Four SNPs associated with CHD across breeds
The raw P-values, the empirical P-values from the permutation procedure, and odds ratios (OR) from the across-breed CMH test as implemented in PLINK [14] are shown in Table 1. Only the markers with a significant association to CHD, and which also passed a test of homogeneity of OR across the breeds (see Methods), are reported. A total of four SNPs associated significantly with CHD in the across-breed analysis (Table 1). The markers are located on CFA1, CFA14, CFA26, and CFA37. Of these, the SNP on CFA1 is from our previous study of the FCI hip score on German Shepherds [13]. The variant ss7212922135 on CFA14 was originally associated with the FCI hip score in Bernese Mountain dogs [6]. On CFA26, ss7212922151 originally associated with the FCI hip score in German Shepherds [7], as did ss7212922122 on CFA33 [8]. The variant ss7212922139 on CFA37 originally associated with OA in a multi-breed analysis [4]. In contrast to the other four markers, ss7212922139 in CFA37 was not significantly associated with CHD in any of the breed-specific analyses.
The definition of cases and controls was the same as it was in the across-breed analysis. We used two methods, basic association analysis by X2-test of allele frequencies and logistic regression, to assess breed-specific association of the SNPs to CHD. The analysis method that demonstrated a better model fit, as evaluated by visual examination of quantile-quantile (Q-Q) plots (see Additional file 3), was chosen for each breed. Logistic regression showed better model fit for five breeds and the basic association test for the other five (see Additional file 3). Some inflation of the expected versus observed P-values was observed in three breeds: Finnish Lapphund, Golden Retriever, and Labrador Retriever (see Additional file 3). All these breeds have at least partially separated breeding lines of herding/working dogs (meaning restricted mixing of the breeding dogs between lines), which may be the source for the inflation.
The within-breed analyses showed multiple significant associations for different SNPs per breed (Table 2). The number of significantly associated SNPs varied between breeds, ranging from none in Bernese Mountain dogs to six significant associations in the Labrador Retrievers (Table 2). In total, 22 SNPs on CFA1, CFA3, CFA8, CFA11, CFA12, CFA14, CFA17, CFA21, CFA24, CFA25, CFA26, CFA33, and CFA34 demonstrated significant associations to CHD (Table 2). Six SNPs were significant in more than one breed (ss7212922122, ss7212922151, ss7212922154, ss7212922155, ss7212922161, ss7212922163 Table 2). These six SNPs are located on CFA33, CFA26, CFA34, CFA11, CFA1, and CFA24. The marker ss7212922155 on CFA11 was significant on three breeds (Table 2).
ss7212922126 and ss7212922153 on CFA1, as well as ss7212922156 and ss7212922152 on CFA8 were in high linkage disequilibrium (r2 > 0.80) and therefore these SNP pairs were interpreted to represent one locus each. Thus, the 22 markers associating with CHD and with OR’s deviating from 1 represent 20 different loci on 13 chromosomes (Table 2).
Our across and within breed analyses highlighted altogether 21 loci on fourteen chromosomes. These loci contain hundreds of candidate genes and we wanted to understand whether they are enriched in any cellular pathways. We performed two analyses using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) searching for the dog genes/proteins [15], including all the positional candidate genes within 1 Mb of the associated SNPs. In the first analysis, no enriched pathways were detected among the 58 positional candidate genes from the across-breed study (see Additional File 4). For the second analysis, we pooled all the 272 positional candidate genes from the across and within breed studies (Additional File 4). An enrichment in the neddylation pathway (Reactome ID: CFA-8951664) was spotted by STRING with a false discovery rate of 0.0314 (12 observed genes / 220 genes in the neddylation-pathway gene set, see Fig. 1 and Table 3).
The protein network of the positional candidate genes from all of the 21 loci. Each circle represents one protein. Red circles indicate proteins that belong to the neddylation pathway. The lines between circles indicate the evidence for the association between proteins. The thicker the line, the stronger the evidence. The total number of nodes is 263. For the complete STRING analysis, see https://version-11-0.string-db.org/cgi/network.pl?networkId=5Sqk4IV9gi5b
Previous efforts by us and others have discovered tens of loci associating with CHD across breeds. However, the significance of these findings has remained vague due to the lack of proper replication because of the variability of the phenotypes, varied study approaches and inadequate sample sizes.
Our replication study in over 1600 dogs across 10 breeds with 52 reported CHD markers validates 21 loci on fourteen chromosomes. Five loci were associated across breeds. Two loci included more than one validated marker. Inclusion of more markers in the study would have made the validation of each locus more robust and possibly allowed further selection of associated loci by the presence of clusters of validated SNPs. Also, after our analyses several interesting markers were published [16].
In total, the replicated loci include over 250 genes with an enrichment of candidate genes in a neddylation pathway. Neddylation contributes to various cellular functions, including inflammation, commonly found in CHD and OA. Collectively, these results highlight the complex genetic background of CHD and provide important insights to the significance of the common and breed-specific loci in CHD. This helps to prioritize loci for further studies to identify causal variants and suggest a novel hypothesis for the possible contribution of the affected neddylation pathway to CHD and OA.
Four loci on CFA1, CFA14, CFA26, and CFA37 associated with CHD across breeds. The variants ss7212922118 and ss7212922120 on CFA1 were reported by us to associate with CHD in German Shepherds [13]. These SNPs locate within and upstream of NADPH Oxidase 3 (NOX3), which is a catalyst for the formation of superoxides and other reactive oxygen species. NADPH oxidases are an essential part of a reaction chain that has been suggested to contribute to the initiation of articular cartilage degradation [17]. However, NOX3 is mainly expressed in the inner ear and foetal tissues, which leaves its role in CHD equivocal.
