The markers of the predictive DNA test for canine hip dysplasia may have a stronger relationship with elbow dysplasia

Abstract

Canine hip and elbow dysplasias, which are prevalent orthopedic conditions rooted in developmental and hereditary factors are yet to be comprehensively assessed. This study aimed to address this gap by exploring the prognostic significance of five markers linked to canine hip dysplasia using available genome-wide association studies (GWAS) data. The influence of these markers on both hip and elbow dysplasia was examined in dogs exposed to standardized environmental conditions. We made a groundbreaking discovery using custom primers, qPCR assays, and evaluation of fluorescent resonance energy transfer (FRET) probes. Three specific SNPs previously associated with the risk of canine hip dysplasia demonstrated a potentially stronger correlation with elbow dysplasia. Notably, the SNP at nucleotide position 22691322, located near the canine CHST3 gene, displayed significance as a marker in multivariable logistic regression analysis. Surprisingly, none of the initially targeted SNPs showed a direct association with hip dysplasia. The genomic positions of these SNPs reside within a region conserved across mammals. In silico analyses suggested that the relevant variant might be positioned in a region linked to bone and muscle structures. Our findings revealed a remarkable relationship between SNP2 genotypes and methylation patterns, shedding light on the underlying mechanism that partially explains the genotype-phenotype correlation in canine CHST3. These groundbreaking findings offer essential insights for future, more extensive investigations into canine orthopedic health. This research significantly contributes to our understanding of the molecular foundations of hip and elbow dysplasia in dogs by charting a course for advancements in veterinary medicine and the overall well-being of canine companions.

Keywords: Dog; Elbow dysplasia; FRET probes; Hip dysplasia; In silico; SNP.

Graphical abstract

Fig. 1

Evaluation of the canine pelvis using conventional ventrodorsal radiographic projections, following the guidelines established by the Fédération Cynologique Internationale (FCI) criteria. The Norberg Angles (NA) for each joint are furnished. (a) Normal hip conformation (category A); 4-year-old female Labrador Retriever; NA-right: 109.9° -left: 108.3° (b) Almost normal hip conformation (category B); 3-year-old male German Shepherd; NA-right: 102.1° -left: 102.7° (c) Mild signs of dysplasia (category C); 2-year-old female Labrador Retriever; NA-right: 102.7° -left: 99.1° (d) Moderate signs of dysplasia (category D); 2-year-old male Labrador Retriever; NA-right: 88.4° -left: 91.4° (e) Severe dysplasia (category E); 2-year-old male Labrador Retriever; NA-right: 97.1° -left: 88.1°. Further details for FCI scoring are presented in the supplementary material and methods.

Fig. 2

Assessment of the canine elbow joint on mediolateral radiographic projections, in accordance with the International Elbow Working Group (IEWG) criteria. (a) Normal elbow conformation (ED0); 2.5-year-old male German Shepherd; no primary lesion related to elbow dysplasia and no signs of arthrosis. (b) Mild arthrosis (ED1); 5.5-year-old male German Shepherd; while there is no primary lesion in the elbow joint, there is new bone formation ≤2 mm (1st degree) around the proccesus anconeus indicated by the white arrow. (c) Moderate arthrosis or suspect primary lesion moderate arthrosis or suspect primary lesion (ED2); 3.5-year-old male German Shepherd; while an ostensibly primary lesion manifests within the demarcated region, concomitant therewith is an insignificantly small extent of ≤2 mm, tantamount to a condition of primary degree arthrosis, encircling the processus anconeus as indicated by the white arrow. (d) Severe arthrosis or evident primary lesion (ED3); 3-year-old female German Shepherd; A primary lesion (ununited anconeal process: UAP) is evident within the demarcated region marked by the white ring, accompanied by a 2–5 mm, or 2nd degree arthrosis, encircling the processus anconeus. Further details for IEWG scoring are presented in the Supplementary Material and Methods.

Fig. 3

Melting peaks for the detection of three genotypes of the SNPs identified in the Real-time PCR analysis with the specific fluorescence resonance energy transfer (FRET) hybridization probes. For each SNP, the initial singular peak corresponds to the wild-type variant, while the subsequent singular peak represents the mutant-type variant. The presence of the heterozygous variant is discerned by the simultaneous presence of two peaks. (a) SNP1: BICF2G630558239, (b) SNP2: BICF2P772455, (c) SNP3: BICF2S230609, (d) SNP4: BICF2G630339806, (e) SNP5: BICF2S2452559.

Fig. 4

The in silico predicted mechanism of action for SNP2 (BICF2P772455). The predicted system indicates that transcription is regulated due to C hydroxymethylation occurring within the promoter region following the mutation.

Fig. 5

Examination of the pertinent SNP sequences within this genomic region and assess its taxonomic significance. (a) common taxonomic trees of the selected species. (b) SNP1: BICF2G630558239, (c) SNP2: BICF2P772455, (d) SNP3: BICF2S230609, (e) SNP4: BICF2G630339806, (f) SNP5: BICF2S2452559.

Fig. 6

Assessment of the methylation status within the genomic region containing SNP2 (BICF2P77245) in the canine CHST3 gene. (a) sample methylation status determination involves assessing the difference in melting temperature (Tm) between methylated (M) and unmethylated (U) primer sets. (b) the association between SNP2 genotypes in the canine CHST3 gene and methylation patterns. One way ANOVA, ***P < 0.001.

Fig. S1

Illustrating the experimental design of the study in sequential steps. All dogs included in this study were sourced from a dog breeding and training center, which accommodates a total of 1000 dogs. These dogs, all of which were purebred, were subjected to identical environmental conditions, exercise routines, and nutritional regimens. We selected dogs that were unrelated up to the grandparent level. Following clinical and orthopedic evaluations, radiographs were acquired. X-rays were assessed based on the FCI scale for hip dysplasia and the IEWG official scale for elbow dysplasia. Each of these disorders is discussed independently. In addition to the primary analyses, phenotypes exhibiting neither disorder and those displaying some degree of both were examined autonomously. Molecular genetic analyses encompass qPCR, Sanger sequencing, in silico analysis, and methylation assay. * Created with BioRender.com (www.biorender.com).

Fig. S2

The positioning of the CpG island within the genomic location of SNP2 in the canine CHST3 gene. Primers specific to this region were subsequently designed for the study.