In sickness and health - a questionnaire based study regarding immune mediated diseases and neoplasia in Swedish Nova Scotia Duck Tolling Retrievers.

BACKGROUND: The Nova Scotia Duck Tolling Retriever (NSDTR) has previously been highlighted as a breed at risk for developing immune mediated diseases and cancer. The immune response is of great importance for the development of neoplastic disease and a dysregulated immune response may predispose to cancer. Two of the commonly seen immune mediated diseases in NSDTRs are immune mediated rheumatic disease (IMRD), which bears similarities to systemic lupus erythematosus (SLE) affecting humans, and steroid-responsive meningitis-arteritis (SRMA), which is a non-infectious inflammation of the meninges and the leptomeningeal vessels. The aim of this survey study was to investigate the lifetime prevalence of immune mediated diseases and tumors among Swedish NSDTRs based on owners' information. The study design was cross-sectional. A questionnaire was sent to 4102 persons who owned or had previously owned a NSDTR. The questions concerned information about the dog and its overall health status as well as specific diseases. RESULTS: The response rate was 30%, including 935 live NSDTRs, corresponding to approximately 20% of the current population registered in Sweden (n = 4564), and 177 dead dogs. The surveyed dogs were spread over different ages and sex and corresponded to the typical demographic profile of the general dog population. Of the 935 individuals that were alive, 28 dogs (3%) were reported as previously diagnosed with IMRD and 33 dogs (3.5%) were reported as previously diagnosed with SRMA, one dog was reported to have been diagnosed with both SRMA and IMRD. There were 129 dogs (14%) reported to have or have had a neoplasia of some kind. For the dead dogs (n = 177), almost 40% of the owners reported neoplasia as the main reason for death/euthanasia. CONCLUSION: This study reports an estimated lifetime prevalence of IMRD and SRMA, in the studied population of Swedish NSDTRs, of 3.0 and 3.5% respectively. In this study, 14% of the living dogs (n = 935) were reported to have a neoplasia of some kind and almost 40% of the deceased dogs (n = 177) were euthanized due to neoplasia or suspicion of it.

Hemangiosarcoma Cells Promote Conserved Host-derived Hematopoietic Expansion.

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors. SIGNIFICANCE: We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation, providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression.

Elevated levels of IL-12/IL-23p40 in Nova Scotia Duck Tolling Retrievers with autoimmune disease and lymphoma.

The Nova Scotia Duck Tolling Retriever (NSDTR) is predisposed to immune mediated rheumatic disease (IMRD), steroid-responsive meningitis-arteritis (SRMA) and certain forms of cancer. Cytokines are the main regulators of the immune system. Interleukin 2 is a cytokine involved in activation of T regulatory cells, playing a role in central tolerance and tumor immunity. Interleukin 12 and interleukin 23 share the same subunit, p40, and are both pro-inflammatory cytokines. The aim of this study was to compare levels of IL-2 in healthy NSDTRs to those with cancer or autoimmune disease and to compare levels of IL-12/IL-23p40 in healthy NSDTRs and beagles versus NSDTRs with cancer or autoimmune disease. 62 dogs were included in the analysis of IL-12/IL-23p40; healthy NSDTRs (n = 16), healthy beagles (n = 16), NSDTRs autoimmune (n = 18) and NDSTRs lymphoma/mastocytoma (n = 12) and 68 dogs for IL-2; healthy (n = 20), autoimmune (n = 36) and lymphoma/mastocytoma/adenocarcinoma (n = 12). NSDTRs with autoimmune disease had higher levels of IL-12/IL-23p40 compared to healthy dogs (p = 0.008). NSDTRs with lymphoma also had higher levels of IL-12/IL-23p40 compared to healthy NSDTRs (p = 0.002). There was no difference in levels of IL-2 between healthy and diseased NSDTR. Statistical analysis was performed using Bonferroni corrections for multiple testing. These findings can contribute to the knowledge of autoimmune disease and cancer in dogs.

