DPF3, A Putative Candidate Gene For Melanoma Etiopathogenesis in Gray Horses.

Melanoma prevalence in gray horses reaches up to 50% and more. Several studies have documented a genetic melanoma predisposition which is referred to the 4.6 kb duplication in intron 6 of STX17 and its surrounding haplotype. However, the genetic background and mechanisms responsible for differences in etiopathogenesis of equine dermal melanomatosis still remain unknown. In the current study, we performed a genome wide association analysis in 141 Lipizzan horses and subsequently identified one candidate gene on chromosome 24 putatively involved in melanoma pathogenesis in gray horses. The associated SNP was located in the intronic region of DPF3, a gene which is involved in humans in cell growth, proliferation, apoptosis and motility of cancer cells. The replication study in 1210 horses from seven breeds demonstrated, that the G/G genotype of the DPF3 associated SNP exhibits putative melanoma suppression effects. As a conclusion DPF3 represents a candidate gene, which might play an essential role for gray horses coping with high genetic melanoma related tumor load.

Equine vitiligo-like depigmentation in grey horses is related to genes involved in immune response and tumor metastasis.

BACKGROUND: In horses, the autoimmune disease vitiligo is characterized by the loss of melanocytes and results in patchy depigmentation of the skin around the eyes, muzzle and the perianal region. Vitiligo-like depigmentation occurs predominantly in horses displaying the grey coat colour and is observed at a prevalence level of 26.0-67.0% in grey horses compared with only 0.8-3.5% in non-grey horses. While the polygenetic background of this complex disease is well documented in humans, the underlying candidate genes for this skin disorder in horses remain unknown. In this study we aim to perform a genome-wide association study (GWAS) for identifying putative candidate loci for vitiligo-like depigmentation in horses. METHODS: In the current study, we performed a GWAS analysis using high-density 670 k single nucleotide polymorphism (SNP) data from 152 Lipizzan and 104 Noriker horses, which were phenotyped for vitiligo-like depigmentation by visual inspection. After quality control 376,219 SNPs remained for analyses, the genome-wide Bonferroni corrected significance level was p < 1.33e-7. RESULTS: We identified seven candidate genes on four chromosomes (ECA1, ECA13, ECA17, ECA20) putatively involved in vitiligo pathogenesis in grey horses. The highlighted genes PHF11, SETDB2, CARHSP1 and LITAFD, are associated with the innate immune system, while the genes RCBTB1, LITAFD, NUBPL, PTP4A1, play a role in tumor suppression and metastasis. The antagonistic pathogenesis of vitiligo in relation to cancer specific enhanced cell motility and/or metastasis on typical melanoma predilection sites underlines a plausible involvement of RCBTB1, LITAFD, NUBPL, and PTP4A1. CONCLUSIONS: The proposed candidate genes for equine vitiligo-like depigmentation, indicate an antagonistic relation between vitiligo and tumor metastasis in a horse population with higher incidence of melanoma. Further replication and expression studies should lead to a better understanding of this skin disorder in horses.

A Genome-Wide Association Analysis in Noriker Horses Identifies a SNP Associated With Roan Coat Color.

The roan coat color in horses is characterized by dispersed white hair and dark points. This phenotype segregates in a broad range of horse breeds, while the underlying genetic background is still unknown. Previous studies mapped the roan locus to the KIT gene on equine chromosome 3 (ECA3). However, this association could not be validated across different horse breeds. Performing a genome-wide association analysis (GWAS) in Noriker horses, we identified a single nucleotide polymorphism (SNP) (ECA3:g.79,543.439 A > G) in the intron 17 of the KIT gene. The G -allele of the top associated SNP was present in other roan horses, namely Quarter Horse, Murgese, Slovenian, and Belgian draught horse, while it was absent in a panel of 15 breeds, including 657 non-roan horses. In further 379 gray Lipizzan horses, eight animals exhibited a heterozygous genotype (A/G). Comparative whole-genome sequence analysis of the KIT region revealed two deletions in the downstream region (ECA3:79,533,217_79,533,224delTCGTCTTC; ECA3:79,533,282_79,533,285delTTCT) and a 3 bp deletion combined with 17 bp insertion in intron 20 of KIT (ECA3:79,588,128_79,588,130delinsTTATCTCTATAGTAGTT). Within the Noriker sample, these loci were in complete linkage disequilibrium (LD) with the identified top SNP. Based upon pedigree information and historical records, we were able to trace back the genetic origin of roan coat color to a baroque gene pool. Furthermore, our data suggest allelic heterogeneity and the existence of additional roan alleles in ponies and breeds related to the English Thoroughbred. In order to study the roan phenotype segregating in those breeds, further association and verification studies are required.

Genome-Wide Homozygosity Patterns and Evidence for Selection in a Set of European and Near Eastern Horse Breeds.

