A Focus on Discovery: Etalon's Discoveries & Publications

Equine Coat Color Discoveries

Dominant White 31 and Dominant White 32 (W31 and W32, Merada White and Scandalous White) (Color)

Patterson Rosa, L., Martin, K., Vierra, M., Foster, G., Lundquist, E., Brooks, S. A., & Lafayette, C. (2021). Two Variants of KIT Causing White Patterning in Stock-Type Horses. The Journal of heredity, esab033. Advance online publication. Access publication here: https://doi.org/10.1093/jhered/esab033

Abstract: Over 30 polymorphisms in the KIT Proto-Oncogene Receptor Tyrosine Kinase (KIT) gene have been implicated in white spotting patterns ranging from small areas to full dermal depigmentation in the horse. We performed a candidate-gene exon sequencing approach on KIT and MITF, 2 known causatives of white spotting patterns, within 2 families of horses of unknown white spotting. Family 1 (Fam1, N = 5) consisted of a Quarter Horse stallion and 4 offspring with white spotting pattern ranging from legs, lower ventral, and head regions with jagged borders, to almost complete white. The second family (Fam2, N = 7) consisted of 6 half-sibling American Paint Horse/Quarter Horse and their dam, demonstrating unpigmented limbs with belly spots and an extensive white patterning on the face. This approach resulted in 2 variants significantly associated with familial phenotypes, where Fam1 variant is an indel leading to a frameshift mutation, and Fam2 a non-synonymous SNP. We validated the variants within an unrelated population of horses (Fam2 variant, P = 0.00271944) as well as for protein functional impact with ExPASy, Protter, Phyre2, SMART, PROVEAN, SIFT, and I-TASSER, confirming the reported associations. Fam1 associated variant, deemed W31, alters the protein sequence, leading to an early stop codon truncating the normal amino acid sequence from 972 to just 115 amino acids. Fam2 associated variant, deemed W32, may have a subtle impact on receptor function or could be in linkage with a non-coding or regulatory change creating the mild spotting pattern observed in this family.

Published: 05 July 2021

Dominant White 30 (W30) (Color)

Martin, K., Patterson Rosa, L., Vierra, M., Foster, G., Brooks, S. A., & Lafayette, C. (2021). De novo mutation of KIT causes extensive coat white patterning in a family of Berber horses. Animal genetics, 52(1), 135–137.

Access publication here: https://doi.org/10.1111/age.13017

Accepted for publication 06 October 2020

Snowdrop (Sno) (Color)

Bisbee, D., Carpenter, M. L., Hoefs-Martin, K., Brooks, S. A., & Lafayette, C. (2020). Identification of a novel missense variant in SLC45A2 associated with dilute snowdrop phenotype in Gypsy horses. Animal genetics, 51(2), 342–343. '

Access publication here: https://doi.org/10.1111/age.12913

First published: 21 January 2020

Pearl (prl) and Sunshine (Sun) (Color)

Holl, H. M., Pflug, K. M., Yates, K. M., Hoefs-Martin, K., Shepard, C., Cook, D. G., Lafayette, C., & Brooks, S. A. (2019). A candidate gene approach identifies variants in SLC45A2 that explain dilute phenotypes, pearl and sunshine, in compound heterozygote horses. Animal genetics, 50(3), 271–274.

Access publication here: https://doi.org/10.1111/age.12790

Abstract: Variations in the SLC45A2 gene are responsible for the dilution phenotypes cream and pearl in domestic horses. Cream dilution is inherited in an incomplete dominant manner, diluting only red in the heterozygous state but both red and black pigments when two alleles are present. The pearl dilution is recessive and dilutes only the red and black pigment in the homozygous state or when paired with a cream allele. Horses that inherit one copy of pearl (Cprl ) and one copy of the dominant cream allele (CC r ) display a dilution phenotype similar to that of homozygous cream, suggesting that pearl is the result of a different variation in the same gene responsible for cream. We sequenced SLC45A2 in two 'false double dilute' horses that appeared phenotypically homozygous cream but tested as possessing only a single CC r allele. We also sequenced one known pearl carrier to screen for putative causal variants. The missense variant ECA21:SLC45A2:c.985G>A; p.Ala329Thr (Cprl ) was present in one false double dilute and the pearl carrier and was also genotyped in an additional 126 horses for statistical evaluation. The genotype matched the expected phenotype in all horses (P-value = 6.5 × 10-41 ) and is identical to a pearl variant found previously. The second false double dilute horse and one non-dilute offspring genotyped as heterozygous for a novel missense variant ECA21:SLC45A2:c.568G>A (p.Gly190Arg), the proposed Csun variant (for the name of the horse). This variant produces a recessive dilution similar to pearl and indicates that multiple alleles of SLC45A2 result in dilution phenotypes in the domestic horse.

First published: 21 April 2019

Splashed White 5 (SW5) (Color)

Henkel, J., Lafayette, C., Brooks, S. A., Martin, K., Patterson-Rosa, L., Cook, D., Jagannathan, V., & Leeb, T. (2019). Whole-genome sequencing reveals a large deletion in the MITF gene in horses with white spotted coat colour and increased risk of deafness. Animal genetics, 50(2), 172–174.