The HLA region in ANCA-associated vasculitis: characterisation of genetic associations in a Scandinavian patient population.

OBJECTIVE: The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are inflammatory disorders with ANCA autoantibodies recognising either proteinase 3 (PR3-AAV) or myeloperoxidase (MPO-AAV). PR3-AAV and MPO-AAV have been associated with distinct loci in the human leucocyte antigen (HLA) region. While the association between MPO-AAV and HLA has been well characterised in East Asian populations where MPO-AAV is more common, studies in populations of European descent are limited. The aim of this study was to thoroughly characterise associations to the HLA region in Scandinavian patients with PR3-AAV as well as MPO-AAV. METHODS: Genotypes of single-nucleotide polymorphisms (SNPs) located in the HLA region were extracted from a targeted exome-sequencing dataset comprising Scandinavian AAV cases and controls. Classical HLA alleles were called using xHLA. After quality control, association analyses were performed of a joint SNP/classical HLA allele dataset for cases with PR3-AAV (n=411) and MPO-AAV (n=162) versus controls (n=1595). Disease-associated genetic variants were analysed for association with organ involvement, age at diagnosis and relapse, respectively. RESULTS: PR3-AAV was significantly associated with both HLA-DPB1*04:01 and rs1042335 at the HLA-DPB1 locus, also after stepwise conditional analysis. MPO-AAV was significantly associated with HLA-DRB1*04:04. Neither carriage of HLA-DPB1*04:01 alleles in PR3-AAV nor of HLA-DRB1*04:04 alleles in MPO-AAV were associated with organ involvement, age at diagnosis or relapse. CONCLUSIONS: The association to the HLA region was distinct in Scandinavian cases with MPO-AAV compared with cases of East Asian descent. In PR3-AAV, the two separate signals of association to the HLD-DPB1 region mediate potentially different functional effects.

Genome wide association study in Swedish Labrador retrievers identifies genetic loci associated with hip dysplasia and body weight.

Genome wide association studies (GWAS) have been utilized to identify genetic risk loci associated with both simple and complex inherited disorders. Here, we performed a GWAS in Labrador retrievers to identify genetic loci associated with hip dysplasia and body weight. Hip dysplasia scores were available for 209 genotyped dogs. We identified a significantly associated locus for hip dysplasia on chromosome 24, with three equally associated SNPs (p = 4.3 × 10-7) in complete linkage disequilibrium located within NDRG3, a gene which in humans has been shown to be differentially expressed in osteoarthritic joint cartilage. Body weight, available for 85 female dogs, was used as phenotype for a second analysis. We identified two significantly associated loci on chromosome 10 (p = 4.5 × 10-7) and chromosome 31 (p = 2.5 × 10-6). The most associated SNPs within these loci were located within the introns of the PRKCE and CADM2 genes, respectively. PRKCE has been shown to play a role in regulation of adipogenesis whilst CADM2 has been associated with body weight in multiple human GWAS. In summary, we identified credible candidate loci explaining part of the genetic inheritance for hip dysplasia and body weight in Labrador retrievers with strong candidate genes in each locus previously implicated in the phenotypes investigated.

Using evolutionary constraint to define novel candidate driver genes in medulloblastoma.

Current knowledge of cancer genomics remains biased against noncoding mutations. To systematically search for regulatory noncoding mutations, we assessed mutations in conserved positions in the genome under the assumption that these are more likely to be functional than mutations in positions with low conservation. To this end, we use whole-genome sequencing data from the International Cancer Genome Consortium and combined it with evolutionary constraint inferred from 240 mammals, to identify genes enriched in noncoding constraint mutations (NCCMs), mutations likely to be regulatory in nature. We compare medulloblastoma (MB), which is malignant, to pilocytic astrocytoma (PA), a primarily benign tumor, and find highly different NCCM frequencies between the two, in agreement with the fact that malignant cancers tend to have more mutations. In PA, a high NCCM frequency only affects the BRAF locus, which is the most commonly mutated gene in PA. In contrast, in MB, >500 genes have high levels of NCCMs. Intriguingly, several loci with NCCMs in MB are associated with different ages of onset, such as the HOXB cluster in young MB patients. In adult patients, NCCMs occurred in, e.g., the WASF-2/AHDC1/FGR locus. One of these NCCMs led to increased expression of the SRC kinase FGR and augmented responsiveness of MB cells to dasatinib, a SRC kinase inhibitor. Our analysis thus points to different molecular pathways in different patient groups. These newly identified putative candidate driver mutations may aid in patient stratification in MB and could be valuable for future selection of personalized treatment options.

