Rates of Loss of Heterozygosity and Mitotic Recombination in NF2 Schwannomas, Sporadic Vestibular Schwannomas and Schwannomatosis Schwannomas

Oncogene (2010) 29, 6216–6221 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Rates of loss of heterozygosity and mitotic recombination in NF2 schwannomas, sporadic vestibular schwannomas and schwannomatosis schwannomas

KD Hadﬁeld1, MJ Smith1, JE Urquhart1, AJ Wallace1, NL Bowers1, AT King2, SA Rutherford2, D Trump1, WG Newman1 and DG Evans1

1Department of Genetic Medicine, St Mary’s Hospital, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK and 2Department of Neurosurgery, Hope Hospital, Salford, Manchester, UK

Biallelic inactivation of the NF2 gene occurs in the tosis, suggesting that susceptibility to MR varies accord- majority of schwannomas. This usually involves a ing to the disease context. combination of a point mutation or multiexon deletion, Oncogene (2010) 29, 6216–6221; doi:10.1038/onc.2010.363; in conjunction with either a second point mutation or loss published online 23 August 2010 of heterozygosity (LOH). We have performed DNA sequence and dosage analysis of the NF2 gene in a panel Keywords: mitotic recombination; schwannomatosis; of 239 schwannoma tumours: 97 neurofibromatosis type 2 SMARCB1; NF2; vestibular schwannoma (NF2)-related schwannomas, 104 sporadic vestibular schwannomas (VS) and 38 schwannomatosis-related schwannomas. In total, we identified germline NF2 Introduction mutations in 86 out of 97 (89%) NF2 patients and a second mutational event in 77 out of 97 (79%). LOH Schwannomas are benign peripheral nerve sheath was by far the most common form of second hit. A tumours that occur within the context of the tumour combination of microsatellite analysis with either conven- suppressor syndromes, neurofibromatosis type 2 (NF2) tional comparative genomic hybridization (CGH) or and schwannomatosis. They also occur sporadically multiplex ligation-dependent probe amplification (MLPA) within the general population. In most schwannoma identified mitotic recombination (MR) as the cause of tumours, the NF2 gene, located on chromosome LOH in 14 out of 72 (19%) total evaluable tumours. 22q12.2, is biallelically inactivated. In NF2 patients, Among sporadic VS, at least one NF2 mutation was the first mutation is frequently a constitutional point identified by sequence analysis or MLPA in 65 out of 98 mutation or a small deletion. This is followed by a (66%) tumours. LOH occurred in 54 out of 96 (56%) second somatic mutation in the tumour, which is either a evaluable tumours, but MR only accounted for 5 out of 77 second point mutation, or, more commonly, by loss of (6%) tested. LOH was present in 28 out of 34 (82%) the wild-type allele. Mutation within the NF2 gene schwannomatosis-related schwannomas. In all eight pa- promoter or hypermethylation of key CpG dinucleo- tients who had previously tested positive for a germline tides within this region may be an additional mechanism SMARCB1 mutation, this involved loss of the whole, or of inactivation (Kino et al., 2001). This region is not part of the long arm, of chromosome 22. In contrast, 5 out routinely tested and may account for a proportion of of 22 (23%) tumours from patients with no germline patients with unfound mutations. In schwannomas SMARCB1 mutation exhibited MR. High-resolution occurring outside NF2 disease, NF2 mutations are only Affymetrix SNP6 genotyping and copy number (CN) found somatically, occurring in the tumour, but not in analysis (Affymetrix, Santa Clara, CA, USA) were used the germline (MacCollin et al., 2003), suggesting that to determine the chromosomal breakpoint locations in other genes are involved in these cases. tumours with MR. A range of unique recombination sites, The chromatin remodelling gene, SMARCB1, located spanning approximately 11.4 Mb, were identified. This approximately 6 Mb upstream of NF2 at 22q11.23, study shows that MR is a mechanism of LOH in NF2 and was recently found to be mutated in germline DNA SMARCB1-negative schwannomatosis-related schwanno- in approximately 50% of familial schwannomatosis mas, occurring less frequently in sporadic VS. We found patients, and, less commonly, in sporadic schwannoma- no evidence of MR in SMARCB1-positive schwannomatosis (5–10%) (Hulsebos et al., 2007; Boyd et al., 2008; Hadfield et al., 2008; Sestini et al., 2008). Our previous study has shown that these germline SMARCB1 Correspondence: Professor DG Evans, Department of Genetic Medi- mutations are frequently associated with a somatic cine, Manchester Academic Health Sciences Centre, St Mary’s Hospital, NF2 mutation in tumours (Boyd et al., 2008; Hadfield University of Manchester, Manchester, Lancashire M13 9WL, UK. E-mail: [email protected] et al., 2008), and a multihit hypothesis has been Received 23 February 2010; revised and accepted 8 July 2010; published suggested, involving both of these loci (Boyd et al., online 23 August 2010 2008; Hadfield et al., 2008; Sestini et al., 2008). Mitotic recombination in schwannomas KD Hadfield et al 6217 The region 22q11 contains several low copy repeat deletions had LOH as the mechanism of second hit domains, and is known to be a particularly unstable (including 3 with 46 exons deleted), although this did region of the genome (Dunham et al., 1999; Edelmann not occur in the two patients with whole-gene deletions. et al., 1999). Recombination events in this region are MR was not found to be the cause of LOH in any of known