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Histiocytic sarcoma (HS) is a rare but aggressive cancer in both humans and dogs. The spontaneous canine model, which has clinical, epidemiological, and histological similarities with human HS and specific breed predispositions, provides a unique opportunity to unravel the genetic basis of this cancer. In this study, we aimed to identify germline risk factors associated with the development of HS in canine-predisposed breeds. We used a methodology that combined several genome-wide association studies in a multi-breed and multi-cancer approach as well as targeted next-generation sequencing, and imputation We combined several dog breeds (Bernese mountain dogs, Rottweilers, flat-coated retrievers, and golden retrievers), and three hematopoietic cancers (HS, lymphoma, and mast cell tumor). Results showed that we not only refined the previously identified HS risk CDKN2A locus, but also identified new loci on canine chromosomes 2, 5, 14, and 20. Capture and targeted sequencing of specific loci suggested the existence of regulatory variants in non-coding regions and methylation mechanisms linked to risk haplotypes, which lead to strong cancer predisposition in specific dog breeds. We also showed that these canine cancer predisposing loci appeared to be due to the additive effect of several risk haplotypes involved in other hematopoietic cancers such as lymphoma or mast cell tumors as well. This illustrates the pleiotropic nature of these canine cancer loci as observed in human oncology, thereby reinforcing the interest of predisposed dog breeds to study cancer initiation and progression.

Circulating tumor DNA (ctDNA) has become an attractive biomarker in human oncology, and its use may be informative in canine cancer. Thus, we used droplet digital PCR or PCR for antigen receptor rearrangement, to explore tumor-specific point mutations, copy number alterations, and chromosomal rearrangements in the plasma of cancer-affected dogs. We detected ctDNA in 21/23 (91.3%) of histiocytic sarcoma (HS), 2/8 (25%) of oral melanoma, and 12/13 (92.3%) of lymphoma cases. The utility of ctDNA in diagnosing HS was explored in 133 dogs, including 49 with HS, and the screening of recurrent PTPN11 mutations in plasma had a specificity of 98.8% and a sensitivity between 42.8 and 77% according to the clinical presentation of HS. Sensitivity was greater in visceral forms and especially related to pulmonary location. Follow-up of four dogs by targeting lymphoma-specific antigen receptor rearrangement in plasma showed that minimal residual disease detection was concordant with clinical evaluation and treatment response. Thus, our study shows that ctDNA is detectable in the plasma of cancer-affected dogs and is a promising biomarker for diagnosis and clinical follow-up. ctDNA detection appears to be useful in comparative oncology research due to growing interest in the study of natural canine tumors and exploration of new therapies.

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.

Background Metastatic melanoma is one of the most aggressive forms of cancer in humans. Among its types, mucosal melanomas represent one of the most highly metastatic and aggressive forms, with a very poor prognosis. Because they are rare in Caucasian individuals, unlike cutaneous melanomas, there has been fewer epidemiological, clinical and genetic evaluation of mucosal melanomas. Moreover, the lack of predictive models fully reproducing the pathogenesis and molecular alterations of mucosal melanoma makes its treatment challenging. Interestingly, dogs are frequently affected by melanomas of the oral cavity that are characterized, as their human counterparts, by focal infiltration, recurrence, and metastasis to regional lymph nodes, lungs and other organs. In dogs, some particular breeds are at high risk, suggesting a specific genetic background and strong genetic drivers. Altogether, the striking homologies in clinical presentation, histopathological features, and overall biology between human and canine mucosal melanomas make dogs invaluable natural models with which to investigate tumor development, including tumor ætiology, and develop tailored treatments. Methods We developed and characterized two canine oral melanoma cell lines from tumors isolated from dog patients with distinct clinical profiles; with and without lung metastases. The cells were characterized using immunohistochemistry, pharmacology and genetic studies. Results We have developed and immunohistochemically, genetically, and pharmacologically characterized. Two cell lines ( Ocr_OCMM1X & Ocr_OCMM2X ) were produced through mouse xenografts originating from two clinically contrasting melanomas of the oral cavity. Their exhaustive characterization showed two distinct biological and genetic profiles that are potentially linked to the stage of malignancy at the time of diagnosis and sample collection of each melanoma case. These cell lines thus constitute relevant tools with which to perform genetic and drug screening analyses for a better understanding of mucosal melanomas in dogs and humans. Conclusions The aim of this study was to establish and characterize xenograft-derived canine melanoma cell lines with different morphologies, genetic features and pharmacological sensitivities that constitute good predictive models for comparative oncology. These cell lines are relevant tools to advance the use of canine mucosal melanomas as natural models for the benefit of both veterinary and human medicine.

