Canine DVL2 variant contributes to brachycephalic phenotype and caudal vertebral anomalies.

A frameshift deletion variant in the Wnt pathway gene dishevelled 2 (DVL2) is associated with a truncated, kinked tail ("screw tail") in English Bulldogs, French Bulldogs and Boston Terriers. These breeds are also characterized by distinctive morphological traits, including a wide head, flat face and short-limbed dwarfism, which are characteristic of Robinow syndrome in humans, caused by defects in genes such as DVL1 and DVL3. Based on these phenotypic and genetic similarities, it has previously been hypothesized that the canine DVL2 variant results in a syndromic phenotype called the Robinow-like syndrome. In our study, we investigated the distribution of the DVL2 variant in 1954 dogs from 15 breeds, identifying breeds with allele variation and enabling the dissection of the genotype-phenotype correlation for the first time. With CT examinations in American Staffordshire Terriers, we confirmed that the DVL2 allele is associated with caudal vertebral malformations and a brachycephalic phenotype. We also hypothesize that the variant may be linked to additional health conditions, including brachycephalic obstructive airway syndrome and congenital heart defects. Altogether, our study strengthens the role of DVL2 as one of the contributors to the "bulldog type" morphology and features on the spectrum of human Robinow syndrome.

A 50-kb deletion disrupting the RSPO2 gene is associated with tetradysmelia in Holstein Friesian cattle.

BACKGROUND: Tetradysmelia is a rare genetic disorder that is characterized by an extremely severe reduction of all limb parts distal of the scapula and pelvic girdle. We studied a Holstein Friesian backcross family with 24 offspring, among which six calves displayed autosomal recessive tetradysmelia. In order to identify the genetic basis of the disorder, we genotyped three affected calves, five dams and nine unaffected siblings using a Bovine Illumina 50 k BeadChip and sequenced the whole genome of the sire. RESULTS: Pathological examination of four tetradysmelia cases revealed a uniform and severe dysmelia of all limbs. Applying a homozygosity mapping approach, we identified a homozygous region of 10.54 Mb on chromosome 14 (Bos taurus BTA14). Only calves that were diagnosed with tetradysmelia shared a distinct homozygous haplotype for this region. We sequenced the whole genome of the cases' sire and searched for heterozygous single nucleotide polymorphisms (SNPs) and small variants on BTA14 that were uniquely present in the sire and absent from 3102 control whole-genome sequences of the 1000 Bull Genomes Project, but none were identified in the 10.54-Mb candidate region on BTA14. Therefore, we subsequently performed a more comprehensive analysis by also considering structural variants and detected a 50-kb deletion in the targeted chromosomal region that was in the heterozygous state in the cases' sire. Using PCR, we confirmed that this detected deletion segregated perfectly within the family with tetradysmelia. The deletion spanned three exons of the bovine R-spondin 2 (RSPO2) gene, which encode three domains of the respective protein. R-spondin 2 is a secreted ligand of leucine-rich repeats containing G protein-coupled receptors that enhance Wnt signalling and is involved in a broad range of developmental processes during embryogenesis. CONCLUSIONS: We identified a 50-kb deletion on BTA14 that disrupts the coding sequence of the RSPO2 gene and is associated with bovine tetradysmelia. To our knowledge, this is the first reported candidate causal mutation for tetradysmelia in a large animal model. Since signalling pathways involved in limb development are conserved across species, the observed inherited defect may serve as a model to further elucidate fundamental pathways of limb development.

Detection of atypical porcine pestivirus in Swedish piglets with congenital tremor type A-II.

