Prime editor-mediated correction of a pathogenic mutation in purebred dogs.

Canine hip dysplasia (HD) is a multifactorial disease caused by interactions between genetic and environmental factors. HD, which mainly occurs in medium- to large-sized dogs, is a disease that causes severe pain and requires surgical intervention. However, the procedure is not straight-forward, and the only way to ameliorate the situation is to exclude individual dogs with HD from breeding programs. Recently, prime editing (PE), a novel genome editing tool based on the CRISPR-Cas9 system, has been developed and validated in plants and mice. In this study, we successfully corrected a mutation related to HD in Labrador retriever dogs for the first time. We collected cells from a dog diagnosed with HD, corrected the mutation using PE, and generated mutation-corrected dogs by somatic cell nuclear transfer. The results indicate that PE technology can potentially be used as a platform to correct genetic defects in dogs.

Correlation of distraction index with arthroscopic findings in juvenile dogs with hip dysplasia.

OBJECTIVE: To compare distraction index (DI) with hip arthroscopy findings in juvenile dogs with hip dysplasia undergoing unilateral or bilateral double pelvic osteotomy (DPO). STUDY DESIGN: Retrospective case series. ANIMALS: Twenty dogs (36 hip joints). METHODS: Medical records from dogs undergoing unilateral or bilateral DPO (2011-2016) were reviewed. Distraction index was measured by using distraction view radiographs. Arthroscopic images were analyzed. Cartilage was graded by using the modified Outerbridge scale (0 = arthroscopically normal, 1 = chondromalacia, 2 = surface fibrillation, 3 = deep fissuring, 4 = full-thickness cartilage loss, and 5 = eburnation of exposed subchondral bone). Dorsal acetabular rim, acetabular labrum, the ligament of head of the femur, and synovitis were graded by using a four-point scale (normal, mild, moderate, severe). Spearman and Pearson correlations were used to compare DI between grade groups and age. RESULTS: Distraction index ranged from 0.4 to 1.0 (mean 0.69 ± 0.16). Most of the hips had an arthroscopically normal femoral head and acetabulum in most regions, except for the region at the insertion of the ligament of the head of the femur. There was a low positive correlation between synovitis and DI (Spearman ρ = 0.35, P = .0346). No other differences in DI were identified between grade or age groups. CONCLUSION: There was a low positive correlation between synovitis and DI. No other differences in DI were identified. CLINICAL SIGNIFICANCE: Arthroscopic evaluation of the coxofemoral joint prior to DPO can allow detection of joint pathology that might not be appreciated by using traditional radiographic screening criteria.

Evolution of Radiographic Parameters of Canine Passive Hip Laxity at 4, 6 and 12 months: A Study of 306 Dogs.

OBJECTIVES: This article evaluates the variation in the distraction index (DI) and Norberg angle measurement with the femur in a neutral position and the hips distracted (distraction Norberg angle [DNA]) between 4, 6 and 12 months of age. METHODS: Radiographic data from dogs (n = 306) line-bred for favourable characteristics, including hip conformation, were reviewed. Dogs underwent stress radiographs, using a Vezzoni distractor, at 4, 6 and 12 months of age. The DI and DNA were calculated, and their evolution was studied for the total population and the sub-category of hips with a DI > 0.7. RESULTS: For the total population, the DI showed a significant increase from 4 to 6 months and a significant decrease at 12 months. For the hips with a DI > 0.7 at 4 months, DI at 6 and 12 months showed a significant decrease (0.14 and 0.26, respectively, on average). For the total population, the DNA followed the same pattern as the DI, and the values are significantly associated (p < 0.001). CLINICAL SIGNIFICANCE: The DI varies significantly from 4 to 12 months in dogs, and the time of radiographic evaluation of hip laxity may strongly influence the value of the DI. The DNA is strongly related to the DI, and its use as a more convenient indicator of hip laxity in dogs should be further investigated.

Prospective evaluation of a patented DNA test for canine hip dysplasia (CHD).

