Antibody Response to Paramyxoviruses in Paget's Disease of Bone.

Paget's disease of bone (PDB) is a common skeletal disorder characterised by focal abnormalities of increased and disorganised bone turnover. Genetic factors play a central role in the pathogenesis of PDB but environmental factors also contribute. Measles virus (MV), respiratory syncytial virus (RSV) and canine distemper virus (CDV) have all been implicated as potential disease triggers but the data are conflicting. Since chronic paramyxovirus infection with measles is known to be accompanied by increased production of antiviral antibodies, we have analysed circulating concentrations of antibodies to MV, CDV, and RSV as well as mumps, rubella and varicella zoster virus (VZV) in 463 patients with PDB and 220 aged and gender-matched controls. We also studied the relation between viral antibody concentrations and various markers of disease severity and extent in 460 PDB patients. A high proportion of cases and controls tested positive for antiviral antibodies but there was no significant difference in circulating antibody concentrations between PDB cases and controls for MV, CDV, RSV, rubella or VZV. However, mumps virus antibody levels were significantly higher in the PDB cases (mean ± SD = 3.1 ± 0.84 vs. 2.62 ± 0.86. p < 0.001). There was no association between disease severity and circulating antibody concentrations to any of the viruses. In conclusion, we found no evidence to suggest that PDB is associated with abnormalities of immune response to measles or other paramyxoviruses, although there was evidence of a greater antibody response to mumps. The results do not support that hypothesis that PDB is associated with a persistent infection with measles or other paramyxoviruses.

Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function.

SQSTM1 mutations are common in patients with Paget disease of bone (PDB), with most affecting the C-terminal ubiquitin-associated (UBA) domain of the SQSTM1 protein. We performed structural and functional analyses of two UBA domain mutations, an I424S mutation relatively common in UK PDB patients, and an A427D mutation associated with a severe phenotype in Southern Italian patients. Both impaired SQSTM1's ubiquitin-binding function in pull-down assays and resulted in activation of basal NF-κB signalling, compared to wild-type, in reporter assays. We found evidence for a relationship between the ability of different UBA domain mutants to activate NF-κB signalling in vitro and number of affected sites in vivo in 1152 PDB patients from the UK and Italy, with A427D-SQSTM1 producing the greatest level of activation (relative to wild-type) of all PDB mutants tested to date. NMR and isothermal titration calorimetry studies were able to demonstrate that I424S is associated with global structural changes in the UBA domain, resulting in 10-fold weaker UBA dimer stability than wild-type and reduced ubiquitin-binding affinity of the UBA monomer. Our observations provide insights into the role of SQSTM1-mediated NF-κB signalling in PDB aetiology, and demonstrate that different mutations in close proximity within loop 2/helix 3 of the SQSTM1 UBA domain exert distinct effects on protein structure and stability, including indirect effects at the UBA/ubiquitin-binding interface.

Common susceptibility alleles and SQSTM1 mutations predict disease extent and severity in a multinational study of patients with Paget's disease.

Paget's disease of bone (PDB) has a strong genetic component. Here, we investigated possible associations between genetic variants that predispose to PDB and disease severity. Allelic variants identified as predictors of PDB from genome-wide association studies were analyzed in 1940 PDB patients from the United Kingdom, Italy, Western Australia, and Spain. A cumulative risk allele score was constructed by adding the variants together and relating this to markers of disease severity, alone and in combination with SQSTM1 mutations. In SQSTM1-negative patients, risk allele scores in the highest tertile were associated with a 27% increase in disease extent compared with the lowest tertile (p < 0.00001) with intermediate values in the middle tertile (20% increase; p = 0.0007). The effects were similar for disease severity score, which was 15% (p = 0.01) and 25% (p < 0.00001) higher in the middle and upper tertiles, respectively. Risk allele score remained a significant predictor of extent and severity when SQSTM-positive individuals were included, with an effect size approximately one-third of that observed with SQSTM1 mutations. A genetic risk score was developed by combining information from both markers, which identified subgroups of individuals with low, medium, and high levels of severity with a specificity of 70% and sensitivity of 55%. Risk allele scores and SQSTM1 mutations both predict extent and severity of PDB. It is possible that with further refinement, genetic profiling may be of clinical value in identifying individuals at high risk of severe disease who might benefit from enhanced surveillance and early intervention.

Mutations of SQSTM1 are associated with severity and clinical outcome in paget disease of bone.

Paget disease of bone (PDB) is a common disorder characterized by increased bone turnover at one of more sites throughout the skeleton. Genetic factors play an important role in the pathogenesis of PDB, and the most important predisposing gene is SQSTM1, which is mutated in about 10% of patients. Here we investigated the relationship between SQSTM1 mutation status, disease severity, and clinical outcome in 737 patients who took part in a randomized study of two different management strategies for the disease. Mutations of SQSTM1 were detected in 80 of 737 (10.9%) patients. Mutation carriers had an earlier age at diagnosis (59.4 ± 11.5 versus 65.0 ± 10.4 years, p < .0001) and a greater number of affected bones (3.2 ± 1.2 versus 2.1 ± 1.2, p < .001) and more commonly required orthopedic surgery (26.2% versus 16.1%, p = .024) and bisphosphonate therapy (86.3% versus 75.2%, p = .01) than those without mutations. Quality of life, as assessed by the short-form-36 (SF36) physical summary score, was significantly reduced in carriers (34.0 ± 11.3 versus 37.1 ± 11.4, p = .036). During the study, fractures were more common in carriers (12.5% versus 5.3%, p = .011), although most of these occurred in unaffected bone. This study demonstrates that SQSTM1 mutations are strongly associated with disease severity and complications of PDB. Genetic testing for SQSTM1 mutations may be of value in identifying individuals at risk of developing severe disease, but further studies will be required to determine if a program of genetic testing and early intervention in these individuals would be cost-effective or be of benefit in preventing these complications.

Assessment of removal of human cytomegalovirus from blood components by leukocyte depletion filters using real-time quantitative PCR.

To assess removal of cytomegalovirus (CMV) by leukocyte depletion (LD) filters, we developed a spiking model of latent virus using peripheral blood mononuclear cells (PBMCs) infected by coculture with CMV-infected human fibroblasts. Infected PBMCs were purified by dual magnetic column selection and then spiked into whole blood units or buffy coat pools prior to LD by filtration. CMV load and fibroblast contamination were assessed using quantitative CMV DNA real-time PCR and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA encoding the fibroblast-specific splice variant of prolyl-4-hydroxylase, respectively. After correcting for fibroblast-associated CMV, the mean CMV load was reduced in whole blood by LD from 7.42 x 10(2) to 1.13 copies per microliter (2.81(10)log reduction) and from 3.8 x 10(2) to 4.77 copies per microliter (1.9(10)log reduction) in platelets. These results suggest that LD by filtration reduces viral burden but does not completely remove CMV from blood components.