BACKGROUND: Congenital tremor (CT) type A-II is a neurological disorder characterized by tremor of the head and body of newborn piglets. The suggested causative agent of the disease is the recently found atypical porcine pestivirus (APPV). The virus has been detected in piglets suffering from congenital tremor in central Europe, South and North America and in China but no studies has so far been performed in the Nordic countries. The overarching goal of this study was to investigate if APPV is present in the brain tissue of Swedish piglets suffering from congenital tremor. From June 2017 - June 2018, 15 piglets from four Swedish farms with ongoing outbreaks of congenital tremor and 13 piglets with splay leg originating from four different farms, were investigated for presence of APPV RNA in brain tissue. Matched healthy control piglets (n = 8) were also investigated. Two APPV-specific RT-qPCR methods targeting the NS3 and NS5B region, respectively, were used. A retrospective study was performed on material from Swedish piglets with congenital tremor sampled in 2004 (n = 11) and 2011/2012 (n = 3) using the described APPV-specific RT-qPCR methods. The total number of piglets with signs of CT in this study was 29. RESULTS: Atypical porcine pestivirus-RNA was detected in 93% (27/29) of the piglets suffering from congenital tremor. All piglets with congenital tremor from 2004 (n = 11) and 2012 (n = 3) were PCR-positive with respect to APPV, whereas, all of the healthy controls (n = 11) were negative. The piglets with congenital tremor sampled 2017-2018 had an odds ratio of 91.8 (95% CI 3.9128 to 2153.7842, z = 2.807, P = 0.0050) to test positive for APPV by qRT-PCR compared to the healthy piglets (Fishers exact test p < 0.0001). These findings make it interesting to continue investigating APPV in the Swedish pig-population. CONCLUSION: This is the first description of atypical porcine pestivirus in piglets suffering from congenital tremor type A-II in Sweden and the Nordic countries. The virus has been present in the Swedish pig population since at least 2004.

VACTERL Association in a Female Pig (Sus scrofa domesticus).

VACTERL/VATER association is a condition defined by the presence of at least three of the following congenital malformations: vertebral defects (V), anal atresia (A), cardiac defects (C), tracheo-oesophageal fistula (TE), renal anomalies (R) and limb abnormalities (L). We describe a stillborn female piglet with cardiac anomalies, renal defects, vertebral anomalies, anal atresia and a single umbilical artery (SUA), which are the main features of VACTERL association. In addition, the piglet had a unilateral abdominal wall defect. This was the only affected animal in a litter of 16 piglets. The molecular inductive mechanisms of this disorder are discussed, as well as the comparative and embryological implications.

A missense variant in the NSDHL gene in a Chihuahua with a congenital cornification disorder resembling inflammatory linear verrucous epidermal nevi.

Congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome in humans is a genodermatosis characterized by inflammatory linear verrucous epidermal nevi (ILVEN), often showing a striking lateralization pattern. It is caused by variants in the NSDHL gene encoding a 3ß-hydroxysteroid dehydrogenase involved in the cholesterol biosynthesis pathway. In the present study, we investigated a female Chihuahua, which showed clinical and histological signs of ILVEN. We performed a candidate gene analysis in the affected animal. This analysis revealed a single missense variant in the NSDHL gene in the affected dog (XM_014111859.2:c.700G>A). The variant is predicted to cause a non-conservative amino acid change from glycine to arginine, XP_013967334.1:p.(Gly234Arg). The mutant allele was absent from WGS data of 594 genetically diverse dogs and eight wolves. Sanger sequencing confirmed that the variant was heterozygous in the affected dog and absent from 22 control Chihuahuas. Based on the knowledge about the functional impact of NSDHL variants in dogs and other species, c.700G>A is probably pathogenic and a convincing candidate causative variant for the observed skin lesions in the affected Chihuahua.

Detection of atypical porcine pestivirus genome in newborn piglets affected by congenital tremor and high preweaning mortality1.