to be involved in several diseases, occurring these tumours. both constitutionally (for example, cat eye syndrome, Of the 41 NF2 patients tested for MR, 27 were velocardiofacial syndrome, DiGeorge syndrome) and mosaic, 13 were non-mosaic and 1 was uncertain. MR somatically (for example, chronic myelogenous leukae- was the cause of LOH in seven of the non-mosaic mia Burkitt’s lymphoma, Ewing’s sarcoma) (Kaplan patients (age range: 7–40 years; average: 23 years). These et al., 1987). seven patients developed a total of 191 spinal and Mitotic recombination (MR) is known to be an peripheral nerve schwannomas, with an average of 27 important mutational mechanism in neurofibromatosis tumours per person. Sixteen non-mosaic NF2 patients type 1 and other tumour syndromes. Loss of hetero- with no evidence of MR, including those with no LOH zygosity (LOH) of 17q is a common somatic hit in (age range: 9–57 years; average: 32 years), had a total of neurofibromas lacking a germline microdeletion muta- 113 schwannomas, with an average of seven tumours per tion, and is frequently the result of MR (Serra et al., person. This may suggest that the MR mechanism 2001; De Raedt et al., 2006). As LOH is also a common functions as a modifier for NF2, leading to a more event in tumours of patients with NF2, as well as with severe schwannoma phenotype, although the difference sporadic vestibular schwannomas (VS) and schwanno- in tumour burden did not reach statistical significance matosis, and chromosome 22 is prone to a variety of by the two-tailed t-test (P ¼ 0.129). Furthermore, 5 out recombinational events, we analysed a panel of tumours of 7 MR patients had an underlying truncating from each of these groups to determine rates of LOH (nonsense/frameshift) mutation, compared with only 5 and MR with respect to the diagnosis and to the out of 16 non-mosaic patients. Out of the 27 mosaic initial genetic event. The Genome-Wide Human SNP patients, seven (six out of seven not identifiable in Array 6.0 (Affymetrix) platform was used to confirm the blood) showed MR and, strikingly, all seven developed results and refine identified breakpoints, as the combi- spinal and/or peripheral nerve schwannomas (three out nation of high density of small-nucleotide polymorph- of seven aged o20 years); one as young as 10 years of ism (SNP) genotyping and high density copy number age, despite the mutation being undetectable in the (CN) probes provides a more accurate and sensitive way lymphocyte DNA. Of 21 mosaic patients without to determine both CN and homozygosity than do evidence of MR (16 out of 21 not identifiable in blood), conventional methods. only three developed a spinal or peripheral nerve schwannoma o20 years and five had no evidence of either spinal or cutaneous schwannomas. There was no evidence that these effects were primarily due to the Results underlying mutation: 6 out of 7 mosaic MR patients and 16 out of 21 mosaic non-MR patients had the more Mutation spectrum of NF2, sporadic VS and severe truncating mutations. schwannomatosis tumours Among sporadic VS, we identified at least one point We have performed DNA sequence and dosage analysis mutation in the NF2 gene in 65 out of 98 (66%) of the NF2 gene in a panel of 239 schwannoma tumours: tumours, with a higher incidence of frameshift (34%) 97 tumours from 97 patients with NF2, 104 sporadic VS than nonsense mutations (17%) in comparison with and 38 tumours from 34 patients with schwannomatosis NF2-related tumours. Splice mutations (10%) and (Table 1). We identified germline mutations in NF2 in missense mutations (1%) occurred in similar propor- 89% (86 out of 97) of NF2 patients. Out of these, 70 tions to NF2 tumours. MLPA identified a single exon- were found by direct sequence analysis and consisted 11 deletion. LOH occurred in 54 out of 96 (56%), but predominantly of nonsense mutations (36%) and frame- MR only accounted for 5 out of 35 evaluable tumours, shift mutations (21%). The remainder consisted of splice with an overall rate of 5 out of 7 (6%). The difference mutations (13%), a single missense mutation and an between MR rates in evaluable tumours between in-frame deletion of a single amino acid. In all, 16 NF2 and sporadic VS is significant by w2-test additional tumours harboured single or multiexon (P-value ¼ 0.0161) if we extrapolate our results to deletions found by multiplex ligation-dependent probe calculate the predicted overall number of tumours in amplification (MLPA). A second mutational event was each group by first extrapolating the predicted propor- found in 77 (79%). This was a second point mutation in tion of LOH hits in the total number of tumours and 13, MLPA deletion of a part of NF2 in 2 and LOH in 62. then extrapolating the proportion of LOH hits due to MR was the cause of LOH in 14 out of 41 evaluable MR. This suggests that 22 out of 97 NF2 tumours and 8 tumours. Excluding tumours with LOH, but not tested out of 104 sporadic VS have been caused by MR in our for MR, this leads to an overall rate of 14 out of 72 cohort. This proportion is also significant by w2-test, (19%). Analysis was not possible in some tumours, adjusting for the lower rate of LOH, in addition to MR because of insufficient DNA or because of poor quality in sporadic VS (P ¼ 0.010). of some DNA samples extracted from paraffin blocks. As expected, no germline NF2 mutations were found In total, 7 of 15 NF2 patients with large multiexon in any schwannomatosis patients. However, germline