Leveraging base-pair mammalian constraint to understand genetic variation and human disease.

Thousands of genomic regions have been associated with heritable human diseases, but attempts to elucidate biological mechanisms are impeded by an inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function, agnostic to cell type or disease mechanism. Single-base phyloP scores from 240 mammals identified 3.3% of the human genome as significantly constrained and likely functional. We compared phyloP scores to genome annotation, association studies, copy-number variation, clinical genetics findings, and cancer data. Constrained positions are enriched for variants that explain common disease heritability more than other functional annotations. Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Leveraging Base Pair Mammalian Constraint to Understand Genetic Variation and Human Disease.

Although thousands of genomic regions have been associated with heritable human diseases, attempts to elucidate biological mechanisms are impeded by a general inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function that is agnostic to cell type or disease mechanism. Here, single base phyloP scores from the whole genome alignment of 240 placental mammals identified 3.5% of the human genome as significantly constrained, and likely functional. We compared these scores to large-scale genome annotation, genome-wide association studies (GWAS), copy number variation, clinical genetics findings, and cancer data sets. Evolutionarily constrained positions are enriched for variants explaining common disease heritability (more than any other functional annotation). Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Multiple Genetic Loci Associated with Pug Dog Thoracolumbar Myelopathy.

Pug dogs with thoracolumbar myelopathy (PDM) present with a specific clinical phenotype that includes progressive pelvic limb ataxia and paresis, commonly accompanied by incontinence. Vertebral column malformations and lesions, excessive scar tissue of the meninges, and central nervous system inflammation have been described. PDM has a late onset and affects more male than female dogs. The breed-specific presentation of the disorder suggests that genetic risk factors are involved in the disease development. To perform a genome-wide search for PDM-associated loci, we applied a Bayesian model adapted for mapping complex traits (BayesR) and a cross-population extended haplotype homozygosity test (XP-EHH) in 51 affected and 38 control pugs. Nineteen associated loci (harboring 67 genes in total, including 34 potential candidate genes) and three candidate regions under selection (with four genes within or next to the signal) were identified. The multiple candidate genes identified have implicated functions in bone homeostasis, fibrotic scar tissue, inflammatory responses, or the formation, regulation, and differentiation of cartilage, suggesting the potential relevance of these processes to the pathogenesis of PDM.

PIK3CA is recurrently mutated in canine mammary tumors, similarly to in human mammary neoplasia.

Biological features of neoplastic disease affecting mammary gland tissue are shared between canines and humans. Research performed in either species has translational value and early phase clinical trials performed in canines with spontaneous disease could be informative for human trials. The purpose of this study was to investigate the somatic genetic aberrations occurring in canine mammary neoplasia by exome capture and next generation sequencing. Based on 55 tumor-normal pairs we identified the PIK3CA gene as the most commonly mutated gene in canine mammary tumors, with 25% of samples carrying mutations in this gene. A recurrent missense mutation was identified, p.H1047R, which is homologous to the human PIK3CA hotspot mutation found in different types of breast neoplasia. Mutations homologous to other known human mutation hotspots such as the PIK3CA p.E545K and the KRAS p.G12V/D were also identified. We identified copy number aberrations affecting important tumor suppressor and oncogenic pathways including deletions affecting the PTEN tumor suppressor gene. We suggest that activation of the KRAS or PIK3CA oncogenes or loss of the PTEN suppressor gene may be important for mammary tumor development in dogs. This data endorses the conservation of cancer across species and the validity of studying cancer in non-human species.

