Shwachman-Diamond Syndrome is Associated with Defective Osteoclastogenesis

Objective: Shwachman-Diamond syndrome (SDS) is a multisystem disorder with exocrine pancreatic dysfunction, haematological, skeletal and dental abnormalities. SDS results from mutations in the SBDS gene. Osteoclast formation (osteoclastogenesis) requires the differentiation, subsequent migration and fusion of monocytic precursor cells to form mature, multinucleated cells capable of resorbing bone. It follows that defects in migration and/or precursor cell fusion may cause reduced osteoclastogenesis, resulting in impaired or altered resorption/remodelling of bone. Methods: Using total bone marrow cells from SBDS null and wild type (WT) mice, monocytes were isolated by ficoll gradient and stimulated to undergo osteoclastogenesis with the addition of the cytokines RANKL (100ng) and M-CSF (20ng). Osteoclastogenesis was evaluated using two criteria: number of osteoclasts per field of view, and osteoclast precursor migration through transwell chambers towards a chemoattractant (M-CSF). In addition, Rho GTPase levels were evaluated to investigate a possible cause of reduced migration and osteoclastogenesis. Results: When measuring number of osteoclasts/field of view (FOV) on plastic 6 well plates SBDS null cells exhibited reduced osteoclastogenesis at the following sizes: 3-4 nuclei (4.4±1.1 /FOV), 5-7 nuclei (1.3±0.4/FOV) and ≥8 nuclei (0.2±0.1/FOV) compared to WT (15.3±1.5/FOV, 8.1±1.8/FOV and 6.8±2.0/FOV respectively). While WT precursor cells showed a dose-response increase in migration to M-CSF (up to 100ng), SBDS null cells showed no response to increasing concentrations of M-CSF, and ultimately migration was less in SBDS null cells than WT cells at 100ng M-CSF. Rac2 levels were significantly reduced in SBDS null cells. Conclusion: Bone marrow monocytes isolated from SBDS null mice showed decreased osteoclastogenesis, monocyte migration, and Rac2 levels. It is likely that the reduced osteoclastogenesis observed, and possible subsequent pathological findings in SDS patients are associated with the decreased migration and Rac2 levels.