Premature termination of SMARCB1 translation may be followed by reinitiation in schwannomatosis-associated schwannomas, but results in absence of SMARCB1 expression in rhabdoid tumors

Abstract

In schwannomatosis, germline SMARCB1 mutations predispose to the development of multiple schwannomas, but not vestibular schwannomas. Many of these are missense or splice-site mutations or in-frame deletions, which are presumed to result in the synthesis of altered SMARCB1 proteins. However, also nonsense and frameshift mutations, which are characteristic for rhabdoid tumors and are predicted to result in the absence of SMARCB1 protein via nonsense-mediated mRNA decay, have been reported in schwannomatosis patients. We investigated the consequences of four of the latter mutations, i.e. c.30delC, c.34C>T, c.38delA, and c.46A>T, all in SMARCB1-exon 1. We could demonstrate for the c.30delC and c.34C>T mutations that the respective mRNAs were still present in the schwannomas of the patients. We hypothesized that these were prevented from degradation by translation reinitiation at the AUG codon encoding methionine at position 27 of the SMARCB1 protein. To test this, we expressed the mutations in MON cells, rhabdoid cells without endogenous SMARCB1 protein, and found that all four resulted in synthesis of the N-terminally truncated protein. Mutation of the reinitiation methionine codon into a valine codon prevented synthesis of the truncated protein, thereby confirming its identity. Immunohistochemistry with a SMARCB1 antibody revealed a mosaic staining pattern in schwannomas of the patients with the c.30delC and c.34C>T mutations. Our findings support the concept that, in contrast to the complete absence of SMARCB1 expression in rhabdoid tumors, altered SMARCB1 proteins with modified activity and reduced (mosaic) expression are formed in the schwannomas of schwannomatosis patients with a germline SMARCB1 mutation.

The molecular pathogenesis of schwannomatosis, a paradigm for the co-involvement of multiple tumour suppressor genes in tumorigenesis

“…In accord with this postulate are the findings of Hulsebos et al (2014a). These authors analysed four schwannomatosis-associated SMARCB1 mutations that were located in the 5′ region of the gene and were predicted to introduce a premature translational termination codon (PTC).…”

“…This finding was confirmed by transient overexpression of a c.34C > T-containing expression vector in a cell line without endogenous SMARCB1 protein and subsequent detection of the shortened SMARCB1 protein by Western blotting. Similar overexpression experiments indicated that the mutations c.30delC, c.38delA, and c.46A > T also lead to truncated SMARCB1 proteins which may well be partially functional (Hulsebos et al 2014a). …”

“…A cyclin D1 repression assay has shown that mutant SMARCB1 proteins, derived from expression plasmids harbouring the same missense and splice-site mutations noted in patients with schwannomatosis, were capable of suppressing cyclin D1 activity in a similar manner to the wild-type SMARCB1 protein (Smith et al 2012c). Taken together, these observations suggest that in schwannomatosis, germline SMARCB1 mutations encode proteins with some residual functionality (Smith et al 2012c; Hulsebos et al 2014a). By contrast, SMARCB1 mutations in rhabdoid tumours and patients with RTPS1 are almost invariably either nonsense mutations or frameshifts, even complete gene deletions, and hence are associated with a loss-of-function (Kordes et al 2010; Bourdeaut et al 2011; reviewed by Biegel et al 2014).…”

“…The other two SMARCB1 mutations, c.34C > T (p.Gln12*) and c.46A > T (p.Lys16*), directly generate PTCs at their respective positions. Although transcripts containing these PTCs may reasonably be expected to be degraded by nonsense-mediated mRNA decay (NMD), stable transcripts were detected by Hulsebos et al (2014a) in schwannoma tissue obtained from two patients harbouring either c.34C > T or c.30delC. Furthermore, Western blot analysis using frozen tumour tissue from the individual with c.30delC indicated the occurrence of a shortened protein owing to downstream translational reinitiation.…”

“…Since, in schwannomas of patients with schwannomatosis, the wild-type SMARCB1 allele is often lost by deletion or monosomy 22, the SMARCB1 protein detected in schwannoma cells must be encoded by the mutant allele. The inability to detect mutant proteins in all tumour cells by immunostaining is likely to be a consequence of the instability of mutant SMARCB1 proteins (Hulsebos et al 2014a). This instability results in immunologically nonreactive SMARCB1 protein degradation products in a proportion of the schwannoma cells.…”