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Mammary tumors in dogs hold great potential as naturally occurring breast cancer models in translational oncology, as they share the same environmental risk factors, key histological features, hormone receptor expression patterns, prognostic factors, and genetic characteristics as their human counterparts. We aimed to develop in vitro tools that allow functional analysis of canine mammary tumors (CMT), as we have a poor understanding of the underlying biology that drives the growth of these heterogeneous tumors. We established the long-term culture of 24 organoid lines from 16 dogs, including organoids derived from normal mammary epithelium or benign lesions. CMT organoids recapitulated key morphological and immunohistological features of the primary tissue from which they were derived, including hormone receptor status. Furthermore, genetic characteristics (driver gene mutations, DNA copy number variations, and single-nucleotide variants) were conserved within tumor-organoid pairs. We show how CMT organoids are a suitable model for in vitro drug assays and can be used to investigate whether specific mutations predict therapy outcomes. Specifically, certain CMT subtypes, such as PIK3CA mutated, estrogen receptor-positive simple carcinomas, can be valuable in setting up a preclinical model highly relevant to human breast cancer research. In addition, we could genetically modify the CMT organoids and use them to perform pooled CRISPR/Cas9 screening, where library representation was accurately maintained. In summary, we present a robust 3D in vitro preclinical model that can be used in translational research, where organoids from normal, benign as well as malignant mammary tissues can be propagated from the same animal to study tumorigenesis.

Bovine spastic syndrome (SS) is a progressive, adult-onset neuromuscular disorder (NMD). SS is inherited but the mode of inheritance is unclear. The aim of this study was to characterize the phenotype and to identify a possible genetic cause of SS by whole-genome sequencing (WGS) and focusing on protein-changing variants. Seven affected unrelated Holstein cattle of both sexes were referred for SS at a mean age of 5.3 years (S.D.±1.1) showing intermittent spasm of the skeletal muscles of the pelvic girdle. Assuming monogenic recessive inheritance, analysis of the WGS data did not reveal any private variants common to all cases. Searching for homozygous rare variants considering each case individually, allowed the identification of a rare recessive likely pathogenic missense variant in TOR3A for one case with an allele frequency of 1.69% in a global Holstein population. In the remaining six SS cases, we identified seven potentially dominant de novo mutations or inherited alleles as private heterozygous, mostly missense, variants of uncertain significance involving seven different NMD candidate genes: MPEG1 , LHX8 , WHAMM , NGRN , TTN , ATP1A1 , PCDH1 . All eight candidate causal variants identified were predicted to be deleterious. This study describes for the first time WGS findings in confirmed cases of bovine SS and provides evidence for a heterogeneous genetic cause of SS in cattle.

Background Chiari malformation type II (CMII) was originally reported in humans as a rare disorder characterized by the downward herniation of the hindbrain and towering cerebellum. The congenital brain malformation is usually accompanied by spina bifida, a congenital spinal anomaly resulting from incomplete closure of the dorsal aspect of the spinal neural tube, and occasionally by other lesions. A similar disorder has been reported in several animal species, including cattle, particularly as a congenital syndrome. A cause of congenital syndromic Chiari-like malformation (CSCM) in cattle has not been reported to date. We collected a series of 14 CSCM-affected Holstein calves (13 purebred, one Red Danish Dairy F1 cross) and performed whole-genome sequencing (WGS). WGS was performed on 33 cattle, including eight cases with parents (trio-based; group 1), three cases with one parent (group 2), and three single cases (solo-based; group 3). Results Sequencing-based genome-wide association study of the 13 Holstein calves with CSCM and 166 controls revealed no significantly associated genome region. Assuming a single Holstein breed-specific recessive allele, no region of shared homozygosity was detected suggesting heterogeneity. Subsequent filtering for protein-changing variants that were only homozygous in the genomes of the individual cases allowed the identification of two missense variants affecting different genes, SHC4 in case 4 in group 1 and WDR45B in case 13 in group 3. Furthermore, these two variants were only observed in Holstein cattle when querying WGS data of > 5,100 animals. Alternatively, potential de novo mutational events were assessed in each case. Filtering for heterozygous private protein-changing variants identified one DYNC1H1 frameshift variant as a candidate causal dominant acting allele in case 12 in group 3. Finally, the presence of larger structural DNA variants and chromosomal abnormalities was investigated in all cases. Depth of coverage analysis revealed two different partial monosomies of chromosome 2 segments in cases 1 and 7 in group 1 and a trisomy of chromosome 12 in the WDR45B homozygous case 13 in group 3. Conclusions This study presents for the first time a detailed genomic evaluation of CSCM in Holstein cattle and suggests an unexpected genetic and allelic heterogeneity considering the mode of inheritance, as well as the type of variant. For the first time, we propose candidate causal variants that may explain bovine CSCM in a certain proportion of affected calves. We present cattle as a large animal model for human CMII and propose new genes and genomic variants as possible causes for related diseases in both animals and humans.