Long non-coding RNAs (lncRNAs) are a family of heterogeneous RNAs that play major roles in multiple biological processes. We recently identified an extended repertoire of more than 10,000 lncRNAs of the domestic dog however, predicting their biological functionality remains challenging. In this study, we have characterised the expression profiles of 10,444 canine lncRNAs in 26 distinct tissue types, representing various anatomical systems. We showed that lncRNA expressions are mainly clustered by tissue type and we highlighted that 44% of canine lncRNAs are expressed in a tissue-specific manner. We further demonstrated that tissue-specificity correlates with specific families of canine transposable elements. In addition, we identified more than 900 conserved dog-human lncRNAs for which we show their overall reproducible expression patterns between dog and human through comparative transcriptomics. Finally, co-expression analyses of lncRNA and neighbouring protein-coding genes identified more than 3,400 canine lncRNAs, suggesting that functional roles of these lncRNAs act as regulatory elements. Altogether, this genomic and transcriptomic integrative study of lncRNAs constitutes a major resource to investigate genotype to phenotype relationships and biomedical research in the dog species.

Bernese mountain dogs are a large dog breed formed in the early 1900s in Switzerland. While originally farm dogs that were used for pulling carts, guarding, and driving cattle, today they are considered multi-purpose companion and family dogs. The breed is predisposed to several complex diseases, such as histiocytic sarcoma, degenerative myelopathy, or hip dysplasia. Using whole-genome sequencing (WGS) data, we assessed the genomic architecture of 33 unrelated dogs from four countries: France, Sweden, Switzerland, and the United States. Analysis of runs of homozygosity (ROH) identified 12,643 ROH with an average length of 2.29 Mb and an average inbreeding coefficient of 0.395. Multidimensional scaling analysis of the genetic relatedness revealed limited clustering of European versus USA dogs, suggesting exchanges of breeding stock between continents. Furthermore, only two mtDNA haplotypes were detected in the 33 studied dogs, both of which are widespread throughout multiple dog breeds. WGS-based ROH analyses revealed several fixed or nearly fixed regions harboring discreet morphological trait-associated as well as disease-associated genetic variants. Several genes involved in the regulation of immune cells were found in the ROH shared by all dogs, which is notable in the context of the breed’s strong predisposition to hematopoietic cancers. High levels of inbreeding and relatedness, strongly exaggerated in the last 30 years, have likely led to the high prevalence of specific genetic disorders in this breed.

White coat color in mammals has been selected several times during the domestication process. Numerous dog breeds are fixed for one form of white coat color that involves darkly pigmented skin. The genetic basis of this color, due to the absence of pigment in the hairs, was suggested to correspond to extreme dilution of the phaeomelanin, by both the expression of only phaeomelanin (locus E) and its extreme dilution (locus I). To go further, we performed genome-wide association studies (GWAS) using a multiple breed approach. The first GWAS, using 34 white dogs and 128 non-white dogs, including White Shepherds, Poodles, Cotons de Tulear and Bichons allowed us to identify two significantly associated loci on the locus E and a novel locus on chromosome 20. A second GWAS using 15 other breeds presenting extreme phaeomelanin dilution confirmed the position of locus I on the chromosome 20 (position 55 Mb pcorrected = 6 × 10−13). Using whole-genome sequencing, we identified a missense variant in the first exon of MFSD12, a gene recently identified to be involved in human, mouse and horse pigmentation. We confirmed the role of this variant in phaeomelanin dilution of numerous canine breeds, and the conserved role of MFSD12 in mammalian pigmentation.