The SNP on CFA14 (ss7212922135) was originally reported to associate with CHD in Bernese Mountain dogs [6]. This SNP did not associate with CHD in our Bernese Mountain dog cohort, which may result from different case definition between the current and the earlier study. The variant ss7212922135 originated from a study cohort where all cases (N = 33) had mild CHD (FCI score C) [6], while our Bernese Mountain dog cases represented mild-to-severe CHD based on FCI hip scoring (Ncases = 88; 42 with mild (FCI score C), 40 with moderate (FCI score D), and six with severe (FCI score E) CHD). However, ss7212922135 was significant in the across-breed analysis. This SNP lies within the ninth intron of Cortactin Binding Protein 2 encoding gene (CTTNBP2) [6]. CTTNBP2 participates in brain development and the regulation of synapse organisation. Although no obvious connection was found between this gene and CHD in the original study [6], some more recent phenotype associations have been listed in the GWAS catalogue (https://www.ebi.ac.uk/gwas/home) for CTTNBP2 that are worth noting: juvenile idiopathic arthritis and idiopathic osteonecrosis of the femoral head. Furthermore, body mass index adjusted waist-hip ratio, a measure for storage fat in humans, is listed in the GWAS catalogue [18] for ST7, WNT2, ASZ1, and CFTR, all within ±1 Mb of ss7212922135. Obesity is a known environmental risk factor for hip dysplasia and OA in both dogs and humans [19], although the mechanism is still poorly understood. Finally, we want to highlight the gene encoding Wnt Family Member 2 (WNT2) ~ 401 kb downstream ss7212922135. Wnt signalling pathways have been shown to participate in joint development, cartilage maintenance homeostasis, and the development and progression of OA [20].
Variant ss7212922151 on CFA26 was the third SNP, which demonstrated significant association to CHD in the across-breed analysis. This SNP originates from an association study of CHD in German Shepherds and locates within the fifth intron of Kinase Suppressor Of Ras 2 encoding gene (KSR2) [7]. KSR2, a scaffolding protein in the Ras-Raf-MEK-ERK pathway, has been associated with obesity in mice and humans [21, 22]. Although the non-SMAD-dependent TGF-β/BMP signalling in the osteoblastic lineage involves MKK3/6 and p38 [23], we are not aware of any studies reporting on their interaction with KSRs. Another gene of interest in the same locus is Nitric Oxide Synthase 1 (NOS1; ~ 262 kb away from ss7212922151). A large variety of different phenotypes have been reported in Nos1 murine models, including abnormal skeletal and skeletal muscle phenotypes for Nos1tm1Plh-mutants [24].
ss7212922122 on CFA33 locates within the gene encoding PEST Proteolytic Signal Containing Nuclear Protein (PCNP), and was originally found to associate with CHD in German Shepherds [8]. PCNP expression is omnipresent in different tissues, and with its ubiquitination partner Np95/ICBP90-like RING finger protein (NIRF) it may be involved in a signalling pathway of cell cycle regulation and/or genome stability [25, 26]. ss7212922122 is also about 469 kb upstream from ABI Family Member 3 Binding Protein (ABI3BP), which is a collagen and glycosaminoglycan binding molecule and an extracellular matrix structural component. Moreover, an intron variant of ABI3BP (rs9828061) has been associated with joint hypermobility measurement in humans [27].
Lastly, ss7212922139 on CFA37 locates within the first intron of Neuropilin 2 (NRP2). It was the only marker that despite not being significantly associated with CHD in any particular breed was still significantly associated with CHD in the across-breed analysis. Indeed, this SNP originated from a study, which utilised association and linkage populations of multiple breeds and their crosses [4]. In our study the marker had OR < 1, indicating protective effect. The original study did not report ORs, and the SNP associated with OA [4]. Although the current study data did not contain the direct OA phenotypes, OA is nevertheless assessed and considered when determining the FCI score. Considering the original and current study, ss7212922139 could represent a genuine “across-breed locus” for CHD and OA. Zhou et al. (2010) suggested Par-3 Family Cell Polarity Regulator Beta (PARD3B) as a candidate gene due to its association with OA of the knee in humans [4, 28]. In addition to PARD3B, NRP2 could also be a plausible candidate for OA. Neuropilin 2 is as a co-receptor for vascular endothelial growth factors (VEGFs) [29]. Increased VEGF expression has been indicated to associate with increased severity of OA [30], and a functional study demonstrated that VEGF injections into knee joints of mice induced OA [31]. Moreover, VEGF and its receptors have been studied as targets for treatment of OA [32], and an experimental study of a VEGF antibody (rhu-Mab-VEGF; Bevacizumab or Avastin®) has shown that Bevacizumab could offer a potential therapy for OA [33]. However, it remains unknown how NRP2-VEGF interaction could affect the development and progression of OA in dogs.
The current within-breed analyses revealed a multitude of loci that associated with CHD, strengthening the hypothesis that CHD has a complex genetic architecture and distinct genetic backgrounds in different breeds. The highest number of associated SNPs was found with Labrador Retriever (6), while none of the tested markers associated with CHD in our Bernese Mountain dog cohort. The associated markers and loci varied between breeds. Some SNPs demonstrated association to CHD in more than one breed and ss7212922151 on CFA26 was also significant in the across-breed analysis. We want to acknowledge here that the results in the Finnish Lapphund, Golden Retriever and Labrador Retriever breeds are inflated due to possible population stratification (as evidenced by the Q-Q plots, see Additional file 3). These breeds have both show and working/herding lines, which we could not account for in this study due to the missing line information. Thus, the breed-specific results for these three breeds should be regarded with some caution.
Some of the significant markers from the within-breed analyses had notably higher or lower ORs (Tables 1 and 2), which is probably due to higher across-breed variation for these markers. Also, worth noting was that some breeds had ORs of over 5 for certain markers (Table 2), which indicates relatively strong association to the disease outcome for such a complex disorder. Future studies should concentrate on these loci for breeds such as the Great Dane that had OR of 5.07 for ss7212922133 on CFA14, and had no marked inflation observed (see Additional file 3, plots M and N).
