Characterization of the canine CLCN3 gene and evaluation as candidate for late-onset NCL

BACKGROUND: The neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of inherited progressive neurodegenerative diseases in different mammalian species. Tibetan Terrier and Polish Owczarek Nizinny (PON) dogs show rare late-onset NCL variants with autosomal recessive inheritance, which can not be explained by mutations of known human NCL genes. These dog breeds represent animal models for human late-onset NCL. In mice the chloride channel 3 gene (Clcn3) encoding an intracellular chloride channel was described to cause a phenotype similar to NCL. RESULTS: Two full-length cDNA splice variants of the canine CLCN3 gene are reported. The current canine whole genome sequence assembly was used for gene structure analyses and revealed 13 coding CLCN3 exons in 52 kb of genomic sequence. Sequence analysis of the coding exons and flanking intron regions of CLCN3 using six NCL-affected Tibetan terrier dogs and an NCL-affected Polish Owczarek Nizinny (PON) dog, as well as eight healthy Tibetan terrier dogs revealed 13 SNPs. No consistent CLCN3 haplotype was associated with NCL. CONCLUSION: For the examined animals we excluded the complete coding region and adjacent intronic regions of canine CLCN3 to harbor disease-causing mutations. Therefore it seems to be unlikely that a mutation in this gene is responsible for the late-onset NCL phenotype in these two dog breeds

Evaluation of an investigative model in dairy herds with high calf perinatal mortality rates in Switzerland

peer-reviewedThe objective of this study was to evaluate an investigative model which encompassed the risk factors, incidence, timing and causes of perinatal mortality (PM) (0–48 h) on high risk dairy farms (PM of >5% in the previous year) in Switzerland. This pilot-study was carried out on 47 predominantly Holstein PM calves from 21 dairy farms, between September 2016 and January 2018. Gross pathological examinations of calves and placentae as well as histopathological examinations of internal organs and placental tissue were performed. Further investigations included microbiological examinations: broad-spectrum bacterial and fungal culture, detection of Chlamydia abortus, Coxiella burnetii, pathogenic Leptospira spp. and Neospora caninum by real-time PCR (qPCR) and of bovine viral diarrhoea virus (BVDV) by Ag-ELISA. Maternal blood samples were used for serology of bovine herpesvirus 1 (BHV-1), Brucella abortus, Chlamydia abortus, Coxiella burnetii and nine pathogenic leptospiral serovars and the evaluation of trace element status. A questionnaire was completed with the farmer, which included general farm characteristics and case-related data. Inbreeding coefficients (IC) were calculated for pure-bred matings. At the farm-level, the PM rate was 10.0% (5.3–28.2%) and at the cow-level, 11.5%. These values, from high-risk farms, were approximately five-times higher than the contemporary national bovine PM rate (2.3%) in Switzerland. The risk factors associated with these high PM rates were the self-selection of high risk herds, the high proportion of primiparae in these herds (45%) and the evidence of widespread pathogenic infections on these farms (exposure: 67% of herds, 53% of dams; infection: 57% of herds, 45% of calves). The majority (68.1%) of calves died intrapartum. The most commonly diagnosed initiating/ultimate cause of death (UCOD) was infection (34%) of which Coxiella burnetii was the most frequently detected pathogen, by antigen. The most frequently diagnosed proximate cause of death (PCOD) was asphyxia (44.7%), though multiple PCOD was also common (21.3%). This study was the first detailed investigation of bovine PM in Switzerland. Infectious causes were diagnosed more frequently than expected. While the findings from these high PM Swiss herds may have limited external validity, the investigative model adopted and the detailed research methodologies employed can be replicated and re-evaluated, respectively, in future studies on PM internationally

A Missense Change in the ATG4D Gene Links Aberrant Autophagy to a Neurodegenerative Vacuolar Storage Disease

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10(-136)) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to themacroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.Peer reviewe

Selection Signatures in Worldwide Sheep Populations

The diversity of populations in domestic species offers great opportunities to study genome response to selection. The recently published Sheep HapMap dataset is a great example of characterization of the world wide genetic diversity in sheep. In this study, we re-analyzed the Sheep HapMap dataset to identify selection signatures in worldwide sheep populations. Compared to previous analyses, we made use of statistical methods that (i) take account of the hierarchical structure of sheep populations, (ii) make use of linkage disequilibrium information and (iii) focus specifically on either recent or older selection signatures. We show that this allows pinpointing several new selection signatures in the sheep genome and distinguishing those related to modern breeding objectives and to earlier post-domestication constraints. The newly identified regions, together with the ones previously identified, reveal the extensive genome response to selection on morphology, color and adaptation to new environments

Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep

Stefan Hiendleder is a member of the International Sheep Genomics ConsortiumIn sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal’s health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization–time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.Michael P. Heaton, Theodore S. Kalbfleisch, Dustin T. Petrik, Barry Simpson, James W. Kijas, Michael L. Clawson, Carol G. Chitko-McKown, Gregory P. Harhay, Kreg A. Leymaster, the International Sheep Genomics Consortiu

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

Dog colour patterns explained by modular promoters of ancient canid origin

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.Peer reviewe