A mutation in the gene encoding osteoprotegerin causes the severe bone disease Idiopathic Hyperphosphatasia

Abstract

Genetic analysis of a large family with Familial Idiopathic Hyperphosphatasia, a rare autosomal recessive bone disease, identified linkage to a region of chromosome 8q that contains the gene for osteoprotegerin (OPG), a key regulator of bone remodelling. Sequencing of this gene in members of the family identified an in-frame 3 base pair deletion resulting in the deletion of an aspartate residue at position 182, within the critical TNF-receptor-like region. Wild-type and mutant OPG cDNA were cloned into pcDNA3.1(-)/myc-His vector and expressed in HEK293 cells. Conditioned media samples containing the recombinant proteins were tested for their ability to inhibit bone resorption in a neonatal murine calvarial organ culture. Conditioned medium containing wild type OPG (wtOPG) inhibited bone resorption, whereas the conditioned medium containing the mutant protein (OPG∆D182) had no effect. Subsequently, the peptides were tested in a murine bone marrow osteoclastogenesis system. In this assay, OPG∆D182 had significantly lower activity than wtOPG. These results indicate the deletion of aspartate 182 impairs the ability of OPG to inhibit osteoclast formation and activation. The ability of wtOPG and OPG∆D182 to bind RANKL has been investigated using a rRANKL-GST pulldown system. These experiments demonstrated the OPG∆D182 had reduced ability to bind RANKL compared to wtOPG. A kinetic study using the surface plasmon resonance technology of BIAcore™ revealed the RANKL binding affinity of OPG∆D182 is lower than that of wtOPG. Altered processing of OPG∆D182 causes hyperglycosylation in comparison to wtOPG. Glycosylation was shown to be important for the function of full-length OPG, probably due to a requirement of glycosylation for correct folding. Site-directed mutagenesis identified two glycosylation sites that contribute to the hyperglycosylation of OPG∆D182. Glycosylation at one of these sites has potential structural implications by preventing disulphide bond formation and correct protein domain folding. The research highlights the effects of a single amino acid deletion on the structure and function of OPG. The deletion of aspartate 182 is a detrimental mutation that results in the severe phenotype seen in the patients, confirming the crucial role of OPG in normal bone physiology in humans.