SMARCA4-associated schwannomatosis

Schwannomatosis is a disorder characterized by a predisposition to multiple benign spinal, peripheral and intracranial nerve sheath tumours called schwannomas. The established molecular mechanism of schwannomatosis involves several genes located on Chromosome 22q (Chr22q), typically a germline SMARCB1 or LZTR1 pathogenic variant (PV), loss of heterozygosity (LOH) of Chr22q, and finally a somatic NF2 PV which collectively lead to the inactivation of SMARCB1 or LZTR1, and NF2 [7]. Other Chr22q genes such as DGCR8 also appear to be implicated, albeit rarely, in schwannomatosis [11, 12]. Here, we describe a familial occurrence of schwannomatosis where the proband (daughter) had a painful to the touch C2/C3 spinal schwannoma at 30 years(y) and the proband’s mother had 4 painless peripheral schwannomas (left and right arms) at 50y and a glioblastoma (GBM) at 54y. Additionally, the proband’s maternal grandfather died of a malignant peripheral nerve sheath tumour (MPNST) at 79y. No other family member has been evaluated for schwannomas or undergone genetic testing. Clinical whole genome sequencing (WGS) of the daughter’s blood DNA detected no pathogenic or likely pathogenic variants (PV or LPV) in the coding, non-coding and regulatory regions of the known Chr22q schwannomatosis genes, but instead, revealed a germline LPV in SMARCA4 [c.1752_1755del, p.(Lys585Argfs*27)](ClinVar ID:873514) (Online Resource [OR] Fig. S1). In the schwannomas, immunohistochemical (IHC) staining showed loss of BRG1 (SMARCA4) expression in 80–90% of cells and loss of INI1 (SMARCB1) in the complementary 10–20% of cells in all 5 schwannomas but complete retention of BRG1 and INI1 in the GBM (Fig. 1a and OR Fig. S2). Furthermore, the grandfather’s MPNST showed retention of BRG1 in 95% of cells. In schwannomas, mosaic loss of INI1 expression is relatively common but loss of BRG1 expression has not been reported before [13]. Whole exome sequencing (WES) of DNA extracted from the daughter’s blood, mother’s normal skin, all 5 schwannomas and the GBM, confirmed the presence of the truncating SMARCA4 LPV in all samples. LOH was observed at the SMARCA4 locus, extending to 12–23 Mb of Chromosome 19p (Chr19p) in all schwannomas, but not in the GBM. No LOH of Chr22q was detected in the schwannomas (Fig. 1b). Other than the SMARCA4 deletion, no known PV or LPV within a list of 2154 genes