Intensive artificial and natural selection have shaped substantial variation among European horse breeds. Whereas most equine selection signature studies employ divergent genetic population structures in order to derive specific inter-breed targets of selection, we screened a total of 1476 horses originating from 12 breeds for the loss of genetic diversity by runs of homozygosity (ROH) utilizing a 670,000 single nucleotide polymorphism (SNP) genotyping array. Overlapping homozygous regions (ROH islands) indicating signatures of selection were identified by breed and similarities/dissimilarities between populations were evaluated. In the entire dataset, 180 ROH islands were identified, whilst 100 islands were breed specific, all other overlapped in 36 genomic regions with at least one ROH island of another breed. Furthermore, two ROH hot spots were determined at horse chromosome 3 (ECA3) and ECA11. Besides the confirmation of previously documented target genes involved in selection for coat color (MC1R, STX17, ASIP), body size (LCORL/NCAPG, ZFAT, LASP1, HMGA2), racing ability (PPARGC1A), behavioral traits (GRIN2B, NTM/OPCML) and gait patterns (DMRT3), several putative target genes related to embryonic morphogenesis (HOXB), energy metabolism (IGFBP-1, IGFBP-3), hair follicle morphogenesis (KRT25, KRT27, INTU) and autophagy (RALB) were highlighted. Furthermore, genes were pinpointed which might be involved in environmental adaptation of specific habitats (UVSSA, STXBP4, COX11, HLF, MMD).

High-resolution population structure and runs of homozygosity reveal the genetic architecture of complex traits in the Lipizzan horse.

BACKGROUND: The sample ascertainment bias due to complex population structures remains a major challenge in genome-wide investigations of complex traits. In this study we derived the high-resolution population structure and levels of autozygosity of 377 Lipizzan horses originating from five different European stud farms utilizing the SNP genotype information of the high density 700 k Affymetrix Axiom™ Equine genotyping array. Scanning the genome for overlapping runs of homozygosity (ROH) shared by more than 50% of horses, we identified homozygous regions (ROH islands) in order to investigate the gene content of those candidate regions by gene ontology and enrichment analyses. RESULTS: The high-resolution population network approach revealed well-defined substructures according to the origin of the horses (Austria, Slovakia, Croatia and Hungary). The highest mean genome coverage of ROH (SROH) was identified in the Austrian (SROH = 342.9), followed by Croatian (SROH = 214.7), Slovakian (SROH = 205.1) and Hungarian (SROH = 171.5) subpopulations. ROH island analysis revealed five common islands on ECA11 and ECA14, hereby confirming a closer genetic relationship between the Hungarian and Croatian as well as between the Austrian and Slovakian samples. Private islands were detected for the Hungarian and the Austrian Lipizzan subpopulations. All subpopulations shared a homozygous region on ECA11, nearly identical in position and length containing among other genes the homeobox-B cluster, which was also significantly (p < 0.001) highlighted by enrichment analysis. Gene ontology terms were mostly related to biological processes involved in embryonic morphogenesis and anterior/posterior specification. Around the STX17 gene (causative for greying), we identified a ROH island harbouring the genes NR4A3, STX17, ERP44 and INVS. Within further islands on ECA14, ECA16 and ECA20 we detected the genes SPRY4, NDFIP1, IMPDH2, HSP90AB1, whereas SPRY4 and HSP90AB1 are involved in melanoma metastasis and survival rate of melanoma patients in humans. CONCLUSIONS: We demonstrated that the assessment of high-resolution population structures within one single breed supports the downstream genetic analyses (e.g. the identification of ROH islands). By means of ROH island analyses, we identified the genes SPRY4, NDFIP1, IMPDH2, HSP90AB1, which might play an important role for further studies on equine melanoma. Furthermore, our results highlighted the impact of the homeobox-A and B cluster involved in morphogenesis of Lipizzan horses.

Population Networks Associated with Runs of Homozygosity Reveal New Insights into the Breeding History of the Haflinger Horse.

Within the scope of current genetic diversity analyses, population structure and homozygosity measures are independently analyzed and interpreted. To enhance analytical power, we combined the visualization of recently described high-resolution population networks with runs of homozygosity (ROH). In this study, we demonstrate that this approach enabled us to reveal important aspects of the breeding history of the Haflinger horse. We collected high-density genotype information of 531 horses originating from 7 populations which were involved in the formation of the Haflinger, namely 32 Italian Haflingers, 78 Austrian Haflingers, 190 Noriker, 23 Bosnian Mountain Horses, 20 Gidran, 33 Shagya Arabians, and 155 Purebred Arabians. Model-based cluster analysis identified substructures within Purebred Arabian, Haflinger, and Noriker that reflected distinct genealogy (Purebred Arabian), geographic origin (Haflinger), and coat color patterns (Noriker). Analysis of ROH revealed that the 2 Arabian populations (Purebred and Shagya Arabians), Gidran and the Bosnian Mountain Horse had the highest genome proportion covered by ROH segments (306-397 Mb). The Noriker and the Austrian Haflinger showed the lowest ROH coverage (228, 282 Mb). Our combined visualization approach made it feasible to clearly identify outbred (admixture) and inbred (ROH segments) horses. Genomic inbreeding coefficients (FROH) ranged from 10.1% (Noriker) to 17.7% (Purebred Arabian). Finally it could be demonstrated, that the Austrian Haflinger sample has a lack of longer ROH segments and a deviating ROH spectrum, which is associated with past bottleneck events and the recent mating strategy favoring out-crosses within the breed.