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Functional, biochemical and genetic studies have over the past decade identified many causative genes in the osteoclast diseases osteopetrosis and Paget's disease of bone. Here, we outline all osteoclast diseases and their genetic associations and then focus specifically on those diseases caused by mutations in the critical osteoclast molecule Receptor Activator of Nuclear factor Kappa B (RANK). Both loss and gain-of-function mutations have been found in humans leading to osteopetrosis and high bone turnover phenotypes, respectively. Osteopetrosis-associated RANK mutations are widely distributed over the RANK molecule. It is likely that some negatively affect ligand binding, whereas others preclude appropriate association of RANK with downstream signalling molecules. In the Paget-like disorders, familial expansile osteolysis, early onset Paget's disease and expansile skeletal hyperphosphatasia, heterozygous insertion mutations are found in the RANK signal peptide. These prevent signal peptide cleavage, trapping the protein translated from the mutated allele in the endoplasmic reticulum. Whole animal studies replicate the hyperactive osteoclast phenotype associated with these disorders and present only with heterozygous expression of the mutation, suggesting an as yet unexplained effect of the mutant allele on normal RANK function. We discuss the cell biological studies and animal models that help us to understand the nature of these different RANK defects and describe how careful dissection of these conditions can help understand critical pathways in osteoclast development and function. We highlight areas that require further study, particularly in light of the pharmacological interest in targeting the RANK signalling pathway to treat diseases caused by excessive bone resorption.

Paget's disease of bone (PDB) is a late-onset disorder characterised by focal areas of increased bone turnover containing enlarged hyperactive osteoclasts. The disease has a strong genetic predisposition and mutations in SQSTM1 have been associated with familial and sporadic disease in up to 40% of cases. Additional genetic loci have been associated in other cases, but genes are yet to be identified. Earlier-onset conditions with similar bone pathology (familial expansile osteolysis, expansile skeletal hyperphosphatasia and early-onset PDB) are caused by mutations in TNFRSF11A (RANK). The syndrome of inclusion body myositis, Paget's disease and frontotemporal dementia is caused by mutations in VCP. Despite the increased knowledge about genes involved in PDB and related disorders, the etiology of the diseases remains puzzling. Presence of inclusion bodies appears to link Pagetic diseases mechanistically to diseases associated with presence of misfolded proteins or abnormalities in the ubiquitin-proteasomal, or autophagy pathways. Juvenile PDB, caused by osteoprotegerin deficiency, appears mechanistically distinct from the other Pagetic diseases. This review will discuss evidence from recent studies, including new animal models for Pagetic diseases.

Unlabelled: Conflicting results have been reported on the detection of paramyxovirus transcripts in Paget's disease, and a possible explanation is differences in the sensitivity of RT-PCR methods for detecting virus. In a blinded study, we found no evidence to suggest that laboratories that failed to detect viral transcripts had less sensitive RT-PCR assays, and we did not detect measles or distemper transcripts in Paget's samples using the most sensitive assays evaluated., Introduction: There is conflicting evidence on the possible role of persistent paramyxovirus infection in Paget's disease of bone (PDB). Some workers have detected measles virus (MV) or canine distemper virus (CDV) transcripts in cells and tissues from patients with PDB, but others have failed to confirm this finding. A possible explanation might be differences in the sensitivity of RT-PCR methods for detecting virus. Here we performed a blinded comparison of the sensitivity of different RT-PCR-based techniques for MV and CDV detection in different laboratories and used the most sensitive assays to screen for evidence of viral transcripts in bone and blood samples derived from patients with PDB., Materials and Methods: Participating laboratories analyzed samples spiked with known amounts of MV and CDV transcripts and control samples that did not contain viral nucleic acids. All analyses were performed on a blinded basis., Results: The limit of detection for CDV was 1000 viral transcripts in three laboratories (Aberdeen, Belfast, and Liverpool) and 10,000 transcripts in another laboratory (Manchester). The limit of detection for MV was 16 transcripts in one laboratory (NIBSC), 1000 transcripts in two laboratories (Aberdeen and Belfast), and 10,000 transcripts in two laboratories (Liverpool and Manchester). An assay previously used by a U.S.-based group to detect MV transcripts in PDB had a sensitivity of 1000 transcripts. One laboratory (Manchester) detected CDV transcripts in a negative control and in two samples that had been spiked with MV. None of the other laboratories had false-positive results for MV or CDV, and no evidence of viral transcripts was found on analysis of 12 PDB samples using the most sensitive RT-PCR assays for MV and CDV., Conclusions: We found that RT-PCR assays used by different laboratories differed in their sensitivity to detect CDV and MV transcripts but found no evidence to suggest that laboratories that previously failed to detect viral transcripts had less sensitive RT-PCR assays than those that detected viral transcripts. False-positive results were observed with one laboratory, and we failed to detect paramyxovirus transcripts in PDB samples using the most sensitive assays evaluated. Our results show that failure of some laboratories to detect viral transcripts is unlikely to be caused by problems with assay sensitivity and highlight the fact that contamination can be an issue when searching for pathogens by sensitive RT-PCR-based techniques.