Keywords Schwannomatosis · SMARCA4 · SWI/SNF · BRG1 · Chromosome 22q Schwannomatosis is a disorder characterized by a predisposition to multiple benign spinal, peripheral and intracranial nerve sheath tumours called schwannomas. The established molecular mechanism of schwannomatosis involves several genes located on Chromosome 22q (Chr22q), typically a germline SMARCB1 or LZTR1 pathogenic variant (PV), loss of heterozygosity (LOH) of Chr22q, and finally a somatic NF2 PV which collectively lead to the inactivation of SMARCB1 or LZTR1, and NF2 [7]. Other Chr22q genes such as DGCR8 also appear to be implicated, albeit rarely, in schwannomatosis [11,12].
Here, we describe a familial occurrence of schwannomatosis where the proband (daughter) had a painful to the touch C2/C3 spinal schwannoma at 30 years(y) and the proband's mother had 4 painless peripheral schwannomas (left and right arms) at 50y and a glioblastoma (GBM) at 54y. Additionally, the proband's maternal grandfather died of a malignant peripheral nerve sheath tumour (MPNST) at 79y. No other family member has been evaluated for schwannomas or undergone genetic testing. Clinical whole genome sequencing (WGS) of the daughter's blood DNA detected no pathogenic or likely pathogenic variants (PV or LPV) in the coding, non-coding and regulatory regions of the known Chr22q schwannomatosis genes, but instead, revealed a germline LPV in SMARCA4 [c.  Fig. S2). Furthermore, the grandfather's MPNST showed retention of BRG1 in 95% of cells. In schwannomas, mosaic loss of INI1 expression is relatively common but loss of BRG1 expression has not been reported before [13].
Whole exome sequencing (WES) of DNA extracted from the daughter's blood, mother's normal skin, all 5 schwannomas and the GBM, confirmed the presence of the truncating SMARCA4 LPV in all samples. LOH was observed at the SMARCA4 locus, extending to 12-23 Mb of Chromosome 19p (Chr19p) in all schwannomas, but not in the GBM. No LOH of Chr22q was detected in the schwannomas (Fig. 1b). Other than the SMARCA4 deletion, no known PV or LPV within a list of 2154 genes associated with cancer were shared amongst all schwannomas, including Chr22q alterations (OR Tables S1-2 and Fig. S3). Furthermore, CytoScan and OncoScan copy-number analyses performed on the daughter's samples verified the somatic loss of Chr19p and ruled out any Chr22q imbalances at a resolution of > 50 kb and > 500 kb in the germline and tumour, respectively (OR Fig. S4). A MUTYH PV (p.Gly396Asp) was detected in the mother's samples only, but there was no second hit in any of the tumours and therefore is unlikely to play a role in the family's schwannoma development.
The germline SMARCA4 variant was also detected in the grandfather's MPNST in a heterozygous state. BRG1 retention (IHC) and heterozygosity for the inherited variant in both the MPNST and GBM suggest that they were not caused by SMARCA4, unlike for the schwannomas.
To further characterize the SMARCA4-schwannomas, their DNA methylation profiles were analysed and compared to Chr22q associated-schwannomas and rhabdoid tumours.  Table S3) [5,10,12] were included in the analyses. The SMARCA4-associated schwannomas clustered away from the SCCOHTs and ATRTs and grouped together with the other schwannomas. However, within the schwannoma cluster, the SMARCA4 cases appear to form a small sub-group (Fig. 1c, OR Figs. S5-6).
Taken together, our results suggest that SMARCA4mutated schwannomas form a new subtype of does not seem to be involved in either of the family's tumours. Green segments represent regions of allelic imbalance. c UMAP of the top 10,000 most variably methylated CpG sites in the SMARCA4 schwannomas and GBM, LZTR1/SMARCB1/negative schwannomas [10,12] SCCOHTs and ATRTs [5]. The colours represent the different tumour subtypes. The dashed oval encapsulates the schwannomas from the family reported here familial schwannomatosis that is distinct from Chr22q-associated schwannomatosis. Since SMARCA4 lies on Chr19p, the well-known loss of Chr22q mechanism does not apply in this case and implies the possibility of a novel schwannomatosis mechanism. The link between Chr22q and SMARCA4 schwannomas may pertain to the shared molecular characteristics of SMARCB1 and SMARCA4, rather than the loci of the genesthey are both key members of the SWI/SNF chromatin remodelling complex, truncating variants in these genes cause aggressive malignant rhabdoid tumours (ATRT and/or SCCOHT) while missense variants tend to cause a milder phenotype in the form of Coffin-Siris Syndrome (CSS), a developmental disorder [6]. It is plausible that SWI/SNF genes are involved in both schwannomatosis and CSS. Further studies to understand the interplay of SWI-SNF gene family members in the etiology of familial schwannomatosis is warranted.
Because a germline truncating variant was identified in this family, carriers might be at risk for SCCOHT, in addition to schwannomas. This would be analogous to cases where a SMARCB1 germline variant was reported to lead to malignant rhabdoid tumours and schwannomas within the same family or in the same person [8] and to a case where a nonsense SMARCA4 germline variant caused SCCOHT and CSS simultaneously in a young girl [4]. Due to this, the proband (daughter) underwent risk-reducing bilateral salpingo-oophorectomy as well as a hysterectomy at age 35y. Of note, histopathology and peritoneal washings were negative for malignancy at the time of surgery.
Our findings lead us to propose SMARCA4 as a candidate schwannomatosis gene. Recent publications have demonstrated the expansion of the phenotypic spectrum associated with SMARCA4 GPVs [2,3,9] and our familial case suggests that schwannomatosis may well be an addition to the list.

Data availability
The datasets generated during and/or analysed during this study will be deposited to the European Genome-phenome Archive under accession number EGAS00001006987.