In horses, graying with age is an autosomal dominant trait associated with a high incidence of melanoma and vitiligo-like depigmentation. Here we show that the Gray phenotype is caused by a 4.6-kb duplication in intron 6 of STX17 (syntaxin-17) that constitutes a cis-acting regulatory mutation. Both STX17 and the neighboring NR4A3 gene are overexpressed in melanomas from Gray horses. Gray horses carrying a loss-of-function mutation in ASIP (agouti signaling protein) had a higher incidence of melanoma, implying that increased melanocortin-1 receptor signaling promotes melanoma development in Gray horses. The Gray horse provides a notable example of how humans have cherry-picked mutations with favorable phenotypic effects in domestic animals.

Deciphering the genomic regulatory code of enhancers is a key challenge in biology as this code underlies cellular identity. A better understanding of how enhancers work will improve the interpretation of non-coding genome variation, and empower the generation of cell type specific drivers for gene therapy. Here we explore the combination of deep learning and cross-species chromatin accessibility profiling to build explainable enhancer models. We apply this strategy to decipher the enhancer code in melanoma, a relevant case study due to the presence of distinct melanoma cell states. We trained and validated a deep learning model, called DeepMEL, using chromatin accessibility data of 26 melanoma samples across six different species. We demonstrate the accuracy of DeepMEL predictions on the CAGI5 challenge, where it significantly outperforms existing models on the melanoma enhancer of IRF4. Next, we exploit DeepMEL to analyse enhancer architectures and identify accurate transcription factor binding sites for the core regulatory complexes in the two different melanoma states, with distinct roles for each transcription factor, in terms of nucleosome displacement

Summary Reasons for performing study: Equine melanomas occur most commonly in grey horses at age 5 years or more. Generally, benign and malignant melanomas are distinguished by microscopy, but a more distinct classification would be helpful. Objectives: The objectives of this study were to gain further evidence concerning the occurrence of melanotic tumours, and to evaluate the impact of heredity on melanoma development. Methods: A clinical study was conducted on a defined population of 296 grey horses of Lipizzaner breed. Individuals were classified according to theirstage of disease using a 0–5 scale. Heritability was estimated on a sample of 296 grey horses with pedigrees traced back as far as 32 generations. Results: Of the 296 horses, dermal melanomas were present in 148 horses (50%), 68 of which were more than age 15 years; 51 of these were melanoma‐bearing. In 75.6% of cases, melanotic tumours were detected underneath the tail. Although melanoma‐bearing grey horses were encountered up to stage 4, none of the affected individuals suffered any severe clinical effect or was handicapped in performance. Statistical analysis revealed highly significant effects of stud and age (P<0.0001), explaining 28% of the total variability. Conclusions: In contrast to melanomas in solid‐coloured horses characterised by early metastases, melanomas in grey horses showed less malignancy. Affected individuals often had encapsulated nodules or structures similar to human blue nevi. Grey horse‐specific genetic factors inhibiting metastatic processes may be responsible for this phenomenon. Potential clinical relevance: Although the obtained heritability estimate of 0.36 with a standard error of 0.11 indicates a strong genetic impact on the development of melanoma in ageing grey horses, a possible influence of the genes with large effects was also suggested. Therefore, further analysis is required of melanoma development in the ageing grey horse.

Equine melanoma shows striking features particularly with regard to clinical development in grey horses: in contrast to malignant melanoma in humans and in solid coloured horses that are characterized by early onset of metastasis, pigment cell tumours display almost benign clinical features in ageing grey horses. Through evolution, grey horses appear to be in a favourable position in regard to the biological behaviour of melanomas. Yet unknown factors inhibiting or retarding early melanoma metastasis may be responsible for this phenomenon. In this study, immunostaining profiles and histopathologic patterns of equine vs. human melanotic tumours were compared. In addition, the expression of melanoma markers currently used in human melanoma detection and characterization were evaluated for their applicability in equine melanoma diagnosis. Immunohistopathologic investigations revealed that benign grey horse melanomas share common features with human blue nevi and with human malignant desmoplastic melanomas, whereas their resemblance to other types of human cutaneous malignant melanomas is less pronounced. Our data equally underline that S-100, proliferating cell nuclear antigen (PCNA), HMB-45, Ki-67, T-311 and CD44 can serve as reliable markers for horse melanomas. Further investigations aiming at identifying factors retarding metastasis in affected grey horses are needed, as they may contribute to the development of novel treatment strategies for human malignant melanoma.

