Abstract
BackgroundMutations in the SQSTM1/p62 gene have been reported in Paget’s disease of bone (PDB), but they are not sufficient to induce the pagetic osteoclast (OC) phenotype. We hypothesized that specific RNA isoforms of OC-related genes may contribute to the overactivity of pagetic OCs, along with other genetic predisposing factors.MethodsAlternative splicing (AS) events were studied using a PCR-based screening strategy in OC cultures from 29 patients with PDB and 26 healthy donors (HD), all genotyped for the p62P392L mutation. Primer pairs targeting 5223 characterized AS events were used to analyze relative isoform ratios on pooled cDNA from samples of the four groups (PDB, PDBP392L, HD, HDP392L). Of the 1056 active AS events detected in the screening analysis, 192 were re-analyzed on non-amplified cDNA from each subject of the whole cohort.ResultsThis analysis led to the identification of six AS events significantly associated with PDB, but none with p62P392L. The corresponding genes included LGALS8, RHOT1, CASC4, USP4, TBC1D25, and PIDD. In addition, RHOT1 and LGALS8 genes were upregulated in pagetic OCs, as were CASC4 and RHOT1 genes in the presence of p62P392L. Finally, we showed that the proteins encoded by LGALS8, RHOT1, USP4, TBC1D25, and PIDD were expressed in human OCs.ConclusionThis study allowed the identification of hitherto unknown players in OC biology, and our findings of a differential AS in pagetic OCs may generate new concepts in the pathogenesis of PDB.
Highlights
Mutations in the SQSTM1/p62 gene have been reported in Paget’s disease of bone (PDB), but they are not sufficient to induce the pagetic osteoclast (OC) phenotype
We studied OC cultures from a cohort of patients (PDB) and healthy donors (HD), all genotyped for the p62P392L mutation, and representing four distinct groups: HDwt (n = 16, mean age 61 years; 39–84), healthy donors carrying the p62P392L mutation (HDP392L) (n = 10, mean age 51.3 years; 33–70; of which three were under 52 years old), PDBwt (n = 17, mean age 70.7 years; 45–86), PDBP392L (n = 12, mean age 75 years; 59–85)
Using a screening strategy that has been applied to cancer research in our institution [16], we found alternative splicing (AS) events significantly associated with PDB, but not with p62P392L, in six genes: LGALS8, RHOT1, CASC4, USP4, TBC1D25, and PIDD
Summary
Mutations in the SQSTM1/p62 gene have been reported in Paget’s disease of bone (PDB), but they are not sufficient to induce the pagetic osteoclast (OC) phenotype. Pagetic OCs are both larger and more numerous than those in healthy individuals They are overactive and hypersensitive to osteoclastogenic factors [4], and to PDB located at the 1p13 (CSF1), 7q33 (CNOT4, NUP205, SLC13A4), 8q22 (TM7SF4), 10p13 (OPTN), 14q32 (RIN3), 15q24 (PML, GOLGA6A), and 18q21 (TNFRSF11A) loci [10,11]. This implies that genes other than SQSTM1 may contribute to the pathogenesis of PDB, so far only disease-causing mutations in SQSTM1 have been identified. Our objective was to look for PDB-specific AS events in these cells, and to investigate the impact of the p62P392L mutation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
