Background: The role of germline and somatic SMARCB1 gene mutations in malignant rhabdoid tumour (MRT) predisposition is well known. Germline SMARCB1 mutations have also recently been identified in a subset of individuals with schwannomatosis. Surprisingly, MRT predisposition and schwannomatosis have never been reported to co-occur in a family. The correlation between genotype and phenotype for mutations in SMARCB1 has not been determined.
Results: We have identified a germline 2631 bp duplication that includes exon 6 of SMARCB1 in a unique family with a four generation history of MRT predisposition and schwannomatosis. This duplication segregates with disease in individuals affected with both conditions, linking MRT predisposition and schwannomatosis as components of the same syndrome in this family.
Conclusion: The unique combination of tumours that result from the duplication described in this report may provide important clues about the mechanisms that influence the phenotype associated with a given SMARCB1 mutation.
Statistics from Altmetric.com
Malignant rhabdoid tumour (MRT) predisposition (OMIM 609322) and schwannomatosis (OMIM 162091) are unique conditions that predispose to distinct tumour types. Autosomal dominant inheritance of each condition has been reported, although both most frequently occur sporadically.1–5 MRT is an aggressive paediatric malignancy with a poor prognosis that commonly occurs in kidney, brain and soft tissue. In the central nervous system, it is termed atypical teratoid/rhabdoid tumour (AT/RT) reflecting the mixed cellular elements present in the tumours.6 7 MRT predisposition results from germline loss of function mutations in the SMARCB1 (also known as INI1, hSNF5 and BAF47) tumour suppressor gene at 22q11.23.8 The Smarcb1 protein is a component of the SWI/SNF complex, which regulates gene transcription through remodelling chromatin.9 Somatic inactivation of SMARCB1 also occurs in the majority of sporadic MRTs.10 11 In contrast, individuals with schwannomatosis have multiple schwannomas, but lack the vestibular schwannomas characteristic of neurofibromatosis type 2 (NF2).12 Significantly, germline loss of function SMARCB1 mutations have also recently been identified in a subset of familial and sporadic cases of schwannomatosis.13–16 Based on these initial studies of mutations as well as studies of genetic linkage and tumours from schwannomatosis families that implicate the chromosomal region encompassing SMARCB1,3 17 such mutations seem to be a frequent cause of schwannomatosis. Since seemingly similar germline SMARCB1 mutations can result in either schwannomatosis or MRT, it is puzzling that there have been no reports of families in which both conditions have occurred.
The family we describe (fig 1) was referred following the death of IV.2 from AT/RT (see below) and the resection of a tumour from the hand of her father (III.1) that proved to be an epithelioid schwannoma. Within the extended family, individuals I.2 (by report), II.1 and III.4 also had multiple skin lumps consistent with schwannomas. The lesions of II.1 first appeared at age 15 years, are tender to palpation, but they have not been biopsied. III.4 first developed skin lesions, described as “painful,” at age 16–17; one resected scalp tumour was confirmed to be an epithelioid schwannoma. III.1 developed painful lumps at age 16. Examination of individuals II.1, III.1, and III.4 by one of the authors (MSW) revealed multiple (at least 10 in each) subcutaneous lesions. The number increased with age, so II.1 had the most lesions. The growths were firm to palpation and some of them were painful when palpated. The majority were located on the back or extremities with the exception of the scalp tumour that had been removed in III.4. None were associated with any local neurologic deficits. The age of onset, pattern of distribution and pain is consistent with other reported individuals.12 No other cutaneous abnormalities, including café-au-lait macules, axillary or inguinal freckling, or other pigmentary changes were detected. No Lisch nodules were evident.
No imaging was performed looking for visceral lesions, nor did any of the evaluated patients have deep or referred pain that suggested a symptomatic lesion. III.4 underwent magnetic resonance imaging (MRI) of the brain and cranial base with gadolinium enhancement, specifically looking for vestibular schwannoma, that was normal at age 23, with the exception of the incidental finding of a Chiari I malformation. Vestibular schwannoma was not clinically diagnosed in any other individual although none were evaluated by MRI. None had symptoms of hearing loss or vestibular dysfunction, and neurologic evaluation by the author targeting cranial nerve VIII was unremarkable in all individuals. All of the children of II.1 had different fathers. III.4 meets the proposed research criteria12 for definite schwannomatosis while III.1 meets the criteria for presumptive schwannomatosis. Other family members have not had imaging or biopsy; however, given the autosomal dominant inheritance pattern in this family, the phenotype in these affected individuals is consistent with hereditary schwannomatosis. Interestingly, the epithelioid variant of schwannoma has not previously been reported in schwannomatosis; however, the two cases with pathology were reviewed by one of the authors (CMC) who concurred with the diagnoses (fig 2).
