The roan coat color is described as the dispersion of white hairs within an otherwise solid background color coat. This phenotype is primarily expressed on the body of the horse, with the head and legs exhibiting few to no white hairs. Previous studies mapped the locus for roan to the KIT region and observed linked variants in a small number of breeds. However, utilizing those linked markers to determine the roan genotype in other breeds has seen limited success. In this communication we identify a second roan allele (RN2) which, in conjunction with a previously observed roan allele (RN1) discovered in previous studies, accounts for approximately 74%, or 188 horses, out of a sample size totaling 257 roan horses. These two alleles were present in the non-roan population (N = 3212) at less than 1%, only in horses with light coat color and dilution alleles, likely obscuring the roan phenotype. Future work is required to identify additional alleles responsible for additional roan-type horse coat color phenotypic variation.

This paper reports three novel KIT variants likely responsible for previously unexplained white patterning phenotypes observed in three groups of horses. White spots and markings may have substantial consequences on the value and health of domesticated horses. This study aims to elucidate the genetic mechanisms underlying depigmented coat colors to aid in producing prosperous herds. Aligned whole genome sequences were manually screened to identify three polymorphisms in a family of Anglo-Arabian horses (N = 7), a family of Warmblood horses (N = 5), and a single stock-type mare with unexplained white markings. Sanger sequencing confirmed the presence of the variants, and in silico predictive programs were used to predict the functional impacts of each. We propose to term the novel variants W37, W38, and W39, respectively, per convention. The W37 polymorphism was always observed in the presence of one W35 allele, suggesting complete linkage. All three variants were predicted to alter or remove the KIT protein active domain, repressing typical protein folding and impacting pathways that upregulate pigmentation. The severe predicted impact on biological function suggests that these variants may cause increased white spotting, providing a possible explanation for the depigmentation phenotypes observed in affected individuals.

Roan coat color is described as the dispersion of white hairs within an otherwise solid background-color coat. This phenotype is primarily expressed on the body of the horse, with the head and legs exhibiting few or no white hairs. Previous studies mapped the locus for roan to the KIT region and observed linked variants in a small number of breeds. Recently, we reported evidence for two independent haplotypes, RN1 and RN2, in the KIT region, which account for approximately 38% and 36% of roan horses, respectively. In the current report, using whole genome sequencing for unknown roan samples. We present a third novel haplotype, RN3, found in American Quarter Horses, that accounts for an additional 30% of American Quarter Horses negative for RN1 and RN2 that display the roan phenotype. Within this haplotype, we observe a variant chr3:79656505 (G > A), which we believe is a founder allele for the RN3 haplotype. In our sample set of horses, these three haplotypes account for more than 95% of the American Quarter Horse population studied and about 50-60% of roan horses in other breeds. Using whole genome sequencing of distantly related animals with a particular phenotype, together with a larger number of control horses, improves the odds of finding linked and/or causative variants.

The influence of a horse’s appearance on health, sentimental and monetary value has driven the desire to understand the etiology of coat color. White markings on the coat define inclusion for multiple horse breeds, but they may disqualify a horse from registration in other breeds. In domesticated horses (Equus caballus), 35 KIT alleles are associated with or cause depigmentation and white spotting. It is a common misconception among the general public that a horse can possess only two KIT variants. To correct this misconception, we used BEAGLE 5.4-phased NGS data to identify 15 haplotypes possessing two or more KIT variants previously associated with depigmentation phenotypes. We sourced photos for 161 horses comprising 12 compound genotypes with three or more KIT variants and employed a standardized method to grade depigmentation, yielding average white scores for each unique compound genotype. We found that 7 of the 12 multi-variant haplotypes resulted in significantly more depigmentation relative to the single-variant haplotypes (ANOVA). It is clear horses can possess more than two KIT variants, and future work aims to document phenotypic variations for each compound genotype.

Depigmentation is a common phenotype in Equus caballus (horse); over 50 identified variants cause white spotting on the skin and coat. HPS5 may influence melanocyte proliferation and mutations in this gene cause depigmentation in humans, mice, and zebrafish. Here we report three equine HPS5 mutations, found in most major breed groups and associated with white spotting. We used a standardized method to quantitatively grade depigmentation, creating a White Score for each horse. We identified associations to each of three variants in HPS5 by comparing the average white score for carriers to that of 41 control horses possessing none of the studied variants. We estimated the functional impact of each mutation using SIFT, GenScan, I-TASSER and cross-species conservation. The first mutation, S1013C termed Eden White 1 (EDXW1), is a missense mutation found to increase average depigmentation by 5.56 points (p=6.47E-6, n=70, t-test). Eden White 2 (EDXW2), a missense mutation L560F, increased depigmentation by 9.5 points for individuals with one copy of the variant, but we were unable to phenotype homozygotes (p=5.55E-16, n=47, t-test). Eden White 3 (EDXW3), a putative donor splice site mutation at exon 4+4, increased white by 5.59 points on average (p=9.56E-6, n=101). EDXW1 and EDXW3 are epistatically influenced by MC1R, the gene that controls black vs red coat color in horses. Individuals possessing the dominant E allele produce black pigment, and this allele was associated with higher levels of depigmentation than individuals with the recessive chestnut genotype (e/e) (p=1.50E-5, p=1.26E-5 respectively). We tested for epistatic interactions between EDXW1 and Dominant White 20 and found that horses with one copy of each allele had increased average white spotting by 8 points with respect to EDXW1/n or W20/n horses. Compound heterozygotes were significantly more white than horses with one copy of either mutation (p=7.33E-5), but interactions between other white spotting alleles and EDXW alleles remain to be tested. We propose the convention Eden White (EDXW) for HPS5 mutations in the domestic horse.

