An Inflammatory Myopathy in the Dutch Kooiker Dog.

The Dutch Kooiker dog (het Nederlandse Kooikerhondje) is one of nine Dutch dog breeds. As of 1960, a number of heritable diseases have been noted in this breed. One is an inflammatory myopathy that emerged in 1972, with numbers of affected dogs gradually increasing during the last few decades. The objective of this paper is to describe clinical signs, laboratory results, electromyography and histopathology of the muscle biopsies of the affected dogs. Method: Both retrospectively as well as prospectively affected Kooiker dogs were identified and categorized using a Tiered level of Confidence. Results: In total, 160 Kooiker dogs-40 Tier I, 33 Tier II and 87 Tier III-were included. Clinical signs were (1) locomotory problems, such as inability to walk long distances, difficulty getting up, stiff gait, walking on eggshells; (2) dysphagia signs such as drooling, difficulty eating and/or drinking; or (3) combinations of locomotory and dysphagia signs. CK activities were elevated in all except for one dog. Histopathology revealed a predominant lymphohistiocytic myositis with a usually low and variable number of eosinophils, neutrophils and plasma cells. It is concluded that, within this breed, a most likely heritable inflammatory myopathy occurs. Further studies are needed to classify this inflammatory myopathy, discuss its treatment, and unravel the genetic cause of this disease to eradicate it from this population.

The Histopathological Characteristic of Gastric Carcinoma in the Belgian Tervueren and Groenendael Dog: A Comparison of Two Classification Methods.

Gastric carcinoma is generally considered to be a rare disease in dogs, carrying a grave prognosis. However, in the Tervueren and Groenendael varieties of the Belgian Shepherd dog breed, the disease is highly prevalent. While histopathology is the gold standard for diagnosing gastric carcinoma, there is no general consensus on the methods for histological classification in these cases. Biopsies of a group of 61 dogs with confirmed gastric carcinoma (45 Tervueren and 16 Groenendael) were examined and classified according to World Health Organization (WHO) and Laurén classifications. Kaplan-Meier curves were used to compare survival between the different subtypes and simple and multiple linear regression were used to analyse the association between age of onset and breed variant, sex, neuter status, location of the tumour, inflammation score, and Laurén and WHO classifications. Mean age at diagnosis was significantly different in Groenendael (10.1 ± 2.01) and Tervueren dogs (8.5 ± 1.90). The Laurén classification resulted in 29 (48%) diffuse- and 32 (52%) intestinal-type tumours. Applying the WHO classification resulted in 30 (49%) tubular carcinoma growth patterns and 31 (51%) others. Median survival time was significantly reduced for the diffuse type as compared to the intestinal type according to the Laurén classification, with the same median survival time results for tubular compared to non-tubular subtypes according to the WHO classification (median survival time of 61 vs. 182 days, respectively). Using the WHO and Lauren classification on tumour biopsies may help the practising clinician in the prognostication of gastric carcinoma in Tervueren and Groenendael dogs.

Corrigendum: Phenotypic characterization of idiopathic epilepsy and epilepsy of unknown cause in Irish Setters.

[This corrects the article DOI: 10.3389/fvets.2022.1066094.].

Phenotypic characterization of idiopathic epilepsy and epilepsy of unknown cause in Irish Setters.

Canine epileptic seizures are common neurological symptom presenting to veterinary practice. Idiopathic epilepsy (IE) with a suspected genetic background has been reported in several dog breeds. Although it has been reported in the Irish Setter (IS), the phenotypic characteristics have not yet been described. The aim of this study was to characterize the phenotype of IE in this breed and to trace its mode of inheritance. Owners of IS were requested to fill in a questionnaire via the Dutch Irish Setter Club concerning the epileptic seizures in their dogs. The data was assessed retrospectively using descriptive statistics. Forty-eight privately owned IS dogs fulfilling tier I criteria for IE according to the International Veterinary Epilepsy Task Force of both sexes were included in the study. The mean age of seizure onset was 41 months. Five of the dogs included in the study had an onset of seizures >6 years of age. These dogs were classified with epilepsy of unknown cause (EUC). Primary generalized tonic-clonic seizures were the most common type of seizure and were seen in almost all dogs. Cluster seizures were reported in 54% of the studied population. Most owners reported pre- (56%) and post-ictal (97%) signs in their dogs. A pedigree analysis of one subpopulation was performed and traced the lineage of 13 affected IS. A segregation analysis of this population rejected a simple autosomal recessive inheritance pattern. The present study supports the occurrence of IE and EUC in the IS. The results provide clinical insight into epileptic seizures in this breed and may be a starting point for further, including genetic, analysis.