UNLABELLED: ABSTRACT: BACKGROUND: Histiocytic malignancies in both humans and dogs are rare and poorly understood. While canine histiocytic sarcoma (HS) is uncommon in the general domestic dog population, there is a strikingly high incidence in a subset of breeds, suggesting heritable predisposition. Molecular cytogenetic profiling of canine HS in these breeds would serve to reveal recurrent DNA copy number aberrations (CNAs) that are breed and/or tumor associated, as well as defining those shared with human HS. This process would identify evolutionarily conserved cytogenetic changes to highlight regions of particular importance to HS biology. METHODS: Using genome wide array comparative genomic hybridization we assessed CNAs in 104 spontaneously occurring HS from two breeds of dog exhibiting a particularly elevated incidence of this tumor, the Bernese Mountain Dog and Flat-Coated Retriever. Recurrent CNAs were evaluated further by multicolor fluorescence in situ hybridization and loss of heterozygosity analyses. Statistical analyses were performed to identify CNAs associated with tumor location and breed. RESULTS: Almost all recurrent CNAs identified in this study were shared between the two breeds, suggesting that they are associated more with the cancer phenotype than with breed. A subset of recurrent genomic imbalances suggested involvement of known cancer associated genes in HS pathogenesis, including deletions of the tumor suppressor genes CDKN2A/B, RB1 and PTEN. A small number of aberrations were unique to each breed, implying that they may contribute to the major differences in tumor location evident in these two breeds. The most highly recurrent canine CNAs revealed in this study are evolutionarily conserved with those reported in human histiocytic proliferations, suggesting that human and dog HS share a conserved pathogenesis. CONCLUSIONS: The breed associated clinical features and DNA copy number aberrations exhibited by canine HS offer a valuable model for the human counterpart, providing additional evidence towards elucidation of the pathophysiological and genetic mechanisms associated with histiocytic malignancies. Extrapolation of data derived from canine histiocytic disorders to human histiocytic proliferation may help to further our understanding of the propagation and cancerization of histiocytic cells, contributing to development of new and effective therapeutic modalities for both species.

In humans, hereditary sensory neuropathies (HSN), also known as hereditary sensory and autonomic neuropathies (HSAN), constitute a clinically and genetically heterogeneous group of disorders characterized by progressive sensory loss, often accompanied by chronic skin ulcerations and nail dystrophic changes. To date, although around 20 genes have already been discovered, they do not explain the genetic causes of all patients. In dogs, similar neuropathies are also diagnosed, several breeds being predisposed to specific forms of the disease. Indeed, the breed specificity of most canine genetic diseases is due to the small numbers of founders and high levels of inbreeding. Recent knowledge and tools developed to study the canine genome efficiently allows deciphering the genetic bases of such diseases. To date, a dozen breeds are recognized to develop specific HSN. For the Border collie and hunting dog breeds, the genes involved have recently been discovered. Other affected breeds thus constitute potential genetic models, with new genes to be found in dogs that can be considered as candidate genes for human HSAN/HSN. Here, we review the different forms of human and canine HSAN/HSN and we present a novel form in Fox terrier cases, highlighting the advantages of the dog model for such rare human diseases.

Mucosal melanomas (MM) are rare aggressive cancers in humans, and one of the most common forms of oral cancers in dogs. Similar biological and histological features are shared between MM in both species, making dogs a powerful model for comparative oncology studies of melanomas. Although exome sequencing recently identified recurrent coding mutations in canine MM, little is known about changes in non-coding gene expression, and more particularly, in canine long non-coding RNAs (lncRNAs), which are commonly dysregulated in human cancers. Here, we sampled a large cohort (n = 52) of canine normal/tumor oral MM from three predisposed breeds (poodles, Labrador retrievers, and golden retrievers), and used deep transcriptome sequencing to identify more than 400 differentially expressed (DE) lncRNAs. We further prioritized candidate lncRNAs by comparative genomic analysis to pinpoint 26 dog–human conserved DE lncRNAs, including SOX21-AS, ZEB2-AS, and CASC15 lncRNAs. Using unsupervised co-expression network analysis with coding genes, we inferred the potential functions of the DE lncRNAs, suggesting associations with cancer-related genes, cell cycle, and carbohydrate metabolism Gene Ontology (GO) terms. Finally, we exploited our multi-breed design to identify DE lncRNAs within breeds. This study provides a unique transcriptomic resource for studying oral melanoma in dogs, and highlights lncRNAs that may potentially be diagnostic or therapeutic targets for human and veterinary medicine.