Oncogene Mitotic recombination in schwannomas KD Hadﬁeld et al 6218 Table 1 Mutational analysis results for the NF2 gene in NF2-related schwannomas, sporadic vestibular schwannomas and schwannomatosis- related schwannomas NF2 Sporadic VS SMARCB1 mutation SMARCB1 mutation þ ve schwannomatosis –ve schwannomatosis

Total tumours analyzed 97 104 8 30 Tumours evaluated for NF2 by sequence analysis 97 98 6 30 Tumours with at least one mutation identiﬁed by sequence analysis 86 65 6 18 Tumours with two mutations identiﬁed by sequence analysis 13 10 0 1 Tumours with intragenic deletions 14a 10 0 Tumours evaluated for LOH 93 96 8 26 Tumours with LOH 62 54 8 20 % of tumours with LOH 62/93 (67%) 54/96 (56%) 8/8 (100%) 20/26 (77%) Tumours with LOH evaluated for MR 41 35 8 16 Tumours with MR 14 5 0 5 MR as % of total evaluated tumours 14/72 (19%) 5/77 (6%) 0/8 (0%) 5/22 (23%) Extrapolated MR as a proportion of all tumoursb 22/97 8/104 NA 7/30

Abbreviations: LOH, loss of heterozygosity; NA, not applicable; NF2, neuroﬁbromatosis type 2; MR, mitotic recombination; VS, vestibular schwannomas; %, percentage. aTwelve of these deletions were constitutional and two were somatic. bLOH was ﬁrst extrapolated as a proportion of the total number of tumours. MR was then extrapolated as a total of the extrapolated LOH and, hence, of the total number of tumours.