Prevalence of Echocardiographic Evidence of Trace Mitral and Aortic Valve Regurgitation in 50 Clinically Healthy, Young Adult Labrador Retrievers without Heart Murmur.

Background-Though physiologic regurgitation of the right-sided cardiac valves is well recognized in dogs and other mammals, the prevalence of trace insufficiency of the mitral and aortic valves in clinically healthy, young adult dogs is unknown. Methods-In this observational cross-sectional study, 50 clinically healthy, young adult Labrador retrievers without an audible heart murmur were enrolled. All dogs were bred and owned by a single organization. Cardiac screening was requested for all dogs that were intended for breeding. These dogs underwent a cardiac auscultation and transthoracic echocardiography by a veterinary cardiology specialist. If mitral or aortic valve regurgitation was noticed, the jet size was subjectively assessed on color Doppler echocardiography. Pedigree analysis was performed to reveal a possible hereditary background of mitral valve regurgitation. Results-The prevalence of trivial mitral valve regurgitation was 52% with no significant predisposition to gender (p = 0.86) or haircoat color (p = 0.68). The prevalence of aortic valve regurgitation was 4%. Pedigree analysis for mitral valve regurgitation showed familial clustering, suggesting a hereditary background of the trait. Conclusions-The prevalence of silent trace mitral valve regurgitation in young adult Labrador retrievers was high. Because the regurgitant jet was trivial in all dogs, it is probably physiologic.

Multi-omic analyses in Abyssinian cats with primary renal amyloid deposits.

The amyloidoses constitute a group of diseases occurring in humans and animals that are characterized by abnormal deposits of aggregated proteins in organs, affecting their structure and function. In the Abyssinian cat breed, a familial form of renal amyloidosis has been described. In this study, multi-omics analyses were applied and integrated to explore some aspects of the unknown pathogenetic processes in cats. Whole-genome sequences of two affected Abyssinians and 195 controls of other breeds (part of the 99 Lives initiative) were screened to prioritize potential disease-associated variants. Proteome and miRNAome from formalin-fixed paraffin-embedded kidney specimens of fully necropsied Abyssinian cats, three affected and three non-amyloidosis-affected were characterized. While the trigger of the disorder remains unclear, overall, (i) 35,960 genomic variants were detected; (ii) 215 and 56 proteins were identified as exclusive or overexpressed in the affected and control kidneys, respectively; (iii) 60 miRNAs were differentially expressed, 20 of which are newly described. With omics data integration, the general conclusions are: (i) the familial amyloid renal form in Abyssinians is not a simple monogenic trait; (ii) amyloid deposition is not triggered by mutated amyloidogenic proteins but is a mix of proteins codified by wild-type genes; (iii) the form is biochemically classifiable as AA amyloidosis.

A CNTNAP1 Missense Variant Is Associated with Canine Laryngeal Paralysis and Polyneuropathy.

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.

Investigation of Genetic Modifiers of Copper Toxicosis in Labrador Retrievers.

