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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.

Coat color and type are essential characteristics of domestic dog breeds. Although the genetic basis of coat color has been well characterized, relatively little is known about the genes influencing coat growth pattern, length, and curl. We performed genome-wide association studies of more than 1000 dogs from 80 domestic breeds to identify genes associated with canine fur phenotypes. Taking advantage of both inter- and intrabreed variability, we identified distinct mutations in three genes, RSPO2, FGF5, and KRT71 (encoding R-spondin-2, fibroblast growth factor-5, and keratin-71, respectively), that together account for most coat phenotypes in purebred dogs in the United States. Thus, an array of varied and seemingly complex phenotypes can be reduced to the combinatorial effects of only a few genes.

Lethal acrodermatitis (LAD) is a genodermatosis with monogenic autosomal recessive inheritance in Bull Terriers and Miniature Bull Terriers. The LAD phenotype is characterized by poor growth, immune deficiency, and skin lesions, especially at the paws. Utilizing a combination of genome wide association study and haplotype analysis, we mapped the LAD locus to a critical interval of ~1.11 Mb on chromosome 14. Whole genome sequencing of an LAD affected dog revealed a splice region variant in the MKLN1 gene that was not present in 191 control genomes (chr14:5,731,405T>G or MKLN1:c.400+3A>C). This variant showed perfect association in a larger combined Bull Terrier/Miniature Bull Terrier cohort of 46 cases and 294 controls. The variant was absent from 462 genetically diverse control dogs of 62 other dog breeds. RT-PCR analysis of skin RNA from an affected and a control dog demonstrated skipping of exon 4 in the MKLN1 transcripts of the LAD affected dog, which leads to a shift in the MKLN1 reading frame. MKLN1 encodes the widely expressed intracellular protein muskelin 1, for which diverse functions in cell adhesion, morphology, spreading, and intracellular transport processes are discussed. While the pathogenesis of LAD remains unclear, our data facilitate genetic testing of Bull Terriers and Miniature Bull Terriers to prevent the unintentional production of LAD affected dogs. This study may provide a starting point to further clarify the elusive physiological role of muskelin 1 in vivo.

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.

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.

The purebred dog population consists of >300 partially inbred genetic isolates or breeds. Restriction of gene flow between breeds, together with strong selection for traits, has led to the establishment of a unique resource for dissecting the genetic basis of simple and complex mammalian traits. Toward this end, we present a comprehensive radiation hybrid map of the canine genome composed of 3,270 markers including 1,596 microsatellite-based markers, 900 cloned gene sequences and ESTs, 668 canine-specific bacterial artificial chromosome (BAC) ends, and 106 sequence-tagged sites. The map was constructed by using the RHDF5000-2 whole-genome radiation hybrid panel and computed by using Multimap and TSP/Concorde. The 3,270 markers map to 3,021 unique positions and define an average intermarker distance corresponding to 1 Mb. We also define a minimal screening set of 325 highly informative well spaced markers, to be used in the initiation of genome-wide scans. The well defined synteny between the dog and human genomes, established in part as a function of this work by the identification of 85 conserved fragments, will allow follow-up of initial findings of linkage by selection of candidate genes from the human genome sequence. This work continues to define the canine system as the method of choice in the pursuit of the genes causing mammalian variation and disease.

In canine genetics, the impact of population structure on whole genome association studies is typically addressed by sampling approximately equal numbers of cases and controls from dogs of a single breed, usually from the same country or geographic area. However one way to increase the power of genetic studies is to sample individuals of the same breed but from different geographic areas, with the expectation that independent meiotic events will have shortened the presumed ancestral haplotype around the mutation differently. Little is known, however, about genetic variation among dogs of the same breed collected from different geographic regions. In this report, we address the magnitude and impact of genetic diversity among common breeds sampled in the U.S. and Europe. The breeds selected, including the Rottweiler, Bernese mountain dog, flat-coated retriever, and golden retriever, share susceptibility to a class of soft tissue cancers typified by malignant histiocytosis in the Bernese mountain dog. We genotyped 722 SNPs at four unlinked loci (between 95 and 271 per locus) on canine chromosome 1 (CFA1). We showed that each population is characterized by distinct genetic diversity that can be correlated with breed history. When the breed studied has a reduced intra-breed diversity, the combination of dogs from international locations does not increase the rate of false positives and potentially increases the power of association studies. However, over-sampling cases from one geographic location is more likely to lead to false positive results in breeds with significant genetic diversity. These data provide new guidelines for association studies using purebred dogs that take into account population structure.

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.

Mucosal melanoma (MM) is a rare, aggressive clinical cancer. Despite recent advances in genetics and treatment, the prognosis of MM remains poor. Canine MM offers a relevant spontaneous and immunocompetent model to decipher the genetic bases and explore treatments for MM. We performed an integrative genomic and transcriptomic analysis of 32 canine MM samples, which identified two molecular subgroups with a different microenvironment and structural variant (SV) content. The overexpression of genes related to the microenvironment and T-cell response was associated with tumors harboring a lower content of SVs, whereas the overexpression of pigmentation-related pathways and oncogenes, such as TERT, was associated with a high SV burden. Using whole-genome sequencing, we showed that focal amplifications characterized complex chromosomal rearrangements targeting oncogenes, such as MDM2 or CDK4, and a recurrently amplified region on canine chromosome 30. We also demonstrated that the genes TRPM7, GABPB1, and SPPL2A, located in this CFA30 region, play a role in cell proliferation, and thus, may be considered as new candidate oncogenes for human MM. Our findings suggest the existence of two MM molecular subgroups that may benefit from dedicated therapies, such as immune checkpoint inhibitors or targeted therapies, for both human and veterinary medicine.

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.