The long (and winding) road to gene discovery for canine hip dysplasia

Abstract

Hip dysplasia is a common inherited trait of dogs that results in secondary osteoarthritis. In this article the methods used to uncover the mutations contributing to this condition are reviewed, beginning with hip phenotyping. Coarse, genome-wide, microsatellite-based screens of pedigrees of greyhounds and dysplastic Labrador retrievers were used to identify linked quantitative trait loci (QTL). Fine-mapping across two chromosomes (CFA11 and 29) was employed using single nucleotide polymorphism (SNP) genotyping. Power analyses and preferential selection of dogs for ongoing SNP-based genotyping is described with the aim of refining the QTL intervals to 1-2 megabases on these and several additional chromosomes prior to candidate gene screening. The review considers how a mutation or a genetic marker such as a SNP or haplotype of SNPs might be combined with pedigree and phenotype information to create a 'breeding value' that could improve the accuracy of predicting a dog's hip conformation.

Fig. 1

Photographs of hip radiographs illustrating: (A) the extended hips of a normal Labrador retriever used to derive the extended-hip (OFA) radiographic score and to measure the Norberg angle; (B) The hips of a Labrador retriever with dysplasia taken in the same position; (C) greyhound hips with excellent conformity used to measure the dorsolateral subluxation score; (D) hips of a Labrador retriever with dysplasia in a similar position to (C) with subluxation of the femoral head from the medial acetabular wall; (E) the ‘tight’ hip of a greyhound taken in the distraction position to measure the maximum distraction laxity as the distraction index (DI); (F) a ‘loose-hipped’ Labrador retriever in the distraction position.

Fig. 1

Photographs of hip radiographs illustrating: (A) the extended hips of a normal Labrador retriever used to derive the extended-hip (OFA) radiographic score and to measure the Norberg angle; (B) The hips of a Labrador retriever with dysplasia taken in the same position; (C) greyhound hips with excellent conformity used to measure the dorsolateral subluxation score; (D) hips of a Labrador retriever with dysplasia in a similar position to (C) with subluxation of the femoral head from the medial acetabular wall; (E) the ‘tight’ hip of a greyhound taken in the distraction position to measure the maximum distraction laxity as the distraction index (DI); (F) a ‘loose-hipped’ Labrador retriever in the distraction position.

Fig. 1

Photographs of hip radiographs illustrating: (A) the extended hips of a normal Labrador retriever used to derive the extended-hip (OFA) radiographic score and to measure the Norberg angle; (B) The hips of a Labrador retriever with dysplasia taken in the same position; (C) greyhound hips with excellent conformity used to measure the dorsolateral subluxation score; (D) hips of a Labrador retriever with dysplasia in a similar position to (C) with subluxation of the femoral head from the medial acetabular wall; (E) the ‘tight’ hip of a greyhound taken in the distraction position to measure the maximum distraction laxity as the distraction index (DI); (F) a ‘loose-hipped’ Labrador retriever in the distraction position.

Fig. 1

Photographs of hip radiographs illustrating: (A) the extended hips of a normal Labrador retriever used to derive the extended-hip (OFA) radiographic score and to measure the Norberg angle; (B) The hips of a Labrador retriever with dysplasia taken in the same position; (C) greyhound hips with excellent conformity used to measure the dorsolateral subluxation score; (D) hips of a Labrador retriever with dysplasia in a similar position to (C) with subluxation of the femoral head from the medial acetabular wall; (E) the ‘tight’ hip of a greyhound taken in the distraction position to measure the maximum distraction laxity as the distraction index (DI); (F) a ‘loose-hipped’ Labrador retriever in the distraction position.

Fig. 1

Photographs of hip radiographs illustrating: (A) the extended hips of a normal Labrador retriever used to derive the extended-hip (OFA) radiographic score and to measure the Norberg angle; (B) The hips of a Labrador retriever with dysplasia taken in the same position; (C) greyhound hips with excellent conformity used to measure the dorsolateral subluxation score; (D) hips of a Labrador retriever with dysplasia in a similar position to (C) with subluxation of the femoral head from the medial acetabular wall; (E) the ‘tight’ hip of a greyhound taken in the distraction position to measure the maximum distraction laxity as the distraction index (DI); (F) a ‘loose-hipped’ Labrador retriever in the distraction position.