Copper toxicosis is a complex genetic disorder in Labrador retrievers characterized by hepatic copper accumulation eventually leading to liver cirrhosis. The variation of hepatic copper levels in Labrador retrievers has been partly explained by mutations in ATP7A c.980C>T and ATP7B c.4358G>A. To further elucidate the genetic background of this disease, we used targeted Next Generation Sequencing (NGS) in a cohort of 95 Labrador retrievers to analyze 72 potential modifier genes for variations associated with hepatic copper levels. Variants associated with copper levels were subsequently evaluated in a replication cohort of 144 Labrador retrievers. A total of 44 variants in 25 different genes were identified, of which four showed significant association with copper levels. Of the four variants found associated with hepatic copper levels in the NGS cohort, one was validated in the replication cohort. The non-reference allele of the variant NC_006602.3.g.52434480C>T in RETN resulting in amino-acid change p.Leu7Phe was associated with decreased hepatic copper levels. In humans, resistin is associated with severity of non-alcoholic fatty liver disease, fibrosis, cirrhosis and mitochondrial dysfunction in hepatocytes. Further studies are needed to investigate the biological function of RETN p.Leu7Phe in the development of copper toxicosis in Labrador retrievers.

Mitochondrial PCK2 Missense Variant in Shetland Sheepdogs with Paroxysmal Exercise-Induced Dyskinesia (PED).

Four female Shetland Sheepdogs with hypertonic paroxysmal dyskinesia, mainly triggered by exercise and stress, were investigated in a retrospective multi-center investigation aiming to characterize the clinical phenotype and its underlying molecular etiology. Three dogs were closely related and their pedigree suggested autosomal dominant inheritance. Laboratory diagnostic findings included mild lactic acidosis and lactaturia, mild intermittent serum creatine kinase (CK) elevation and hypoglycemia. Electrophysiological tests and magnetic resonance imaging of the brain were unremarkable. A muscle/nerve biopsy revealed a mild type II fiber predominant muscle atrophy. While treatment with phenobarbital, diazepam or levetiracetam did not alter the clinical course, treatment with a gluten-free, home-made fresh meat diet in three dogs or a tryptophan-rich, gluten-free, seafood-based diet, stress-reduction, and acetazolamide or zonisamide in the fourth dog correlated with a partial reduction in, or even a complete absence of, dystonic episodes. The genomes of two cases were sequenced and compared to 654 control genomes. The analysis revealed a case-specific missense variant, c.1658G>A or p.Arg553Gln, in the PCK2 gene encoding the mitochondrial phosphoenolpyruvate carboxykinase 2. Sanger sequencing confirmed that all four cases carried the mutant allele in a heterozygous state. The mutant allele was not found in 117 Shetland Sheepdog controls and more than 500 additionally genotyped dogs from various other breeds. The p.Arg553Gln substitution affects a highly conserved residue in close proximity to the GTP-binding site of PCK2. Taken together, we describe a new form of paroxysmal exercise-induced dyskinesia (PED) in dogs. The genetic findings suggest that PCK2:p.Arg553Gln should be further investigated as putative candidate causal variant.

Association of the canine ATP7A and ATP7B with hepatic copper accumulation in Dobermann dogs.

BACKGROUND: Hepatic copper accumulation causes chronic hepatitis in dogs. Mutations in the copper transporters ATP7A and ATP7B were, respectively, associated with attenuation and enhancement of hepatic copper concentrations in Labrador Retrievers. There is a predisposition of Dobermanns to hepatitis with increased hepatic copper concentrations. OBJECTIVES: To investigate whether the ATP7A:c.980C>T and ATP7B:c.4358G>A mutations identified in Labrador Retrievers were associated with hepatic copper concentrations in Dobermanns. ANIMALS: Dobermanns from the Netherlands (n = 122) and the United States (n = 78). METHODS: In this retrospective study, mutations in ATP7A and ATP7B were investigated as risk factors for hepatic copper accumulation in Dobermanns. Liver biopsies of 200 Dobermanns were evaluated by histochemical copper staining, quantitative copper measurement, or both modalities. ATP7A and ATP7B genotypes were obtained by Kompetitive Allele Specific PCR. A linear regression model was used to investigate an association between genotype and hepatic copper concentrations. RESULTS: The ATP7A:c.980C>T was identified in both Dutch (2 heterozygous individuals) and American Dobermanns. In the American cohort, the minor allele frequency of the mutation was low (.081) and a possible effect on hepatic copper concentrations could not be established from this data set. A significant association of the ATP7B:c.4358G>A variant with increased hepatic copper concentrations in Dobermanns was observed. CONCLUSIONS AND CLINICAL IMPORTANCE: The ATP7B:c.4358G>A variant could be a contributor to hepatic copper accumulation underlying the risk of development of copper-associated hepatitis in breeds other than the Labrador Retriever.