Table 2 Chromosomal positions of recombination breakpoints on chromosome 22q, relevant to the pathogenesis of determined by Affymetrix SNP6 array analysis schwannomas. We used Affymetrix SNP6.0 arrays to Patient Diagnosis Genomic position SNP ID at identify recombination breakpoints in a subset of of breakpoint (Chr 22) breakpoint location tumour samples that were previously found to have undergone MR by a combination of dosage and 1 NF2 26 560 066 rs16985600 heterozygosity analysis, consisting of either conven- 2 NF2 23 061 070 rs5760352 3 NF2 19 448 306 rs4822497 tional CGH and microsatellite analysis or MLPA and 4 NF2 28 018 632 rs3761426 microsatellite analysis. Genomic DNA was available 5i NF2 16 615 305 rs181408 for analysis from seven NF2-related schwannomas, one 5ii NF2 16 965 064 rs467814 sporadic VS and two SMARCB1 mutation-negative 6 Sporadic VS 16 754 150 rs2075453 7 Schw 23 283 227 rs2298386 schwannomatosis-related schwannomas, along with 8 Schw 18 335 375 rs5993889 their respective matched blood DNAs. The data confirmed that MR was unique to chromo- Abbreviations: Chr, chromosome; NF2, neurofibromatosis type 2; some 22 in each sample, as all other chromosomes Schw, schwannoma; SNP, single nucleotide polymorphism; VS, showed normal CN and comparable heterozygosity in vestibular schwannomas. blood and tumour. We determined the breakpoint locations on chromosome 22q in six tumours obtained from five patients with NF2, one sporadic VS and two SMARCB1 mutations were found in eight patients (six SMARCB1 mutation-negative schwannomatosis-related from three different families and two sporadic patients) schwannomas (Table 2). Examples of the SNP array and somatic NF2 point mutations were found in 24 results are shown in Figure 1. tumours (Hadfield et al., 2008). The proportions of NF2 NF2 tumours showed a range of unique breakpoints frameshift mutations (37%) and splice mutations (11%) from 16 615 305 (SNP ID: rs181408) to 28 018 632 (SNP were similar to those found in sporadic VS, but with ID: rs3761426) on chromosome 22 (Figure 2a). Two fewer nonsense mutations (5%) and a greater number of tumours from the same patient (5i and 5ii) showed missense (8%) mutations. relatively close, but non-identical breakpoints, approxi- LOH was present in 28 out of 38 (74%) schwanno- mately 350 000 bases apart. For one further NF2-related matosis-related schwannomas. In all eight tumours from sample, the combined genotyping and CN data analysis schwannomatosis patients who tested positive for a determined that the LOH previously identified as MR germline SMARCB1 mutation, this involved loss of the by a combination of microsatellite analysis and conven- whole chromosome 22 or part of the long arm. In tional comparative genomic hybridization (CGH) was in contrast, 5 out of 22 (23%) evaluable tumours from fact due to loss of genetic material. 19 patients who had tested negative for SMARCB1 A breakpoint was determined in a single sporadic VS mutations exhibited MR. after 16 754 150 (SNP ID: rs2075453). Recombination breakpoints were determined for both SMARCB1 mutation-negative sporadic schwannomato- Determination of mitotic recombination breakpoints sis tumours tested. Interestingly, the breakpoint in one To investigate the potential mechanisms leading to MR, of these tumours occurred at 23 283 227 (SNP ID: we sought to define a critical region of recombination rs2298386), which is downstream of the SMARCB1

Oncogene Mitotic recombination in schwannomas KD Hadﬁeld et al 6219

Blood 8 identify by conventional methods. In the nine remaining tumours, MR was conﬁrmed as the cause of LOH. Tumour 8 The low copy repeat (LCR22) domains within the 22q11.2 region are distributed across an approximately

Blood 8 6.3 Mb region. The constitutional breakpoints responsible for deletions in the velocardiofacial syndrome only tend to occur within three of these (Edelmann et al., Patient 8 Tumour 8 1999), whereas the somatic translocation events that occur in chronic myelogenous leukaemia are found in Blood 1 the breakpoint cluster region (BCR) gene, in a separate low copy repeat domain. In our study, seven of the nine Tumour 1 approximate breakpoints that we were able to determine were distributed across region 22q11.2. However, each

Blood 1 breakpoint was unique and found outside of the LCR22 sequences (Figure 2b). The distribution of breakpoints

