Background

A highly co-ordinated and regulated process known as bone remodelling maintains the structural integrity and mineral density of bone. During bone remodelling, worn or damaged surfaces of trabecular and cortical bone are resorbed by osteoclast cells and replaced by osteoblast cells 1,2. The extent to which bone resorption (osteolysis) and synthesis (osteogenesis) occur are dependent on the interaction of the following three principle cytokines: tumour necrosis factor ligand superfamily member receptor activator of nuclear factor-cob (NF-kB) ligand (RANKL); its cellular receptor, receptor activator of NF-kB (RANK), and the decoy receptor, osteoprotegerin (OPG) 3.

Osteolysis is initiated by RANKL/RANK signalling. RANKL is secreted by osteoblastic cells and specifically recognised and bound by RANK receptors of osteoclastic cells. RANKL binding catalyses the formation, activation and maintenance of multinucleated osteoclasts (osteoclastogenesis), which attach to and resorb bone 3. Bone resorption is in turn regulated by a third cytokine called OPG. OPG is a soluble decoy receptor that binds to and sequesters RANKL, thereby limiting the activation of RANK and reducing osteoclastogenesis and osteolysis3 4. As a result the ratio of RANKL/OPG is an important determinant of bone mineral density (BMD) and skeletal integrity. Inherent perturbations of this ratio contribute to a variety of pathological conditions ranging from osteoporosis (low BMD) to osteopetrosis (high BMD) 4. Recently allelic variants of Rankl and Opg have been correlated with varying gene expression profiles and bone mineral density in patients with osteoporosis 5 6. These observations highlight the potential effects allelic variation may have on RANKL/OPG ratios and BMD.

Our research group has recently completed a genome wide association study based on tibial pQCT scans obtained in ALSPAC children at age 15. We identified several allelic variants which are located within and adjacent to Rankl which show associations with cortical BMD at genome-wide significance levels We hypothesise that these allelic variants may influence the steady state levels of RANKL sufficiently to alter the bone remodelling process thereby influencing BMD.

Aims

1. To quantify the steady state levels of serum RANKL in a cohort of 200 ALSPAC subjects who are homozygous and heterozygous for a specific Rankl marker.

2. To determine if genotypic variation at a single Rankl marker accounts for variation in RANKL steady state level and BMD.

Methods

A small sub-group of children (~200 individuals) from ALSPAC will be selected on the basis of being homozygous for the common and rare Rankl allele which is most strongly associated with cortical BMD (as identified in our recent genome-wide association scan). It is proposed that these individuals will have their RANKL protein levels quantified using the ampli-sRANKL enzyme immuno assay kit (Biomedica, Geneva) at Professor J Holly's laboratory at Southmead Hospital. Statistical association between RANKL protein levels and Rankl genotypes will be tested. Association will also be tested between RANKL protein levels and bone mineral density (as assessed by DXA and pQCT measurements).

Literature cited

1. Teitelbaum, S.L. & Ross, F.P. Genetic regulation of osteoclast development and function. Nat. Rev. Genet 4, 638-649(2003).

2. Martin, T.J. & Sims, N.A. Osteoclast-derived activity in the coupling of bone formation to resorption. Trends in Molecular Medicine 11, 76-81(2005).

3. Boyce, B.F. & Xing, L. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch Biochem Biophys. 473, 139-146(2008).

4. Simonet, W. et al. Osteoprotegerin: A Novel Secreted Protein Involved in the Regulation of Bone Density. Cell 89, 309-319(1997).

5. Hsu, Y. et al. Variation in genes involved in the RANKL/RANK/OPG bone remodelling pathway are associated with bone mineral density at different skeletal sites in men. Human Genetics 118, 568-577(2006).

6. Takacs, I. et al. Allelic variations of RANK/RANKL/OPG signaling system is related to bone mineral density and in vivo gene expression. Bone 44, S116(2009).