This study replicated altogether 21 loci with over 250 potential candidate genes raising an interesting question of the possible relationship and enrichment of the candidate genes in the associated loci predisposing to CHD. The STRING analysis revealed that the candidate genes were enriched in a single pathway, neddylation, which is a ubiquitination-like conserved post-translational protein modification process [34]. The key actor in neddylation is NEDD8, which is conjugated to its substrates with the help of enzymes E1 (activation of NEDD8; NAE1-UBA3 heterodimer), E2 (conjugation; UBE2M or UBE2F), and E3 (ligase; RBX1 or RBX2) [35]. Neddylation modifies the biochemical properties of the target substrates [34], such as many members of the cullin-family, p53, or EGFR [35, 36]. Neddylation is essential for cell cycle progression [37, 38], and it has been linked to many pathologies, especially to human cancers [36, 37]. Neddylation was recently linked to inflammatory arthritis via increased NF-κB activation, although increased expression of neddylation-related genes (NEDD8 and CUL1) were observed only in the synovium of the rheumatic arthritis and not in the controls with non-inflammatory OA [39].
Interestingly, searching the STRING database with the 12 neddylation pathway associated candidate genes from the current study (Table 3) and with 14 genes (CYBA, MAPK14, MMP2, MMP9, NCF1, NCF2, NCF4, NOX3, NOXA1, NTN1, RAC1, RAC1, TRIO, VCAM1) highlighted in our previous studies on CHD on German Shepherds [13, 40] produced two gene clusters that shared 12 genes associated with Class I MHC mediated antigen processing & presentation (R-CFA-983169, https://version-11-0b.string-db.org/cgi/network?networkId=bPwtieGk6WuV, and Additional file 5).
There is cumulating evidence that inflammatory mechanisms are active in OA [41, 42]. Neddylation is demonstrated to participate in regulation of i.a. T-cell and macrophage functions during inflammation [39, 43, 44]. Both T-cell and macrophage mediated inflammatory responses have been observed in OA [42, 45], and therefore, the possible role of neddylation-pathway in the immune base of OA should be explored further.
We replicate 21 previously reported CHD-associated loci on fourteen chromosomes and identify neddylation as a novel candidate pathway for CHD and OA. We identify common and breed-specific loci and highlight the complex genetic architecture of CHD. Identification of the causal genes and variants in the associated loci remains as an important future task to better understand the molecular pathogenesis of CHD and its subtraits towards improved treatment and diagnostic options.
We used SNP genotyping to validate 52 SNPs on several chromosomes in a large cohort comprising of ten breeds. Our cohort consisted of 1607 dogs with FCI hip scores (Table 4), which were used to categorize dogs into cases (hip score C or worse on both joints, N = 772) and controls (hip score A/A, N = 835). It is worth noting that the FCI scoring does not represent a quantitative phenotype. Furthermore, the distribution of phenotype categories of the cohort is very skewed and does not meet the assumptions required for the analysis of datasets in the ordinary scale. Because of these reasons, only case-control analysis was possible. To minimize phenotype ambiguity, dogs with hips scored B were excluded. Although score B is considered normal, it nevertheless represents a borderline normal phenotype.
The participating breeds were (in order of number of samples per breed): Finnish Lapphund, Golden Retriever, Lagotto Romagnolo, Bernese Mountain dog, Samoyed, Spanish Water dog, Great Dane, Labrador Retriever, Karelian Bear dog, and Finnish Hound. These breeds were chosen for the project because the prevalence of CHD in them is at least moderate. FKC collects FCI hip scoring data into an open-access breeding database from which the phenotypes were gathered for this study [2, 46]. We investigated the prevalence of CHD in the above-mentioned breeds as the mean of observed yearly prevalences including all cases from FCI hip score C to E, measured in an eleven-year period (2006–2016, year of birth) [2, 46]. The lowest mean prevalence was observed for Labrador Retriever (19%) and the Finnish Hound (28%) [46]. Finnish Lapphund, Golden Retriever, Samoyed, Spanish Water dog, and Great Dane all had a mean prevalence of CHD between 30 and 40% during this time period [46]. The highest mean prevalence was observed for the Karelian Bear dog (41%), Bernese Mountain dog (43%), and Lagotto Romagnolo (45%) [46].
To minimise possible genomic stratification and subsequent inflation of the test statistics we were careful not to incorporate close relatives and included only one individual from all core-families during the initial data collection. Nevertheless, more distant relatedness may exist within the breeds. Also, Finnish Lapphund, Golden Retriever and Labrador Retriever are breeds that have working/herding breeding lines, which are at least partially separate from the rest of the breeding dogs. In the current study we did not have the breeding line information for these dogs and could not account for it in the analyses. This may cause additional inflation of the test statistics due to stratification within the breed. The breed-specific results for these three breeds must therefore be interpreted with some caution.
Agena MassARRAY® iPLEX was used to genotype 52 SNPs (Additional files 1 and 2) in 1607 dogs from ten breeds. Six of the SNPs were chosen for the project from our own study in German Shepherds [13]. Ten markers from CFA1, CFA5, CFA8, CFA20, and CFA25 were selected based on investigations by Wisdom Health. These markers are included in a patent (Patent no.: US10150998B2 [12]) and five of them were also in Lavrijsen et al. (2014) [9]. The rest of the markers were chosen from earlier studies of Zhou et al. (2010) [4], Friedenberg et al. (2011) [5], Pfahler and Distl (2012) [6], Bartolome et al. (2015) [11], Fels and Distl (2014) [7], Fels et al. (2014) [8], and Sanchez-Molano et al. (2014) [10] to evaluate whether results were replicable in our data set. Data from the previous studies (breed, size of cohort and reported raw and corrected P-values) are summarized for each marker in Additional file 1.