Background Genetic skeletal disorders are a heterogeneous group of syndromic or non-syndromic diseases characterized by abnormal bone, joint or cartilage development. These disorders generally occur sporadically in ruminants. Although a genetic etiology is often suspected, only a limited number of causal variants have been identified and no comprehensive genetic analyses of a cohort of bovine and ovine skeletal developmental defects have been published. The aims of our study were (1) to propose a nosology of genetic skeletal disorders in cattle and sheep and (2) to contribute to the nosology with a number of novel genomically characterized cases. Results Based on a literature review, the proposed nosology of skeletal disorders in cattle and sheep with a confirmed molecular cause was found to comprise 43 different disorders associated with 45 different genes. In addition, horn traits were also included. The disorders were grouped into 21 categories based on the human medical nosology. Thirty novel bovine and nine ovine cases of congenital skeletal disorders were investigated. These represented 19 different disorders, which were grouped into 9 categories. Whole-genome sequencing (WGS) data were generated based on sample availability for either complete trios, affected paternal halfsiblings or isolated single cases. We identified 21 SNVs or small indels for 12 skeletal disorders. Of these, 17 were considered candidate variants affecting 16 different genes, including 11 that were classified as pathogenic and six as likely pathogenic. Additionally, the remaining 4 SNVs were of uncertain significance. Two aneuploidies (trisomy and partial monosomy) were the cause of two different disorders. For eight cases affected by six disorders no variant could be identified. Different modes of inheritance were detected, including spontaneous dominant de novo mutations, autosomal recessive alleles, an X-linked dominant allele, as well as aneuploidies. The overall molecular genetic diagnostic rate was 64%. Conclusions Genomic analysis revealed considerable heterogeneity of the described phenotypes in terms of mode of inheritance, affected genes, and variant type. We propose, for the first time in veterinary medicine, a nosology of genetic skeletal disorders in ruminants that may be useful for more precise differential clinicopathological diagnosis. We emphasize the potential of WGS to enhance genetic disease diagnosis and the importance of adopting a nosology for disease categorization.

Background Leonberger is a giant dog breed formed in the 1850s in Germany. Its post-World War II popularity has resulted in a current global population of ~ 30,000 dogs. The breed has predispositions to neurodegenerative disorders and cancer, which is likely due in large part to limited genetic diversity. However, to date there is no scientific literature on the overall demography and genomic architecture of this breed. Results We assessed extensive pedigree records, SNP array genotype data, and whole-genome sequences (WGS) on 142,072, 1203 and 39 Leonberger dogs, respectively. Pedigree analyses identified 22 founder animals and revealed an apparent popular sire effect. The average pedigree-based inbreeding coefficient of 0.29 and average kinship of 0.31 show a dramatic loss of genetic diversity. The observed average life span decreased over time from 9.4 years in 1989 to 7.7 years in 2004. A global health survey confirmed a high prevalence of cancer and neurological disorders. Analysis of SNP-based runs of homozygosity (ROH) identified 125,653 ROH with an average length of 5.88 Mb, and confirmed an average inbreeding coefficient of 0.28. Genome-wide filtering of the WGS data revealed 28 non-protein-changing variants that were present in all Leonberger individuals and a list of 22 potentially pathogenic variants for neurological disorders of which 50% occurred only in Leonbergers and 50% occurred rarely in other breeds. Furthermore, one of the two mtDNA haplogroups detected was present in one dog only. Conclusions The increasing size of the Leonberger population has been accompanied by a considerable loss of genetic diversity after the bottleneck that occurred in the 1940s due to the intensive use of popular sires resulting in high levels of inbreeding. This might explain the high prevalence of certain disorders; however, genomic data provide no evidence for fixed coding variants that explain these predispositions. The list of candidate causative variants for polyneuropathy needs to be further evaluated. Preserving the current genetic diversity is possible by increasing the number of individuals for breeding while restricting the number of litters per sire/dam. In addition, outcrossing would help optimize long-term genetic diversity and contribute to the sustainability and health of the population.

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.

