Molecular characterization of rare forms of canine neurological diseases as potential models for similar human diseases

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

A large deletion in the COL2A1 gene expands the spectrum of pathogenic variants causing bulldog calf syndrome in cattle.

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

Correction to: Genomic diversity and population structure of the Leonberger dog breed.

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

Multiple FGF4 retrocopies recently derived within canids

Two transcribed retrocopies of the fibroblast growth factor 4 (FGF4) gene have previously been described in the domestic dog. An FGF4 retrocopy on chr18 is associated with disproportionate dwarfism, while an FGF4 retrocopy on chr12 is associated with both disproportionate dwarfism and intervertebral disc disease (IVDD). In this study, whole-genome sequencing data were queried to identify other FGF4 retrocopies that could be contributing to phenotypic diversity in canids. Additionally, dogs with surgically confirmed IVDD were assayed for novel FGF4 retrocopies. Five additional and distinct FGF4 retrocopies were identified in canids including a copy unique to red wolves (Canis rufus). The FGF4 retrocopies identified in domestic dogs were identical to domestic dog FGF4 haplotypes, which are distinct from modern wolf FGF4 haplotypes, indicating that these retrotransposition events likely occurred after domestication. The identification of multiple, full length FGF4 retrocopies with open reading frames in canids indicates that gene retrotransposition events occur much more frequently than previously thought and provide a mechanism for continued genetic and phenotypic diversity in canids

Genomic Diversity and Runs of Homozygosity in Bernese Mountain Dogs

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

Whole Genome Sequencing Indicates Heterogeneity of Hyperostotic Disorders in Dogs

Craniomandibular osteopathy (CMO) and calvarial hyperostotic syndrome (CHS) are proliferative, non-neoplastic disorders affecting the skull bones in young dogs. Different forms of these hyperostotic disorders have been described in many dog breeds. However, an incompletely dominant causative variant for CMO affecting splicing of SLC37A2 has been reported so far only in three Terrier breeds. The purpose of this study was to identify further possible causative genetic variants associated with CHS in an American Staffordshire Terrier, as well as CMO in seven affected dogs of different breeds. We investigated their whole-genome sequences (WGS) and filtered variants using 584 unrelated genomes, which revealed no variants shared across all affected dogs. However, filtering for private variants of each case separately yielded plausible dominantly inherited candidate variants in three of the eight cases. In an Australian Terrier, a heterozygous missense variant in the COL1A1 gene (c.1786G>A; p.(Val596Ile)) was discovered. A pathogenic missense variant in COL1A1 was previously reported in humans with infantile cortical hyperostosis, or Caffey disease, resembling canine CMO. Furthermore, in a Basset Hound, a heterozygous most likely pathogenic splice site variant was found in SLC37A2 (c.1446+1G>A), predicted to lead to exon skipping as shown before in SLC37A2-associated canine CMO of Terriers. Lastly, in a Weimaraner, a heterozygous frameshift variant in SLC35D1 (c.1021_1024delTCAG; p.(Ser341ArgfsTer22)) might cause CMO due to the critical role of SLC35D1 in chondrogenesis and skeletal development. Our study indicates allelic and locus heterogeneity for canine CMO and illustrates the current possibilities and limitations of WGS-based precision medicine in dogs

Whole Genome Sequencing Indicates Heterogeneity of Hyperostotic Disorders in Dogs

Craniomandibular osteopathy (CMO) and calvarial hyperostotic syndrome (CHS) are proliferative, non-neoplastic disorders affecting the skull bones in young dogs. Different forms of these hyperostotic disorders have been described in many dog breeds. However, an incompletely dominant causative variant for CMO affecting splicing of SLC37A2 has been reported so far only in three Terrier breeds. The purpose of this study was to identify further possible causative genetic variants associated with CHS in an American Staffordshire Terrier, as well as CMO in seven affected dogs of different breeds. We investigated their whole-genome sequences (WGS) and filtered variants using 584 unrelated genomes, which revealed no variants shared across all affected dogs. However, filtering for private variants of each case separately yielded plausible dominantly inherited candidate variants in three of the eight cases. In an Australian Terrier, a heterozygous missense variant in the COL1A1 gene (c.1786G>A; p.(Val596Ile)) was discovered. A pathogenic missense variant in COL1A1 was previously reported in humans with infantile cortical hyperostosis, or Caffey disease, resembling canine CMO. Furthermore, in a Basset Hound, a heterozygous most likely pathogenic splice site variant was found in SLC37A2 (c.1446+1G>A), predicted to lead to exon skipping as shown before in SLC37A2-associated canine CMO of Terriers. Lastly, in a Weimaraner, a heterozygous frameshift variant in SLC35D1 (c.1021_1024delTCAG; p.(Ser341ArgfsTer22)) might cause CMO due to the critical role of SLC35D1 in chondrogenesis and skeletal development. Our study indicates allelic and locus heterogeneity for canine CMO and illustrates the current possibilities and limitations of WGS-based precision medicine in dogs

Independent COL17A1 Variants in Cats with Junctional Epidermolysis Bullosa.

Epidermolysis bullosa (EB), characterized by defective adhesion of the epidermis to the dermis, is a heterogeneous disease with many subtypes in human patients and domestic animals. We investigated two unrelated cats with recurring erosions and ulcers on ear pinnae, oral mucosa, and paw pads that were suggestive of EB. Histopathology confirmed the diagnosis of EB in both cats. Case 1 was severe and had to be euthanized at 5 months of age. Case 2 had a milder course and was alive at 11 years of age at the time of writing. Whole genome sequencing of both affected cats revealed independent homozygous variants in COL17A1 encoding the collagen type XVII alpha 1 chain. Loss of function variants in COL17A1 lead to junctional epidermolysis bullosa (JEB) in human patients. The identified splice site variant in case 1, c.3019+1del, was predicted to lead to a complete deficiency in collagen type XVII. Case 2 had a splice region variant, c.769+5G>A. Assessment of the functional impact of this variant on the transcript level demonstrated partial aberrant splicing with residual expression of wildtype transcript. Thus, the molecular analyses provided a plausible explanation of the difference in clinical severity between the two cases and allowed the refinement of the diagnosis in the affected cats to JEB. This study highlights the complexity of EB in animals and contributes to a better understanding of the genotype-phenotype correlation in COL17A1-related JEB

A living biobank of canine mammary tumor organoids as a comparative model for human breast cancer.

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

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