reduced haplotypic diversity in domestic animal populations to gain insight into the genomic interactions underlying complex phenotypes. White face and leg markings are examples of complex traits in horses where little is known of the underlying genetics. In this study, Franches-Montagnes (FM) horses were scored for the occurrence of white facial and leg markings using a standardized scoring system. A genome-wide association study (GWAS) was performed for several white patterning traits in 1,077 FM horses. Seven quantitative trait loci (QTL) affecting the white marking score with p-values p≤10(-4) were identified. Three loci, MC1R and the known white spotting genes, KIT and MITF, were identified as the major loci underlying the extent of white patterning in this breed. Together, the seven loci explain 54% of the genetic variance in total white marking score, while MITF and KIT alone account for 26%. Although MITF and KIT are the major loci controlling white patterning, their influence varies according to the basic coat color of the horse and the specific body location of the white patterning. Fine mapping across the MITF and KIT loci was used to characterize haplotypes present. Phylogenetic relationships among haplotypes were calculated to assess their selective and evolutionary influences on the extent of white patterning. This novel approach shows that KIT and MITF act in an additive manner and that accumulating mutations at these loci progressively increase the extent of white markings