Patient 1 showed no particular clustering and in each case the Tumour 1 recombinant region extended from the breakpoint to the chromosome terminus, suggesting an alternative trigger for this type of recombination. The two remaining recombination breakpoints occurred outside Figure 1 Examples of genotyping and copy number (CN) analysis of the q11.2 region, in q12.1 (patient 1) and q12.2 in tumours displaying mitotic recombination. Results from paired (patient 4). Both of these were from NF2 patients and blood and tumour DNA from each patient were compared side by side to identify regions of homozygosity and changes in CN. Allele occurred upstream of the NF2 gene. difference plots show homozygous single-nucleotide polymorphism Four schwannomas from our panel (from patients 1, (SNP) calls in tracks þ 1 and À1 and heterozygous SNP calls at 0. 2, 4 and 7) harboured a recombination breakpoint Loss of heterozygous SNP calls,without loss of the entire between the SMARCB1 and NF2 loci (Figure 2a). One chromosome and in conjunction with a CN of 2, indicates a mitotic recombination event. of these was a SMARCB1 mutation-negative schwannomatosis-related tumour. The breakpoint for this tumour causes disruption of the NF2 gene by effecting locus. Together, the results show a wide range of homozygosity of a somatic mutation identified in recombination breakpoints, spanning approximately the tumour, but may also have a positional effect on 11.4 Mb with no shared recombination sites (Figure 2a). the SMARCB1 gene, despite occurring downstream of this locus. Interestingly, none of the LOH in SMARCB1 mutation-positive schwannomas was due to MR. It is Discussion possible that the initiating genetic event may influence the type of second hit that occurs in the tumour, as has We have found that mitotic recombination events are been suggested for the difference in occurrence of LOH responsible for the LOH found in a proportion of in neurofibromatosis type 1 neurofibromas with and schwannoma tumours. These events are particularly without germline microdeletions (De Raedt et al., 2006). important for SMARCB1 mutation-negative schwanno- In our cohort of NF2 patients, the most common matosis and NF2-related tumours, in which 23 and 19% sequence of mutation events was a constitutional point of evaluable tumours tested positive for MR, respec- mutation, followed by a somatic LOH event, whereas tively. A lower proportion of patients with sporadic VS the two patients with whole-gene deletions as a first hit also seem to be susceptible to MR (6%). However, had a point mutation as the second hit. although all schwannomatosis patients who tested In SMARCB1-positive schwannomatosis, the initiat- positive for a germline SMARCB1 mutation also ing event is a germline SMARCB1 mutation with the harboured LOH in their tumour, none of this loss was due second proposed event being an LOH deletion of 22q to an MR event, suggesting that tumours from this group of that removes the wild-type SMARCB1 and an allelic patients develop through an alternative mechanism. copy of NF2 (Hulsebos et al., 2007; Boyd et al., 2008; We used high-density SNP analysis to refine the Hadfield et al., 2008; Sestini et al., 2008). The third hit breakpoints and identify any common regions of would be an NF2 mutation on the remaining ipsilateral recombination in 10 tumours. This method provides allele to the germline SMARCB1 mutation. MR would much more information than the combination of not have a role in this three-hit hypothesis, as microsatellite analysis and conventional CGH or MLPA reduplication of the mutant allele would only be possible used previously. In this study, one NF2 tumour, if the ipsilateral NF2 mutation occurred second. It is originally identified with somatic LOH caused by MR, possible that loss of SMARCB1 and a dosage loss of was shown to be due to loss of one chromosome 22. One half of NF2 would cause schwann cell proliferation, thus reason for a false-positive result in the original analysis driving the third mutation. is the mixed population of normal and tumour cell DNA Aberrant methylation of three CpG islands in the in some tumour samples, which is more difficult to NF2 gene promoter has been shown to downregulate its

Oncogene Mitotic recombination in schwannomas KD Hadﬁeld et al 6220

Tumour recombination 68324 7 1 breakpoints: 5i 5ii

SMARCB1 NF2 Chr22 p13p11.2 q11.21 q11.23 q12.1 q12.2q12.3 q13.1q13.2 q13.31 0 10,000,00020,000,000 30,000,000 40,000,000

Recombination 6 Breakpoints: 5i 5ii 8 3 2 7 patients 1 + 4 2 3a 3b 4 5 678 LCR22s

17,000,00018,000,000 19,000,000 20,000,000 21,000,000 22,000,000 23,000,000 Figure 2 Schematic representation of the chromosomal locations of recombination breakpoints found in tumours, relative to SMARCB1 and NF2 genes (a), and a higher resolution schematic of the 22q11.2 region, showing breakpoint locations relative to low copy repeat (LCR) domains (b).

