Attenuated clinical and osteoclastic phenotypes of Paget's disease of bone linked to the p.Pro392Leu/SQSTM1 mutation by a rare variant in the DOCK6 gene.

BACKGROUND: We identified two families with Paget's disease of bone (PDB) linked to the p.Pro392Leu mutation within the SQSTM1 gene displaying a possible digenism. This study aimed at identifying this second genetic variant cosegregating with the p.Pro392Leu mutation and at characterizing its impact on the clinical and cellular phenotypes of PDB. METHODS: Whole exome sequencing was performed in one patient per family and two healthy controls. We compared clinical characteristics of PDB in 14 relatives from the two families. The osteoclastic phenotype was compared in in vitro differentiated osteoclasts from 31 participants carrying the DOCK6 and/or SQSTM1 variants. Tridimensional models of SQSTM1 and DOCK6 proteins were generated to evaluate the impact of these variants on their stability and flexibility. Statistical analyses were performed with Graphpad prism. RESULTS: Whole-exome sequencing allowed us to identify the p.Val45Ile missense variant in the DOCK6 gene in patients. In both families, the mean age at PDB diagnosis was delayed in pagetic patients carrier of the p.Val45Ile variant alone compared to those carrying the p.Pro392Leu mutation alone (67 vs. 44 years, P = 0.03). Although both p.Val45Ile and p.Pro392Leu variants gave rise to a pagetic phenotype of osteoclast versus healthy controls, the p.Val45Ile variant was found to attenuate the severity of the osteoclastic phenotype of PDB caused by the p.Pro392Leu mutation when both variants were present. The DOCK6 mRNA expression was higher in carriers of the p.Val45Ile variant than in pagetic patients without any mutations and healthy controls. Structural bioinformatics analyses suggested that the p.Pro392Leu mutation might rigidify the UBA domain and thus decrease its possible intramolecular interaction with a novel domain, the serum response factor-transcription factor (SRF-TF)-like domain, whereas the p.Val45Ile variant may decrease SRF-TF-like activity. CONCLUSION: The p.Val45Ile variant may attenuate the severity of the clinical phenotype of PDB in patient carriers of both variants. In vitro, the rare variant of the DOCK6 may have a modifier effect on the p.Pro392Leu mutation, possibly via its effect on the SRF-TF-like.

Clinical phenotype of adult offspring carriers of the p.Pro392Leu mutation within the SQSTM1 gene in Paget's disease of bone.

Paget's disease of bone (PDB) is a common chronic bone disorder. In the French-Canadian population, the p.Pro392Leu mutation within the SQSTM1 gene is involved in 46% of familial forms. In New Zealand, the emergence of PDB in offspring inheriting SQSTM1 mutations was reported to be delayed by a decade compared to their parents. We aimed at assessing the clinical phenotype of offspring carriers of this mutation in our French-Canadian cohort. We reviewed research records from adult offspring carriers of this mutation aged <90 years and their affected parents. In parents, we collected data on sex, age at diagnosis, number of affected bones, total serum alkaline phosphatase levels (tALPs) at diagnosis. In offspring, PDB extended phenotype assessment relying on tALPs, bone specific alkaline phosphatase levels (bALPs), procollagen type 1 amino-terminal propeptide (P1NP), whole body bone scan and skull and pelvis radiographs, was performed at inclusion from 1996 to 2009 and updated in 2016 to 2018, if not done during the past 8 years. The results showed that among the 36 offspring with an updated phenotype, four of them developed a clinical phenotype of PDB characterized by monostotic or polyostotic increased bone uptake associated with typical radiographic lesions in the affected sites, representing an incidence of 1.83 per 1000 person-years. Moreover, the age at PDB diagnosis was delayed by at least 10 years in the adult offspring carriers of the p.Pro392Leu mutation versus their affected parents. Our findings support the utility of a regular monitoring of the adult offspring without PDB but carriers of this mutation.

Gene-environment interactions in Paget's disease of bone.

OBJECTIVES: This study explored the role of outdoor and indoor air pollutants in Paget's disease of bone (PDB). METHODS: We performed a survey in 140 French-Canadian patients with PDB, including 39 carriers of p.Pro392Leu mutation (SQSTM1 gene) and 113 healthy not mutated controls. The survey covered outdoor air pollution near the residence and indoor air pollutants by focusing on heating fuels and exposure to tobacco smoke. In a subgroup of patients, urinary concentrations of 17 heavy metals and 11 polycyclic aromatic hydrocarbons were measured by mass spectrometry. In light of what we learned from the survey and urinary assays, we explored the in vitro effects of certain toxics on osteoclasts in PDB. We conducted in vitro monocytes differentiation from peripheral blood of more than 40 participants, whose osteoclasts were treated with or without the toxic. The morphology of osteoclasts, their bone resorption abilities, gene and protein expression levels, and cellular oxidative stress levels were assayed. RESULTS: An inhibitory effect of cigarette smoke condensate and heavy metals was observed on morphology and bone resorption activity of patients' osteoclasts. SQSTM1 gene expression was upregulated in osteoclasts from patients with PDB versus healthy controls in presence of cadmium, and SQSTM1 protein expression was upregulated in presence of bismuth and tobacco smoke condensates, in particular in osteoclasts from carriers of the SQSTM1 mutation. Furthermore, high levels of oxidative stress in patients' osteoclasts were observed. CONCLUSIONS: Our in vitro experiments suggest an interaction between SQSTM1 gene and exposure to cadmium and tobacco smoke condensates.