Rapid-throughput skeletal phenotyping of 100 knockout mice identifies 9 new genes that determine bone strength

Abstract

Osteoporosis is a common polygenic disease and global healthcare priority but its genetic basis remains largely unknown. We report a high-throughput multi-parameter phenotype screen to identify functionally significant skeletal phenotypes in mice generated by the Wellcome Trust Sanger Institute Mouse Genetics Project and discover novel genes that may be involved in the pathogenesis of osteoporosis. The integrated use of primary phenotype data with quantitative x-ray microradiography, micro-computed tomography, statistical approaches and biomechanical testing in 100 unselected knockout mouse strains identified nine new genetic determinants of bone mass and strength. These nine new genes include five whose deletion results in low bone mass and four whose deletion results in high bone mass. None of the nine genes have been implicated previously in skeletal disorders and detailed analysis of the biomechanical consequences of their deletion revealed a novel functional classification of bone structure and strength. The organ-specific and disease-focused strategy described in this study can be applied to any biological system or tractable polygenic disease, thus providing a general basis to define gene function in a system-specific manner. Application of the approach to diseases affecting other physiological systems will help to realize the full potential of the International Mouse Phenotyping Consortium.

Figure 1. Faxitron x-ray microradiography and micro-CT.

A, Faxitron femur images from WT and Sparc mice (arrows indicate location for cortical bone thickness measurement, bar = 1 mm). B, Bone mineral content (BMC) in WT and Sparc mice. Pseudo-colored images in which lower BMC is in green and yellow and higher BMC is red and purple. C, Cumulative frequency histograms of BMC in n = 77 female, 16 week-old WT (mean ±2.0SD reference range in grey) and Sparc mice (red line). The median grey level is indicated by the dotted line. Graphs showing mean (solid line), 1.0SD (dotted line) and 2.0SD (grey box) for D, median grey level BMC, E, bone length and F, cortical thickness in WT (n = 77) mice. Values for Sparc (n = 2) in red. Micro-CT tibia images from G, WT and H, Sparc mice (bar = 1 mm). Graphs showing mean, 1.0SD and 2.0SD for I, BV/TV, J, trabecular number (Tb.N) and K, trabecular thickness (Tb.Th) in WT mice. Values for Sparc in red.

Figure 2. Biomechanical analysis.

A, Load-displacement curve from a WT tibia showing yield load, maximum load, fracture load and gradient of the linear elastic phase (stiffness). B, Curves from WT and Sparc. C, Graphs showing mean (solid line), 1.0SD (dotted line) and 2.0SD (grey box) for yield load, maximum load, fracture load and stiffness of WT (n = 77) mice. Values for Sparc in red. D, Energy dissipated prior to maximum load (DE_ML, purple) and elastic stored energy at maximum load (ESE_ML, yellow). E, Graph showing mean ±1.0SD and 2.0SD for the proportion DE_ML/(DE_ML+ESE_ML) prior to maximum load for WT mice. Value for Sparc in red. F, Energy dissipated prior to fracture (DE_F, purple) and elastic stored energy at fracture (ESE_F, yellow). G, Graph showing mean ±1.0SD and 2.0SD for the proportion DE_F/(DE_F+ESE_F) prior to fracture for WT mice. Value for Sparc in red. y-axis scale reflects angular transformation to normalize data distribution.

Figure 3. Knockout strains with abnormal skeletal phenotypes.

Venn diagram showing strains with at least one outlier structural parameter >2.0SD from the C57BL/6 reference mean determined by Faxitron, micro-CT, Mahalanobis distance calculation or primary phenotype screening. Strains with at least one outlier biomechanical parameter in blue. 10 strains with major phenotypes are highlighted in boxes.

Figure 4. Functional classification of bone structure.

A, Load-displacement curve from WT tibia showing 2.0SD distribution of C57BL/6 reference range in grey. B, Curves from Bbx, Cadm1 and Fam73b mice with weak but flexible bones and low bone mineral content (BMC). C, Curves from Sparc, Prpsap2, and Slc38a10 mice with weak and brittle bones and low BMC. D, Curves from Asxl1, Trim45, Spns2 and Setdb1 mice with strong but brittle bones and high BMC. E, Proportion of energy dissipated prior to fracture (DE_F/DE_F+ESE_F) versus maximum load. The y-axis scale reflects angular transformation to normalise data distribution. Strains with major phenotypes in red and individual WT mice in black. The plot separates four functional categories of bone structure that include normal bone which is strong and flexible with normal BMC and the three abnormal categories in B, C, and D.

Figure 5. Knockout strains with major phenotypes affecting bone structure and strength.

A, Digital radiographs of femurs from WT mice and each of the 10 knockout strains with major phenotypes (bar = 1 mm). B, Magnified images of mid-diaphysis, the region where cortical thickness was determined (bar = 1 mm). C, Grey-scale images pseudo-coloured using a 16-colour palette in which lower BMC is in green and yellow and higher BMC is red and purple (bar = 1 mm). D, Cumulative frequency histograms of whole femur BMC in WT mice and knockout strains: Bbx, Cadm1 and Fam73b mice with weak but flexible bones and low BMC (left); Prpsap2, Slc38a10 and Sparc mice with weak and brittle bones and low BMC (middle); and Asxl1, Setdb1, Spns2 and Trim45 mice with strong but brittle bones and high BMC (right). E, Transverse sections of tibias from WT and knockout mice imaged by micro-CT (bar = 1 mm). F, Mid-sagittal longitudinal sections of tibias from WT and knockout mice imaged by micro-CT (bar = 1 mm).

Figure 6. Relationship between mid-diaphyseal cortical bone diameter and strength.

A, Graphs showing mid-diaphyseal cortical bone diameter mean (solid line), 1.0SD (dotted line) and 2.0SD (grey box) in mutant strains with weak but flexible, weak and brittle, and strong but brittle bones. B, Relationship between fracture load and mid-diaphyseal cortical bone diameter. Strains with major phenotypes in red and individual WT mice in black. The 2.0SD reference range for each variable is represented by the grey box. The plot separates four functional categories of bone structure that include normal bone which is strong and flexible with normal BMC and the three abnormal categories weak but flexible (low BMC, green), weak and brittle (low BMC, purple) and strong but brittle (high BMC, orange). C, Relationship between energy dissipated prior to fracture (DE_F/(DE_F+ESE_F)) and cortical bone diameter. The y-axis scale reflects angular transformation to normalize data distribution. The same functional categories of bone structure are separated by this plot.