Klinck Roscoe, Laberge Gino, Bisson Martine, McManus Stephen, Michou Laëtitia, Brown Jacques P, Roux Sophie
BMC Med Genet. 2014 Aug 14;15:98. doi: 10.1186/s12881-014-0098-1.
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 hypothesized that specific RNA isoforms of OC-related genes may contribute to the overactivity of pagetic OCs, along with other genetic predisposing factors.
Alternative 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.
This 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.
This 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.
骨Paget病（PDB）中已报道了SQSTM1/p62基因的突变，但这些突变不足以诱导Paget破骨细胞（OC）表型。我们推测，OC相关基因的特定RNA异构体可能与其他遗传易感因素一起，导致Paget OC的过度活跃。
采用基于PCR的筛选策略，研究了29例PDB患者和26例健康供体（HD）的OC培养物中的可变剪接（AS）事件，所有样本均进行了p62P392L突变基因分型。使用针对5223个已表征AS事件的引物对，分析四组样本（PDB、PDBP392L、HD、HDP392L）混合cDNA上的相对异构体比例。在筛选分析中检测到的1056个活跃AS事件中，对整个队列中每个受试者的未扩增cDNA重新分析了192个。
该分析鉴定出6个与PDB显著相关的AS事件，但与p62P392L均无关。相应基因包括LGALS8、RHOT1、CASC4、USP4、TBC1D25和PIDD。此外，LGALS8和RHOT1基因在Paget OC中上调，在存在p62P392L的情况下，CASC4和RHOT1基因也上调。最后，我们表明LGALS8、RHOT1、USP4、TBC1D25和PIDD编码的蛋白在人OC中表达。
本研究鉴定出了OC生物学中迄今未知的因素，我们在Paget OC中发现的差异AS可能为PDB的发病机制带来新的概念。