Genetic testing has been propagated as a suitable means to specify individual risks for canine hip dysplasia (CHD). However, the current lack of validation of most genetic CHD tests has left dog owners and breeders in the dark about their practical utility. Therefore, the Society for German Shepherd Dogs (Verein für Deutsche Schäferhunde, SV) initiated a prospective study of 935 animals to assess independently the value of a genetic CHD test (European Patent Specification EP 2 123 777 B1) that was developed by Distl et al. (2009) on the basis of the SV animal stock. Dogs were followed-up for 3 years after birth, classified regarding their CHD phenotype using the scheme of the Fédération Cynologique Internationale, and genotyped for the 17 single nucleotide polymorphisms (SNPs) constituting the CHD test in question. Individual SNP genotypes were combined into animal-specific genomic breeding values (GBVs), calculated as the weighted sum of SNP-wise scores as laid down in the patent specification. Logistic regression analysis revealed that, unexpectedly, the odds ratio for CHD decreased, rather than increased, by a factor of 0.98 per unit increase of the GBV. Nevertheless, since this effect was not statistically significant (95% CI: 0.93-1.03), and the area-under-curve of the test was only 0.523, it must be concluded that the genetic test patented by Distl et al. (2009) is unsuitable for individual CHD risk assessment.

[Nerve distribution and density in the canine hip joint capsule. Comparison of healthy and dysplastic hip joints]. / Nervenverteilung und -verteilungsdichte in der Hüftgelenkskapsel des Hundes. Vergleichende Untersuchung gesunder und dysplastischer Hüftgelenke.

OBJECTIVE: The hip-joint capsule is exposed to increased tension forces during canine hip dysplasia, resulting in inflammation of the capsular tissue. It has been postulated that inflammation is associated with an increased nerve-distribution density. Therefore, it could be supposed that the nerve-distribution density in the hip-joint capsule is higher in dogs with dysplastic hip compared to healthy dogs. MATERIAL AND METHODS: In 16 Labrador Retriever dogs that had been euthanised due to unrelated reasons, the hip joints were classified as normoplastic (group 1, n = 18) or dysplastic (group 2, n = 14) based on radiography. Following staining of the capsular nerve fibres by the Sihler method, histological specimens of the hip-joint capsules were scanned. By subdividing each specimen into 10 quadrants numbered from dorsomedial (Q01) to craniodorsolateral (Q10), the ratio of black to white pixels was calculated digitally for each specimen and each quadrant by using a semiautomatic image analysis. Statistical analysis was performed using an independent t-test. RESULTS: Comparison of the mean values of each quadrant showed a significantly higher (p < 0.03) nerve distribution density for the craniodorsolateral quadrant (Q10) in group 2 when compared to group 1. Mean nerve-distribution density for all quadrants combined was not significantly different between the two groups. CONCLUSION: The increase in nerve-distribution density of the craniodorsal region of the hip-joint capsule in dogs with dysplastic hip could be the result of increased tension forces on this area following hip-joint dysplasia. The craniodorsal region of the hip-joint capsule is an important origin of pain and coxarthrosis in canine hip dysplasia. CLINICAL RELEVANCE: The results provide the pathophysiological basis for the efficacy of hip-joint denervation. Denervation of the cranial region of the acetabular rim is essential to reduce capsular inflammation and joint-related pain in canine hip dysplasia.

Asymmetrical lumbosacral transitional vertebrae in dogs may promote asymmetrical hip joint development.

OBJECTIVES: This study examines the relationship between the morphology of the lumbosacral transitional vertebra (LTV) and asymmetrical development of the hip joints in dogs. METHODS: A total of 4000 dogs which had been consecutively scored for canine hip dysplasia were checked for the presence of a LTV. A LTV was noted in 138 dogs and classified depending on the morphology of the transverse processes and the degree of contact with the ilium. RESULTS: In dogs with an asymmetrical LTV, the hip joint was significantly more predisposed to subluxation and malformation on the side of the intermediate or sacral-like transverse process (p <0.01), on the side of the elevated pelvis (p <0.01), or when an asymmetrical LTV resulted in pelvic rotation on its long axis (p <0.01), whereas hip joint conformation was less affected on the side featuring a free transverse process (p <0.01). CLINICAL SIGNIFICANCE: The results support our hypothesis that an asymmetrical LTV favours pelvic rotation over its long axis, resulting in inadequate femoral head coverage by the acetabulum on one side. Inadequate coverage of the femoral head favours subluxation, malformation of the hip joint, and secondary osteoarthritis. Asymmetrical hip conformation may therefore be the sequela of a LTV and mask or aggravate genetically induced canine hip dysplasia.