In contrast, the phenotype in three other affected family members was consistent with hereditary MRT predisposition. III.3 died before 2 years of age from a malignant posterior fossa tumour (consistent with AT/RT, but pathology was unavailable), while IV.1 and IV.2 each died before 2 years of age from tumours confirmed to be AT/RTs (fig 2). Finally, two family members have no evidence of symptoms. IV.3 was examined clinically at the age of 7 years. MRI of the brain and spine (for evidence of AT/RT) were normal, and no cutaneous abnormalities were apparent. However, schwannomatosis commonly presents at a later age in this family and in other reported families.12 Individual III.5 has not been examined, but she has no reported cutaneous abnormalities.
This is the first family in which schwannomatosis and MRT predisposition have been reported to co-occur, a significant finding given that SMARCB1 mutations are causal for both conditions. As such, analysis of the germline mutation in this family might provide important clues about the molecular mechanisms that underlie both disorders. Following informed consent, the family, K18853, was enrolled in the University of Utah Clinical Genetics Research Program for study.
We initially evaluated genomic DNA samples from three family members affected with schwannomatosis (II.1, III.1 and III.4) for a germline point mutation in SMARCB1. Polymerase chain reaction (PCR) products amplified from each exon (primer sequences available upon request) were bidirectionally sequenced; however, no variants were detected in the coding sequence or splice sites of the gene. Two heterozygous single nucleotide polymorphisms (SNPs) in intron 5 (rs2070456 and rs2070457), detected in III.1 and III.4, allowed us to exclude a complete deletion of the gene. Next, a panel of eight microsatellite markers that spanned the region from 22q11.1 to 22q13.1 was used to confirm that III.1 and III.4 likely shared a maternal haplotype in the region encompassing SMARCB1, consistent with the presence of an undetected mutation in the gene. We subsequently submitted DNA from II.1 to NimbleGen for high resolution array comparative genomic hybridisation (array CGH) using a chromosome 22 specific array with 65 bp median probe spacing. The results, interpreted using NimbleGen SignalMap software and the University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu), revealed that a segment within SMARCB1 was increased in copy number (fig 3A). Independent sets of PCR primers designed to amplify the junction of a direct duplication in this region (primer sequences available upon request) each specifically amplified products from all three family members that did not amplify from control individuals (fig 4). Upon sequencing the products, family members proved to carry a direct duplication of 2,631 bp (chr22:g.22,488,596_22,491,226 dup2631 (NCBI Build 36.1)) that included exon 6 of SMARCB1 (fig 3B–D). Exon 6 contains 167 bp, so its direct duplication in the mRNA is predicted to result in a frameshift and protein truncation (p.Leu266fs). Short imperfect direct repeats were present at the duplication breakpoints, consistent with occurrence through slipped strand mispairing18 (fig 3B). We amplified and sequenced exon 6 independently from the centromeric and telomeric duplicated segments and confirmed that it was present and unaltered in both segments (fig 3D,E).
Based on the diagnosis of AT/RT in IV.1 and IV.2, molecular genetic analysis of the tumours had previously been performed, as reported.19 An inactivating somatic mutation of SMARCB1 was detected in each tumour (a full gene deletion resulting from monosomy 22 in IV.1, and c.578_585dup8 in IV.2). To confirm that the duplication of exon 6 was present and represented the germline mutation in these individuals, we extracted DNA from paraffin embedded tissue blocks and successfully amplified a duplication specific PCR product from each sample (fig 4). Thus, the same germline mutation in SMARCB1 is associated with both schwannomatosis and MRT predisposition in the family. IV.3 was subsequently also found to carry the duplication; although the risk of MRT is low by his age (7 years), he will be followed clinically for MRT predisposition and schwannomatosis.
Immunohistochemical staining (BAF47 antibody, BD Transduction Laboratories, BD Biosciences, San Jose, California, USA) of the two AT/RTs as well as the resected schwannomas from III.1 and III.4 confirmed that Smarcb1 was absent from the neoplastic cells of all four tumours (fig 2). Positive staining non-neoplastic cells provided good internal controls in each specimen; normal tissue from tonsil was used as an external positive control. Although the schwannomas contained immunopositive stromal cells and inflammatory cells, the mosaic staining pattern previously reported in schwannomas associated with schwannomatosis13 20 was not observed in neoplastic cells from either tumour.