Focal epilepsy with fear-related behavior as primary presentation in Boerboel dogs.

BACKGROUND: Focal seizures with fear as a primary ictal manifestation, their diagnostic challenges, and impact on quality of life are well described in human medicine. Reports focusing on ictal fear-like behavior in animals are scarce. OBJECTIVE: To describe the clinical and histopathological characteristics of a novel focal epilepsy in Boerboel dogs. ANIMALS: Five client-owned Boerboel littermates presented for evaluation of sudden episodes of severe fear-related behavior. METHODS: Clinical examination, complete blood cell count, routine blood biochemistry, and urinalysis were performed in all dogs. Magnetic resonance imaging (MRI) scans of the brain were performed in 3 affected Boerboels. In addition, in 2 affected Boerboels, metabolic screening, cerebrospinal fluid (CSF) analysis, and necropsy were performed. RESULTS: Onset of signs was 3 months of age in all affected Boerboels. All Boerboels howled loudly, had an extremely fearful facial expression and trembled during seizures. All affected Boerboels also had autonomic or motor signs. Results of laboratory investigations, diagnostic imaging, and metabolic screening were generally unremarkable. Histopathology showed moderate numbers of single large vacuoles in the perikaryon of neurons throughout the brain, specifically in the deeper cerebral cortical regions. Family history, pedigree analysis, and the homogenous phenotype were suggestive of autosomal recessive inheritance. CONCLUSIONS AND CLINICAL IMPORTANCE: The observed paroxysmal fear-related behavior represents a newly recognized hereditary focal epilepsy in dogs with distinctive clinical and histopathologic features. Veterinarians should be aware that sudden episodes of unusual behavior can represent focal epilepsy.

Current insights into the molecular genetic basis of dwarfism in livestock.

Impairment of bone growth at a young age leads to dwarfism in adulthood. Dwarfism can be categorised as either proportionate, an overall size reduction without changes in body proportions, or disproportionate, a size reduction in one or more limbs, with changes in body proportions. Many forms of dwarfism are inherited and result from structural disruptions or disrupted signalling pathways. Hormonal disruptions are evident in Brooksville miniature Brahman cattle and Z-linked dwarfism in chickens, caused by mutations in GH1 and GHR. Furthermore, mutations in IHH are the underlying cause of creeper achondroplasia in chickens. Belgian blue cattle display proportionate dwarfism caused by a mutation in RNF11, while American Angus cattle dwarfism is caused by a mutation in PRKG2. Mutations in EVC2 are associated with dwarfism in Japanese brown cattle and Tyrolean grey cattle. Fleckvieh dwarfism is caused by mutations in the GON4L gene. Mutations in COL10A1 and COL2A1 cause dwarfism in pigs and Holstein cattle, both associated with structural disruptions, while several mutations in ACAN are associated with bulldog-type dwarfism in Dexter cattle and dwarfism in American miniature horses. In other equine breeds, such as Shetland ponies and Friesian horses, dwarfism is caused by mutations in SHOX and B4GALT7. In Texel sheep, chondrodysplasia is associated with a deletion in SLC13A1. This review discusses genes known to be involved in these and other forms of dwarfism in livestock.

FEELnc: a tool for long non-coding RNA annotation and its application to the dog transcriptome.