A genetic predictive model for canine hip dysplasia: integration of Genome Wide Association Study (GWAS) and candidate gene approaches.

Canine hip dysplasia is one of the most prevalent developmental orthopedic diseases in dogs worldwide. Unfortunately, the success of eradication programs against this disease based on radiographic diagnosis is low. Adding the use of diagnostic genetic tools to the current phenotype-based approach might be beneficial. The aim of this study was to develop a genetic prognostic test for early diagnosis of hip dysplasia in Labrador Retrievers. To develop our DNA test, 775 Labrador Retrievers were recruited. For each dog, a blood sample and a ventrodorsal hip radiograph were taken. Dogs were divided into two groups according to their FCI hip score: control (A/B) and case (D/E). C dogs were not included in the sample. Genetic characterization combining a GWAS and a candidate gene strategy using SNPs allowed a case-control population association study. A mathematical model which included 7 SNPs was developed using logistic regression. The model showed a good accuracy (Area under the ROC curve = 0.85) and was validated in an independent population of 114 dogs. This prognostic genetic test represents a useful tool for choosing the most appropriate therapeutic approach once genetic predisposition to hip dysplasia is known. Therefore, it allows a more individualized management of the disease. It is also applicable during genetic selection processes, since breeders can benefit from the information given by this test as soon as a blood sample can be collected, and act accordingly. In the authors' opinion, a shift towards genomic screening might importantly contribute to reducing canine hip dysplasia in the future. In conclusion, based on genetic and radiographic information from Labrador Retrievers with hip dysplasia, we developed an accurate predictive genetic test for early diagnosis of hip dysplasia in Labrador Retrievers. However, further research is warranted in order to evaluate the validity of this genetic test in other dog breeds.

Canine hip dysplasia: phenotypic scoring and the role of estimated breeding value analysis.

Canine hip dysplasia (CHD) is a developmental orthopaedic disease of the coxofemoral joints with a multifactorial mode of inheritance. Multiple gene effects are influenced by environmental factors; therefore, it is unlikely that a simple genetic screening test with which to identify susceptible individuals will be developed in the near future. In the absence of feasible methods for objectively quantifying clinical CHD, radiographic techniques have been developed and widely used to identify dogs for breeding which are less affected by the disease. A hip-extended ventrodorsal view of the pelvis has been traditionally used to identify dogs with subluxation and/or osteoarthritis of the coxofemoral joints. More recently, there has been emphasis on the role of coxofemoral joint laxity as a determinant of CHD and methods have been developed to measure passive hip laxity. Though well-established worldwide, the effectiveness of traditional phenotypic scoring schemes in reducing the prevalence of CHD has been variable. The most successful implementation of traditional CHD scoring has occurred in countries or breeding colonies with mandatory scoring and open registries with access to pedigree records. Several commentators have recommended that for quantitative traits like CHD, selection of breeding stock should be based on estimated breeding values (EBV) rather than individual hip score/grade. The EBV is a reflection of the genetic superiority of an animal compared to its counterparts and is calculated from the phenotype of an individual and its relatives and their pedigree relationship. Selecting breeding stock on the basis of a dog's genetic merit, ideally based on a highly predictive phenotype, will confer the breeder with greater selection power, accelerate genetic improvement towards better hip conformation and thus more likely decrease the prevalence of CHD.

Genetic evaluation of the total hip score of four populous breeds of dog, as recorded by the New Zealand Veterinary Association Hip Dysplasia Scheme (1991-2011).

