Factor IX-Deficient Mice Have Decreased Skeletal Health
Authors: Larson Emily A , Taylor Jason A 
Published: October 2019

Abstract
Background:
We have previously shown that factor VIII deficiency decreases skeletal health in persons with hemophilia and animal mouse models; however, it is uncertain if this is unique to factor VIII deficiency or persists in factor IX (FIX) deficiency as well. Animal models can eliminate genetic and environmental factors of a complex trait like osteoporosis, and offer more invasive testing beyond bone mass density (BMD), studying bone properties using microtomography (μCT), histomorphometry and biomechanics. Decreased skeletal health can be caused by altered signaling between cells that are responsible for bone remodeling, osteoblasts (build bone) and osteoclasts (absorb bone). Understanding mechanisms of bone disease present in factor deficient mice can impact clinically how it is diagnosed and treated in the future.
Methods:
FIX-deficient and wild-type (WT) littermates were scanned using peripheral dual energy X-ray absorptiometry (DEXA) to obtain measurements for whole body BMD and femoral BMD at 10 and 20 weeks of age. Cortical bone at the midshaft of the femur was analyzed using microtomography (μCT) and biomechanical testing determined by three-point bending. Three-dimensional high resolution μCT analysis of a tibia is analyzed for bone histomorphometry. Important biomarkers of bone remodeling are analyzed on collected mouse serum and include measuring alkaline phosphatase (ALP) activity spectrophotometrically, osteoprotegrin (OPG), receptor activator of nuclear factor kappa-B Ligand (RANKL) and osteocalcin by commercially available ELISA kits.
Results:
FIX-deficient mice have decreased skeletal health compared to their wild-type (WT) littermate controls at both 10 and 20 weeks of age, without statistical differences in weight or body length.
At 10 weeks, FIX-deficient mice (n=11) have lower whole body BMD (p=0.006) and femoral BMD (p=0.015) compared to WT littermates (n=12). Femoral μCT analysis shows parameters related to bone size, including cortical area (p=0.078) and marrow area (p=0.001) are decreased, and overall cross-sectional area (p=0.002). Three-point bending of the bones have decreased in ultimate failure (p=0.042) and stiffness (p=0.039). High resolution μCT of the tibia show decreased total bone volume (p=0.004) and at the midshaft the cortical bone (p=0.004), marrow area (p=0.029), and the total area (p=0.004).
At 20 weeks, FIX-deficient mice (n=14) show decreased whole body BMD (p=0.044) and femoral BMD (p=0.053) trends from their WT littermates (n=15). Femoral μCT analysis shows decreased cortical area (p=0.038). Ultimate failure (p=0.053) and stiffness (p=0.002) differences are present in this group. No differences in cortical bone are seen with high resolution μCT, but do have distinct decreased cancellous bone in the tibia's metaphysis. The FIX-deficient bones show decreased bone volume (p=0.05) and structural differences with decreased trabecular number (p=0.03) and trabecular connectivity (p=0.01), and increased trabecular space (p=0.03).
Receptor activator of nuclear factor kappa-B ligand (RANKL) located on osteoblasts plays a direct role in bone metabolism with osteoclasts, by binding to RANK causing precursor osteoclasts to mature and begin bone resorption. At 10 weeks of age RANKL was not detectable in our assays. At 20 weeks of age, RANKL was found to be significantly decreased in FIX-deficient mice over WT littermates (p=2.2E-06).
OPG is a receptor decoy that inhibits bone resorption by binding to RANKL on osteoblasts. At 10 weeks, the FIX-deficient mice were found to have lower levels of OPG compared to WT controls (p=0.012); however, at 20 weeks, there was a marked increase in OPG levels of the FIX-deficient mice (p=5.7E-05). Bone building biomarkers, ALP activity and osteocalcin showed no measurable differences in serum at 10 or 20 weeks of age (p>0.5) and (p>0.2) respectively.
Conclusions:
FIX deficent mice have bone disease suggesting that alteration of the clotting cascade has a direct impact on skeltal health. The mechanism behind of how factor deficiency alters bone health is unclear, but the RANKL/OPG data is consistent with aberrant osteoblast-osteoclast signaling. Our laboratory is actively investigating the mechanism of decreased skeletal health associated with factor deficiencies.

Disclosures
Taylor: CSL Behring: Consultancy, Research Funding; Kedrion: Research Funding; Baxalta/Shire: Consultancy, Research Funding; Novo Nordisk: Research Funding.