Whole transcriptome sequencing (RNA-seq) has become a standard for cataloguing and monitoring RNA populations. One of the main bottlenecks, however, is to correctly identify the different classes of RNAs among the plethora of reconstructed transcripts, particularly those that will be translated (mRNAs) from the class of long non-coding RNAs (lncRNAs). Here, we present FEELnc (FlExible Extraction of LncRNAs), an alignment-free program that accurately annotates lncRNAs based on a Random Forest model trained with general features such as multi k-mer frequencies and relaxed open reading frames. Benchmarking versus five state-of-the-art tools shows that FEELnc achieves similar or better classification performance on GENCODE and NONCODE data sets. The program also provides specific modules that enable the user to fine-tune classification accuracy, to formalize the annotation of lncRNA classes and to identify lncRNAs even in the absence of a training set of non-coding RNAs. We used FEELnc on a real data set comprising 20 canine RNA-seq samples produced by the European LUPA consortium to substantially expand the canine genome annotation to include 10 374 novel lncRNAs and 58 640 mRNA transcripts. FEELnc moves beyond conventional coding potential classifiers by providing a standardized and complete solution for annotating lncRNAs and is freely available at https://github.com/tderrien/FEELnc.

Epidemiology, presentation and population genetics of patent ductus arteriosus (PDA) in the Dutch Stabyhoun dog.

BACKGROUND: Patent ductus arteriosus (PDA) is one of the most common congenital heart defects in dogs and is considered to be a complex, polygenic threshold trait for which a female sex predisposition has been described. Histological studies in dogs suggest that smooth muscle hypoplasia and asymmetry of the ductus tissue is the major cause of PDA. The Stabyhoun population is small and a predisposition for PDA has been suggested. The aims of this study were to describe the incidence, presentation from a clinical and histopathological perspective, and the population genetics of PDA in the Dutch Stabyhoun population. RESULTS: Forty-six cases were identified between 2000 and 2013. Between 2009 and 2012 the birth incidence of PDA in the Stabyhoun breed was 1.05 %. We estimated this to be 7-13 times higher than expected in the general dog population. Twelve of the 46 cases were part of a litter in which more than one sibling was affected. There was no sex predilection in our case cohort. Dogs diagnosed in adulthood showed severe cardiomegaly. The mean inbreeding coefficient of the reference population of Stabyhoun dogs was 31.4 % and the actual and effective numbers of founders were 14 and 6.5, respectively. The heritability of PDA was 0.51 (±0.09) for the reference population and 0.41 (±0.10) for the phenotyped population. Histopathology of sections of the PDA from two dogs showed findings similar to those described in other breeds although the smooth muscle of the ductus adjacent to the pulmonary artery appeared more hypoplastic than that in the ductus adjacent to the aorta. CONCLUSIONS: The Stabyhoun breed shows a strong predisposition for PDA. Apart from the absence of a higher incidence in females, no other significant features distinguish PDA in Stabyhouns from the condition in other dog breeds. Heritability and the mean inbreeding coefficient are both very high making the Dutch Stabyhoun breed particularly suited to the study of inherited risk factors for PDA.

Canine Models for Copper Homeostasis Disorders.

Copper is an essential trace nutrient metal involved in a multitude of cellular processes. Hereditary defects in copper metabolism result in disorders with a severe clinical course such as Wilson disease and Menkes disease. In Wilson disease, copper accumulation leads to liver cirrhosis and neurological impairments. A lack in genotype-phenotype correlation in Wilson disease points toward the influence of environmental factors or modifying genes. In a number of Non-Wilsonian forms of copper metabolism, the underlying genetic defects remain elusive. Several pure bred dog populations are affected with copper-associated hepatitis showing similarities to human copper metabolism disorders. Gene-mapping studies in these populations offer the opportunity to discover new genes involved in copper metabolism. Furthermore, due to the relatively large body size and long life-span of dogs they are excellent models for development of new treatment strategies. One example is the recent use of canine organoids for disease modeling and gene therapy of copper storage disease. This review addresses the opportunities offered by canine genetics for discovery of genes involved in copper metabolism disorders. Further, possibilities for the use of dogs in development of new treatment modalities for copper storage disorders, including gene repair in patient-derived hepatic organoids, are highlighted.

The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders.