Mer-tyrosine kinase: a novel susceptibility gene for SLE related end-stage renal disease.

OBJECTIVE: Lupus nephritis (LN) is a common and severe manifestation of SLE. The genetic risk for nephritis and progression to end-stage renal disease (ESRD) in patients with LN remains unclear. Herein, we aimed to identify novel genetic associations with LN, focusing on subphenotypes and ESRD. METHODS: We analysed genomic data on 958 patients with SLE (discovery cohort: LN=338) with targeted sequencing data from 1832 immunological pathway genes. We used an independent multiethnic cohort comprising 1226 patients with SLE (LN=603) as a replication dataset. Detailed functional annotation and functional epigenomic enrichment analyses were applied to predict functional effects of the candidate variants. RESULTS: A genetic variant (rs56097910) within the MERTK gene was associated with ESRD in both cohorts, meta-analysis OR=5.4 (2.8 to 10.6); p=1.0×10-6. We observed decreased methylation levels in peripheral blood cells from SLE patients with ESRD, compared with patients without renal SLE (p=2.7×10-4), at one CpG site (cg16333401) in close vicinity to the transcription start site of MERTK and located in a DNAse hypersensitivity region in T and B cells. Rs56097910 is linked to altered MERTK expression in kidney tissue in public eQTL databases. Two loci were replicated for association with proliferative LN: PRDM1 (rs6924535, pmeta=1.6×10-5, OR=0.58) and APOA1BP (NAXE) (rs942960, pmeta=1.2×10-5, OR=2.64). CONCLUSION: We identified a novel genetic risk locus, MERTK, associated with SLE-ESRD using the data from two large SLE cohorts. Through DNA methylation analysis and functional annotation, we showed that the risk could be mediated through regulation of gene expression. Our results suggest that variants in the MERTK gene are important for the risk of developing SLE-ESRD and suggest a role for PRDM1 and APOA1BP in proliferative LN.

Canine Oral Melanoma Genomic and Transcriptomic Study Defines Two Molecular Subgroups with Different Therapeutical Targets.

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.

Correction: The genetic consequences of dog breed formation-Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels.

[This corrects the article DOI: 10.1371/journal.pgen.1009726.].

Identification and functional characterization of a novel susceptibility locus for small vessel vasculitis with MPO-ANCA.

OBJECTIVE: To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV). METHODS: Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay. RESULTS: PR3-ANCA+ AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 × 10-61, odds ratio (OR) 0.10; rs9277341, P = 1.5 × 10-44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 × 10-10, OR 2.9). MPO-ANCA+ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 × 10-25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 × 10-7, OR 3.0), the latter a novel susceptibility locus for MPO-ANCA+ granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele. CONCLUSION: We identified a novel susceptibility locus for MPO-ANCA+ AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types.

Genome-Wide Analyses for Osteosarcoma in Leonberger Dogs Reveal the CDKN2A/B Gene Locus as a Major Risk Locus.

Dogs represent a unique spontaneous cancer model. Osteosarcoma (OSA) is the most common primary bone tumor in dogs (OMIA 001441-9615), and strongly resembles human forms of OSA. Several large- to giant-sized dog breeds, including the Leonberger, have a greatly increased risk of developing OSA. We performed genome-wide association analysis with high-density imputed SNP genotype data from 273 Leonberger cases with a median age of 8.1 [3.1-13.5] years and 365 controls older than eight years. This analysis revealed significant associations at the CDKN2A/B gene locus on canine chromosome 11, mirroring previous findings in other dog breeds, such as the greyhound, that also show an elevated risk for OSA. Heritability (h2SNP) was determined to be 20.6% (SE = 0.08; p-value = 5.7 × 10-4) based on a breed prevalence of 20%. The 2563 SNPs across the genome accounted for nearly all the h2SNP of OSA, with 2183 SNPs of small effect, 316 SNPs of moderate effect, and 64 SNPs of large effect. As with many other cancers it is likely that regulatory, non-coding variants underlie the increased risk for cancer development. Our findings confirm a complex genetic basis of OSA, moderate heritability, and the crucial role of the CDKN2A/B locus leading to strong cancer predisposition in dogs. It will ultimately be interesting to study and compare the known genetic loci associated with canine OSA in human OSA.