Access publication here: https://doi.org/10.1111/age.12762

Abstract: White spotting phenotypes in horses are highly valued in some breeds. They are quite variable and may range from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for white spotting phenotypes in horses. For the present study, we investigated an American Paint Horse family segregating a phenotype involving white spotting and blue eyes. Six of eight horses with the white-spotting phenotype were deaf. We obtained whole-genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~63-kb deletion spanning exons 6–9 of the MITF gene (chr16:21 503 211–21 566 617). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. PCR-based genotyping revealed that all eight available affected horses from the family carried the deletion. The finding of an MITF variant fits well with the syndromic phenotype involving both depigmentation and an increased risk for deafness and corresponds to human Waardenburg syndrome type 2A. Our findings will enable more precise genetic testing for depigmentation phenotypes in horses.

First published: 15 January 2019

Dominant White 22 (W22, Airdrie Apache) (Color)

Dürig, N., Jude, R., Holl, H., Brooks, S. A., Lafayette, C., Jagannathan, V., & Leeb, T. (2017). Whole genome sequencing reveals a novel deletion variant in the KIT gene in horses with white spotted coat colour phenotypes. Animal genetics, 48(4), 483–485.

Access publication here: https://doi.org/10.1111/age.12556

Abstract: White spotting phenotypes in horses can range in severity from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for such phenotypes in horses. For the present study, we re-investigated a large horse family segregating a variable white spotting phenotype, for which conventional Sanger sequencing of the candidate genes’ individual exons had failed to reveal the causative variant. We obtained whole genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~1.9-kb deletion spanning exons 10–13 of the KIT gene (chr3:77,740,239_77,742,136del1898insTATAT). In continuity with previously named equine KIT variants we propose to designate the newly identified deletion variant W22. We had access to 21 horses carrying the W22 allele. Four of them were compound heterozygous W20/W22 and had a completely white phenotype. Our data suggest that W22 represents a true null allele of the KIT gene, whereas the previously identified W20 leads to a partial loss of function. These findings will enable more precise genetic testing for depigmentation phenotypes in horses.

First published: 26 April 2017

Dominant White 23 (W23) and homozygous W15 (Khartoon Khlassic) (Color)

Holl, H. M., Brooks, S. A., Carpenter, M. L., Bustamante, C. D., & Lafayette, C. (2017). A novel splice mutation within equine KIT and the W15 allele in the homozygous state lead to all white coat color phenotypes. Animal genetics, 48(4), 497–498.

Access publication here: https://doi.org/10.1111/age.12554

First published: 05 April 2017

White 30 (discovered 2020)

De novo mutation of KIT causes extensive coat white patterning in a family of Berber horses

Access the publication here: https://www.researchgate.net/publication/345340553_De_novo_mutation_of_KIT_causes_extensive_coat_white_patterning_in_a_family_of_Berber_horses

Health, Performance & Ancestry Composition Discoveries

Is HERDA Always a Death Sentence? Horse Discovered Homozygous for HERDA with Minimal Signs (Health)

Laura Patterson Rosa, Tyler W. Troop, Katie Martin, Micaela Vierra, Gabe Foster, Erica Lundquist, Samantha A. Brooks, Christa Lafayette (2021). Hereditary Equine Regional Dermal Asthenia homozygote adult working horse with mild signs - A Case Report, Journal of Equine Veterinary Science, 103756, ISSN 0737-0806.

Access Publication here: https://doi.org/10.1016/j.jevs.2021.103756.

Abstract: Hereditary Equine Regional Dermal Asthenia (HERDA) is an autosomal recessive condition present in the American Quarter Horse and other related breeds. Resulting from a mutation in the peptidyl-prolyl cis-trans isomerase B (PPIB) gene, HERDA is homologous to Ehlers-Danlos syndrome in humans. Characterized by fragile, hyperelastic, skin, HERDA affected horses often present first with slow-healing wounds usually on the dorsum, and resulting in atrophic scars, seromas, and ulcers. As there is no treatment for the condition affected horses are typically reported to be unrideable, and if persistent wounds are sufficiently severe, may require euthanasia. This case report describes clinical presentation and genetic diagnostics of HERDA in an 8-year-old horse with notably mild clinical signs, previously undiagnosed. On recommendation from the referring veterinarian, the horse owners pursued genetic diagnostics for HERDA following development of painful dorsal skin lesions under the saddle area during a riding clinic. The individual was confirmed homozygous for HERDA c.115G>A missense mutation in the PPIB gene by commercial testing service (Etalon Diagnostics Inc.). Further objective studies on the severity and clinical presentation of HERDA are necessary to evaluate complex elements of this disease. Furthermore, mildly affected individuals may be underdiagnosed as a result of not demonstrating the clinical signs that commonly encourage genetic testing.

DMRT3 in Stock Horses (Performance/Ancestry Composition)

Patterson Rosa, L., Staiger, E. A., Martin, K., Vierra, M., Foster, G., Lundquist, E., Brooks, S. A., & Lafayette, C. (2021). Stock-type equine disciplines Hunter, Reining and Roping are associated with the A allele at the DMRT3 locus for gait phenotypes in the horse. Animal genetics, 10.1111/age.13110. Advance online publication.

