Study of RIN3 : a susceptibility gene for Paget’s disease of bone

Paget’s disease of bone (PDB) shows a strong genetic component and mutations in SQSTM1 (Sequestosome 1) are observed in about 10% of sporadic PDB patients. My PhD investigated the RIN3 gene (Ras and Rab interactor protein 3), previously implicated in the pathogenesis of PDB by GWAS. The RIN3 gene encodes a guanine exchange factor (GEF), involved in the activation of GTPases which are crucial in osteoclast activity. It also has a role in endocytosis and recycling of tyrosine kinase receptor. The role of RIN3 in bone remodelling is unclear, however some investigations revealed some associations with bone: RIN3 has been associated with high lower limb bone mineral density in children in a meta-analysis of GWAS studies, and was shown to be expressed in primary calvarial osteoblasts. The expression of RIN3 was down regulated during human primary osteoclast differentiation, and also in iliac bone biopsies from osteoporotic patients compared to healthy postmenopausal donors (Kemp et al, 2014).   In Chapter 1, I present normal bone structure, composition and remodelling before detailing PDB and its genetics. I then introduce RIN3 as a candidate gene for PDB. In Chapter 2, I describe all methods performed and materials used for the completion of this project. This includes primary cell cultures, RNA and protein work, immunostaining, immunochemistry and phenotype analysis on Rin3-/- mice. Chapter 3 presents the fine mapping of RIN3 using Sanger and next generation sequencing performed on PDB cases and controls. 18 variants were detected and one common variant (p.R279C) showed a strong association with PDB. Rare variants were also over-represented in cases, and many were shown to be on the same haplotype as p.R279C. Chapter 4 details the association study performed on a UK cohort and includes the investigation of the clinical phenotype severity in patients against the RIN3 mutations. Chapter 5 presents the expression pattern of RIN3 in bone cells and bone microenvironment. Important variations of RIN3 mRNA and protein were detected during the differentiation of bone marrow derived osteoclasts. Protein levels of RIN3 were also found in osteoclasts from human osteoclastoma, human osteosarcoma, PDB patients, giant cell tumour (GCT) and healthy controls. Within all the mouse tissues analysed, Rin3 mRNA was expressed the highest in bone after lung. Chapter 6 focuses on the work performed on mice deficient of the Rin3 gene. They showed a higher trabecular bone volume and a smaller active resorption surface occupied by osteoclasts in trabecular bone. In conclusion, the combined in vitro and in vivo analyses have uncovered that RIN3 plays a role in bone metabolism and is a strong gene candidate for PDB

Pattern of SQSTM1 Gene Variants in a Hungarian Cohort of Paget’s Disease of Bone

Paget’s disease of bone (PDB) is characterized by focal or multifocal increase in bone turnover. One of the most well-established candidate genes for susceptibility to PDB is Sequestosome 1 (SQSTM1). Mutations in SQSTM1 have been documented among Western-European, British and American patients with PDB. However, there is no information on SQSTM1 mutation status in PDB patients from the Central- and Eastern-European regions. In this study, we conducted a mutation screening for SQSTM1 gene variants in 82 PDB patients and 100 control participants in Hungary. Mutations of SQSTM1 were detected in 18 PDB patients (21.95%); associations between genotype and clinical characteristics were also analyzed. Altogether, six different exonic alterations, including two types of UTR variants in the SQSTM1 gene, wereobserved in our PDB patients. Similarly, to previous genetic studies on Paget’s disease, our most commonly detected variant was the c.1175C > T (p.Pro392Leu) in nine cases (four in monostotic and five in polyostotic form). We have surveyedthe germline SQSTM1 variant distribution among Hungarian patients with PDB. We also highlighted that the pattern of the analyzed disease-associated pathophysiological parameters could partially discriminate PDB patients with normal or mutantSQSTM1 genotype. However, our findings also underline and strengthen that not solely SQSTM1 stands in the background of the complex PDB etiolog

Targeted sequencing of the Paget's disease associated 14q32 locus identifies several missense coding variants in RIN3 that predispose to Paget's disease of bone

Paget's disease of bone (PDB) is a common disorder with a strong genetic component characterized by increased but disorganized bone remodelling. Previous genome-wide association studies identified a locus on chromosome 14q32 tagged by rs10498635 which was significantly associated with susceptibility to PDB in several European populations. Here we conducted fine-mapping and targeted sequencing of the candidate locus to identify possible functional variants. Imputation in 741 PDB patients and 2699 controls confirmed that the association was confined to a 60 kb region in the RIN3 gene and conditional analysis adjusting for rs10498635 identified no new independent signals. Sequencing of the RIN3 gene identified a common missense variant (p.R279C) that was strongly associated with the disease (OR = 0.64; P = 1.4 × 10(−9)), and was in strong linkage disequilibrium with rs10498635. A further 13 rare missense variants were identified, seven of which were novel and detected only in PDB cases. When combined, these rare variants were over-represented in cases compared with controls (OR = 3.72; P = 8.9 × 10(−10)). Most rare variants were located in a region that encodes a proline-rich, intrinsically disordered domain of the protein and many were predicted to be pathogenic. RIN3 was expressed in bone tissue and its expression level was ∼10-fold higher in osteoclasts compared with osteoblasts. We conclude that susceptibility to PDB at the 14q32 locus is mediated by a combination of common and rare coding variants in RIN3 and suggest that RIN3 may contribute to PDB susceptibility by affecting osteoclast function