The genotyping was performed by the Institute of Molecular Medicine Finland (FIMM) Technology Centre, University of Helsinki. The genotyping was done in two separate batches; the first batch included samples from breeds Labrador Retriever, Golden Retriever, and Bernese Mountain Dog; the second batch included samples from breeds Spanish Water Dog, Karelian Bear Dog, Lagotto Romagnolo, Finnish Hound, Samoyed, Finnish Lapphund, and Great Dane. Initial quality control of data was carried out in FIMM. In the first batch one sample was discarded due to success rates lower than 70%, and four SNP assays were discarded due to unreliable or no results. In the second batch, five samples were discarded due to success rates lower than 70%, and two SNP assays were discarded due to unreliable or no results. The resulting data were delivered to us as map and ped files. The map file was based on the CanFam3.1. reference, Annotation Release 104.
We carried out the quality control in stages for within-breed and across-breed association analyses. Initially there were 1607 samples and 52 SNPs before the QC steps. The QC thresholds for the breed-specific data were: 0.90 for per ID and per SNP call rates, 0.05 for the minor allele frequency (MAF), and 0.0001 for the cut-off p-value in check for Hardy-Weinberg Equilibrium (HWE) (done in controls only). The resulting breed-specific data is described in Table 4. SNP-specific failures to meet the QC criteria are presented in Additional file 1.
The QC for the across-breed data was done in two steps in PLINK. The first QC step was done at the breed-level before merging, with the following thresholds: 0.90 for per ID and per SNP call rates, 0.0001 for the cut-off p-value in check for HWE check in controls. MAF cut-off level was set to zero in this initial step, because some SNPs that might not pass the MAF threshold within a breed, might however pass it in the across-breed cohort. Subsequently, the SNPs and individuals that passed this initial QC step were merged into the across-breed data set. The final QC with the MAF cut-off threshold at 0.05 was then executed over the whole across-breed data, which left us 46 SNPs and 1572 samples. However, one Bernese Mountain dog and one Golden Retriever had hip scores of B/D (left hip/right hip) and they were excluded from the analysis, because we could not rule out the possibility of unilateral CHD induced by an injury or other environmental factor. Therefore, we finally had 1570 dogs in our analyses, of which 751 were cases, 819 were controls, and 666 were males and 904 were females.
Before the analyses we tested the quality-controlled SNP data for possible batch effects in R with the glm-function. This was done because the genotyping was executed in two batches. We did not observe any significant batch effects. The within-breed analyses were carried out with the –assoc (X2-test of allele frequencies) or with –logistic (logistic regression) functions in PLINK, with age at radiographing as covariate in the logistic model (–assoc cannot use covariates).
As the SNPs in the study are a strongly selected and small subset of all the SNPs in the genome, the quantification of inflation is a challenge. QQ-plots were used to assess the overall fit of the alternative models and to select between them (Additional file 3). The QQ-plot is a more representative proxy for the fit of the model than the lambda value by PLINK which is based on the ratio of single (median) value of the test variable.
Some inflation of the test statistics in both analysis methods was observed in three breeds (see Additional file 3, plots A–D and O–P): Finnish Lapphund, Golden Retriever, and Labrador Retriever. We did not attempt to quantify or control the stratification in these breeds by PCA as it is not expected to work when the number of markers is small and the effect of any single marker is expected to be low [47] as is the case in our study.
Odds ratios and their 95% confidence intervals were calculated in PLINK using the default settings and the function –ci. PLINK assigns the less frequent allele as the minor allele that increases the risk when the odds ratio is greater than one.
We used 2x2xK (K = 11) Cochran-Mantel-Haenszel (CMH) statistics for the across-breed analysis. CMH is a standard test for a stratified case-control analysis. This was carried out in PLINK with the function –mh and with breed clusters defined with the –within function. Odds ratios and the respective 95% confidence intervals were automatically calculated by PLINK. The CMH test assumes homogeneity of the odds ratios between strata (breeds in our case), and violation of this assumption may lead to false positive associations [48]. Therefore, we used –homog function in PLINK to check if any of the SNPs demonstrating association in the CMH test, would violate the homogeneity assumption; all associated SNPs passed the homogeneity test. All permutation analyses (using 10,000 permutations) within and across breeds were executed with the max(T) permutation procedure in PLINK with the function — mperm 10,000. We used a fixed seed (–seed 873,051,416) generated in Unix shell with “date +%N” to ensure reproducible results in all of the permutation analyses.
We used STRING (Search tool for retrieval for interacting genes/proteins) (V11.0) [15] to carry out a pathway analysis of the candidate gene sets, which we acquired from our association analyses. All genes from within 1 Mb from the variant that demonstrated significant association to CHD were listed and then used as an input for the STRING Multiple proteins search. The used candidate gene sets are listed in the Additional file 4. The STRING database was queried with 272 canine genes but seven genes (TMEM244, STRA6L, FOXE1, RPS13, SERGEF, ESPNL, and FAB172B) were not found. In addition, U6 and CNKSR3 were not recovered in the expected chromosome and were discarded. Thus, the STRING network for the positional candidate genes consisted 263 nodes (https://version-11-0.string-db.org/cgi/network.pl?networkId=5Sqk4IV9gi5b). We also performed an additional search with a combined set of neddylation pathway associated genes from Tables 3 and 14 genes highlighted in our previous studies (https://version-11-0b.string-db.org/cgi/network?networkId=bPwtieGk6WuV).
The datasets generated and analysed during this study are available in the FIGSHARE repository: https://doi.org/10.6084/m9.figshare.11369511. The data was anonymised to protect the privacy of the dog owners.
