title: Novel UBA domain mutations of SQSTM1 in Paget's disease of bone: genotype phenotype correlation, functional analysis, and structural consequences.
abstract: Three novel missense mutations of SQSTM1 were identified in familial PDB, all affecting the UBA domain. Functional and structural analysis showed that disease severity was related to the type of mutation but was unrelated to the polyubiquitin-binding properties of the mutant UBA domain peptides.
  
  
  BACKGROUND
  Mutations affecting the ubiquitin-associated (UBA) domain of Sequestosome 1 (SQSTM1) gene have recently been identified as a common cause of familial Paget's disease of bone (PDB), but the mechanisms responsible are unclear. We identified three novel SQSTM1 mutations in PDB, conducted functional and structural analyses of all PDB-causing mutations, and studied the relationship between genotype and phenotype.
  
  
  METHODS
  Mutation screening of the SQSTM1 gene was conducted in 70 kindreds with familial PDB. We characterized the effect of the mutations on structure of the UBA domain by protein NMR, studied the effects of the mutant UBA domains on ubiquitin binding, and looked at genotype-phenotype correlations.
  
  
  CONCLUSIONS
  Three novel missense mutations affecting the SQSTM1 UBA domain were identified, including a missense mutation at codon 411 (G411S), a missense mutation at codon 404 (M404V), and a missense mutation at codon 425 (G425R). We also identified a deletion leading to a premature stop codon at 394 (L394X). None of the mutations were found in controls. Structural analysis showed that M404V and G425R involved residues on the hydrophobic surface patch implicated in ubiquitin binding, and consistent with this, the G425R and M404V mutants abolished the ability of mutant UBA domains to bind polyubiquitin chains. In contrast, the G411S and P392L mutants bound polyubiquitin chains normally. Genotype-phenotype analysis showed that patients with truncating mutations had more extensive PDB than those with missense mutations (bones involved = 6.05 +/- 2.71 versus 3.45 +/- 2.46; p < 0.0001). This work confirms the importance of UBA domain mutations of SQSTM1 as a cause of PDB but shows that there is no correlation between the ubiquitin-binding properties of the different mutant UBA domains and disease occurrence or extent. This indicates that the mechanism of action most probably involves an interaction between SQSTM1 and a hitherto unidentified protein that modulates bone turnover.
authors.affiliation: Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, United Kingdom.
authors.forename: Lynne J
authors.initials: LJ
authors.lastname: Hocking
authors.forename: Gavin J A
authors.initials: GJ
authors.lastname: Lucas
authors.forename: Anna
authors.initials: A
authors.lastname: Daroszewska
authors.forename: Tim
authors.initials: T
authors.lastname: Cundy
authors.forename: Geoff C
authors.initials: GC
authors.lastname: Nicholson
authors.forename: Judit
authors.initials: J
authors.lastname: Donath
authors.forename: John P
authors.initials: JP
authors.lastname: Walsh
authors.forename: Catriona
authors.initials: C
authors.lastname: Finlayson
authors.forename: James R
authors.initials: JR
authors.lastname: Cavey
authors.forename: Barbara
authors.initials: B
authors.lastname: Ciani
authors.forename: Paul W
authors.initials: PW
authors.lastname: Sheppard
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Ralston
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D004252:DNA Mutational Analysis; D005260:Female; D005820:Genetic Testing; D006801:Humans; D008297:Male; D020125:Mutation, Missense / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010001:Osteitis Deformans / Q000175:diagnosis / Q000235:genetics*; D010641:Phenotype; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D011506:Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D025801:Ubiquitin