Fig. 1

Photographs of hip radiographs illustrating: (A) the extended hips of a normal Labrador retriever used to derive the extended-hip (OFA) radiographic score and to measure the Norberg angle; (B) The hips of a Labrador retriever with dysplasia taken in the same position; (C) greyhound hips with excellent conformity used to measure the dorsolateral subluxation score; (D) hips of a Labrador retriever with dysplasia in a similar position to (C) with subluxation of the femoral head from the medial acetabular wall; (E) the ‘tight’ hip of a greyhound taken in the distraction position to measure the maximum distraction laxity as the distraction index (DI); (F) a ‘loose-hipped’ Labrador retriever in the distraction position.

Fig. 2

Flow diagram illustrating the investigative progression from identifying an inherited trait to the identification of the underlying polymorphisms and mutations. * SNP = single nucleotide polymorphism, * QTL = quantitative trait locus.

Fig. 3

Variance ratio profile plot of the left hip Norberg angle across canine chromosome CFA11 following a genome-wide, microsatellite-based screen on 159 Labrador retriever/greyhound crossbreed dogs. The variance ratio is shown on the Y-axis for each position where the presence of a quantitative trait locus (QTL) for the left Norberg angle was tested at various recombination fractions between every two microsatellite flanking markers across the chromosome (X-axis in centimorgans [cM]). The log of the odds (LOD) score at the peak position was 2.8 in this example. The chromosome-wide thresholds for the F ratio statistic at * (P < 0.01) and ** (P < 0.05) are drawn across the graph. QTL Express software was used for modeling.

Fig. 4

Association tests between the Norberg angle and 40 simulated single nucleotide polymorphism (SNP) markers spaced 2 cM apart across an 80 centimorgan (cM) length chromosome. The strength of association was measured as the log (1/probability [P]) on the Y-axis for each individual SNP genotype and the haplotype of two adjacent markers. The haplotype model considers the contribution of flanking marker genotypes beginning with the two markers flanking the quantitative trait locus (QTL) and eventually including the haplotype over multiple markers. Advantages of haplotype analysis are that interactions among QTLs are revealed and the power of detection for QTLs that are not superimposed on markers is increased. The threshold for the level of significance would be determined based on the number of association tests. The simulated QTL was located at 50 cM where the peak is revealed. The additional power to detect the QTL with the flanking haplotypes compared to the single SNP genotype is clear. A centimorgan (cM) is equivalent to a megabase (Mb) over short intervals. The cM is the unit of measurement for a linkage study because cM is a measure of recombination distance. The Mb is the physical genetic distance used in a true association study of unrelated dogs where recombinations are not observed.

Fig. 5

Distribution of P values under the null hypotheses of no association between the quantitative trait locus (QTL) and the marker genotype. A broken straight diagonal line drawn in black is expected if there is no association with the lowest type I error rate, i.e. calling the presence of a QTL when in fact the null hypothesis of no association holds. The mixed model with the relationship matrix based on markers (Marker) is the closest to the expected relationship. The model excluding background QTLs (Fixed) is the worst relationship. The mixed model with relationship based on the true Cornell pedigrees (Pedigree) is intermediate. All the analyses were carried out using the Statistical Analysis System (SAS) procedures HAPLOTYPE, MIXED and SAS. The SAS procedure HAPLOTYPE was used to estimate the haplotype probability by maximum likelihood using the expectation maximization (EM) algorithm. The procedure MIXED tests for both fixed and random effects (Littell et al., 1996). To make the polygenic effect a correlated random effect, the variance and covariance structure was constructed before using the procedure. A computer program LORG in the form of a SAS macro was developed to facilitate the applications. The macro automatically constructs a SAS dataset that defines the variance and covariance structure of genetically correlated random effects. The SAS dataset can be imported by the SAS procedure MIXED with the option of GDATA or LDATA.

Fig. 6

Effect of single nucleotide polymorphism (SNP) genotypes on the distraction index (DI) at 19.6 Mb on CFA11 according to the mixed linear model association analysis. The AA genotype increases hip laxity (distraction index is indicated on the Y-axis) and the CC genotype is protective (decreases the DI).

Fig. 7

The impact of a sampling approach based on inbreeding coefficient (A) and kinship (B) for pairs of dogs sampled. In the inbreeding coefficient graph (A), the light histograms (sent) were the result of human efforts to maximize phenotype diversity in 82 dogs selected for single nucleotide polymorphism (SNP) genotyping. The dark histograms (computer-aided) resulted from the strategy to maximize phenotypic and genetic diversity of 82 dogs sampled from the same population based on a computer algorithm. The sample from human efforts to maximize diversity resulted in 37 dogs with a 0 inbreeding coefficient. The computer algorithm sample resulted in 58 dogs with a 0 inbreeding coefficient. The kinship graph (B) shows kinship plotted as a function of each pair of 82 dogs. Kinship is the probability that two individuals will share an identical allele by descent. For 82 dogs, 3,321 [(82*81)/2] pairs of dogs are possible. The human effort and computer-aided approaches are shaded light and dark respectively. It is clear that the kinship and inbreeding coefficients were greatly reduced using the computer-aided approach.