Traits such as shape, size, and color often influence the economic and sentimental value of a horse. Around the world, horses are bred and prized for the colors and markings that make their unique coat patterns stand out from the crowd. The underlying genetic mechanisms determining the color of a horse’s coat can vary greatly in their complexity. For example, only two genetic markers are used to determine a horse’s base coat color, whereas over 50 genetic variations have been discovered to cause white patterning in horses. Some of these white-causing mutations are benign and beautiful, while others have a notable impact on horse health. Negative effects range from slightly more innocuous defects, like deafness, to more pernicious defects, such as the lethal developmental defect incurred when a horse inherits two copies of the Lethal White Overo allele. In this review, we explore, in detail, the etiology of white spotting and its overall effect on the domestic horse to Spot the Pattern of these beautiful (and sometimes dangerous) white mutations.

Mutations in KIT, a gene that influences melanoblast migration and pigmentation, often result in mammalian white spotting. As of February 2023, over 30 KIT variants associated with white spotting were documented in Equus caballus (horse). Here we report an association of increased white spotting on the skin and coat with a variant in the 5'UTR of KIT (rs1149701677: g.79,618,649A>C). Horses possessing at least one alternate allele demonstrate phenotypic characteristics similar to other KIT mutations: clear borders around unpigmented regions on the body, face, and limbs. Using a quantitative measure of depigmentation, we observed an average white score of 10.70 among individuals with rs1149701677, while the average score of the control, homozygous reference sample was 2.23 (P = 1.892e-11, n = 109, t-test). The rs1149701677 site has a cross-species conservation score of 3.4, one of the highest scores across the KIT 5'UTR, implying regulatory importance for this site. Ensembl also predicted a "moderately impactful" functional effect for the rs1149701677 variant. We propose that this single nucleotide variant likely alters the regulation of KIT, which in turn may disrupt melanoblast migration causing an increase in white spotting on the coat. Alternatively, the rs1149701677 variant may be in linkage with another nearby variant with an as-yet-undiscovered functional impact. We propose to term this new allele "Holiday White" or W35 based on conventional nomenclature. (c) 2023 Elsevier Inc. All rights reserved.

Sophie Visvikis-Siest*, Maria G. Stathopoulou, Raute Sunder-Plassmann, Behrooz Z. Alizadeh, Robert Barouki, Ekaterina Chatzaki67, Georges Dagher891011†, George Dedoussis, Panagiotis Deloukas, Alexander Haliassos, Brigitte Boisson Hiegel, Vangelis Manolopoulos615, Christine Masson, Guillaume Paré, Markus Paulmichl, Alexandros M. Petrelis118, Csilla Sipeky, Belgin Süsleyici, Georges Weryha, Alex Chenchik, Paul Diehl, Robin E. Everts, Alexander Haushofer, John Lamont, Ruth Mercado, Heiko Meyer, Herna Munoz-Galeano, Helena Murray, Ferrier Nhat, Charity Nofziger, Wolfgang Schnitzel and Stavroula Kanoni*

Mutations causing depigmentation are relatively common in Equus caballus (horse). Over 40 alleles in multiple genes are associated with increased white spotting (as of February 2023). The splashed white phenotype, a coat spotting pattern described as appearing like the horse has been splashed with white paint, was previously associated with variants in the PAX3 and MITF genes . Both genes encode transcrip-tion factors known to control melanocyte migration and pigmentation. We report two novel mutations, a stop-gain mutation in PAX3 (XM_005610643.3:c.927C > T, ECA6:11,196,181, EquCab3.0) and a missense mu-tation in a binding domain of MITF (NM_001163874.1:c.993A > T, ECA16:21,559,940, EquCab3.0), each with a strong association with increased depigmentation in Pura Raza Espanola horses ( P = 1.144E-11, N = 30, P = 4.441E-16, N = 39 respectively). Using a quantitative method to score depigmentation, the PAX3 and MITF mutations were found to have average white scores of 25.50 and 24.45, respectively, compared to the average white coat spotting score of 1.89 in the control set. The functional impact for each mutation was predicted to be moderate to extreme (I-TASSER, SMART, Variant Effect Predictor, SIFT). We propose to designate the MITF mutant allele as Splashed White 9 and the PAX3 mutant allele as Splashed White 10 per convention. & COPY; 2023 Elsevier Inc. All rights reserved.

