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Organoid cultures could constitute a valuable in vitro model to explore new treatments for canine (c) medullary thyroid carcinoma (MTC). The study's objectives were to establish and characterize 3D organoid cultures of cMTC using histology and immunohistochemistry (IHC) and to evaluate the effect of antitumor drugs on organoids' viability. Five cMTC tissue samples were used to develop organoid cultures of which one organoid line, named cMTC N°2, could be passaged for an extended period. This cMTC N°2 organoid line was further compared to the primary tumor regarding morphology and IHC expression of thyroid transcription factor-1 (TTF-1), thyroglobulin, calcitonin, synaptophysin, vimentin, Ki-67, cyclooxygenase-2 (COX-2), P-glycoprotein and vascular endothelial growth factor (VEGF). Quality control of the cMTC N°2 organoid line was achieved by a single nucleotide polymorphism (SNP) array of the organoids, primary tumor and healthy blood cells of the same dog. The effect of carboplatin, meloxicam and toceranib phosphate (TOC) on cMTC N°2 organoids' viability was evaluated. The cMTC N°2 organoid line was cultured for 94 days and showed similar histological features with the primary tumor. Immunolabeling for TTF-1, thyroglobulin, calcitonin and VEGF was similar between the primary tumor and cMTC N°2 organoids. Compared to the primary tumor, organoids showed higher immunolabeling for vimentin and Ki-67, and lower immunolabeling for synaptophysin, COX-2 and P-glycoprotein. The SNP genotype was similar for each chromosome between healthy blood cells, primary tumor and cMTC N°2 organoids. Carboplatin, meloxicam and TOC had no effect on cMTC N°2 organoid cell viability within achievable in vivo concentration range. In conclusion, the cMTC N°2 organoid line is a promising first milestone towards an established in vitro organoid model to explore pathophysiology and new treatment modalities in cMTC. This article is protected by copyright. All rights reserved.

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.

Distinctive colour patterns in dogs are an integral component of canine diversity. Colour pattern differences are thought to have arisen from mutation and artificial selection during and after domestication from wolves but important gaps remain in understanding how these patterns evolved and are genetically controlled. In other mammals, variation at the ASIP gene controls both the temporal and spatial distribution of yellow and black pigments. Here, we identify independent regulatory modules for ventral and hair cycle ASIP expression, and we characterize their action and evolutionary origin. Structural variants define multiple alleles for each regulatory module and are combined in different ways to explain five distinctive dog colour patterns. Phylogenetic analysis reveals that the haplotype combination for one of these patterns is shared with Arctic white wolves and that its hair cycle-specific module probably originated from an extinct canid that diverged from grey wolves more than 2 million years ago. Natural selection for a lighter coat during the Pleistocene provided the genetic framework for widespread colour variation in dogs and wolves., Dogs exhibit remarkable variation in colour patterns. Here, the authors identify structural variants of independent regulatory modules for ventral and hair cycle expression of the ASIP gene that explain five distinctive dog colour patterns and trace back the origin of one colour pattern to an extinct canid.