We next extracted DNA from the paraffin embedded schwannomas of III.1 and III.4 to evaluate them for evidence of somatic loss of the wild-type (WT) SMARCB1 allele. A PCR product that contained one of the heterozygous SNPs (rs2070457), located within the duplicated region, was amplified from each tumour and sequenced. The peak heights of the SNP alleles were compared between sequence of products from tumour and products from leucocyte DNA of the same individual. We did not anticipate a complete loss of the WT allele in the tumour DNA due to the intermixed stromal and inflammatory cells (fig 2). In the schwannoma from III.1, we observed a 47% reduction (averaged from sequence of both strands of the PCR product) in the height of the allele on the WT chromosome with respect to the height of the allele on the mutant chromosome. (Since the SNP was located within the duplication, the peak height of the allele on the mutant chromosome was derived from two copies of the same allele.) This result is consistent with loss of the WT allele of SMARCB1 in a population of cells within the tumour. No loss of the WT allele was detected in the schwannoma from III.4 (results not shown).
There is some knowledge of the pathways through which Smarcb1 suppresses neoplasia. As a core component of the chromatin remodelling SWI/SNF complex, it is involved in regulating gene transcription. Smarcb1 suppression of MRT involves increasing the expression of p16INK4a and repressing the expression of cyclin D1.21–24 The pathways involved in schwannomatosis remain to be defined, although somatic biallelic inactivation of the NF2 gene (mutated in NF2) at 22q12.2 is a frequent finding in schwannomas from SMARCB1 mutation carriers.14–16
The mechanisms by which SMARCB1 germline mutations predispose to schwannomatosis versus MRT are unknown. In MRT, SMARCB1 acts as a classic tumour suppressor gene that is subject to biallelic inactivation in tumours.8 11 Interestingly, SMARCB1 appears to function in a similar fashion in schwannomatosis.13–16 It is, therefore, significant that MRT has not previously been reported in families with schwannomatosis, and unaffected SMARCB1 mutation carriers in the three previously reported families with MRT predisposition have not been diagnosed with schwannomatosis.2 4 5 Accordingly, MRT predisposition and schwannomatosis could result from different types of mutations in SMARCB1. Although no clear genotype–phenotype correlation has yet emerged, schwannomatosis cases may have an increased frequency of missense mutations and mutations in exon 1.13 15 Additional factors must be involved, however, because a number of reported mutations associated with schwannomatosis, not all of which lie in exon 1, are predicted to result in complete loss of gene function.14–16 It has been proposed that an early developmental window exists during which an SMARCB1 mutation carrier is predisposed to MRT, and survivors might subsequently develop schwannomatosis.4 13 Thus, mutant SMARCB1 alleles that retain partial function might have low penetrance for MRT, yet still cause schwannomatosis. The fact that schwannomatosis was not detected in unaffected mutation carriers from previously reported MRT families could reflect the incomplete penetrance and variable expressivity that is a feature of hereditary schwannomatosis.3 It is also possible that common co-inherited variants associated with nearby genes, such as NF2, could play some role in the phenotype conferred by a mutation. In fact, the MRT from IV.1 was also deleted for NF2 as a result of monosomy 22. Finally, since some sporadic cases of apparent schwannomatosis are known to result from mosaicism for NF2 mutations,17 mosaicism for SMARCB1 mutations may be responsible for other cases. In such a circumstance, the tissue distribution of a mutation will, no doubt, influence the associated tumour spectrum.
How the germline duplication described in this report results in predisposition to both MRT and schwannomatosis in the family is still unknown. The duplicated SMARCB1 exon could be variably spliced into the mRNA transcript, allowing some residual or altered gene function. Alternatively, a unique co-inherited variant in a neighbouring gene could be influencing the phenotype. We are currently examining the splicing of mutant SMARCB1 mRNA in members of the family, and we plan to study NF2 and other nearby genes as possible modifiers of the phenotype. It is also intriguing that the schwannomas studied in this report differ from those in other reported schwannomatosis families in two aspects: first, they are of the epithelioid variant; second, they are fully negative, rather than mosaic, for Smarcb1 immunostaining in neoplastic cells. We will evaluate other tumours from this family, as they are resected, to determine if these individuals have a unique subtype of schwannomatosis or if the unusual characteristics are confined to a subset of tumours.
This is the first report of a family with inherited predisposition to both MRT and schwannomatosis. Analysis of the germline duplication in SMARCB1 that is responsible for this unique combination of conditions may allow for a greater understanding of the molecular bases of the disorders associated with this gene.
We thank the family for their participation in this study. We thank C Miller and A Openshaw for critical reading of the manuscript, J McCowen-Rose for assistance with figure preparation, S Tripp for assistance with IHC, B Clifford for assistance with sample preparation, and L Tooke for technical assistance.
Funding: This work was supported in part by a grant from the NIH (CA46274) to JAB.
Competing interests: None.
Ethics approval: The University of Utah Clinical Genetics Research Program was approved by the University of Utah Institutional Review Board
Patient consent: Obtained.
CMC’s present affiliation: Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.