The deleterious effects of a disrupted copper metabolism are illustrated by hereditary diseases caused by mutations in the genes coding for the copper transporters ATP7A and ATP7B. Menkes disease, involving ATP7A, is a fatal neurodegenerative disorder of copper deficiency. Mutations in ATP7B lead to Wilson disease, which is characterized by a predominantly hepatic copper accumulation. The low incidence and the phenotypic variability of human copper toxicosis hamper identification of causal genes or modifier genes involved in the disease pathogenesis. The Labrador retriever was recently characterized as a new canine model for copper toxicosis. Purebred dogs have reduced genetic variability, which facilitates identification of genes involved in complex heritable traits that might influence phenotype in both humans and dogs. We performed a genome-wide association study in 235 Labrador retrievers and identified two chromosome regions containing ATP7A and ATP7B that were associated with variation in hepatic copper levels. DNA sequence analysis identified missense mutations in each gene. The amino acid substitution ATP7B:p.Arg1453Gln was associated with copper accumulation, whereas the amino acid substitution ATP7A:p.Thr327Ile partly protected against copper accumulation. Confocal microscopy indicated that aberrant copper metabolism upon expression of the ATP7B variant occurred because of mis-localization of the protein in the endoplasmic reticulum. Dermal fibroblasts derived from ATP7A:p.Thr327Ile dogs showed copper accumulation and delayed excretion. We identified the Labrador retriever as the first natural, non-rodent model for ATP7B-associated copper toxicosis. Attenuation of copper accumulation by the ATP7A mutation sheds an interesting light on the interplay of copper transporters in body copper homeostasis and warrants a thorough investigation of ATP7A as a modifier gene in copper-metabolism disorders. The identification of two new functional variants in ATP7A and ATP7B contributes to the biological understanding of protein function, with relevance for future development of therapy.

Central Hypothyroidism in Miniature Schnauzers.

BACKGROUND: Primary hypothyroidism is a common endocrinopathy in dogs. In contrast, central hypothyroidism is rare in this species. OBJECTIVES: The objective of this article is to describe the occurrence and clinical presentation of central hypothyroidism in Miniature Schnauzers. Additionally, the possible role of the thyroid-stimulating hormone (TSH)-releasing hormone receptor (TRHR) gene and the TSHß (TSHB) gene was investigated. ANIMALS: Miniature Schnauzers with proven central hypothyroidism, based on scintigraphy, and the results of a 3-day-TSH-stimulation test, or a TSH-releasing hormone (TRH)-stimulation test or both, presented to the Department of Clinical Sciences of Companion Animals at Utrecht University or the Department of Medicine and Clinical Biology of Small Animals at Ghent University from 2008 to 2012. METHODS: Retrospective study. Pituitary function tests, thyroid scintigraphy, and computed tomography (CT) of the pituitary area were performed. Gene fragments of affected dogs and controls were amplified by polymerase chain reaction (PCR). Subsequently, the deoxyribonucleic acid (DNA) sequences of the products were analyzed. RESULTS: Central hypothyroidism was diagnosed in 7 Miniature Schnauzers. Three dogs had disproportionate dwarfism and at least one of them had a combined deficiency of TSH and prolactin. No disease-causing mutations were found in the TSHB gene and the exons of the TRHR gene of these Schnauzers. CONCLUSIONS AND CLINICAL IMPORTANCE: Central hypothyroidism could be underdiagnosed in Miniature Schnauzers with hypothyroidism, especially in those of normal stature. The fact that this rare disorder occurred in 7 dogs from the same breed suggests that central hypothyroidism could have a genetic background in Miniature Schnauzers.

Reference genes for reverse transcription quantitative PCR in canine brain tissue.

BACKGROUND: In the last decade canine models have been used extensively to study genetic causes of neurological disorders such as epilepsy and Alzheimer's disease and unravel their pathophysiological pathways. Reverse transcription quantitative polymerase chain reaction is a sensitive and inexpensive method to study expression levels of genes involved in disease processes. Accurate normalisation with stably expressed so-called reference genes is crucial for reliable expression analysis. RESULTS: Following the minimum information for publication of quantitative real-time PCR experiments precise guidelines, the expression of ten frequently used reference genes, namely YWHAZ, HMBS, B2M, SDHA, GAPDH, HPRT, RPL13A, RPS5, RPS19 and GUSB was evaluated in seven brain regions (frontal lobe, parietal lobe, occipital lobe, temporal lobe, thalamus, hippocampus and cerebellum) and whole brain of healthy dogs. The stability of expression varied between different brain areas. Using the GeNorm and Normfinder software HMBS, GAPDH and HPRT were the most reliable reference genes for whole brain. Furthermore based on GeNorm calculations it was concluded that as little as two to three reference genes are sufficient to obtain reliable normalisation, irrespective the brain area. CONCLUSIONS: Our results amend/extend the limited previously published data on canine brain reference genes. Despite the excellent expression stability of HMBS, GAPDH and HRPT, the evaluation of expression stability of reference genes must be a standard and integral part of experimental design and subsequent data analysis.

