Accelerating functional gene discovery in osteoarthritis

Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease.

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Replication Randomization Data Availability Statement Source data are provided with this paper. All datasets generated and/or analyzed during the current study are included in Supplementary and Source Data, and available from the corresponding authors on reasonable request or as detailed below. Figures 1, 3-7, and Supplementary Figures 1-8  There are no restrictions on data availability.
Sample sizes were based on power calculations performed using the coefficient of variance (for parameters that were normally-distributed in a wild-type population of 100 animals) or the percentage median absolute deviation from the median (median absolute deviation from the median (MAD)/median) for non-normally distributed parameters.
For analysis of 50 unselected mouse knockout lines generated by the International Mouse Phenotyping Consortium (IMPC), no samples were excluded.
For analysis of 100 wild-type samples, 13 samples were excluded based on pre-determined criteria including iatrogenic damage to the joint that occurred during sample preparation.
For histological analysis of mice undergoing surgical provocation of osteoarthritis, one mouse was excluded due to unsuccessful surgery. 4 mice were excluded due to surgery damaging ligaments in the knee that were not the target ligaments of the surgery. These exclusion criteria were pre-determined based on published accounts of the surgical method and included detection of subchondral bone erosion and calcification of the cruciate ligaments (Glasson, Osteoarthritis and Cartilage, 2007. A repeatability study was performed for the new methods described. To determine the repeatability of each method, 7 wild-type and 6 mutant samples were selected that covered the full spectrum of phenotype severity. Samples were blinded and analyzed five times in a random order, and in a different random order for each method. Mean, standard error of the mean, number of standard deviations from baseline mean, absolute precision error (standard deviation; PE(SD), and precision error as percentage of coefficient of variation (PE(%CV) and two-way mixed-model intraclass correlation coefficients (absolute agreement) with 95% confidence intervals were calculated for repeated analyses by a single rater.
For analysis of 50 unselected mouse knockout strains generated by the IMPC, 7 samples were available for each genotype. In cases where fewer than 7 samples were analysed, samples were randomly selected for analysis.
To generate the wild-type reference range, 100 wild-type samples were randomly selected for analysis.
For surgical provocation of osteoarthritis in wild-type male mice, 2 mice per cage of 4 were randomly allocated for analysis by either rapid