Bart Broeckx

Development and performance of a targeted whole exome sequencing enrichment kit for the dog (Canis Familiaris Build 3.1)

Whole exome sequencing is a technique that aims to selectively sequence all exons of protein-coding genes. A canine whole exome sequencing enrichment kit was designed based on the latest canine reference genome (build 3.1.72). Its performance was tested by sequencing 2 exome captures, each consisting of 4 pre-capture pooled, barcoded Illumina libraries on an Illumina HiSeq 2500. At an average sequencing depth of 102x, 83 to 86% of the target regions were completely sequenced with a minimum coverage of five and 90% of the reads mapped on the target regions. Additionally, it is shown that the reproducibility within and between captures is high and that pooling four samples per capture is a valid option. Overall, we have demonstrated the strong performance of this WES enrichment kit and are confident it will be a valuable tool in future disease association studies.

Improved canine exome designs, featuring ncRNAs and increased coverage of protein coding genes

By limiting sequencing to those sequences transcribed as mRNA, whole exome sequencing is a cost-efficient technique often used in disease-association studies. We developed two target enrichment designs based on the recently released annotation of the canine genome: the exome-plus design and the exome-CDS design. The exome-plus design combines the exons of the CanFam 3.1 Ensembl annotation, more recently discovered protein-coding exons and a variety of non-coding RNA regions (microRNAs, long non-coding RNAs and antisense transcripts), leading to a total size of ≈152 Mb. The exome-CDS was designed as a subset of the exome-plus by omitting all 3’ and 5’ untranslated regions. This reduced the size of the exome-CDS to ≈71 Mb. To test the capturing performance, four exome-plus captures were sequenced on a NextSeq 500 with each capture containing four pre-capture pooled, barcoded samples. At an average sequencing depth of 68.3x, 80% of the regions and well over 90% of the targeted base pairs were completely covered at least 5 times with high reproducibility. Based on the performance of the exome-plus, we estimated the performance of the exome-CDS. Overall, these designs provide flexible solutions for a variety of research questions and are likely to be reliable tools in disease studies.

Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores.

Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca(2+) signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca(2+) signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca(2+) signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca(2+) stores, thereby leaving less Ca(2+) available for release upon IP3- and ryanodine-receptor activation. The Ca(2+)-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca(2+) release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression.

Intra‐ and Interobserver Agreement on Radiographic Phenotype in the Diagnosis of Canine Hip Dysplasia

OBJECTIVE To investigate the repeatability and reproducibility of the presence of a circumferential femoral head osteophyte (CFHO), a curvilinear caudolateral osteophyte (CCO), osteosclerosis of the cranial acetabular edge (Scler CrAE), degenerative joint disease (DJD), and the diagnosis of suspected canine hip dysplasia (CHD) in different groups of experienced observers. STUDY DESIGN Cross-sectional study. SAMPLE POPULATION Standard hip extended radiographs (n = 50). METHODS Nine experienced observers were divided into 3 groups: surgeons (DECVS), radiologists (DECVDI), and non-board certified observers (NBC) and 2 subgroups (academics and non-academics). Cohens kappa (κ) was calculated for CFHO, CCO, Scler CrAE, DJD, and suspected CHD, and weighted κ was calculated for DJD score to determine inter- and intraobserver agreement. RESULTS Intraobserver agreement on CFHO, CCO, Scler CrAE, DJD, and suspected CHD ranged from slight to almost perfect, but was not significantly different between NBC, DECVS, and DECVDI. Radiologists and non-board certified observers had a more uniform scoring than surgeons on the overall DJD score, as did academics versus non-academics. Interobserver agreement for NBC was more uniform than that of radiologists and surgeons on CCO and DJD. NBC and radiologists scored more uniformly than surgeons on CFHO, and radiologists scored more uniformly than NBC and surgeons on Scler CrAE. Academics scored more uniformly than non-academics, but only significantly for Scler CrAE. CONCLUSIONS Recognition of specific radiographic markers is only fairly reliable within and between experienced observers. Therefore, care must be taken to apply these traits in official screening, surgical decision-making and scientific research.