Ortolani sign hip dysplasia test in dogs
Mário Ginja,1 Ana Rita Gaspar,1 Catarina Ginja,2,3 1Department of Veterinary Sciences-CITAB, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; 2Ce3C – Centro de Ecologia, Evolução e Alterações Ambientais, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal; 3CIBIO-InBIO – Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Vairão, Portugal Abstract: Canine hip dysplasia (CHD) is the most common inherited polygenic orthopedic trait in dogs with the phenotype influenced also by environmental factors. This trait was described in the dog in 1935 and leads to a debilitating secondary hip osteoarthritis. The diagnosis is confirmed radiographically by evaluating signs of degenerative joint disease, incongruence, and/or passive hip joint laxity. There is no ideal medical or surgical treatment so prevention based on controlled breeding is the optimal approach. The definitive CHD diagnosis based on radiographic examination involves the exposure to ionizing radiation under general anesthesia or heavy sedation but the does not reveal the underlying genetic quality of the dog. Phenotypic expression of CHD is modified by environmental factors and dogs with a normal phenotype can be carriers of some mutations and transmit these genes to their offspring. Programs based on selection of dogs with better individual phenotypes for breeding are effective when strictly applied but remain inferior to the selection of dogs based on estimation of breeding values. Molecular studies for dissecting the genetic basis of CHD are ongoing, but progress has been slow. In the future, the recommended method to improve hip quality in controlled breeding schemes, which will allow higher selection pressure, would be based on the estimation of the genomic breeding value. Since 2012, a commercial DNA test has been available for Labrador Retrievers using a blood sample and provides a probability for development of CHD but we await evidence that this test reduces the incidence or severity of CHD. Keywords: canine hip dysplasia, phenotype, breeding stock, GWAS, screening, diagnosis
Canine hip dysplasia (CHD) is the most common inherited polygenic orthopedic trait with the phenotype influenced by environmental factors.1 This trait was described in the dog in 1935, in the USA, and leads to a debilitating secondary hip osteoarthritis.2 Heritability estimates for CHD vary from 0.1 to 0.83,3,4 due to different pedigrees, methods used to calculate the heritability, and the hip phenotypes analyzed.5 CHD is more prevalent in large and giant breeds of dogs often resulting in mild or no clinical signs.1,6 However, for some dogs clinical signs can be severe and resistant to medical management needing aggressive and expensive surgical treatments.1,7 The definitive diagnosis of CHD is made if characteristic radiographic signs are evident on a standard or stressed ventrodorsal view of the pelvis, occurring along a gradual scale from nearly normal to severely affected.1 This is a crucial aspect of CHD as the radiographic diagnosis has been essential for the selection of breeding stock.1 Studies attempting to find genetic markers for CHD diagnosis are now frequent.8–11 The sequencing and annotation of the canine genome has resulted in renewed interest in research of the genetic underpinnings of canine orthopedic disorders, particularly those of a multifactorial etiology, such as CHD.12 Recently, the first commercial CHD diagnostic genetic test for Labrador Retrievers appeared,13 but, the imaging diagnosis continues to be of major importance for disease screening and treatment. Humans are also affected by hip dysplasia and both conditions have phenotypic similarities of joint subluxation and the development of osteoarthritis.14 However, the main medical approach in humans is different, being mainly based on the preventive management and with good results.1,15 Currently, molecular CHD studies are considered useful for the understanding of the genetic basis of analogous conditions in humans, mainly because the heterogeneity in human populations and the complexity of this disorder makes the genetic dissection of human hip osteoarthritis more difficult.12 The main purpose of this review is to present and discuss medical aspects of CHD for which knowledge is incomplete and have therefore merited major current research efforts.
CHD continues to be a common trait mainly in large and giant breeds, in pet and working dogs, with prevalence higher than 50% in some breeds.2 The clinical presentation of the disease is not correlated with the radiographic changes.2,6 Clinical signs of CHD are more evident in dogs younger than 1 year of age due to hip instability, or in adult dogs with chronic pain from osteoarthritis.7,16 Chronic hip alterations, such as fibrosis and thickening of the joint capsule, result in joint stability and improvement of limb function, masking the clinical signs and functional limitations in middle-aged animals.6,17 Clinical signs warrant medical and/or surgical treatment.18,19 Preventive conservative or surgical management could be indicated in puppies at risk of developing CHD,19,20 but early intervention is hampered because there are no pathognomonic clinical signs for CHD. Common clinical signs are: slight to moderate lameness; gait and running abnormalities, such as shortened stride length and bunny hopping; difficulty in rising and reluctance to climb stairs.21
Information about the conformation of the hip joint can be obtained using clinical or diagnostic imaging tests.21,22 These medical tests are usually performed on sedated or anesthetized animals and are separated into two main categories: to evaluate hip joint laxity (HJL), mainly used on young animals; to detect clinical or radiographic signs of osteoarthritis, as crepitation and reduced range of motion on joint palpation or degenerative joint disease (DJD) signs on radiographs.1,21 However, it would be very helpful to develop medical CHD screening techniques for fully conscious young animals, similar to hip dysplasia screening examination of human neonates.1 The imaging diagnosis of CHD has been the main area of research of CHD in the last 50 years, for the purposes of reproductive control. Clearly, in terms of human medicine the main focus has been different, trying to refine diagnostic accuracy and preventive management.1,15
The Ortolani test is the most common and popular physical maneuver that is used in veterinary medicine to diagnose HJL in young dogs (4–12 months of age).23,24 Other clinical tests are described, like the Barlow’s and Barden tests for puppies younger than 4 months of age but their clinical accuracy is more questionable.1,17 The Ortolani test is performed with the dog awake, sedated or anesthetized with the patient in lateral or dorsal recumbency.23 The test has two steps, first apply proximal force to the stifle joint on the non-dependent limb, with the hip at a normal weight-bearing angle, and while still applying this force, slowly abduct the joint. In hips with abnormal laxity, the dysplastic femoral head, may be displaced dorsally beyond the dorsal acetabular rim (in the first step) and then the limb abduction promotes its reduction back into the acetabulum (in the second step) which elicits a typical palpable and/or audible clunk, of variable magnitude, commonly called a positive Ortolani sign (Figure 1).1,23 Advanced stages of CHD with destruction of the acetabular rim or in dogs younger than 4 months of age with an inadequate acetabular ossification can result in false negative cases based on the Ortolani maneuver even though HJL may be present.17,24,25 The Ortolani test showed an excellent sensitivity in prediction of CHD when used in dogs younger than 1 year of age that later developed moderate or severe CHD.24
Radiography is the reference technique for the definitive diagnosis of CHD from its first description in 1935. This imaging technique uses different radiographic views of the hip joint for genetic screening purposes or for diagnosis and treatment of dogs with clinical CHD. All these radiographic techniques should be performed under anesthesia or heavy sedation, which facilitates accurate positioning and elicitation of passive HJL.1,3,7,19 Given the complexity of the topic and the objectives of this review, we will cover particularly the radiographic studies used for genetic screening of CHD, which are used to detect HJL, the major risk factor for CHD, or signs of DJD.