Canis lupus familiaris, the domestic dog, possesses a huge variability in traits such as size, conformation, coat color, or character, which reflects the generations of targeted human selection after the dog's domestication thousands of years ago. The phenotypic differences naturally reflect the underlying, often breed-specific, genetic variation. While heterogeneity between breeds is large, at the same time, the individuals within one breed are usually very homogeneous. This specific population structure of modern dog breeds with low genetic diversity favors the propagation of spontaneous occurrences of genetic mutations that might lead to the development of diseases, and thus makes the dog a valuable animal model. Inherited neurological disorders in animals as well as in human patients are incurable, often severe, and result in progressively worsening quality of life; early diagnosis is therefore beneficial for managing the disease development. DNA-based precision medicine using state-of-the-art methods, e.g. whole-genome sequencing (WGS) has been successfully utilized in recent years for routine diagnosis of rare diseases in human as well as in veterinary medicine. Identification of disease-causing variants allows dog breeders to avoid the spread of such variants in the affected dog breed, ultimately improving the health of the whole population through better breeding management, as well as to advance the understanding of the molecular mechanisms involved in corresponding human disease, and may be useful for the development of novel therapeutic strategies. In this thesis, I took part in the analysis of seven specific canine neurological phenotypes applying different genetic mapping methods, candidate gene analysis, and WGS. I also generated and analyzed extensive genealogical and genomic data on the worldwide Leonberger dog population in regards to its diversity and disease prevalence. Despite its increasing size in recent years, the population lost considerable genetic diversity due to a bottleneck in the last century. The heavy use of popular sires led to high relatedness among the breeding dogs and thus to high inbreeding rates. This facilitated undesirable genetic traits to spread within the gene pool of the Leonberger breed. A private homozygous frameshift variant in the GJA9 gene was identified in Leonbergers with an adult-onset form of polyneuropathy using genome-wide association study (GWAS) and WGS. The GJA9 gene encodes a connexin gap junction family protein, which are important components of peripheral myelinated nerve fibers; this discovery for the first time adds GJA9 to the list of candidate genes for similar human conditions. During a study of additional forms of polyneuropathy and/or laryngeal paralysis, I found a missense variant in the CNTNAP1 gene in Leonbergers and Saint Bernards showing early signs of laryngeal paralysis. CNTNAP1 encodes a contactin-associated protein important for the organization of myelinated axons and has been implicated in various forms of human neurological diseases. Interestingly, this variant was seen in several other unrelated dog breeds and most likely predates modern breed establishment. A similar approach revealed two independent variants in the NAPEPLD gene in Leonberger and Rottweiler dogs affected by leukoencephalomyelopathy providing evidence for allelic heterogeneity of this disorder and the first description of NAPEPLD-associated inherited defects in the endocannabinoid system associated with myelin disorders. In another study, a form of canine neuroaxonal dystrophy occurred in young adult Rottweiler dogs. WGS data of two cases revealed a homozygous missense variant in the VPS11 gene, encoding a member of VPS class C complex, a key factor of the endosome-autophagosome-lysosome pathway, previously associated with an infantile-onset neurological syndrome in humans. In a family of Alpine dachsbracke dogs, I used linkage analysis and homozygosity mapping to discover an autosomal recessive variant in the puppies affected by spinocerebellar ataxia, which affects the SCN8A gene. The gene encodes a subunit of a channel important for sodium ion transport to neurons in the central nervous system and was previously implicated in human neurogenetic conditions. By in-depth pedigree analysis, I found a common ancestor of two geographically separated families of Saluki dogs in which puppies suffering from succinic semialdehyde dehydrogenase deficiency causing neurological abnormalities were observed. GWAS and subsequent filtering of WGS data of two affected Saluki cases identified a causative variant in the ALDH5A1 gene encoding an essential enzyme of the gamma-aminobutyric acid neurotransmitter metabolic pathway. Finally, the underlying genetics of a previously described Leigh-like subacute necrotizing encephalopathy in Yorkshire terriers was solved by discovering a perfectly associated loss-of-function indel variant in the SLC19A3 gene encoding thiamine transporter 2, which is important in brain development, and its disruption was previously seen in similar human neurometabolic disease. In conclusion, the discovery of the herein described likely pathogenic DNA variants enabled systematic genetic testing of breeding dogs, and selection against the corresponding disorders to improve the health and welfare of the respective populations. This thesis provides molecular descriptions of several canine neurological conditions and presents additional physiologically relevant models of corresponding human diseases. Apparently, species- and site-specific differences in pathological phenotypes for mutations within the same gene exist as seen, e.g. in canine VPS11-related neuroaxonal dystrophy. All these spontaneous canine models closely resemble rare human syndromes and provide physiologically relevant models to better understand poorly characterized gene functions, e.g. in defects of the endocannabinoid system related to NAPEPLD; and provide potential new candidate genes for corresponding human forms of diseases with yet unsolved genetic etiology, e.g. GJA9-associated polyneuropathy. Therefore, this thesis demonstrates that genomic studies of domestic animal species such as the dog improve the understanding of rare complex and heterogeneous groups of neurodegenerative disorders.

Laryngeal paralysis associated with a generalized polyneuropathy (LPPN) most commonly exists in geriatric dogs from a variety of large and giant breeds. The purpose of this study was to discover the underlying genetic and molecular mechanisms in a younger-onset form of this neurodegenerative disease seen in two closely related giant dog breeds, the Leonberger and Saint Bernard. Neuropathology of an affected dog from each breed showed variable nerve fiber loss and scattered inappropriately thin myelinated fibers. Using across-breed genome-wide association, haplotype analysis, and whole-genome sequencing, we identified a missense variant in the CNTNAP1 gene (c.2810G>A; p.Gly937Glu) in which homozygotes in both studied breeds are affected. CNTNAP1 encodes a contactin-associated protein important for organization of myelinated axons. The herein described likely pathogenic CNTNAP1 variant occurs in unrelated breeds at variable frequencies. Individual homozygous mutant LPPN-affected Labrador retrievers that were on average four years younger than dogs affected by geriatric onset laryngeal paralysis polyneuropathy could be explained by this variant. Pathologic changes in a Labrador retriever nerve biopsy from a homozygous mutant dog were similar to those of the Leonberger and Saint Bernard. The impact of this variant on health in English bulldogs and Irish terriers, two breeds with higher CNTNAP1 variant allele frequencies, remains unclear. Pathogenic variants in CNTNAP1 have previously been reported in human patients with lethal congenital contracture syndrome and hypomyelinating neuropathy, including vocal cord palsy and severe respiratory distress. This is the first report of contactin-associated LPPN in dogs characterized by a deleterious variant that most likely predates modern breed establishment.