Access publication here: https://doi.org/10.1111/age.13110

Abstract: A mutation in the double-sex and mab-3-related transcription factor 3 (DMRT3) gene leading to an early stop (DMRT3_Ser301STOP) has been previously correlated with changes in the locomotion pattern of several equine breeds. We genotyped 11463 horses representing more than 80 breeds recognized worldwide, using the commercial genetic testing service of Etalon Diagnostics (Etalon Diagnostic Inc., Menlo Park, CA). We evaluated the allelic frequency of the mutation (A allele) and its statistical correlation with sports disciplines of Quarter Horse, Paint Horse and Appaloosa breeds. Despite the low frequency of the mutant A allele in these breeds, we determined a significant association between the C/A genotype and the Hunter and Reining categories (P = 1.3 x 10-5 and P = 5.34 x 10-7, respectively) and Roping with the A/A genotype (P = 1.32 x 10-6). The disciplines of Lesson Horse and Roping also demonstrate selective breeding effects (Hardy Weinberg Equilibrium, mid-p = 0.0204 and mid-p = 0.0312, respectively). Future studies evaluating the relationship between carriers of the variant with “popular sire” effects or changes in the locomotion phenotype are needed to better understand the implications of this DMRT3 allele.

First published: 12 July 2021

Fragile Foal Syndrome (Health)

Martin, K., Brooks, S., Vierra, M., Lafayette, W. T., McClure, S., Carpenter, M., & Lafayette, C. (2020). Fragile Foal Syndrome (PLOD1 c.2032G>A) occurs across diverse horse populations. Animal genetics, 52(1), 137–138.

Access publication here: https://doi.org/10.1111/age.13020

Accepted for publication 12 October 2020

Kissing Spines (Health)

Whitaker, B. and Brooks, S.A. (2020) Identification of Genomic Loci Associated with Performance-Limiting Kissing Spines in Quarter Horses and Warmbloods. S0706 Talk - AAEP Virtual Convention 2020. Phase I of the study has been published. Genomic loci associated with performance limiting equine overriding spinous processes (kissing spines). Research in Veterinary Science, vol 150, December 5, 2022, 65-71

Access publication here: https://www.sciencedirect.com/science/article/abs/pii/S0034528822001606?via%3Dihub

Parentage Testing through SNPs in the Horse (Parentage)

Holl, H. M., Vanhnasy, J., Everts, R. E., Hoefs-Martin, K., Cook, D., Brooks, S. A., Carpenter, M. L., Bustamante, C. D., & Lafayette, C. (2017). Single nucleotide polymorphisms for DNA typing in the domestic horse. Animal genetics, 48(6), 669–676.

Access publication here: https://doi.org/10.1111/age.12608

Abstract: 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.

First published: 13 September 2017

International Discoveries

Disciplinas esportivas em cavalos de raças de trabalho são associadas ao alelo A no locus DMRT3.

Gaspar, M.C., Araújo, F., Staiger, E.A., Martin, K., Vierra, M., Foster, G., Lundquist. E., Brooks, S.A., Patterson Rosa, L. and Lafayette, C. (2021) Brazilian Journal of Equine Medicine. In press.

Resumo: Uma mutação no gene double-sex and mab-3-related transcription factor 3 (DMRT3) levando a um stop precoce (DMRT3_Ser301STOP) foi previamente correlacionada a alterações no padrão de locomoção de diversas raças equinas. Genotipamos 11463 cavalos representando mais de 80 raças reconhecidas mundialmente pelo serviço comercial de testes genéticos da Etalon Diagnostics (Etalon Diagnostic Inc., Menlo Park, CA). Avaliamos a frequência alélica da mutação (alelo A) e sua correlação estatística com disciplinas esportivas das raças Quarto de Milha, Paint Horse e Appaloosa. Apesar do alelo mutante A se encontrar em baixa frequência nestas raças, determinamos a associação entre o genótipo C/A e as categorias de Hunter e Rédeas (P = 1.3 x 10-5 e P = 5.34 x 10-7, respectivamente) e “Laço” com o genótipo A/A (P = 1.32 x 10-6). As disciplinas de “Cavalo de Escolinha” e “Laço” também demonstram efeitos de seleção (Equilíbrio de Hardy Weinberg, mid-p = 0.0204 e mid-p = 0.0312, respectivamente). Futuros estudos avaliando a relação entre portadores da variante com efeitos de “popular sire” ou alterações no fenótipo de locomoção se fazem necessários para compreender as implicações deste alelo no DMRT3.

Ancestry of a Popular Mangalarga Sire, Turbante JO (Ancestry)

Patterson Rosa, L. & Campos, Fellipe & Martin, Katie & Vierra, Micaela & Foster, Gabriel & Lundquist, Erica & Brooks, Samantha & Lafayette, Christa. (2021). Debunking the genetic origins of the Mangalarga through Turbante J.O. International Society for Animal Genetics (ISAG) 2021 Conference, July 2021.