The radiographic information on HJL is obtained using radiographic techniques such as PennHIP,26 dorsolateral subluxation (DLS),27 Flückiger28 and half-axial position methods.22 Signs of DJD are evaluated using the standard ventrodorsal hip-extended view (SVDV).2,29,30
In this group of methods, the PennHIP was the pioneer and is the most popular. It was developed at the University of Pennsylvania in the 1980s with the main purpose of CHD breeding control.26 One of the main advantages of this procedure is its precocity, being performed with accuracy on dogs at 16 weeks of age, compared with 1 or 2 years of age for previous screening systems.26,29,30 The PennHIP method requires certified members and is performed with three hip radiographic views: hip-extended, compression, and distraction.26 The distraction view is used to measure HJL. It is performed with the dog in dorsal recumbency, with the hips at a neutral position and the PennHIP distractor between hind limbs acting as a fulcrum lateralizing the femoral heads under examiner force.26 The radiographs are sent to the PennHIP Analysis Center at University of Pennsylvania for an official report, and the dogs are placed in rank-order with the other dogs of the breed in the database. The HJL is evaluated in the distraction view calculating the distraction index (DI), which measures the relative degree of femoral head displacement from the acetabulum.3,26 The DI ranges from 0 to >1, with 0 representing a tight hip and 1 a loose hip.26
The DLS is also a passive stress radiographic or tomographic imaging technique; the HJL is measured as the DLS score.27 The hip stress is caused by weight bearing. The DLS score has a strong correlation with DI. This method was reported in dogs at 4 and 8 months of age to evaluate the chondro-osseous acetabular and femoral head structure as an indicator of functional joint stability.27,31
In the Flückiger method, the HJL is estimated with the subluxation index in a similar manner to the DI.28 The stress in hip joints is caused by the dorsocranial force exerted by the examiner, with the dog in dorsal recumbency.28 No follow-up studies were published and the research was performed in adult animals together with the SVDV to better assess the quality of the hips.
The half-axial position method was used by positioning the dog and performing the hip stress similar to the PennHIP, using a trapezoidal-shaped distractor.22 The HJL measures using this method are performed mainly with the purpose of early CHD diagnosis and treatment, using the juvenile pubic symphysiodesis (JPS).
The DJD is evaluated using the SVDV, a universal radiographic view used in dogs older than 1 (Fédération Cynologique Internationale’s [FCI] system) or 2 years (Orthopaedic Foundation for Animals [OFA] system) of age.1,29 This view has been used since the 1960s.32 The dog is placed in dorsal recumbency on the X-ray table, with hind limbs extended parallel to each other with the stifles internally rotated.1,32 Many international systems are used to evaluate DJD, including the FCI,1 OFA,29 British Veterinary Association/Kennel Club (BVA/KC),30 and the Flückiger33 method with more influence in continental European countries, USA, UK and Australia, and Switzerland, respectively. As the main guideline of all of these scoring methods is based on the degree of subluxation, joint congruence and remodeling of the femoral head and acetabulum, these scoring systems may be equal (Table 1). However, these direct comparisons between grades and schemes are considered speculative, due to their subjective nature.34 These scoring systems have some particularities: FCI requires a minimum age of 12 months in medium breeds and the OFA method 24 months; FCI scrutinizers are not certified; FCI, OFA, and BVA/KC are voluntary screening schemes. The SVDV is not strongly evaluative of HJL. It is underestimated. The parallel hip-extended positioning and the internal rotation of stifles twist the hip soft tissues, tightening the tensile elements of the joint capsule and may reduce some degree of luxation.26
Ultrasonography in human neonates is the reference technique for the definitive diagnosis of developmental hip dysplasia.35 However, the use of ultrasound in puppies for the confirmation of CHD is not recommended, as the acetabulum cannot be evaluated after 8 weeks of age because femoral head ossification and acetabular chondro-osseous alterations are only evident after this age.17 Increased synovial fluid volumes in hip joints detected by magnetic resonance imaging in 8-week-old puppies were correlated with later HJL and CHD.17 Dynamic ultrasonography was used in puppies at 8–16 weeks of age, to quantify HJL.36 HJL and osseous acetabular structure can be evaluated confidently using computed tomography.27
Some preventive conservative and surgical treatments have been proposed for young dogs with clinical predisposition for CHD.7,19 The main conservative management recommendations are based on limiting food consumption and controlled weight-bearing activity to prevent obesity and develop muscular tissues.1,37 Disease-modifying osteoarthritis drugs given by injection are recommended, as they retard breakdown and may promote the synthesis of cartilage matrix and reduce pain and inflammation.20 Analgesic or anti-inflammatory medications are effective to manage pain and lameness but should only be used for the short term due to their undesirable side effects. However, the long-term effectiveness of this conservative treatment is questionable, since their ability to prevent the development and progression of osteoarthritis is at best limited.7,19
JPS is a surgical treatment used on puppies at 14–20 weeks of age and at risk of developing CHD,19,38 with greater improvements achieved when surgery is performed at 15 weeks of age.38 JPS is a minimally invasive procedure based on induction of thermal necrosis in chondrocytes of the growth plate of the pubis.19,38 The pubic growth plate undergoes premature closure resulting in an underdeveloped ventral pelvis and normal dorsal development.7 This modified pelvic growth results in an increase in acetabular coverage of the femoral head and reduction of subluxation forces.38 This technique has been recommended in puppies with slight to moderate signs of CHD but not in animals with severe signs of CHD. In the JPS, acetabular ventroversion occurs slowly and in severe cases of CHD the femoral head continues to slide laterally, the dorsal acetabular edge becomes round and femoral head stability is never obtained.19
Triple pelvic osteotomy is a reasonable surgical treatment option for CHD being used in animals between 5 and 12 months old, without radiographic signs of DJD and with minimal or clinical signs of CHD.7 However, triple pelvic osteotomy is more effective in preventing the development of DJD when used in dogs younger than 7 months of age.7 The pelvis is cut in the pubis, ischium, and ilium, rotated and the ilium fixed with a surgical plate. This surgical procedure results in ventrolateral rotation of the acetabulum and provides immediate increased femoral head stability. However, the hips of dogs with extant osteoarthritic changes or with a high HJL continue to deteriorate and have a less favorable outcome.7 When osteoarthritis is already at an advanced stage, treatment should be performed to alleviate pain and maintain joint function.18,39
Femoral head and neck excision reduces the pain produced by abnormal bone to bone hip joint contact, but it does not effectively maintain the full range of hip motion and limb function. The total hip replacement is the best treatment to preserve long-term limb functionality.