Recently, piglets from a high-health status farm began exhibiting congenital tremors, high preweaning mortality and incidence of splayed legs. Postmortem histological examination identified a small number of scattered white matter vacuoles in the cerebellum and underlying brainstem of affected piglets. Presence of potential viral sources associated with this neurologic condition was initially infirmed using quantitative PCR for atypical porcine pestivirus (APPV), porcine teschovirus, and porcine sapelovirus. Using metagenomic analysis, APPV was identified as the main microbial species in serum obtained from piglets affected by congenital tremor. These piglets had higher preweaning mortality rates (46.4% vs. 15.3%) and incidence of splayed legs (33.0% vs. 0.8 %) compared to unaffected piglets. Piglets affected by congenital tremor had higher viral titer (P < 0.15) and larger birth weights (P < 0.05) compared to normal litter mates. Whole-genome sequencing and genome assembly of the novel APPV strain (MK728876) was carried out using Oxford Nanopore and related bioinformatics pipelines. Phylogenic analysis demonstrated that this strain along with other completely sequenced APPV strains were grouped into 2 clades, both including strains-inducing congenital tremor. Strains appear to cluster based on region but there were still significant differences within regions. Future research needs to address potential underdiagnosis due to genetic diversity but also to understand mode of transmission, variation in virulence, and the role of host genetics in APPV susceptibility.

Three-dimensional computer-assisted corrective osteotomy with a patient-specific surgical guide for an antebrachial limb deformity in two dogs.

INTRODUCTION: We describe patient-specific surgical guide prototyping and surgical treatment of a complex antebrachial deformity in two skeletally mature dogs presented with chronic lameness. Computer-assisted surgery was elected to increase accuracy in the correction of the complex deformity. Radiographs and computed tomography (CT) scans revealed a biplane deformity with valgus, procurvatum and external torsion of the right radius in both cases. The pre-surgical planning started from the quantification of the angular deformity, followed by computer simulated correction and to end up with a rehearsal surgery on 3D printed bone models. During the surgery, the custom-made osteotomy guides closely fitted the bone, allowing for a precise corrective osteotomy, that was stabilized with two locking plates. Postoperative radiographs showed the successful correction of the deformity. Eight and 12 weeks postoperative follow up examinations showed improved lameness, weight-bearing and progression of bone healing in both dogs. Patient-specific surgical guides allowed for a satisfactory correction of the antebrachial deformity. Additional benefits of using customized surgical devices include standardization and reduced surgical time.

INTRODUCTION: Nous décrivons le prototypage d'une procédure chirurgicale spécifique au patient et le traitement chirurgical d'une déformation antébrachiale complexe chez deux chiens ayant atteint leur maturité squelettique et présentant une boiterie chronique. La chirurgie assistée par ordinateur a été choisie pour accroître la précision de la correction de la déformation complexe. Les radiographies et la tomodensitométrie (TDM) ont révélé une déformation dans deux plans avec valgus, procurvatum et torsion externe du radius droit dans les deux cas. La planification préopératoire a commencé par la quantification de la déformation angulaire, suivie par une correction simulée sur ordinateur et a abouti à une opération de répétition sur des modèles d'os imprimés en 3D. Pendant l'intervention, des guides d'ostéotomie sur mesure ont ajusté l'os de manière exacte, permettant ainsi une ostéotomie correctrice précise, stabilisée avec deux plaques de verrouillage. Les radiographies postopératoires ont montré la réussite de le correction de la déformation. Les examens de suivi postopératoires effectués à huit et douze semaines ont montré une amélioration de la boiterie et de la mise en charge ainsi que la progression de la cicatrisation des os chez les deux chiens. Les guides chirurgicaux spécifiques au patient ont ­permis une correction satisfaisante de la déformation antébrachiale. L'utilisation de matériel chirurgical personnalisé comporte d'autres avantages, tels que la standardisation et la réduction du temps de l'intervention chirurgicale.

Polymelia in a chimeric Simmental calf: nociceptive withdrawal reflex, anaesthetic and analgesic management, anatomic and genetic analysis.

