Margaret Vernier-Singer

Quantitative trait loci for hip dysplasia in a crossbreed canine pedigree

Canine hip dysplasia is a common developmental inherited trait characterized by hip laxity, subluxation or incongruity of the femoral head and acetabulum in affected hips. The inheritance pattern is complex and the mutations contributing to trait expression are unknown. In the study reported here, 240 microsatellite markers distributed in 38 autosomes and the X chromosome were genotyped on 152 dogs from three generations of a crossbred pedigree based on trait-free Greyhound and dysplastic Labrador Retriever founders. Interval mapping was undertaken to map the QTL underlying the quantitative dysplastic traits of maximum passive hip laxity (the distraction index), the dorsolateral subluxation score, and the Norberg angle. Permutation testing was used to derive the chromosome-wide level of significance at p < 0.05 for each QTL. Chromosomes 4, 9, 10, 11 (p < 0.01), 16, 20, 22, 25, 29 (p < 0.01), 30, 35, and 37 harbor putative QTL for one or more traits. Successful detection of QTL was due to the crossbreed pedigree, multiple-trait measurements, control of environmental background, and marked advancement in canine mapping tools.

Corticosteroids alter the differentiated phenotype of articular chondrocytes

Experimental evidence suggests that recommended dosages of some corticosteroids used clinically as antiinflammatory agents for treating arthropathies damage articular cartilage, but low dosages may be chondroprotective. The purpose of this study was to evaluate how different concentrations of methylprednisolone affect chondrocyte function and viability. Articular cartilage and chondrocytes were obtained from young adult horses, 1.5–3.5 years of age. Corticosteroid‐induced changes in collagen expression were studied at the transcriptional level by Northern blot analyses and at the translational level by measuring [3H]‐proline incorporation into [3H]‐hydroxyproline. Fibronectin mRNA splicing patterns were evaluated with ribonuclease protection assays. Cytotoxicity was studied using erythrosin B dye exclusion. Steady‐state levels of type II procollagen mRNA decreased without concurrent changes in type I procollagen expression as the medium methylprednisolone concentrations were increased from 1 × 101 to 1 × 108 pg/ml, dropping below 10% of control values by 1 × 105 pg/ml. Cytotoxicity occurred as methylprednisolone levels were increased further from 1 × 108 to 1 × 109 pg/ml. Changes in total collagen (protein) synthesis were less pronounced, but also demonstrated significant suppression between 1 × 104 and 1 × 108 pg/ml. Corticosteroid‐induced changes in fibronectin isoform levels were evaluated in articular cartilage samples without in vitro culture. The cartilage‐specific (V + C)− isoform was suppressed in both normal and inflamed joints by a single intraarticular injection (0.1 mg/kg) of methylprednisolone. Combined, these data indicate that methylprednisolone suppresses matrix protein markers of chondrocytic differentiation. Decreased and altered chondrocyte expression of matrix proteins likely contributes to the pathogenesis of corticosteroid‐induced cartilage degeneration.

Evaluation of a fibrillin 2 gene haplotype associated with hip dysplasia and incipient osteoarthritis in dogs

OBJECTIVE To determine whether a mutation in the fibrillin 2 gene (FBN2) is associated with canine hip dysplasia (CHD) and osteoarthritis in dogs. ANIMALS 1,551 dogs. Procedures-Hip conformation was measured radiographically. The FBN2 was sequenced from genomic DNA of 21 Labrador Retrievers and 2 Greyhounds, and a haplotype in intron 30 of FBN2 was sequenced in 90 additional Labrador Retrievers and 143 dogs of 6 other breeds. Steady-state values of FBN2 mRNA and control genes were measured in hip joint tissues of fourteen 8-month-old Labrador Retriever-Greyhound crossbreeds. RESULTS The Labrador Retrievers homozygous for a 10-bp deletion haplotype in intron 30 of FBN2 had significantly worse CHD as measured via higher distraction index and extended-hip joint radiograph score and a lower Norberg angle and dorsolateral subluxation score. Among 143 dogs of 6 other breeds, those homozygous for the same deletion haplotype also had significantly worse radiographic CHD. Among the 14 crossbred dogs, as the dorsolateral subluxation score decreased, the capsular FBN2 mRNA increased significantly. Those dogs with incipient hip joint osteoarthritis had significantly increased capsular FBN2 mRNA, compared with those dogs without osteoarthritis. Dogs homozygous for the FBN2 deletion haplotype had significantly less FBN2 mRNA in their femoral head articular cartilage. CONCLUSIONS AND CLINICAL RELEVANCE The FBN2 deletion haplotype was associated with CHD. Capsular gene expression of FBN2 was confounded by incipient secondary osteoarthritis in dysplastic hip joints. Genes influencing complex traits in dogs can be identified by genome-wide screening, fine mapping, and candidate gene screening.

Single nucleotide polymorphisms refine QTL intervals for hip joint laxity in dogs

Hip laxity is one characteristic of canine hip dysplasia (CHD), an inheritable disease that leads to hip osteoarthritis. Using a genome-wide screen with 250 microsatellites in a crossbreed pedigree of 159 dysplastic Labrador retrievers and unaffected greyhounds, we previously identified putative (P < 0.01) QTL on canine chromosomes 11 and 29 (CFA11 and CFA29). To refine these QTL locations, we have genotyped 257 dogs including 105 Labrador retrievers, seven greyhounds, four generations of their crossbreed offspring and three German shepherds for 111 and 171 SNPs on CFA11 and CFA29 respectively. The distraction index (DI, a measure of maximum hip laxity) was used as an intermediate phenotype that predicts whether a hip joint will or will not develop osteoarthritis. Using a multipoint linkage analysis, significant evidence (95% posterior probability) was found for QTL contributing to hip laxity in the 16.2-21 cM region on CFA11 that explained 15-18% of the total variance in DI. Evidence for an independent QTL on CFA29 was weaker than that on CFA11. Identification of the causative mutation(s) will lead to better understanding of biochemical pathways in both dogs and humans with hip laxity and dysplasia.

Evaluation of quantitative trait loci for hip dysplasia in Labrador Retrievers

OBJECTIVE To identify the quantitative trait loci (QTL) that contribute to hip dysplasia in dogs. ANIMALS 192 Labrador Retrievers. PROCEDURES Hip dysplasia was measured by use of the Norberg angle (NA), dorsolateral subluxation (DLS) score, and distraction index (DI). Genome-wide screening was conducted by use of 276 unique microsatellites. Linkage analysis was performed with a variance-based linear model. Logarithm of the odds (LOD) scores were reported when values were > 2.0. RESULTS Canis familiaris autosomes (CFAs) 01, 02, 10, 20, 22, and 32 harbored significant QTL at LOD scores > 2.0. Among the 6 QTL, the QTL on CFA02 had not been reported to harbor QTL for hip dysplasia. The highest LOD score of 3.32 on CFA20 contributed to the second principal component of the DLS score and NA of the right hip joint. The QTL that was mapped on CFA01 (LOD score of 3.13 at 55 centimorgans) was located on the same chromosome reported to harbor a QTL for hip dysplasia in Portuguese Water Dogs and German Shepherd Dogs. In this study, CFAs 10, 20, 22, and 32 harbored QTL for hip dysplasia that have been identified in a Labrador Retriever-Greyhound pedigree and in German Shepherd Dogs. CONCLUSIONS AND CLINICAL RELEVANCE Multiple QTL were clearly involved with hip dysplasia. Identification of these QTL will enable fine-resolution mapping and subsequent assessment of candidate genes within the refined intervals to enable researchers to develop genetic screening tests and preventative and novel therapeutic regimens.