AIM: To use estimated breeding value (EBV) analysis to investigate the genetic trend of the total hip score (to assess canine hip dysplasia) in four populous breeds of dogs using the records from the New Zealand Veterinary Association (NZVA) Canine Hip Dysplasia Scheme database (1991 to 2011). METHODS: Estimates of heritability and EBV for the NZVA total hip score of individual dogs from the German Shepherd, Labrador Retriever, Golden Retriever and Rottweiler breeds were obtained using restricted maximum likelihood procedures with a within-breed linear animal model. The model included the fixed effects of gender, birth year, birth season, age at scoring and the random effect of animal. The pedigree file included animals recorded between 1990 and 2011. A total of 2,983 NZVA hip score records, from a pedigree of 3,172 animals, were available for genetic evaluation. Genetic trends of the NZVA total hip score were calculated as the regression coefficient of the EBV (weighted by reliabilities) on year of birth. RESULTS: The estimates of heritability for hip score were 0.32 (SE 0.08) in German Shepherd, 0.37 (SE 0.08) in Labrador Retriever, 0.29 (SE 0.08) in Golden Retriever and 0.52 (SE 0.18) in Rottweiler breeds. Genetic trend analysis revealed that only the German Shepherd breed exhibited a genetic trend towards better hip conformation over time, with a decline of 0.13 (SE 0.04) NZVA total hip score units per year (p<0.001). The genetic trends of total hip score for the remaining three breeds were not significantly different from zero (p>0.1). CONCLUSIONS: Despite moderate heritability of the NZVA total hip score, there has not been substantial improvement of this trait for the four breeds analysed in the study period. CLINICAL RELEVANCE: Greater improvement in reducing the prevalence of canine hip dysplasia may be possible if screening were to be compulsory as a requirement for registration of pedigree breeding stock, greater selection pressure were to be applied and selection of breeding stock made on the basis on an individual's EBV rather than the NZVA total hip score alone.

Radiographic changes of the pelvis in Labrador and Golden Retrievers after juvenile pubic symphysiodesis: objective and subjective evaluation.

OBJECTIVES: The hypothesis of this study was that juvenile pubic symphysiodesis (JPS) results in pelvic changes that can be identified radiographically in adult dogs. METHODS: The medical records at the Clinica Veterinaria Vezzoni were searched for standard ventro-dorsal views of the pelvis of adult Labrador and Golden Retrievers that had undergone JPS or had not undergone surgery. The objective assessment of radiographs included the analysis of various pelvic measurements. Subjective evaluation of radiographs was undertaken by 18 specialists and 21 general practitioners and was based on five criteria relating to 1) the acetabular fossae, 2) the pubic symphysis, 3) the margin of the cranial pubic area, 4) the pubic rami, and 5) the obturator foramen. RESULTS: The radiographs of 42 Labrador Retrievers and 16 Golden Retrievers were evaluated. The most useful criteria were the radiographic measurement of the shape of the obturator foramen and two different ratios of length to width of the pubic rami; these values were significantly smaller in dogs after JPS. The pelvic canal width was the same in both groups. All objective measurements were repeatable within and between evaluators. The most reliable subjective criterion was number 4, followed by number 5 in Golden Retrievers and by 2 in Labrador Retrievers. CONCLUSION: Our objective and subjective evaluations were simple and yielded useful and repeatable results. There was no significant difference between general practitioners and specialists with regard to subjective evaluation, which indicates that these evaluation criteria can be used by small animal clinicians after minimal training.

Associations among exercise duration, lameness severity, and hip joint range of motion in Labrador Retrievers with hip dysplasia.

OBJECTIVE: To evaluate factors associated with lameness severity and hip joint range of motion in dogs with hip dysplasia and to assess the association between hip joint range of motion and degree of lameness. DESIGN: Prospective case series. ANIMALS: 60 client-owned Labrador Retrievers with hip dysplasia. PROCEDURES: Owners completed a questionnaire regarding their dogs' daily exercise duration and type (i.e., low impact vs high impact) and lifestyle. Range of motion of affected hip joints was measured with a transparent plastic goniometer. The presence of subluxation or luxation of hip joints as a consequence of hip dysplasia and the size of the largest osteophytes or enthesophytes of hip joints on ventrodorsal radiographic images of the pelvis were recorded. Multivariate analyses were performed to identify factors associated with lameness, loss of hip joint flexion, and loss of hip joint extension and to identify factors associated with the presence of large osteophytes. RESULTS: Exercise was associated with a decrease in the severity of lameness in dogs with hip dysplasia. The strength of this inverse relationship increased with longer exercise duration. Lameness was more severe in dogs with hip joint luxation than in dogs without luxation. Hip joint extension was 1° lower for each year of age, and osteophyte or enthesophyte size was 1 mm larger with each 3-year increase in age. CONCLUSIONS AND CLINICAL RELEVANCE: Longer daily exercise duration was associated with lower lameness scores in dogs with hip dysplasia. Dogs with hip joint luxation secondary to hip dysplasia had higher lameness scores than did dogs without hip joint luxation.