BACKGROUND: Polymelia is a congenital defect characterized by one or more supernumerary legs. The genetics and aetiology of this condition in cattle have not yet been thoroughly investigated even though several case reports do exist. The model of the nociceptive withdrawal reflex (NWR) has been characterized in several species to study spinal nociceptive processing. It is a polysynaptic spinal reflex that can be elicited by noxious electrical stimulation and recorded by electromyography. Thorough nociceptive examination and preventive analgesic management has not yet been an aspect in the perioperative management of polymelia cases. CASE PRESENTATION: A 4-month-old female Simmental calf was presented with notomelia. The animal was in good health and showed no neurologic deficiencies. Preoperatively, computed tomography was performed to gain more detailed anatomical information. To evaluate the sensitivity of the accessory limb, NWR testing was performed and revealed a connection of the afferent reflex pathway of the accessory limb to the efferent of the normal limb. The accessory limb was surgically removed under general anaesthesia. Intensive care included multimodal pain therapy adapted to the pain intensity scored during regular pain assessment. A gross anatomical dissection as well as a genetic analysis of the accessory limb were performed postoperatively. The calf was identified as a chimera. CONCLUSION: This calf was successfully relieved of its accessory limb. Chimerism has not been described in the congenital defect polymelia. As the accessory limb was pain sensitive and a common nociceptive reflex pathway was identified, thorough perioperative pain management was performed with the intention to prevent chronic neuropathic pain development.

Whole genome variant association across 100 dogs identifies a frame shift mutation in DISHEVELLED 2 which contributes to Robinow-like syndrome in Bulldogs and related screw tail dog breeds.

Domestic dog breeds exhibit remarkable morphological variations that result from centuries of artificial selection and breeding. Identifying the genetic changes that contribute to these variations could provide critical insights into the molecular basis of tissue and organismal morphogenesis. Bulldogs, French Bulldogs and Boston Terriers share many morphological and disease-predisposition traits, including brachycephalic skull morphology, widely set eyes and short stature. Unlike other brachycephalic dogs, these breeds also exhibit vertebral malformations that result in a truncated, kinked tail (screw tail). Whole genome sequencing of 100 dogs from 21 breeds identified 12.4 million bi-allelic variants that met inclusion criteria. Whole Genome Association of these variants with the breed defining phenotype of screw tail was performed using 10 cases and 84 controls and identified a frameshift mutation in the WNT pathway gene DISHEVELLED 2 (DVL2) (Chr5: 32195043_32195044del, p = 4.37 X 10-37) as the most strongly associated variant in the canine genome. This DVL2 variant was fixed in Bulldogs and French Bulldogs and had a high allele frequency (0.94) in Boston Terriers. The DVL2 variant segregated with thoracic and caudal vertebral column malformations in a recessive manner with incomplete and variable penetrance for thoracic vertebral malformations between different breeds. Importantly, analogous frameshift mutations in the human DVL1 and DVL3 genes cause Robinow syndrome, a congenital disorder characterized by similar craniofacial, limb and vertebral malformations. Analysis of the canine DVL2 variant protein showed that its ability to undergo WNT-induced phosphorylation is reduced, suggesting that altered WNT signaling may contribute to the Robinow-like syndrome in the screwtail breeds.

Assessment of overall thoracic limb axial alignment in dogs with antebrachial deformity.