The dominant phenotype of greying with age in horses, caused by a 4.6-kb duplication in intron 6 of STX17, is associated with a high incidence of melanoma and vitiligo-like skin depigmentation. However, the progressive greying and the incidence of melanoma, vitiligo-like depigmentation, and amount of speckling in these horses do not follow a simple inheritance pattern. To understand their inheritance, we analysed the melanoma grade, grey level, vitiligo grade, and speckling grade of 1,119 Grey horses (7,146 measurements) measured in six countries over a 9-year period. We estimated narrow sense heritability (h2), and we decomposed this parameter into polygenic heritability (h2 POLY), heritability due to the Grey (STX17) mutation (h2 STX17), and heritability due to agouti (ASIP) locus (h2 ASIP). A high heritability was found for greying (h2 = 0.79), vitiligo (h2 = 0.63), and speckling (h2 = 0.66), while a moderate heritability was estimated for melanoma (h2 = 0.37). The additive component of ASIP was significantly different from zero only for melanoma (h2 ASIP = 0.02). STX17 controlled large proportions of phenotypic variance (h2 STX17 = 0.18–0.55) and overall heritability (h2 STX17/h2 = 0.28–0.83) for all traits. Genetic correlations among traits were estimated as moderate to high, primarily due to the effects of the STX17 locus. Nevertheless, the correlation between progressive greying and vitiligo-like depigmentation remained large even after taking into account the effects of STX17. We presented a model where four traits with complex inheritance patterns are strongly influenced by a single mutation. This is in line with evidence of recent studies in domestic animals indicating that some complex traits are, in addition to the large number of genes with small additive effects, influenced by genes of moderate-to-large effect. Furthermore, we demonstrated that the STX17 mutation explains to a large extent the moderate to high genetic correlations among traits, providing an example of strong pleiotropic effects caused by a single gene.

Melanoma is a disease with high incidence in gray horses and has limited therapeutic options in metastatic disease. Gene therapy has shown some success in animal models and human patients. A randomized double-blind, placebo-controlled study was conducted to investigate 2 treatment options using cytokine-encoding plasmid DNA in horses with metastatic melanoma to induce immunologic antitumor effects. Adult gray horses with spontaneously occurring metastatic melanoma (n=26) were included in the study. Treatment of 26 gray horses with metastatic melanoma consisted of interleukin-18-encoding plasmid DNA, interleukin-12-encoding plasmid DNA, or empty plasmid DNA (control group), injected intratumorally, respectively. Tumor response was assessed using ultrasound and caliper measurements and histologic assessment of tumor biopsies. Significant tumor regression could be shown in both the treatment groups receiving IL-18 and IL-12-encoding plasmid DNA whereas placebo-treated control patients showed tumor growth over the course of the treatment. In addition, 7 of 10 tumors from horses treated with IL-18 or IL-12 showed peritumoral and/or intratumoral inflammatory infiltrates after treatment compared with 1 of the 6 in the control group. The treatment as assessed by serial blood draws and clinical investigation, was safe and well tolerated. These data suggest that the intratumoral treatment with IL-18 and IL-12-encoding plasmid DNA has antitumor effects, which is well tolerated and thus holds promise for the treatment of patients with metastatic melanoma.

Perineal hernia occurs spontaneously in older male dogs after idiopathic weakening of the pelvic diaphragm. Hernias invariably contain cystic paraprostatic tissues. Castration reduces incidence and recurrence after surgical repair. Although cystic prostatic hypertrophy is a consistent feature in patients with perineal hernia, an endocrine link of the disease to steroid sex hormones has not been demonstrated. Employing immunohistochemistry, we found intense relaxin immunoreactivity in dogs with perineal hernia within the epithelia of hypertrophic prostates and in periprostatic tissues. The prostate of normal dogs exhibited similar but less intense relaxin staining. In neutered dogs with prostatic atrophy, relaxin immunostaining was weak or absent. Periprostatic cysts highly expressed relaxin precursors in the fluid phase as shown by SDS-gel electrophoresis. Relaxin of prostatic origin, therefore, is possibly a local factor in connective tissue weakening and subsequently in perineal hernia formation.

BackgroundGreying with age in horses is an autosomal dominant trait, associated with loss of hair pigmentation, melanoma and vitiligo-like depigmentation. We recently identified a 4.6 kb duplication in STX17 to be associated with the phenotype. The aims of this study were to investigate if the duplication in Grey horses shows copy number variation and to exclude that any other polymorphism is uniquely associated with the Grey mutation.ResultsWe found little evidence for copy number expansion of the duplicated sequence in blood DNA from Grey horses. In contrast, clear evidence for copy number expansions was indicated in five out of eight tested melanoma tissues or melanoma cell lines. A tendency of a higher copy number in aggressive tumours was also found. Massively parallel resequencing of the ~350 kb Grey haplotype did not reveal any additional mutations perfectly associated with the phenotype, confirming the duplication as the true causative mutation. We identified three SNP alleles that were present in a subset of Grey haplotypes within the 350 kb region that shows complete linkage disequilibrium with the causative mutation. Thus, these three nucleotide substitutions must have occurred subsequent to the duplication, consistent with our interpretation that the Grey mutation arose more than 2,000 years before present.ConclusionsThese results suggest that the mutation acts as a melanoma-driving regulatory element. The elucidation of the mechanistic features of the duplication will be of considerable interest for the characterization of these horse melanomas as well as for the field of human melanoma research.