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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An heuristic filtering tool to identify phenotype-associated genetic variants applied to human intellectual disability and canine coat colors.

BACKGROUND: Identification of one or several disease causing variant(s) from the large collection of variants present in an individual is often achieved by the sequential use of heuristic filters. The recent development of whole exome sequencing enrichment designs for several non-model species created the need for a species-independent, fast and versatile analysis tool, capable of tackling a wide variety of standard and more complex inheritance models. With this aim, we developed "Mendelian", an R-package that can be used for heuristic variant filtering. RESULTS: The R-package Mendelian offers fast and convenient filters to analyze putative variants for both recessive and dominant models of inheritance, with variable degrees of penetrance and detectance. Analysis of trios is supported. Filtering against variant databases and annotation of variants is also included. This package is not species specific and supports parallel computation. We validated this package by reanalyzing data from a whole exome sequencing experiment on intellectual disability in humans. In a second example, we identified the mutations responsible for coat color in the dog. This is the first example of whole exome sequencing without prior mapping in the dog. CONCLUSION: We developed an R-package that enables the identification of disease-causing variants from the long list of variants called in sequencing experiments. The software and a detailed manual are available at https://github.com/BartBroeckx/Mendelian.

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.

Selection for growth performance in broiler chickens associates with less diet flexibility.

Global competition for high standard feed-food resources between man and livestock, such as industrial broilers, is a concerning problem. In addition, the low productivity of scavenger chickens in developing countries leaves much to be desired. Changing the ingredients, and therefore, the nutrient composition of feed intake by commercial fed as well as scavenger chickens seems like an obvious solution. In this study, the ability of four broiler chicken breeds to perform on a commercial versus a scavenger diet was tested. The four broiler breeds differed genetically in growth potential. A significant (P < 0.01) negative effect of the scavenger diet on the bodyweight of the fast growing breeds was found and this effect decreased with decreasing growth rate in the other breeds. These differences in bodyweight gain could not be explained by differences in nutrient digestibility but were caused by the lack of ability of the fast growing breeds to increase their feed intake sufficiently.

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). Cohen's 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.

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.

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.

Worldwide screening for canine hip dysplasia: where are we now?

OBJECTIVE: To critically review the different screening systems used for canine hip dysplasia (CHD) and their impact on the prevalence of the disease. STUDY DESIGN: Critical literature review. METHODS: Literature search through PubMed (November 1959-October 2011) and the Orthopedic Foundation for Animals (OFA), Fédération Cynologique Internationale (FCI), British Veterinary Association/Kennel Club (BVA/KC), and Pennsylvania Hip Improvement Program (PennHIP) websites. RESULTS: The OFA, FCI, and BVA/KC screening methods, which use the hip-extended radiographic projection, have had relatively minor success on CHD prevalence. These screening approaches are prone to conflicting data regarding interobserver agreement. The PennHIP and Dorsolateral Subluxation (DLS) systems, both distraction methods, have not reported on prevalence but seem to be important heritable traits in genomic screening of dysplastic dogs. CONCLUSION: A shift towards genome screening yields a promising future combating CHD, although further investigation towards fine-mapping in the search for genes, responsible for CHD, is necessary.

The effect of a technical quality assessment of hip-extended radiographs on interobserver agreement in the diagnosis of canine hip dysplasia.

Experienced and inexperienced observers evaluated the assessability of 50 radiographs (25 dogs) and determined the hip status (dysplasia/nondysplasia and final scoring according Fédération Cynologique Internationale [FCI]-criteria) individually. A radiographic technical quality assessment was performed in a separate reading session. Interobserver agreement in determining dysplasia/nondysplasia and FCI-scoring did not significantly increase with the increasing quality of a radiograph, irrespective whether these observers are experienced or not. There was a significant agreement between the technical quality assessment and assessability (P < 0.0005). Despite the effort to objectify radiographic quality and to present high-quality radiographs to observers, interobserver agreement on dysplasia/nondysplasia and final scoring, remains low, even in the experienced group. Although increased radiographic quality narrows the range of scoring, the range remains unacceptably high.

Interobserver agreement on the assessability of standard ventrodorsal hip-extended radiographs and its effect on agreement in the diagnosis of canine hip dysplasia and on routine FCI scoring.

Insufficient agreement on scoring hip quality might be caused by differences in the assessability of a radiograph (exposure, contrast, positioning, and diagnostic quality). We studied the agreement in assessability of standard ventrodorsal hip-extended radiographs by experienced (nine) and inexperienced (21) observers, using the standard subjective method of quality control, currently applied in screening programs. The effect of assessability on the agreement of scoring hip quality [dysplastic vs. nondysplastic and the final Federation Cinologique International (FCI) score] was also investigated. There was a significant difference (P < 0.0001) in agreement on assessability between the experienced and inexperienced observers. In 68% of evaluations, experienced observers stated that the radiograph was assessable. Inexperienced observers evaluated the radiographs as being assessable in only 46.5% of evaluations. Increased interobserver agreement on assessability of a radiograph did not increase the overall interobserver agreement in the diagnosis of hip dysplasia, nor did it result in consistent scoring of the hip status from that radiograph, despite a significant (P < 0.05) increase in agreement of FCI scoring with an increasing agreement on assessability at a one to five ratio in the experienced group. The inconsistent evaluation of radiographic quality, as well as the inconsistent evaluation of the hip quality, caused differences in diagnosing hip dysplasia and FCI scoring in the same dog ranging from excellent hips to moderate hip dysplasia. Therefore, the credibility of the FCI screening method for canine hip dysplasia, using the standard hip-extended radiographic view, as currently applied in most European countries, is questionable.