The Prevalence of Nine Genetic Disorders in a Dog Population from Belgium, the Netherlands and Germany

The objective of this study was to screen a dog population from Belgium, the Netherlands and Germany for the presence of mutant alleles associated with hip dysplasia (HD), degenerative myelopathy (DM), exercise-induced collapse (EIC), neuronal ceroid lipofuscinosis 4A (NCL), centronuclear myopathy (HMLR), mucopolysaccharidosis VII (MPS VII), myotonia congenita (MG), gangliosidosis (GM1) and muscular dystrophy (Duchenne type) (GRMD). Blood samples (K3EDTA) were collected for genotyping with Kompetitive Allele Specific PCR (n = 476). Allele and genotype frequencies were calculated in those breeds with at least 12 samples (n = 8). Hardy-Weinberg equilibrium was tested. Genetic variation was identified for 4 out of 9 disorders: mutant alleles were found in 49, 15, 3 and 2 breeds for HD, DM, EIC and NCL respectively. Additionally, mutant alleles were identified in crossbreeds for both HD and EIC. For HD, DM, EIC and NCL mutant alleles were newly discovered in 43, 13, 2 and 1 breed(s), respectively. In 9, 2 and 1 breed(s) for DM, EIC and NCL respectively, the mutant allele was detected, but the respective disorder has not been reported in those breeds. For 5 disorders (HMLR, MPS VII, MG, GM1, GRMD), the mutant allele could not be identified in our population. For the other 4 disorders (HD, DM, EIC, NCL), prevalence of associated mutant alleles seems strongly breed dependent. Surprisingly, mutant alleles were found in many breeds where the disorder has not been reported to date.

Phenobarbital or potassium bromide as an add-on antiepileptic drug for the management of canine idiopathic epilepsy refractory to imepitoin

Imepitoin has recently been approved in Europe for the management of dogs with idiopathic epilepsy. Currently, there is no evidence-based information available on the efficacy of antiepileptic drugs used as additions to the therapeutic regimen in dogs with idiopathic epilepsy that are not well controlled with imepitoin. The goal of this study was to evaluate the efficacy of phenobarbital or potassium bromide (KBr) as add-on antiepileptic drugs for controlling dogs refractory to a maximum dose of imepitoin (30 mg/kg twice daily). The study was performed as a prospective, randomised, controlled clinical trial. The efficacy of phenobarbital and KBr was evaluated by comparing monthly seizure frequency (MSF), monthly seizure day frequency (MSDF), the presence of cluster seizures during a retrospective 2-month period with a prospective follow-up of 6 months, and the overall responder rate. Twenty-seven dogs were included in the study, 14 dogs in the phenobarbital group and 13 dogs in the KBr group. Both median MSF and MSDF decreased in the phenobarbital group (both P = 0.001) and in the KBr group (P = 0.004 and P = 0.003, respectively). Overall, the number of dogs with cluster seizures decreased (P = 0.0005). The responder rate was 79% vs. 69% in the phenobarbital and KBr groups, respectively. We conclude that phenobarbital or KBr add-on treatment decreases median MSF and MSDF in epileptic dogs refractory to a maximum dose of imepitoin. Combination therapy was generally well tolerated and resulted in an improvement in seizure management in the majority of the dogs.

Lack of Ubiquitin Specific Protease 8 (USP8) Mutations in Canine Corticotroph Pituitary Adenomas

Purpose Cushing’s disease (CD), also known as pituitary-dependent hyperadrenocorticism, is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumours. Affected humans and dogs have similar clinical manifestations, however, the incidence of the canine disease is thousand-fold higher. This makes the dog an obvious model for studying the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite certain similarities identified at the molecular level, the question still remains whether the two species have a shared oncogenetic background. Recently, hotspot recurrent mutations in the gene encoding for ubiquitin specific protease 8 (USP8) have been identified as the main driver behind the formation of ACTH-secreting pituitary adenomas in humans. In this study, we aimed to verify whether USP8 mutations also play a role in the development of such tumours in dogs. Methods Presence of USP8 mutations was analysed by Sanger and PCR-cloning sequencing in 38 canine ACTH-secreting adenomas. Furthermore, the role of USP8 and EGFR protein expression was assessed by immunohistochemistry in a subset of 25 adenomas. Results None of the analysed canine ACTH-secreting adenomas presented mutations in the USP8 gene. In a subset of these adenomas, however, we observed an increased nuclear expression of USP8, a phenotype characteristic for the USP8 mutated human tumours, that correlated with smaller tumour size but elevated ACTH production in those tumours. Conclusions Canine ACTH-secreting pituitary adenomas lack mutations in the USP8 gene suggesting a different genetic background of pituitary tumourigenesis in dogs. However, elevated nuclear USP8 protein expression in a subset of tumours was associated with a similar phenotype as in their human counterparts, indicating a possible end-point convergence of the different genetic backgrounds in the two species. In order to establish the dog as a useful animal model for the study of CD, further comprehensive studies are needed.