A study of 80 cases of spindle cell thymoma in which the spindle cell component was overshadowed by massive numbers of stromal lymphocytes is presented. The patients were 38 women and 42 men, aged 8 to 81 years (mean=54 y). All tumors presented as an anterior mediastinal mass; 5 patients had myasthenia gravis and one had Good syndrome. The tumors were well-circumscribed, encapsulated, and measured 2.9 to 26.0 cm in greatest diameter (mean=7.3 cm). Using modified Masaoka staging, 66 tumors were stage I, 10 were stage IIa, 2 were stage III and 1 was stage IV. Histologically the tumors were characterized by a predominant lymphocytic population admixed with scattered small spindle epithelial cells. The neoplastic spindle cells in these tumors demonstrated 2 major growth patterns: in 33 cases, the tumors were exclusively composed of dense sheets of lymphocytes containing scattered spindle cells resembling a lymphocyte-rich thymoma (WHO type B1); in the remaining cases the tumors showed admixtures of a predominantly lymphocytic component with areas that were lymphocyte-poor and contained a pure spindle cell population similar to WHO type A. Immunohistochemical stains and electron microscopy corroborated the spindle cell morphology in both types. The GTF2I p.L424H variant was identified in 53 of 63 (84%) cases analyzed. Clinical follow-up in 27 cases showed that most of the tumors behaved in an indolent manner. Our study expands the spectrum of spindle cell thymoma by demonstrating the existence of cases that are predominantly composed of lymphocyte-rich elements and lack areas with a pure (lymphocyte poor) spindle cell morphology.

A study of 80 cases of spindle cell thymoma in which the spindle cell component was overshadowed by massive numbers of stromal lymphocytes is presented. The patients were 38 women and 42 men, aged 8 to 81 years (mean=54 y). All tumors presented as an anterior mediastinal mass; 5 patients had myasthenia gravis and one had Good syndrome. The tumors were well-circumscribed, encapsulated, and measured 2.9 to 26.0 cm in greatest diameter (mean=7.3 cm). Using modified Masaoka staging, 66 tumors were stage I, 10 were stage IIa, 2 were stage III and 1 was stage IV. Histologically the tumors were characterized by a predominant lymphocytic population admixed with scattered small spindle epithelial cells. The neoplastic spindle cells in these tumors demonstrated 2 major growth patterns: in 33 cases, the tumors were exclusively composed of dense sheets of lymphocytes containing scattered spindle cells resembling a lymphocyte-rich thymoma (WHO type B1); in the remaining cases the tumors showed admixtures of a predominantly lymphocytic component with areas that were lymphocyte-poor and contained a pure spindle cell population similar to WHO type A. Immunohistochemical stains and electron microscopy corroborated the spindle cell morphology in both types. The GTF2I p.L424H variant was identified in 53 of 63 (84%) cases analyzed. Clinical follow-up in 27 cases showed that most of the tumors behaved in an indolent manner. Our study expands the spectrum of spindle cell thymoma by demonstrating the existence of cases that are predominantly composed of lymphocyte-rich elements and lack areas with a pure (lymphocyte poor) spindle cell morphology.

Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing.

Genetic markers are important resources for individual identification and parentage assessment. Although short tandem repeats (STRs) have been the traditional DNA marker, technological advances have led to single nucleotide polymorphisms (SNPs) becoming an attractive alternative. SNPs can be highly multiplexed and automatically scored, which allows for easier standardization and sharing among laboratories. Equine parentage is currently assessed using STRs. We obtained a publicly available SNP dataset of 729 horses representing 32 diverse breeds. A proposed set of 101 SNPs was analyzed for DNA typing suitability. The overall minor allele frequency of the panel was 0.376 (range 0.304-0.419), with per breed probability of identities ranging from 5.6 × 10-35 to 1.86 × 10-42 . When one parent was available, exclusion probabilities ranged from 0.9998 to 0.999996, although when both parents were available, all breeds had exclusion probabilities greater than 0.9999999. A set of 388 horses from 35 breeds was genotyped to evaluate marker performance on known families. The set included 107 parent-offspring pairs and 101 full trios. No horses shared identical genotypes across all markers, indicating that the selected set was sufficient for individual identification. All pairwise comparisons were classified using ISAG rules, with one or two excluding markers considered an accepted parent-offspring pair, two or three excluding markers considered doubtful and four or more excluding markers rejecting parentage. The panel had an overall accuracy of 99.9% for identifying true parent-offspring pairs. Our developed marker set is both present on current generation SNP chips and can be highly multiplexed in standalone panels and thus is a promising resource for SNP-based DNA typing.

It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer's patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum, inhibition of Ca signaling associated with decreased phagosome-lysosome fusion as well as phagocytosis inhibition, bias toward Th2 cell immune response accompanied by cell recruitment, cell proliferation and cell differentiation; leading to persistent infection. Contrarily, MAA infection was related to cellular responses associated with activation of molecular pathways that release chemicals and cytokines involved with containment of infection and a strong bias toward Th1 immune response, resulting in a transient infection.

Pharmacogenetic testing is increasingly available from clinical Laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, 1NATI, NAT2, SLC15A2, 5LC22A2, SLC01B1, SLC02B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and Laboratory developed tests. Through consensus verification, we confirmed the presence of at Least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among Laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing.

This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.