Kinematic analysis in healthy and hip-dysplastic German Shepherd dogs.

This study investigated kinematic patterns in clinically normal German Shepherd dogs (GSDs) compared to those with hip dysplasia and with no clinical signs of lameness. Two groups of GSDs, including 10 clinically healthy dogs (G1) and 10 with hip dysplasia (G2), were trotted on a treadmill at a constant speed. Kinematic data were collected by a 3-camera system and analysed by a motion-analysis program. Flexion and extension joint angles and angular velocities were determined for the shoulder, elbow, carpal, hip, stifle, and tarsal joints. Within each group, the differences between the right and left limbs in all kinematic variables were not significant. Minimum angle, angular displacement and minimum angular velocity did not differ between groups. Significant differences were observed in the maximum angular velocity and maximum angle of the hip joint (dysplastic>healthy), and in the maximum angular velocity of the carpal joint (healthy>dysplastic). It was concluded that, when trotting on a treadmill, dysplastic dogs with no signs of lameness may present joint kinematic alterations in the hind as well as the forelimbs.

Chronology of hip dysplasia development in a cohort of 48 Labrador retrievers followed for life.

OBJECTIVE: To determine the chronology of radiographic signs of canine hip dysplasia (CHD), specifically joint laxity and secondary osteoarthritis (OA). STUDY DESIGN: Longitudinal cohort study. ANIMALS: Paired littermates, 48 Labrador retrievers. METHODS: Conventional, ventrodorsal, hip-extended (HE) radiographs were evaluated yearly for CHD according to the subjective criteria of the Orthopedic Foundation for Animals (OFA). PennHIP screening was performed at 2 years of age to assess joint laxity by distraction index (DI). Histopathologic evaluation of coxofemoral joints was performed at the dogs' natural end of life. RESULTS: Coxofemoral subluxation, as identified on the HE radiograph occurred by 2 years of age and not thereafter. Accuracy of OFA-criteria scoring was poor: 55% of dogs scored "normal" at 2 years of age became radiographically dysplastic by the end of life (45% negative predictive value, NPV); 92% of the dogs scored as normal at 2 years of age had histopathologic OA of CHD (8% NPV). The DI predicted all 48 dogs to be susceptible to OA of CHD and 98% had radiographic or histopathologic OA by the end of life. CONCLUSION: OFA-criteria score was profoundly influenced by environmental factors, such as diet restriction and age, reducing its value as a selection criterion. DI measurements were not influenced by dietary treatment suggesting higher trait heritability.

The S-measurement in the diagnosis of canine hip dysplasia.

OBJECTIVE: To propose a direct measure of subluxation of the femoral head (S) in the assessment of hip joint laxity and evaluate it for clinical use. STUDY DESIGN: Method comparison study. ANIMALS: Dogs (n = 51). METHODS: Dogs were sedated or anesthetized for a dorsolateral subluxation (DLS) examination. Two sets of radiographs were acquired, 1 each by a different technologist. A calibrated measuring bar was included on the image at the height of the hip to assess magnification. The DLS was calculated for each hip and different persons unaware of these details measured the "S"-value. One person measured the S-value 3 times over 3 days. Box plots were used to determine a cut-off for the empiric (8 mm) and corrected (4 mm) S-value. RESULTS: Of 51 dogs, 33 were dysplastic based on a DLS score <55%. Magnification and body weight were strongly correlated (r = 0.4922, P = .0006). Both empiric and corrected S measurements showed good agreement with the DLS score (κ = 0.688 and κ = 0.681, respectively). The corrected S measurement produced more false negatives. Bland-Altman analysis showed interobserver and technician variance acceptable for clinical use (limits of agreement < ±3 mm). Intraobserver repeatability was acceptable for the right hip (95% of differences were ≤1.3 mm and 100% ≤ 1.9) but not for the left hip. CONCLUSION: Using a cut-off value of 5 mm, the empirical S measurement can be used to exclude hip dysplasia in young dogs of various body proportions.

Palpation and dorsal acetabular rim radiographic projection for early detection of canine hip dysplasia: a prospective study.