Ultrasound approach to the canine distal tibia and trochlear ridges of the talus.

The aim of the present study was to evaluate to what extent the distal tibia and the trochlear ridges of the talus can be examined with ultrasound (US) in the dog and to establish a protocol for an optimal US examination of these ridges. Six hind limbs of deceased adult mixed-breed dogs were used. In two limbs, needles were placed using US guidance on the trochlea of the talus, just dorsal to and plantar to the distal tibia: one with the tarsal joint in extension and one with the joint in flexion. Then mediolateral (ML) radiographs of both joints were made with the needle in place to determine the percentage of the trochlear ridge of the talus that can be seen using US imaging. An US examination of the tarsal joint was performed on the four other limbs using microconvex (8 MHz) and linear (12 MHz) transducers (Logiq 7) and compound imaging. A three-step protocol was performed including a dorsal approach with the limb extended and the linear transducer (step I), a plantar approach with the limb flexed and the linear transducer (step II), and a plantar approach with the limb flexed and the microconvex transducer (step III). After the US examination, the four limbs were frozen and sectioned, two in a transverse and two in a sagittal plane. Bony structures on the US images were matched with the corresponding anatomic sections. The distal tibia and both trochlear ridges of the talus were easily recognized on the US images using the proposed protocol. When combining the dorsal and plantar approaches, it was possible to visualize up to 75% of the trochlear ridges of the talus in the dog.

Estimates of regional cerebral blood flow and 5-HT2A receptor density in impulsive, aggressive dogs with 99mTc-ECD and 123I-5-I-R91150.

Impulsive aggression in dogs has an important impact on human public health. Better insight into the pathophysiology of this phenomenon could lead to more adequate diagnosis and treatment. Indirect in vivo research on peripheral body fluids and post-mortem studies in impulsive animals and humans indicate a deficient serotonergic system in general and disturbances in the serotonin-2A (5-HT2A) receptor in particular. In this study, brain perfusion and the 5-HT2A receptors were examined in impulsive, aggressive dogs, in comparison with a group of normally behaving animals. In order to decide which dogs to include in this study, owners were asked to describe the general behaviour of the dogs, the circumstances in which aggression occurred and their conduct during aggressive acts. Finally, 19 dogs were retained for this study, showing, according to different behavioural specialists, disinhibited dominance aggression. Functional imaging studies were performed on all these dogs. Single-photon emission tomography (SPET) was used to measure regional brain perfusion using technetium-99m labelled ethyl cysteinate dimer (ECD). The 5-HT2A receptor binding properties were investigated using the selective radioligand iodine-123 labelled 5-I-R91150. A significant increase in uptake of the 5-HT2A radioligand was noted in all cortical areas. No significant alterations were found in regional cortical perfusion, indicating that the increased binding index was not a consequence of increased tracer delivery. This study supports a role for the serotonergic system in canine impulsive aggression.

Effects of aging on brain perfusion and serotonin-2A receptor binding in the normal canine brain measured with single photon emission tomography.

Normal aging is associated with a decrease in number and size of neurons, loss of synapses and neuronal branching and with a reduced functioning neurotransmitter systems, such as the serotonergic system. These structural and functional alterations have important impact on the behavioural, cognitive and affective status of the individual. With the introduction of functional brain imaging in veterinary medicine, the canine brain can be examined in vivo, evaluating changes in perfusion, metabolism and neurotransmitter systems. Since cognitive decline is recognised in the aging dog, it was our aim to investigate whether age related changes concerning cerebral perfusion and binding index of the selective 5-HT2A receptor ligand 123I-5-I-R91150 could be found in the canine brain. A group of twelve normal, aging dogs, older than 96 months, was compared to a normal reference group (n = 12), younger than 96 months. SPET images were obtained, using the radiopharmaceutical 99mTc-N,N''-1,2-ethylene-diylbis-L-cysteine diethylester dihydrochloride (99mTc-ECD) for evaluation of the regional perfusion and the selective radioligand 123I-5-I-R91150 for visualization of the 5-HT2A receptor. Regional decrease of cerebral blood-flow was noted in the fronto- and temporocortical area and in the subcortical region. Age was negatively correlated with perfusion in the left and right fronto-cortical region. The binding index of the neuroreceptor radioligand was decreased in the fronto-cortical region, with a significant negative correlation with age in the right fronto-cortical area. No correlation was found between alteration of perfusion and binding index of the receptor ligand, suggesting that age related perfusion differences do not influence the binding of this radioligand. These results suggest that age related effects should be considered in functional canine brain imaging.