Paget's disease of bone is a common bone disease characterized by increased and disorganized bone remodeling at focal sites throughout the skeleton. The etiology of the disease is unresolved. A persistent viral infection has long been suggested to cause the disease. Antigen and/or nucleic acid sequences of paramyxoviruses (in particular measles virus [MV], canine distemper virus [CDV], and respiratory syncytial virus [RSV]) have been reported in pagetic bone by a number of groups; however, others have been unable to confirm this and so far no virus has been isolated from patients. Here, we reexamined the question of viral involvement in Paget's disease in a study involving 53 patients with established disease recruited from seven centers throughout the United Kingdom. Thirty-seven patients showed clear signs of active disease by bone scan and/or histological assessment of the bone biopsy specimens and 12 of these had not received any therapy before samples were taken. Presence of paramyxovirus nucleic acid sequences was sought in bone biopsy specimens, bone marrow, or peripheral blood mononuclear cells using reverse-transcription polymerase chain reaction (RT-PCR) with a total of 18 primer sets (7 of which were nested), including 10 primer sets (including 3 nested sets) specifically for MV or CDV. For each patient at least one sample was tested with all primer sets by RT-PCR and no evidence for the presence of paramyxovirus RNA was found in any patient. In 6 patients, bone biopsy specimens with clear histological evidence of active disease tested negative for presence of measles and CDV using immunocytochemistry (ICC) and in situ hybridization (ISH). Intranuclear inclusion bodies, similar to those described by others previously, were seen in pagetic osteoclasts. The pagetic inclusions were straight, smooth tubular structures packed tightly in parallel bundles and differed from nuclear inclusions, known to represent MV nucleocapsids, in a patient with subacute sclerosing panencephalitis (SSPE) in which undulating, diffuse structures were found, arranged loosely in a nonparallel fashion. In the absence of amplification of viral sequences from tissues that contain frequent nuclear inclusions and given that identical inclusions are found in other bone diseases with a proven genetic, rather than environmental, etiology, it is doubtful whether the inclusions in pagetic osteoclasts indeed represent viral nucleocapsids. Our findings in this large group of patients recruited from throughout the United Kingdom do not support a role for paramyxovirus in the etiology of Paget's disease.

We believe that the assembled data are consistent with the presence of mRNA species and/or proteins in pagetic bone that are recognized by some paramyxovirus antibodies and nucleic acid probes. The evidence presented so far is insufficient to substantiate claims for the "unequivocal" presence of paramyxovirus sequences in pagetic bone, because the molecular targets for these probes could be endogenous mRNA's and proteins rather than viruses. Positive reports of a viral presence in Paget's disease have so far been confined to two laboratories, both of which have consistently demonstrated evidence for the virus they have worked on most. We argue that independent replication of the aforementioned findings is necessary to conclude that pagetic bone can be considered a site of chronic paramyxovirus infection. For this to be convincing, we would expect to see colocalization of viral antigens, mRNA, and genomic RNA in cells that also show ultrastructural evidence of viral infection. If virus is indeed present, it should, in addition, be possible to clone and characterize extensive tracts of viral cDNA from samples of pagetic tissue. Although we acknowledge that the absence of evidence for viral mRNA in some RT-PCR studies does not constitute evidence of absence, the data implicating paramyxoviruses as causal agents is conflicting and insufficient to prove a cause-effect relationship. In view of this, we believe that the role of paramyxovirus infection as a cause Paget's disease remains uncertain.