Determining the importance of coding variants is an increasing challenge in human molecular genetics, exemplified in the skeletal disorder Pagets disease of bone (PDB) where mutations affecting Q7STM1 are commonly identified. As part of a screening effort to enter asymptomatic adults carrying Q7STM1 mutations with a family history of PDB into the Zoledronate in Prevention of Pagets disease (ZIPP) study, exons 7 and 8 of the gene were sequenced in 1420 probands with PDB. Subsequent sequencing of first degree relatives (predominantly children) of probands found to be positive for Q7STM1 mutations led to the identification of 324 asymptomatic subjects (44%) with mutations, who were invited to enter the trial. The analyses identified one family in the UK carrying a novel Ala390Gly missense mutation and one family in Australia with an Ala390Val mutation, located close to the ubiquitin-binding UBA domain of the Q7STM1/p62 protein. Although mutation pathogenicity prediction programmes indicated both variants may be pathogenic, analysis of protein structural models showed that Ala390 is not directly involved in either dimerisation or ubiquitin-binding of the UBA domain, properties critical to protein function. Protein binding assays showed the Ala390Val variant, but not Ala390Gly, is however associated with a loss of ubiquitin-binding function, which for Ala390Val mirrors that seen for pathogenic Q7STM1 mutations. In contrast, cell-based reporter assays showed both variants strongly activated NF-kB signalling relative to wild-type Q7STM1/p62, indicating both are probably pathogenic. On the basis of the functional analyses the Ala390Val and Ala390Gly positive subjects received genetic counselling to the effect that they were carriers of pathogenic mutations and were offered the chance of participating in the ZIPP study. Our work highlights a disconnect between in silico predictions, protein function analyses and cell-based assays of Q7STM1 sequence variants, and suggest cell-based assays are required to identify those variants which are potentially pathogenic.
Disclosure: The authors declared no competing interests.