OBJECTIVE: To assess overall thoracic limb axial alignment in dogs with and without angular limb deformity (ALD). STUDY DESIGN: Ex vivo and retrospective observational clinical study. ANIMALS: Forty canine thoracic limbs from bilateral normal specimens (10 pairs) and unilateral antebrachial ALD (10 uniapical and 10 biapical deformities). METHODS: Computed tomography images of the entire thoracic limb were collected for multiplanar reconstruction. Baseline limb axis was defined by the humeral anatomic axis. Axial alignment of the distal humerus, proximal and distal radius, and metacarpal bones were obtained and compared among levels and groups. RESULTS: The mean overall thoracic limb rotation of normal dogs was 35.4 ° (95% CI, 27.2 °, 43.6 °). Mean overall limb rotation in uniapical and biapical deformity was 41.6 ° (95% CI, 33.0 °, 50.1 °) and 54.4 ° (95% CI, 45.8 °, 62.9 °), respectively, but was not statistically different (P = .096). Only overall limb rotation in limbs with biapical ALD was different from normal limbs (P = .008). Rotation at the level of the elbow was 77% and 85% of overall limb rotation in normal and deformity limbs, respectively, and was increased from normal in limbs with ALD (both P < .05). Radial torsion did not differ among groups but was moderately correlated with overall limb rotation. CONCLUSION: Rotation arising in the elbow of limbs with ALD was higher than in normal limbs and accounted for the greatest contribution of overall limb rotation. CLINICAL SIGNIFICANCE: Assessment of radial torsion alone is insufficient to understand overall limb axial alignment in dogs with antebrachial bone deformity.

Congenital Malformations Resembling VACTERL Association in a Golden Retriever.

A 2 mo old golden retriever presented with malformation of the left thoracic limb and a small circular indentation of the skin in the cranial thoracic spine. Radiographs showed a cleft between the second and fifth metacarpal bones of the left thoracic limb compatible with ectrodactyly and spina bifida affecting T4 and T5 vertebrae. Magnetic resonance imaging of the thoracic spine showed dorsal reposition of the spinal cord and a tract connecting from the dura mater to the skin. No other malformations were detected. Surgical excision of the tract was performed and histopathological examination diagnosed a dermoid sinus type IV. Dermoid sinus and spina bifida are well-recognized congenital spinal and spinal cord defects, yet association with other congenital malformation are rarely reported in dogs. The here-reported dog had spinal and spinal cord abnormalities with concurrent limb malformation, which are two components of a nonrandom association of birth defects described as VACTERL in people. To the authors' knowledge, this is the first report describing concurrent dermoid sinus type IV, spina bifida, and ectrodactyly in a dog, and highlights the importance of patient examination for occurrence of multiple malformations to provide an appropriate prognosis for an owner.

Habitat quality affects the incidence of morphological abnormalities in the endangered salamander Ambystoma ordinarium.

Identification of early warning signals previous to the occurrence of population decline or extinction is a major challenge for the conservation of animal species. Prevalence of morphological abnormalities in a population can be one of these signals. We registered morphological abnormalities in the salamander Ambystoma ordinarium. We also evaluated the relation between habitat quality and the prevalence of abnormalities in this species. We used scores from rapid bioassessment protocols (RBPs) to assess the habitat quality of streams inhabited by A. ordinarium. A preliminary survey indicated that of 29 streams where this species has been historically registered, 13 might have few or no A. ordinarium. The association between habitat quality and the incidence of morphological abnormalities was evaluated in these 16 streams. Of 502 sampled individuals, 224 (44.62%) had at least one body abnormality. Of the 224 individuals with body abnormalities, 84 (37.5%) presented more than one abnormality. Of a total of 5,522 evaluated morphological characters, 344 (6.74%) were abnormal. Partial loss of gills and missing digits were the most frequent abnormalities. Results of a binomial logistic regression indicated that the probability of a character of an individual to be abnormal was significantly associated with habitat quality; as the levels of the quality of the habitat increased, the prevalence of morphological abnormalities decreased. These results suggest that RBPs are a quick and useful method for assessing the habitat quality of streams inhabited by A. ordinarium. Given that RBPs provide rapid and cost-effective assessments of the ecological health of aquatic ecosystems, it will be important to test if the RBPs protocols can be used to rapidly assess habitat quality for other species of stream amphibians. The negative association between habitat quality and the prevalence of morpohological abnormalities that we found indicates that habitat condition plays an important role in the high number of abnormalities registered in A. ordinarium. Therefore, our results suggest that one of the several negative effects of habitat degradation on amphibians is an increase in the frequency of morphological abnormalities with marked consequences for the survival and general fitness of aquatic amphibians.