Background Many inherited polyneuropathies (PN) observed in dogs have clinical similarities to the genetically heterogeneous group of Charcot-Marie-Tooth (CMT) peripheral neuropathies in humans. The canine disorders collectively show a variable expression of progressive clinical signs and ages of onset, and different breed prevalences. Previously in the Leonberger breed, a variant highly associated with a juvenile-onset PN was identified in the canine orthologue of a CMT-associated gene. As this deletion in ARHGEF10 (termed LPN1) does not explain all cases, PN in this breed may encompass variants in several genes with similar clinical and histopathological features. Results A genome-wide comparison of 173 k SNP genotypes of 176 cases, excluding dogs homozygous for the ARHGEF10 variant, and 138 controls, was carried out to detect further PN-associated variants. A single suggestive significant association signal on CFA15 was found. The genome of a PN-affected Leonberger homozygous for the associated haplotype was sequenced and variants in the 7.7 Mb sized critical interval were identified. These variants were filtered against a database of variants observed in 202 genomes of various dog breeds and 3 wolves, and 6 private variants in protein-coding genes, all in complete linkage disequilibrium, plus 92 non-coding variants were revealed. Five of the coding variants were predicted to have low or moderate effect on the encoded protein, whereas a 2 bp deletion in GJA9 results in a frameshift of high impact. GJA9 encodes connexin 59, a connexin gap junction family protein, and belongs to a group of CMT-associated genes that have emerged as important components of peripheral myelinated nerve fibers. The association between the GJA9 variant and PN was confirmed in an independent cohort of 296 cases and 312 controls. Population studies showed a dominant mode of inheritance, an average age of onset of approximately 6 years, and incomplete penetrance. Conclusions This GJA9 variant represents a highly probable candidate variant for another form of PN in Leonberger dogs, which we have designated LPN2, and a new candidate gene for CMT disease. To date, approximately every third PN-diagnosed Leonberger dog can be explained by the ARHGEF10 or GJA9 variants, and we assume that additional genetic heterogeneity in this condition exists in the breed.

Background Palatoschisis or cleft palate is a known anomaly in pigs resulting in their death. However, little is known about its aetiology. A detailed description of the phenotype was derived from necropsy and by computed tomography revealing that all 20 cases also exhibited hypodontia and renal cysts. Furthermore, a genetic origin was assumed due to dominant inheritance as all 20 recorded cases were confirmed offspring of a single boar. Results Single nucleotide variant (SNV) genotyping data were used to map the defect in the porcine genome and led to the detection of a chromosomal imbalance in the affected offspring. Whole genome sequencing of an affected piglet and a normal full sib was used to identify a chromosomal translocation and to fine map the breakpoints in the genome. Finally, we proved that the boar, which sired the malformed piglets, carried a balanced translocation. The detected translocation of Mb-sized segments of chromosome 8 and 14 had not been previously observed during karyotyping. All affected offspring were shown to be carriers of a partial trisomy of chromosome 14 including the FGFR2 gene, which is associated with various dominant inherited craniofacial dysostosis syndromes in man, and partial monosomy of chromosome 8 containing MSX1 known to be associated with tooth agenesis and orofacial clefts in other species. Conclusions This study illustrates the usefulness of recently established genomic resources in pigs. In this study, the application of genome-wide genotyping and sequencing methods allowed the identification of the responsible boar and the genetic cause of the observed defect. By implementing systematic surveillance, it is possible to identify genetic defects at an early stage and avoid further distribution of congenital disorders.

Background Congenital bovine chondrodysplasia, also known as bulldog calf syndrome, is characterized by disproportionate growth of bones resulting in a shortened and compressed body, mainly due to reduced length of the spine and the long bones of the limbs. In addition, severe facial dysmorphisms including palatoschisis and shortening of the viscerocranium are present. Abnormalities in the gene collagen type II alpha 1 chain ( COL2A1 ) have been associated with some cases of the bulldog calf syndrome. Until now, six pathogenic single-nucleotide variants have been found in COL2A1 . Here we present a novel variant in COL2A1 of a Holstein calf and provide an overview of the phenotypic and allelic heterogeneity of the COL2A1 -related bulldog calf syndrome in cattle. Case presentation The calf was aborted at gestation day 264 and showed generalized disproportionate dwarfism, with a shortened compressed body and limbs, and dysplasia of the viscerocranium; a phenotype resembling bulldog calf syndrome due to an abnormality in COL2A1 . Whole-genome sequence (WGS) data was obtained and revealed a heterozygous 3513 base pair deletion encompassing 10 of the 54 coding exons of COL2A1 . Polymerase chain reaction analysis and Sanger sequencing confirmed the breakpoints of the deletion and its absence in the genomes of both parents. Conclusions The pathological and genetic findings were consistent with a case of “bulldog calf syndrome”. The identified variant causing the syndrome was the result of a de novo mutation event that either occurred post-zygotically in the developing embryo or was inherited because of low-level mosaicism in one of the parents. The identified loss-of-function variant is pathogenic due to COL2A1 haploinsufficiency and represents the first structural variant causing bulldog calf syndrome in cattle. Furthermore, this case report highlights the utility of WGS-based precise diagnostics for understanding congenital disorders in cattle and the need for continued surveillance for genetic disorders in cattle.

An amendment to this paper has been published and can be accessed via the original article.