OBJECTIVE: To determine the value of 2 diagnostic methods: (1) the reduction angle (RA) using the Ortolani maneuver and (2) the dorsal acetabular slope (DAS) from the dorsal acetabular rim (DAR) radiographic projection, to predict osteoarthritis (OA) in dogs with hip dysplasia. STUDY DESIGN: Prospective study. SAMPLE POPULATION: Dogs (n = 73). METHODS: Hip-extended ventrodorsal (VD) radiographic projections, RA, and DAS were evaluated when dogs were 6, 12, and 24 months of age. VD projections were qualitatively scored for OA. RA was determined using the Ortolani maneuver in dorsal recumbency and DAS using the DAR projection. Distraction index (DI) measurements from the compression-distraction radiographic projections (PennHIP method) were also performed at 6 months of age. Statistical analyses were used to establish the range of values of normal and abnormal RA and DAS, to document the temporal variation in RA and DAS, to compare the ability of the different methods to predict coxofemoral OA, to determine the influence of pure passive laxity and of the DAS on the occurrence of an Ortolani sign and on the magnitude of the RA, to establish the relationship between the DAS and subsequent development of passive laxity and coxofemoral OA, and to evaluate the influence of the DAS and RA on the occurrence of coxofemoral OA with severe, moderate, and minimal coxofemoral passive joint laxity, respectively. RESULTS: VD, RA, DAS, and DI methods of coxofemoral joint evaluation correlated significantly with the status of the coxofemoral joints at 2 years of age. The risk of occurrence of coxofemoral OA increased, as the RA, DAS, or DI increased. There was a significant positive linear relationship between RA and DI (P = .015, r(2) = 0.32), RA and DAS (P = .0078, r(2) = 0.38), and DAS and DI (P = .015, r(2) = 0.33). A negative Ortolani sign was at all times significantly predictive of absence of coxofemoral OA at 2 years of age. DAS best predicted coxofemoral OA for DI ≥ 0.7, whereas RA best predicted coxofemoral OA for 0.3 < DI < 0.7; however, RA proved to be the best overall predictor of coxofemoral OA. CONCLUSION: RA measured at 6 months of age in dorsal recumbency was the best predictor of coxofemoral OA at 2 years of age.

Seasonal variation in the hip score of dogs as assessed by the New Zealand Veterinary Association hip dysplasia scheme.

AIM: To determine whether there is a seasonal variation in the phenotypic hip score of dogs born in New Zealand as assessed by the New Zealand Veterinary Association (NZVA) canine hip dysplasia (CHD) scheme. METHODS: Data from dogs born in New Zealand between 1988 and 2009 that have been scored for CHD were retrospectively evaluated for the effect of month of birth on radiographic phenotype. Data included both the total score and the subtotal score, comprising Norberg's angle, the subluxation score and changes to the cranial acetabular edge, for each dog. Datasets were created for all breeds combined and for the four most populous breeds using the scheme (German Shepherd dog, Labrador Retriever, Golden Retriever and Rottweiler) and stratified according to month of birth and season. Due to the skewed nature of the data, a Kruskal-Wallis Rank Sum test was used to test for statistical significance. Additionally, χ² analysis was performed using the median of each dataset (proportion above/below the median). The null hypothesis was that there would be no effect of month of birth, and hence seasonality, on hip phenotype for dogs born and scored in New Zealand by the NZVA. RESULTS: For all breeds combined, month of birth had an effect on total and subtotal NZVA CHD scores (p<0.001) with a lower total hip score in the autumn months of March and April than other months. When individual large breed data were analysed, there was an effect of month of birth on total and subtotal scores for the Labrador Retriever and the Rottweiler (p ≤ 0.05), but not the German Shepherd dog or Golden Retriever breeds. CONCLUSIONS: Being born in the autumn was associated with a protective effect on hip phenotype in some breeds. These results suggest that weather and/or another seasonal factor may have a significant environmental effect on the phenotype of the coxofemoral joint. CLINICAL RELEVANCE: The protective effect of being born in autumn suggests that a decreased level of exercise during subsequent development over winter may positively impact on final coxofemoral joint conformation. Whilst statistically significant, the magnitude of the sparing effect is not likely to be clinically relevant. However, this study, in concert with other studies, may suggest that the effects of exercise can be manipulated to improve hip phenotype.