expression (Kino et al., 2001) and has been suggested as disease (Evans et al., 1992). Another 104 patients had suffered a mechanism for progression to growth of VS (Gonza- only a unilateral VS and had tested negative for germline NF2 lez-Gomez et al., 2003). It is possible that increased rates mutation. of aberrant methylation in sporadic VS may account for There were 38 tumours from 34 schwannomatosis patients; the reduced detection rate of NF2 point mutations in eight patients were from families with multiple affected sporadic VS in comparison with NF2 tumours. As a members who were negative for VS and germline NF2 mutations. The remaining 26 were sporadic patients with at reduction in the levels of methylation has also been least three schwannomas in more than one body segment and proposed as a mechanism of increased occurrence of who were negative for VS and germline NF2 mutations. genomic recombination events (Carbone et al., 2009), it Approval for the study was provided by the Central is also possible that an increased incidence of methyla- Manchester local ethics committee. Informed consent was tion in certain tumours would reduce the likelihood of obtained from patients and their families for publication of MR involvement. Further work is necessary to test this their details in this report. hypothesis. A number of patients have shown MR in more than Mutational analysis one tumour and there seems to be a trend towards an DNA sequence analysis of NF2 and SMARCB1 genes, CN increased schwannoma formation in those patients who analysis of SMARCB1 exon deletions or amplifications by develop schwannomas by this mechanism. We were quantitative RT–PCR (TaqMan) assay and LOH analysis unable to demonstrate a formal statistical significance by microsatellite markers D22S303, D22S310, D22S446, for this, but it remains possible that some individuals D22S449, D22S1174, D22S275, NF2CA3 and D22S268 were with non-mosaic NF2 or SMARCB1-negative schwan- carried out as described by Hadfield et al, 2008. The microsatellite markers spanned a total of approximately nomatosis and an increased susceptibility to MR have a 12.7 Mb of chromosome 22, covering both the NF2 and higher risk of increased tumour burden. SMARCB1 loci. Analysis of the centromeric microsatellite In summary, our study demonstrates a role for marker D22S420, which is found 6.3 Mb upstream of mitotic recombination events in the development of a SMARCB1, was used to confirm that any observed LOH proportion of schwannoma tumours and shows that the was not due to deletion, followed by reduplication of the rates of MR vary, depending on the disease context. The homologous chromosome 22. lack of SMARCB1 mutations in schwannomatosis- Hybridization, ligation and amplification of MLPA probes related schwannomas with MR suggests that there are for exon CN analysis were carried out according to the additional genetic causes of schwannomatosis, one of manufacturer’s instructions (MRC-Holland, Amsterdam, which may involve an increased susceptibility to mitotic The Netherlands). The method for CGH was previously described in (James and Varley, 1996; James et al., 1999; recombination. Further studies will be required to Warren et al., 2003). determine the specific combination of genetic events that initiates tumour growth High-resolution genotyping and CN analysis Matched blood and tumour samples for each patient were genotyped using the Genome-Wide Human SNP Array 6.0 Materials and methods (Affymetrix). Genomic DNA was purified by ethanol pre- cipitation and 500 ng was hybridized to an SNP6 chip Patient tumours according to the manufacturer’s instructions. This array Tumour DNA from a total of 239 patients was included in the contains a total of B1.8 million probes, comprised of study. In total, 97 patients fulfilled the clinical criteria for NF2 906 600 SNP probes and 946 000 CN probes spaced throughout

Oncogene Mitotic recombination in schwannomas KD Hadfield et al 6221 the genome. Staining of the array was carried out on a Fluidics lost in comparison with blood. For these samples, an Station 450 (Affymetrix) and the array was scanned using the approximate recombination breakpoint was determined from GeneChip scanner 3000 7G system (Affymetrix). the allele difference plots. Chromosomal numbering is taken from the NCBI Build Data analysis 36 ¼ hg18. The CN data were generated using the BRLMM-P-Plus algorithm from Affymetrix using reference model files generated from a pool of 56 unaffected genomic DNA control Conflict of interest samples. Genotype and CN analysis were carried out using the Genotyping Console 3.0.2 (Affymetrix). The genotyping no- The authors declare no conflict of interest. call threshold was set to 0.01 and the CN/LOH detection threshold was set to 0.02. Mitotic recombination was demon- strated by tumour-specific, incomplete loss of heterozygosity, Acknowledgements with no change in CN throughout the homozygous region. In tumour samples containing a heterogeneous population of This work was supported by a grant from the Children’s normal and tumour cell DNA, regions of MR appeared as Tumor Foundation. The Department of Genetic Medicine is dual tracks of SNP calls on allele difference plots and were not supported by the NIHR Manchester Biomedical Research called as LOH by the Genotyping Console. However, the copy Centre. We also thank all the patients who provided samples number remained at 2, whereas heterozygous base calls were for the study.