CHD is a complex polygenic disease due to the small additive effect of many genes.4,8 Environmental factors such as sex, age, and body weight can influence the expression and severity of the disease.37 It appears that CHD is not a congenital disease, hips are normal at birth with adequate femoral head and acetabular congruence. The first 60 days of a puppy’s life is thought to be the most critical period in terms of development of the hip joint.16 In this period, the depth of the acetabular cavity and the proximal femoral head and neck conformation are susceptible to modeling according to the stress loading.15
In a normal congruent hip joint, the normal weight bearing force is transmitted between femoral head and acetabulum across the surface of the articular cartilage. The joint incongruence favors the reduction of contact between the cartilaginous surfaces, the early destruction of chondrocytes by increasing pressure and the cyclic cascade of osteoarthritis. Small synovial joint volume and low intracapsular pressure, high pelvic muscle mass, and a reduced level of the hormones that promote soft tissue relaxation maintain stability and prevent the development of CHD signs.26 The HJL is the primary risk factor, well-evaluated in a radiographic study that is associated with CHD development.40 Acetabular and proximal femoral head and neck conformation is directly associated with the magnitude of transmitted hip forces.41 So, genetic factors associated with CHD can be related to hip conformation, cartilage susceptibility to pressure forces, joint soft tissues or even to hormonal factors.
Published heritabilities of CHD traits are variable and commonly range between 0.1 to 0.60.12,42 Differences of heritability estimates depend on the trait used, calculation method, selection, the population and sample used for estimation.5,42 For example, heritability reached as high as 0.83 for passive hip laxity in the Estrela Mountain dog breed from Portugal.3 The genetic improvement used in selection of traits with higher heritabilities and similar selection pressures will be bigger per generation.1
Selection of breeding stock with low hip scores – progress toward reducing incidences of hip dysplasia
Because there is no ideal medical or surgical treatment, veterinarians in practice are at the forefront, and their main focus is on prevention of CHD through reproductive control.43 Selection of breeding stock has clearly been a priority intervention area of veterinary medicine. Control CHD programs based on radiographic phenotype quality of hips were used in some countries as early as the 1960s.44–46 These programs were based on radiographic screening of CHD using the SVDV and scores of hip quality based on DJD signs and hip congruence. Selection of breeding stock was based on an individual dog’s hip phenotype and subjective pedigree evaluation performed by the breeder. The results of phenotype-based genetic screening on CHD prevalence and severity are somewhat disparate in different countries and breeds. When the CHD control schemes are voluntary, as the OFA, FCI, and BVA/KC, only the better hip phenotypes are scrutinized and the studies performed using these databases are biased.2,42 Using the OFA scoring scheme and the best linear unbiased prediction method for the estimation of breeding values and its application in the selection for hip joint conformation, for Labrador Retrievers the total genetic improvement in four decades (1970–2007) corresponded to only ~17% of the total phenotypic standard deviation.12 However, radiographs of dogs with normal-appearing hips are several times more likely to be submitted for evaluation in the OFA system than radiographs of dogs that are severely dysplastic.47 For example, in a closed breeding colony of German Shepherd dogs and Labrador Retrievers, CHD prevalence over five generations of selection decreased from 55% to 24%, and from 30% to 10%, respectively.46 Selective breeding against CHD was less effective in decreasing its prevalence and severity in Sweden.44 In other countries, such as Finland, the general CHD control program was even considered ineffective in reducing the prevalence of CHD in various dog breeds.45 For the PennHIP method, there are no reports of its effectiveness in reducing the prevalence of CHD in different dog populations. Theoretically, it is a promising method since studies have shown that HJL has a higher heritability than the CHD scores based on DJD. The lack of desired success of CHD control programs that rely totally on individual phenotype has been due partly to its sensitivity to environmental factors. Animals with a normal individual radiographic phenotype can still be carriers of CHD genes, which will be transmitted to their offspring and maintained in the population.48 Estimated breeding values (EBV) are commonly used in farm animal selection for complex polygenic traits (phenotype expression is influenced by environmental factors), such as milk yield or growth rate.28,49 So, the phenotypic expressions of these traits are very similar to CHD, determined by heredity and environment. The EBV for CHD is a genetic parameter derived from the hip quality of relatives, and is thus more representative of the dog’s genetic quality,12,44 and allows monitoring of the genetic trends in dog populations,50 being recommended for CHD selection purposes.51,52
With the rapid development of high-throughput sequencing technology and emergence of high-density genome-wide single nucleotide polymorphisms (SNPs) canine arrays, associations between genetic markers in linkage disequilibrium and CHD genes have been discovered.14,42 The molecular genetic information can be applied for CHD selection purposes, particularly if the most informative SNPs are used to estimate the genomic breeding value (GBV) of an individual. Such genomic selection was successfully applied in livestock animal breeding programs and can be used for selection against prevalence of undesired traits with greater genetic improvement.10,42,43,53 In the near future, GBV might become the recommended method to improve hip quality in CHD control schemes.12,43,53 In a particular dog breed, pedigree and phenotypic data can be used to obtain EBV and combined with genomic data to derive a predictive formula for the GBV.12 Then, the genotyping of a puppy for a set of informative SNPs can be combined with radiographic information, and used to determine the susceptibility to CHD and make decisions regarding breed management.