The genetic consequences of dog breed formation-Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels.

Selective breeding for desirable traits in strictly controlled populations has generated an extraordinary diversity in canine morphology and behaviour, but has also led to loss of genetic variation and random entrapment of disease alleles. As a consequence, specific diseases are now prevalent in certain breeds, but whether the recent breeding practice led to an overall increase in genetic load remains unclear. Here we generate whole genome sequencing (WGS) data from 20 dogs per breed from eight breeds and document a ~10% rise in the number of derived alleles per genome at evolutionarily conserved sites in the heavily bottlenecked cavalier King Charles spaniel breed (cKCs) relative to in most breeds studied here. Our finding represents the first clear indication of a relative increase in levels of deleterious genetic variation in a specific breed, arguing that recent breeding practices probably were associated with an accumulation of genetic load in dogs. We then use the WGS data to identify candidate risk alleles for the most common cause for veterinary care in cKCs-the heart disease myxomatous mitral valve disease (MMVD). We verify a potential link to MMVD for candidate variants near the heart specific NEBL gene in a dachshund population and show that two of the NEBL candidate variants have regulatory potential in heart-derived cell lines and are associated with reduced NEBL isoform nebulette expression in papillary muscle (but not in mitral valve, nor in left ventricular wall). Alleles linked to reduced nebulette expression may hence predispose cKCs and other breeds to MMVD via loss of papillary muscle integrity.

3D genomics across the tree of life reveals condensin II as a determinant of architecture type.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

Genomically Complex Human Angiosarcoma and Canine Hemangiosarcoma Establish Convergent Angiogenic Transcriptional Programs Driven by Novel Gene Fusions.

Sporadic angiosarcomas are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic angiosarcomas. In this study, we leveraged RNA-sequencing data from 13 human angiosarcomas and 76 spontaneous canine hemangiosarcomas to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in seven of the 13 human tumors, with two tumors showing mutations of TP53. HRAS and NRAS mutations were found in angiosarcomas without fusions or TP53 mutations. We found 15 novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 in 11 of the 76 canine hemangiosarcomas; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS. In particular, fusion genes and mutations of TP53 cooccurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human angiosarcomas and canine hemangiosarcomas identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data suggest that genome instability induced by TP53 mutations might create a predisposition for fusion events that may contribute to tumor progression by promoting selection and/or enhancing fitness through activation of convergent angiogenic pathways in this vascular malignancy. IMPLICATIONS: This study shows that, while drive events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphologic organization and biological behavior of these tumors in both species.

A novel canine reference genome resolves genomic architecture and uncovers transcript complexity.

We present GSD_1.0, a high-quality domestic dog reference genome with chromosome length scaffolds and contiguity increased 55-fold over CanFam3.1. Annotation with generated and existing long and short read RNA-seq, miRNA-seq and ATAC-seq, revealed that 32.1% of lifted over CanFam3.1 gaps harboured previously hidden functional elements, including promoters, genes and miRNAs in GSD_1.0. A catalogue of canine "dark" regions was made to facilitate mapping rescue. Alignment in these regions is difficult, but we demonstrate that they harbour trait-associated variation. Key genomic regions were completed, including the Dog Leucocyte Antigen (DLA), T Cell Receptor (TCR) and 366 COSMIC cancer genes. 10x linked-read sequencing of 27 dogs (19 breeds) uncovered 22.1 million SNPs, indels and larger structural variants. Subsequent intersection with protein coding genes showed that 1.4% of these could directly influence gene products, and so provide a source of normal or aberrant phenotypic modifications.