Severe oxalate nephropathy has been previously reported in sheep and is mostly associated with excessive oxalate in the diet. However, a rare native Dutch breed (Zwartbles) seems to be predisposed to an inherited juvenile form of primary hyperoxaluria and no causative genetic variant has been described so far. This study aims to characterize the phenotype and genetic etiology of the inherited metabolic disease observed in several purebred Zwartbles sheep. Affected animals present with a wide range of clinical signs including condition loss, inappetence, malaise, and, occasionally, respiratory signs, as well as an apparent sudden unexpected death. Histopathology revealed widespread oxalate crystal deposition in kidneys of the cases. Whole-genome sequencing of two affected sheep identified a missense variant in the ovine AGXT gene (c.584G&gt, A, p.Cys195Tyr). Variants in AGXT are known to cause type I primary hyperoxaluria in dogs and humans. Herein, we present evidence that the observed clinicopathological phenotype can be described as a form of ovine type I primary hyperoxaluria. This disorder is explained by a breed-specific recessively inherited pathogenic AGXT variant. Genetic testing enables selection against this fatal disorder in Zwartbles sheep as well as more precise diagnosis in animals with similar clinical phenotype. Our results have been incorporated in the Online Mendelian Inheritance in Animals (OMIA) database (OMIA 001672-9940).

Additional file 7: Figure S4. Proportion of dogs with a SNP within a ROH on each of the 38 autosomes. Individual Manhattan plots showing the percentage of 1203 dogs genotyped on the SNP array that share a SNP within a ROH for each canine autosome.

Additional file 10: Table S6. List of the potentially pathogenic variants for neurological disorders detected in 39 Leonberger dogs including the predictions of the effect of the variants on the proteins. The table includes detailed description and predictions of the biological consequences of the discovered variants on the corresponding proteins using different in silico prediction tools: PROVEAN [43], the MutPred suite (includes MutPred2 [44], MutPred-Indel [45], MutPred-LOF [46]), and PredictSNP [47].

Entropion is a known congenital disorder in sheep presumed to be heritable but no causative genetic variant has been reported. Affected lambs show a variable inward rolling of the lower eyelids leading to blindness in severe cases. In Switzerland, the Swiss White Alpine (SWA) breed showed a significantly higher prevalence for entropion than other breeds. A GWAS using 150 SWA sheep (90 affected lambs and 60 controls), based on 600k SNP data, revealed a genome-wide significant signal on chromosome 15. The 0.2 Mb associated region contains functional candidate genes, SMTNL1 and CTNND1. Pathogenic variants in human CTNND1 cause blepharocheilodontic syndrome 2, a rare disorder including eyelid anomalies, and SMTNL1 regulates contraction and relaxation of skeletal and smooth muscle. WGS of a single entropion-affected lamb revealed two private missense variants in SMTNL1 and CTNND1. Subsequent genotyping of both variants in 231 phenotyped SWA sheep was performed. The SMTNL1 variant p.(Asp452Asn) affects an evolutionary conserved residue within an important domain and represents a rare allele, which occurred also in controls. The p.(Glu943Lys) variant in CTNND1 represents a common variant unlikely to cause entropion as the mutant allele occurred more frequently in non-affected sheep. Therefore, we propose that these protein-changing variants are unlikely to explain the phenotype. Additionally, WGS of three further disconcordant pairs of full siblings was carried out but revealed no obvious causative variant. Finally, we conclude that entropion represents a more complex disease caused by different non-coding regulatory variants.

IGV snapshot indicating the translocation. Note the reduced coverage on chromosome 8 and the increased coverage on chromosome 14 in the affected piglet. Paired-end sequence reads mapping on two different chromosomes are displayed in different colors. (PDF 2757 kb)

Segregation of SNV alleles. In all 6 examined families for both parents alternative homozygous SNVs (red/blue) were selected to determine their inheritance pattern. Note that in all affected piglets paternal homozygous SNVs show an underrepresentation on chromosome 8 and an overrepresentation on chromosome 14. For SNVs where the dams were homozygous for the alternative allele, an opposite segregation could be observed. (PDF 2183 kb)