Flexural Deformity of the Distal Interphalangeal Joint.

Flexural deformities in young horses are commonly referred to as contracted tendons, which is a term that is not consistent with what is currently understood about their cause. Flexural deformity of the distal interphalangeal joint can be either congenital (present at birth) or acquired (develop at a later stage of growth typically between 1 and 6 months of age). These 2 manifestations are commonly managed differently depending on the cause, age of onset, severity, duration, complicating factors, and owner expectations. Early recognition and appropriate intervention are essential to ensure that it is not performance limiting.

Angular Limb Deformities: Growth Augmentation.

Angular limb deformities are seen in young foals and are defined as lateral or medial deviations of the limb in the frontal plane distal to a particular joint. Several factors can contribute to the development of an angular limb deformity. Early assessment of the level of ossification of the cuboidal bones is critical to avoid complications long term. Although most deviations self-correct with minimal intervention other than modifications in exercise and hoof trimming, some require surgical intervention in the form of growth acceleration or retardation. This article focuses on growth augmentation techniques, such as hemicircumferential transection and elevation.

Angular Limb Deformities: Growth Retardation.

Angular limb deformities are common in foals; however, the importance of the deformity and if treatment is required depend on the degree of deformity relative to normal conformation for stage of growth, the breed and discipline expectations, age, and response to conservative therapies. This article addresses the importance of the foal conformation examination to determine which foals need surgical intervention to correct an angular deformity and when. Techniques for surgical growth retardation include the transphyseal staple, screw and wire transphyseal bridge, and transphyseal screw. Appropriate timing for intervention for each location and complications associated with each procedure are discussed.

Genome-wide association study identifies candidate genes for piglet splay leg syndrome in different populations.

BACKGROUND: Piglet splay leg syndrome (PSL) is one of the most frequent genetic defects, and can cause considerable economic loss in pig production. The present understanding of etiology and pathogenesis of PSL is poor. The current study focused on identifying loci associated with PSL through a genome-wide association study (GWAS) performed with the Illumina Porcine60 SNP Beadchip v2.0. The study was a case/control design with four pig populations (Duroc, Landrace, Yorkshire and one crossbred of Landrace × Yorkshire). RESULT: After quality control of the genotyping data, 185 animals (73 cases, 112 controls) and 43,495 SNPs were retained for further analysis. Principal components (PCs) identified from the genomic kinship matrix were included in the statistical model for correcting the effect of population structure. Seven chromosome-wide significant SNPs were identified on Sus scrofa chromosome 1 (SSC1), SSC2 (2 SNPs), SSC7, SSC15 (2 SNPs) and SSC16 after strict Bonferroni correction. Four genes (HOMER1 and JMY on SSC2, ITGA1 on SSC16, and RAB32 on SSC1) related to muscle development, glycogen metabolism and mitochondrial dynamics were identified as potential candidate genes for PSL. CONCLUSIONS: We identified seven chromosome-wide significant SNPs associated with PSL and four potential candidate genes for PSL. To our knowledge, this is the first pilot study aiming to identify the loci associated with PSL using GWAS. Further investigations and validations for those findings are encouraged.

Flexural Limb Deformities of the Carpus and Fetlock in Foals.

Early recognition and treatment of congenital and acquired flexural deformities of the carpi and fetlocks of foals can lead to conformation correction and an athletic future. Treatment is often based on rigid external coaptation assisted by systemic medical treatment. Foals that readily respond to treatment and correct conformation faults can have normal adult athletic expectations.

Routine Trimming and Therapeutic Farriery in Foals.

Hoof care in the first few months of life is serious business and should never be taken lightly. Farriery plays a vital role in both the development of the hoof and the conformation of the limb. Management of the feet and limbs during this period will often dictate the success of the foal as a sales yearling or mature sound athlete. A sound foot care program is time-consuming, whereas assembly-line trimming is quick and easy, but the former is much more beneficial.