Genetically determined phenotype covariation networks control bone strength

Abstract

To identify genes affecting bone strength, we studied how genetic variants regulate components of a phenotypic covariation network that was previously shown to accurately characterize the compensatory trait interactions involved in functional adaptation during growth. Quantitative trait loci (QTLs) regulating femoral robustness, morphologic compensation, and mineralization (tissue quality) were mapped at three ages during growth using AXB/BXA Recombinant Inbred (RI) mouse strains and adult B6-i(A) Chromosome Substitution Strains (CSS). QTLs for robustness were identified on chromosomes 8, 12, 18, and 19 and confirmed at all three ages, indicating that genetic variants established robustness postnatally without further modification. A QTL for morphologic compensation, which was measured as the relationship between cortical area and body weight, was identified on chromosome 8. This QTL limited the amount of bone formed during growth and thus acted as a setpoint for diaphyseal bone mass. Additional QTLs were identified from the CSS analysis. QTLs for robustness and morphologic compensation regulated bone structure independently (ie, in a nonpleiotropic manner), indicating that each trait may be targeted separately to individualize treatments aiming to improve strength. Multiple regression analyses showed that variation in morphologic compensation and tissue quality, not bone size, determined femoral strength relative to body weight. Thus an individual inheriting slender bones will not necessarily inherit weak bones unless the individual also inherits a gene that impairs compensation. This systems genetic analysis showed that genetically determined phenotype covariation networks control bone strength, suggesting that incorporating functional adaptation into genetic analyses will advance our understanding of the genetic basis of bone strength.

Fig. 1

Schematic showing how sets of mid-diaphyseal traits acquired by an individual arise from variation in subperiosteal expansion defining robustness combined with variation in marrow infilling defining diaphyseal bone mass (cortical area) and mineralization defining tissue quality. These growth processes are highly coordinated, resulting in a population showing a narrow range of trait sets that is predictable based on bone robustness. The dashed diagonal line indicates the trait sets in which stiffness is maximized using minimum mass for a population. The gray value represents the variation in mineralization that accompanies morphologic compensation. The dashed elliptical line indicates the expected range of trait sets for a population showing variation in morphologic compensation.

Fig. 2

Variation in (A) robustness (Tt.Ar/Le) and (B) morphologic compensation (Ct.Ar/BW) among the AXB/BXA RI panel. Mean trait values are shown for female A/J, B6, and F₁ inbred strains. Trait values for AB6F1 and B6AF1 strains were similar and are indicated as an average F₁.

Fig. 3

Path analysis was conducted to determine how variation in Tt.Ar and Le among the RI strains contributed to the variation in robustness (Tt.Ar/Le) when the effects of body weight were taken into consideration. The coefficients next to the curved arrows in the path models are the linear correlations between independent variables, and the coefficients next to the straight arrows are the relative contributions of each trait to Tt.Ar/Le in terms of standardized units (Z-scores). A large path coefficient was observed for Tt.Ar at each age, indicating that most of the variation in robustness was determined by Tt.Ar. Length contributed to the variation in Tt.Ar/Le but to a much lesser extent. The relative contributions were determined after controlling for the effects of body weight, which correlated significantly with Tt.Ar and Le but contributed very little to the variation in Tt.Ar/Le. The analyses were consistent across growth, suggesting that variation in robustness among the RI strains resulted primarily from variation in growth in width.

Fig. 4

Variation in femoral robustness (Tt.Ar/Le) at 16 weeks of age for the panel of female CSSs. Tt.Ar/Le was corrected for body weight by linear regression analysis. Data are shown as mean ± SD. The asterisk indicates significant differences between each CSS and the B6 host (p < .004, t test).

Fig. 5

Interval mapping showed that QTLs for robustness (Tt.Ar/Le) and morphologic compensation (Ct.Ar/BW) measured at 8 weeks of age localized to different regions on chromosome 8. The solid black line represents the LRS level and the solid gray line represents the additive effect. The horizontal solid and dashed black lines represent the suggestive and significant (95th percentile) LRS levels, respectively. The vertical bars are the histograms showing the range of the maximum LRS values.

Fig. 6

Variation in (A) morphologic compensation (Ct.Ar/BW) and (B) ash content at 16 weeks of age for the panel of female CSSs. Data are shown as mean ± SD. The asterisk indicates significant differences between each CSS and the B6 host (p < .004, t test).

Fig. 7

Effect of B6 and A/J genotypes on the trait sets inherited by the AXB/BXA RI strains. The femoral diaphyseal morphologic traits (mean ± SD) were measured at 8 weeks of age. (A) The effect of genotype for Ct.Ar/BW on chromosome 8 and for Tt.Ar/Le on chromosome 18 on diaphyseal morphology. The letters (a, b, c) indicate differences among groups based on a two-way ANOVA. (B) The effect of genotype for Ct.Ar/BW on chromosome 8 and for Tt.Ar/Le on chromosome 19 on diaphyseal morphology.