Fig. 7

The impact of a sampling approach based on inbreeding coefficient (A) and kinship (B) for pairs of dogs sampled. In the inbreeding coefficient graph (A), the light histograms (sent) were the result of human efforts to maximize phenotype diversity in 82 dogs selected for single nucleotide polymorphism (SNP) genotyping. The dark histograms (computer-aided) resulted from the strategy to maximize phenotypic and genetic diversity of 82 dogs sampled from the same population based on a computer algorithm. The sample from human efforts to maximize diversity resulted in 37 dogs with a 0 inbreeding coefficient. The computer algorithm sample resulted in 58 dogs with a 0 inbreeding coefficient. The kinship graph (B) shows kinship plotted as a function of each pair of 82 dogs. Kinship is the probability that two individuals will share an identical allele by descent. For 82 dogs, 3,321 [(82*81)/2] pairs of dogs are possible. The human effort and computer-aided approaches are shaded light and dark respectively. It is clear that the kinship and inbreeding coefficients were greatly reduced using the computer-aided approach.

Fig. 8

The impact of the computer-aided sampling approach on phenotype distribution (X-axis) among the frequency of sampled dogs (Y-axis). The phenotype distribution for the left distraction index (DI) (top), left dorsolateral subluxation (DLS) score (middle), and left Norberg angle (NA) score (bottom) is shown. The dark histograms resulted from human effort to maximize phenotype diversity for 85 dogs while accounting for genetic unrelatedness of the first 85 dogs chosen for dense single nucleotide polymorphism (SNP) genotyping. The light histograms resulted from a computer-aided dog selection method. Intermediate shading indicates overlap. It is clear that the computer-aided approach resulted in greater phenotypic diversity, which should result in maximum mapping power among the dogs available for genotyping. The frequency is the proportion of all dogs with each phenotype group measurement.

Fig. 8

The impact of the computer-aided sampling approach on phenotype distribution (X-axis) among the frequency of sampled dogs (Y-axis). The phenotype distribution for the left distraction index (DI) (top), left dorsolateral subluxation (DLS) score (middle), and left Norberg angle (NA) score (bottom) is shown. The dark histograms resulted from human effort to maximize phenotype diversity for 85 dogs while accounting for genetic unrelatedness of the first 85 dogs chosen for dense single nucleotide polymorphism (SNP) genotyping. The light histograms resulted from a computer-aided dog selection method. Intermediate shading indicates overlap. It is clear that the computer-aided approach resulted in greater phenotypic diversity, which should result in maximum mapping power among the dogs available for genotyping. The frequency is the proportion of all dogs with each phenotype group measurement.

Fig. 8

The impact of the computer-aided sampling approach on phenotype distribution (X-axis) among the frequency of sampled dogs (Y-axis). The phenotype distribution for the left distraction index (DI) (top), left dorsolateral subluxation (DLS) score (middle), and left Norberg angle (NA) score (bottom) is shown. The dark histograms resulted from human effort to maximize phenotype diversity for 85 dogs while accounting for genetic unrelatedness of the first 85 dogs chosen for dense single nucleotide polymorphism (SNP) genotyping. The light histograms resulted from a computer-aided dog selection method. Intermediate shading indicates overlap. It is clear that the computer-aided approach resulted in greater phenotypic diversity, which should result in maximum mapping power among the dogs available for genotyping. The frequency is the proportion of all dogs with each phenotype group measurement.

Fig. 9

The distribution of the breeding value and accuracy on the first principal component calculated from the distraction index, the dorsolateral subluxation score, the Norberg angle, and the Orthopedic Foundation for Animals-type hip score measured on the left and right hips. The breeding values and accuracy (three accuracy groupings of 0.75–1.0, 0.5–0.74 and 0–0.49 are listed at bottom left of the graph) were calculated for 2,716 dogs (1,888 were phenotyped). For the remainder, only the genetic relationships were used in the calculations. The dogs with the highest breeding values had the broadest trait distribution, enabling the best discrimination (highest accuracy) between dogs for breeder selection and purchase.