A nonsense mutation in B3GALNT2 is concordant with hydrocephalus in Friesian horses.

BACKGROUND: Hydrocephalus in Friesian horses is a developmental disorder that often results in stillbirth of affected foals and dystocia in dams. The occurrence is probably related to a founder effect and inbreeding in the population. The aim of our study was to find genomic associations, to investigate the mode of inheritance, to allow a DNA test for hydrocephalus in Friesian horses to be developed. In case of a monogenic inheritance we aimed to identify the causal mutation. RESULTS: A genome-wide association study of hydrocephalus in 13 cases and 69 controls using 29,720 SNPs indicated the involvement of a region on ECA1 (P <1.68 × 10(-6)). Next generation DNA sequence analysis of 4 cases and 6 controls of gene exons within the region revealed a mutation in ß-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2) as the likely cause of hydrocephalus in Friesian horses. The nonsense mutation XM_001491545 c.1423C>T corresponding to XP_001491595 p.Gln475* was identical to a B3GALNT2 mutation identified in a human case of muscular dystrophy-dystroglycanopathy with hydrocephalus. All 16 available cases and none of the controls were homozygous for the mutation, and all 17 obligate carriers (= dams of cases) were heterozygous. A random sample of the Friesian horse population (n = 865) was tested for the mutation in a commercial laboratory. One-hundred and forty-seven horses were carrier and 718 horses were homozygous for the normal allele; the estimated allele frequency in the Friesian horse population is 0.085. CONCLUSIONS: Hydrocephalus in Friesian horses has an autosomal recessive mode of inheritance. A nonsense mutation XM_001491545 c.1423C>T corresponding to XP_001491595 p.Gln475* in B3GALNT2 (1:75,859,296-75,909,376) is concordant with hydrocephalus in Friesian horses. Application of a DNA test in the breeding programme will reduce the losses caused by hydrocephalus in the Friesian horse population.

COLQ variant associated with Devon Rex and Sphynx feline hereditary myopathy.

Some Devon Rex and Sphynx cats have a variably progressive myopathy characterized by appendicular and axial muscle weakness, megaesophagus, pharyngeal weakness and fatigability with exercise. Muscle biopsies from affected cats demonstrated variable pathological changes ranging from dystrophic features to minimal abnormalities. Affected cats have exacerbation of weakness following anticholinesterase dosing, a clue that there is an underlying congenital myasthenic syndrome (CMS). A genome-wide association study and whole-genome sequencing suggested a causal variant for this entity was a c.1190G>A variant causing a cysteine to tyrosine substitution (p.Cys397Tyr) within the C-terminal domain of collagen-like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase (COLQ). Alpha-dystroglycan expression, which is associated with COLQ anchorage at the motor end-plate, has been shown to be deficient in affected cats. Eighteen affected cats were identified by genotyping, including cats from the original clinical descriptions in 1993 and subsequent publications. Eight Devon Rex and one Sphynx not associated with the study were identified as carriers, suggesting an allele frequency of ~2.0% in Devon Rex. Over 350 tested cats from other breeds did not have the variant. Characteristic clinical features and variant presence in all affected cats suggest a model for COLQ CMS. The association between the COLQ variant and this CMS affords clinicians the opportunity to confirm diagnosis via genetic testing and permits owners and breeders to identify carriers in the population. Moreover, accurate diagnosis increases available therapeutic options for affected cats based on an understanding of the pathophysiology and experience from human CMS associated with COLQ variants.