The genetic etiology of CHD has been proven and accepted by the scientific community.54 The first molecular studies for CHD diagnosis were developed by Todhunter et al in the 1990s at Cornell University, who began by searching for molecular genetic markers that were linked to quantitative trait loci (QTL) responsible for different CHD phenotypes.9,55 To optimize the linkage of genes to CHD traits an outcross between breeds with high and low susceptibility to develop CHD, Labrador Retrievers and Greyhounds, respectively, was implemented. Twelve chromosomes were identified to harbor putative QTL for different CHD traits.9 QTL were also associated to the Norberg angle8 and acetabular osteophyte formation.56 Recently, more QTL were associated with other CHD traits.10,57
Pedigree and CHD phenotype analysis showed some evidence of a major QTL (contributing about 20% of variance) associated to CHD in several studies.46,58–60
However, the QTL region may contain hundreds of genes and the identification of genes remains problematic.4 The strategy that is followed by some researchers is to refine the QTL interval using SNPs and across-breed-mapping thus reducing the linkage disequilibrium interval.4 Unrelated affected animals with CHD probably share more common disease alleles than an unaffected dog population.4 The associated SNPs might be physically next to the responsible gene.14 GWAS consider the joint effect of multiple SNPs, being much more effective than individual SNP analysis, in the identification of common genetic variants for complex diseases.14 In eight different dog breeds using GWAS, four SNPs were significantly associated with CHD on CFA3, 11, and 30, and two with osteoarthritis on CFA17 and 37.14 In German Shepherd dogs, 13 SNPs were also associated with CHD on chromosome CFA14 and 37,61 CFA19, 24, 26, and 34,11 and CFA3, 9, 26, 33, and 34.62 In other recent studies on Labrador Retrievers, four SNPs were associated with CHD on chromosome CFA1 and 21,10 and 31 SNPs on CFA1, 5, 8, 15, 20, 25, and 32 positioned within or in the vicinity of 24 different genes (Table 2).63 Candidate genes involved in hypertrophic differentiation of chondrocytes and extracellular matrix integrity of basement membrane and cartilage were located in significantly associated regions on CFA1, 8, 20, and 25.63 These results confirm the complex genetic architecture of CHD, based on many genes with small individual effect, which encourages circumspection about a marker-assisted, accurate CHD diagnostic test in the near future. The immediate importance of CHD molecular diagnosis will probably be their use in genomic (many markers assessed for their combined contribution) selection.
One mutation in the FBN2 gene on CFA11 chromosome was significantly associated with CHD in Labrador Retrievers and other dog breeds.64 However, other genes must be involved in CHD because the FBN2 locus only explains a small part of the genetic trait variation in CHD.64 Studies on QTL and/or SNPs associated with other phenotypes of CHD, such as the passive hip laxity, could provide additional information on the genetic basis of this condition. Passive hip laxity is the highest risk factor for CHD and is the trait associated with the highest heritability.3,40
Genetic studies regarding the developmental hip dysplasia in humans were unable to make much progress, so knowledge on the loci-linked hip dysplasia in humans is still limited.12 Despite recent developments in whole-genome analysis in humans, with the finding of a number of genetic variants associated with this condition in affected patients,65,66 understanding the genetics of hip dysplasia in humans can benefit from similar studies in the dog.
A DNA-based test for CHD is a desirable tool for early identification of dogs susceptible or resistant to the disease. In 2012, such a test was registered by Bioiberia. Called Dysgen, it was the first commercial marker-based DNA test for susceptibility to CHD in the Labrador Retriever breed. This test analyzes blood samples using a DNA kit containing seven SNPs.13 The Dysgen diagnosis is reported as a prediction, classifying the dog into a risk group for developing CHD – minimal, low, moderate, and high. However, the performance of the Dysgen diagnosis test was not independently tested and there are no published studies reporting its success in the control for CHD at the population level. Breeding of dogs with minimal or low risk of developing CHD is recommended by the manufacturer. This is a first step in the molecular diagnosis of CHD, but until all the genes involved in the disease are detected, CHD control programs continue to require the combination of an accurate phenotype screening, EBV, and the information of available genetic tests. Particularly, if the heritability of the trait is low effectiveness of selection will benefit from combining information on major gene genotypes and EBV. Moreover, if we consider that CHD affects a rather large group of distinct dog breeds, from the Alaskan Malamute to the Portuguese Water Dog and which are raised in different environments, we require a deep understanding of the genetics underlying the incidence of this condition at the population level.
Despite phenotypic screening and breeding programs, CHD continues to be one of the most common orthopedic hereditary diseases in dogs. There is no ideal diagnosis or treatment for CHD and reproductive control schemes have been, in the last 50 years, a priority area of veterinary medicine to deal with the disease.
The genetic architecture of CHD is complex, as the many associated genes have small individual effect. This fact makes the development of a marker-assisted accurate CHD diagnosis test difficult, despite intensive research worldwide.
The molecular diagnosis of CHD will be based on genomic selection until all contributing and critical mutations are identified, and may have a significant impact for a better understanding of the genetic basis of similar conditions in humans.
C Ginja received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number PCOFUND-GA-2009-246542 and from the Fundação para a Ciência e a Tecnologia, Portugal, through a Marie Curie/Welcome II fellowship (Ref DFRH/WIIA/15/2011). The authors are grateful to R Todhunter (College of Veterinary Medicine, Cornell University) for expert critical review of the manuscript and helpful comments.
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