SMARCB1/INI1 germline mutations contribute to 10% of sporadic schwannomatosis

Neurofibromatoses (NF) are an heterogeneous group of genetic disorders predisposing to various tumors of the nervous system, divided into two well recognized distinct clinical entities, NF1 and NF2. Patients with multiple non-vestibular schwannomas have been assembled into a particular category within neurofibromatoses called schwannomatosis [1]. The SMARCB1 gene has been found to harbor germline alterations in both familial and sporadic schwannomatosis patients [2, 3], with a greater number of spinal schwannomas in familial cases and the presence of meningiomas [4] although this latter point remains debated [5]. To improve the clinical indications for SMARCB1 molecular screening in medical genetics practice, we evaluated its implication in a series of patients exhibiting non-vestibular schwannomas and no NF2 germline alteration.

From 1992 to 2006, 303 patients were referred from the French outpatient genetic clinics for point mutations and genomic rearrangements analysis of the NF2 gene to investigate the presence of schwannomas. All patients or legal representatives signed an informed consent for genetic analyses related to their disease according to the French law (Public Health Code, article R145-15-4). No specific consent was required for the present study, as the SMARB1 analysis is part of the genetic tests proposed to patients affected with schwannomas. Analyses were performed in a laboratory labeled by the French Biomedicine Agency ABM (lab no. 2008-gen-01). A subgroup of 56 cases exhibiting non-vestibular schwannomas as confirmed by MRI examination at time of diagnosis were selected for SMARCB1 point mutations screening. Medical reports were collected (number and localization of schwannomas, age at diagnosis and family history). Exons and splicing junctions of the SMARCB1 gene (NM_003073, NG_009303.1) were analyzed by sequencing using the SeqScape software (Applied Biosystems, Courtaboeuf France) after PCR amplification (Table 1). Functional consequence of intronic variants was evaluated using NetGene2, GeneSplicer and a splice-site model that accounts for adjacent and nonadjacent pair wise dependencies MaxEnt.

Thirty-one females and 25 males were included in the study. The age of onset ranged from 2 to 70 years. Six patients had a single schwannoma, 11 patients had 2 to 7 and 39 patients more than 10 schwannomas. Two patients had a positive family history. Nine different sequence variants scattered along the sequence of SMARCB1 were found (Table 2). Three were present in 12 patients, 10 in a heterozygous and two in a homozygous state indicating a complete linkage disequilibrium (Fisher exact test, p < 10^-6). Two of them were present in dbSNP as rs2229354 (c.897G > A) and rs5030613 (c.1119-41G > A); the third change (c.1149-41G > A) was also considered as a polymorphism. The remaining 6 variants were unique: c.30del, c.34C > T and c.832C > T were expected to truncate the protein; c.500+5G > T was predicted to suppress the donor splicing site of exon 4, leading to p.Arg121_Cys167delinsSer and lacking the DNA-binding domain. Using the MaxEnt program, maximum entropy of -5.62 was computed compared to 0.87 with the normal sequence. Considering the existence of a rare long isoform, as a 51-bp extension at the 3' end of exon 4, this variant also encoded c.505G > T was expected to produce a p.Gly169X truncated protein lacking the two highly conserved regions Rpt1 and Rpt2 and the coiled-coil domain. The variant c.971_978delinsTGCTACCT implicated the highly conserved Lys₃₂₄ and Tyr₃₂₆ amino-acids, Tyr₃₂₆ being involved in the coiled-coil domain and possibly in the nuclear localization signal and/or the binding of Akt. All five were considered deleterious. The transversion c.501-23T > G was not predicted to affect splicing using NetGene2 and GeneSplicer and was classified as not responsible for the disease. All five patients carrying a SMARCB1 mutation were sporadic patients diagnosed before 35 years of age with multiple non-vestibular schwannomas.

Five mutations were identified through the analysis of the SMARCB1 gene for point mutations in a series of 56 NF2-negative patients affected with non-vestibular schwannomas. Patients carrying a mutation presented with multiple schwannomas at a young age, i.e. 10.2% in schwannomatosis patients [6] and 20% of patients diagnosed before 35 years of age, none with any family history of schwannomas. This proportion remains lower than that described in familial schwannomatosis [3, 7, 8]; however neither somatic mosaicisms nor genomic rearrangements were investigated [2, 7]. Tumor samples were not available thus precluding dosage analyses and the demonstration of SMARCB1 and/or NF2 somatic inactivation [3].

Schwannomatosis is a rare condition characterized by the development of multiple schwannomas and the lack of vestibular nerve involvement. As germline SMARCB1 mutations have never been reported in cases of vestibular schwannomas, the presence of multiple non-vestibular schwannomas appears a good criterion to first investigate the SMARCB1 gene, as reported through several studies (Table 3). From our study, as mutations were found in patients diagnosed before 35 years of age, this aspect might also be considered to enlarge the SMARCB1 analysis first to the search for genomic rearrangements and somatic mosaicisms and second to the NF2 gene screening. On the other hand, the presence of vestibular lesions remains the first indication for an NF2 gene molecular analysis.