Low-Field MRI and Multislice CT for the Detection of Cerebellar (Foramen Magnum) Herniation in Cavalier King Charles Spaniels

Background Cavalier King Charles Spaniels (CKCS) have a high prevalence of Chiari‐like malformation (CM). Herniation of the cerebellum into the foramen magnum is a key diagnostic feature for CM. Midsagittal MR images are the preferred technique for visualizing cerebellar herniation (CH). Objective To investigate whether CT can be used to diagnose CH. Animals Fifteen client‐owned CKCS dogs referred for investigation of the brain and cranial cervical spine on MRI and CT. Methods Two reviewers retrospectively analyzed midsagittal T1WSE and T2WSE MR images and midsagittal pre‐ and postcontrast 2D multiplanar reformatted CT images from each dog for the presence of CH. And, if present, the length (mm, CHL) of the herniation was measured. The results were analyzed statistically. Results There was no significant difference between the different observers and techniques for the detection of CH and measurement of CHL. Overall, the CHL was longer on the CT images. Conclusion and Clinical Importance Both techniques are useful for detecting CH and measuring CHL. Because CHL does not have a known direct impact on the clinical presentation of CM, CT can be used as a diagnostic tool in a routine clinical practice for CM in CKCS when MRI is not available. We emphasize that MRI is the standard screening technique in CKCS for breeding purposes to detect the presence of CM and SM and, at the current time, CT cannot replace MRI.

The effects of positioning, reason for screening and the referring veterinarian on prevalence estimates of canine hip dysplasia

Although the prevalence of canine hip dysplasia (HD) has been the subject of a number of published studies, estimates vary widely. This study evaluated several possible causes for these differences. Sixty Belgian, Dutch and German veterinarians were asked to submit all hip radiographs obtained for screening purposes (irrespective of HD status) over a 2-year period, resulting in a database of 583 dogs. Each set of radiographs was accompanied by information on the reason for screening (breeding soundness examination, clinical complaint, assistance dogs, or other reasons), and dog breed, date of birth and age. Dog positioning exerted an effect at multiple levels. The agreement among different observers regarding correct or incorrect positioning was limited and incorrect positioning itself reduced the inter-observer agreement for radiographic hip conformation. Dysplastic dogs were more commonly positioned incorrectly than non-dysplastic dogs. The clinical complaint population had a high prevalence of dysplastic dogs (>70%) compared with the breeding population (11%) and the assistance dogs (6%). There was a significantly lower prevalence of HD among cases referred by veterinarians who frequently submitted hip-extended radiographs for evaluation (P = 0.002) compared to those who refer less frequently. However, this was likely to be selection bias, as radiographs that were from dogs suspected to be dysplastic were not submitted by frequent senders. The prevalence of dysplastic dogs varied widely between breeds (16.7-71.4%). Dogs diagnosed with dysplasia were significantly older than dogs considered healthy (P = 0.001) and dogs classified as borderline dysplastic (P = 0.035). Inter-observer agreement for hip conformation was moderately low, resulting in >7% variation in prevalence estimates for dysplasia.

Comparison of Three Methods to Quantify Laxity in the Canine Hip Joint

OBJECTIVES  Comparison of PennHIP and a novel method to diagnose hip laxity, called the Vezzoni modified Badertscher distension device technique. METHODS  In a total of 10 dogs, it was first assessed whether the distraction index (DI) from the PennHIP evaluation center could be reproduced by two individual observers. In the next two steps, the DI measurements made by the individual observers and the PennHIP evaluation center were compared with the laxity index (LI) measured on the Vezzoni modified Badertscher distension device view. Finally, the interobserver agreement of the DI, LI and Norberg angle was assessed and compared with classification criteria. RESULTS  The results were similar for the first three comparisons: there was no evidence for bias, the relation between DI and LI was linear and the variability was small. A comparison of the interobserver agreement showed that the measurement variability for the NA was substantial, while the reproducibility for the DI and LI was equal. CLINICAL SIGNIFICANCE  While the standard ventrodorsal hip extended radiograph is most commonly used for diagnosis and screening of canine hip dysplasia, it lacks sensitivity to diagnose laxity. To improve the identification of hip joint laxity, distraction-based radiographic techniques are helpful. The Vezzoni modified Badertscher distension device technique allows for a reliable in-house evaluation of canine hip joint laxity.