References

Boyd C, Smith MJ, Kluwe L, Balogh A, Maccollin M, Plotkin SR. SMARCB1 in familial schwannomatosis. Am J Hum Genet 80: (2008). Alterations in the SMARCB1 (INI1) tumor suppressor gene 805–810. in familial schwannomatosis. Clin Genet 74: 358–366. James L, Varley J. (1996). Preparation, labelling and detection of Carbone L, Harris RA, Vessere GM, Mootnick AR, Humphray S, DNA from archival tissue sections suitable for comparative genomic Rogers J et al. (2009). Evolutionary breakpoints in the gibbon hybridization. Chromosome Res 4: 163–164. suggest association between cytosine methylation and karyotype James LA, Kelsey AM, Birch JM, Varley JM. (1999). Highly evolution. PLoS Genet 5: e1000538. consistent genetic alterations in childhood adrenocortical tumours De Raedt T, Maertens O, Chmara M, Brems H, Heyns I, Sciot R et al. detected by comparative genomic hybridization. Br J Cancer 81: (2006). Somatic loss of wild type NF1 allele in neurofibromas: 300–304. Comparison of NF1 microdeletion and non-microdeletion patients. Kaplan JC, Aurias A, Julier C, Prieur M, Szajnert MF. (1987). Human Genes Chromosomes Cancer 45: 893–904. chromosome 22. J Med Genet 24: 65–78. Dunham I, Shimizu N, Roe BA, Chissoe S, Hunt AR, Collins JE et al. Kino T, Takeshima H, Nakao M, Nishi T, Yamamoto K, Kimura T (1999). The DNA sequence of human chromosome 22. Nature 402: et al. (2001). Identification of the cis-acting region in the NF2 gene 489–495. promoter as a potential target for mutation and methylation- Edelmann L, Pandita RK, Morrow BE. (1999). Low-copy repeats dependent silencing in schwannoma. Genes Cells 6: 441–454. mediate the common 3-Mb deletion in patients with velo-cardio- MacCollin M, Willett C, Heinrich B, Jacoby LB, Acierno JS. (2003). facial syndrome. Am J Hum Genet 64: 1076–1086. Familial schwannomatosis: exclusion of the NF2 locus as the Evans DG, Huson SM, Donnai D, Neary W, Blair V, Newton V et al. germline event. Neurology 60: 1968–1974. (1992). A clinical study of type 2 neurofibromatosis. QJMed84: Serra E, Rosenbaum T, Nadal M, Winner U, Ars E, Estivill X et al. 603–618. (2001). Mitotic recombination effects homozygosity for NF1 Gonzalez-Gomez P, Bello MJ, Alonso ME, Lomas J, Arjona D, germline mutations in neurofibromas. Nat Genet 28: 294–296. Campos JM et al. (2003). CpG island methylation in sporadic and Sestini R, Bacci C, Provenzano A, Genuardi M, Papi L. (2008). neurofibromatis type 2-associated schwannomas. Clin Cancer Res 9: Evidence of a four-hit mechanism involving SMARCB1 and NF2 5601–5606. in schwannomatosis-associated schwannomas. Hum Mutat 29: Hadfield KD, Newman WG, Bowers NL, Wallace A, Bolger C, Colley A 227–231. et al. (2008). Molecular characterisation of SMARCB1 and NF2 in Warren C, James LA, Ramsden RT, Wallace A, Baser ME, Varley JM familial and sporadic schwannomatosis. JMedGenet45: 332–339. et al. (2003). Identification of recurrent regions of chromosome loss Hulsebos TJ, Plomp AS, Wolterman RA, Robanus-Maandag EC, and gain in vestibular schwannomas using comparative genomic Baas F, Wesseling P. (2007). Germline mutation of INI1/ hybridisation. J Med Genet 40: 802–806.

Oncogene