First insight into the somatic mutation burden of neurofibromatosis type 2-associated grade I and grade II meningiomas: a case report comprehensive genomic study of two cranial meningiomas with vastly different clinical presentation
Dewan et al. BMC Cancer
First insight into the somatic mutation burden of neurofibromatosis type 2- associated grade I and grade II meningiomas: a case report comprehensive genomic study of two cranial meningiomas with vastly different clinical presentation
Ramita Dewan 4
Alexander Pemov 3
Amalia S. Dutra 2
Evgenia D. Pak 2
Nancy A. Edwards 4
Abhik Ray-Chaudhury 4
Nancy F. Hansen 7
Settara C. Chandrasekharappa 7
James C. Mullikin 6 7
Ashok R. Asthagiri 5
NISC Comparative Sequencing Program
John D. Heiss 4
Douglas R. Stewart 3
Anand V. Germanwala 0 1
0 Department of Neurological Surgery, Loyola University Stritch School of Medicine , Maywood, IL , USA
1 Department of Otolaryngology, Edward Hines, Jr. VA Hospital , 2160 South First Avenue, Maywood, IL 60153 , USA
2 Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health , Bethesda, MD , USA
3 Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute , Rockville, MD , USA
4 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health , Bethesda, MD , USA
5 Department of Neurological Surgery, University of Virginia School of Medicine , Charlottesville, VA , USA
6 NIH Intramural Sequencing Center, National Human Genome Research Institute, National Institutes of Health , Rockville, MD , USA
7 Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health , Bethesda, MD , USA
Background: Neurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition disorder caused by constitutive inactivation of one of the two copies of NF2. Meningiomas affect about one half of NF2 patients, and are associated with a higher disease burden. Currently, the somatic mutation landscape in NF2associated meningiomas remains largely unexamined. Case presentation: Here, we present an in-depth genomic study of benign and atypical meningiomas, both from a single NF2 patient. While the grade I tumor was asymptomatic, the grade II tumor exhibited an unusually high growth rate: expanding to 335 times its initial volume within one year. The genomes of both tumors were examined by whole-exome sequencing (WES) complemented with spectral karyotyping (SKY) and SNP-array copynumber analyses. To better understand the clonal composition of the atypical meningioma, the tumor was divided in four sections and each section was investigated independently. Both tumors had second copy inactivation of NF2, confirming the central role of the gene in meningioma formation. The genome of the benign tumor closely resembled that of a normal diploid cell and had only one other deleterious mutation (EPHB3). In contrast, the chromosomal architecture of the grade II tumor was highly re-arranged, yet uniform among all analyzed fragments, implying that this large and fast growing tumor was composed of relatively few clones. Besides multiple gains and losses, the grade II meningioma harbored numerous chromosomal translocations. WES analysis of the atypical tumor identified deleterious mutations in two genes: ADAMTSL3 and CAPN5 in all fragments, indicating that the mutations were present in the cell undergoing fast clonal expansion (Continued on next page) © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Conclusions: This is the first WES study of NF2-associated meningiomas. Besides second NF2 copy inactivation, we
found low somatic burden in both tumors and high level of genomic instability in the atypical meningioma.
Genomic instability resulting in altered gene dosage and compromised structural integrity of multiple genes may
be the primary reason of the high growth rate for the grade II tumor. Further study of ADAMTSL3 and CAPN5 may
lead to elucidation of their molecular implications in meningioma pathogenesis.
Neurofibromatosis type 2 (NF2) is an autosomal
dominant tumor syndrome characterized by the growth of
multiple neoplasms within the central nervous system.
Although bilateral vestibular schwannomas are the
hallmark of NF2, meningiomas are the second most
frequent intracranial tumor, and occur in about 52% of
NF2 patients [1, 2]. Benign meningiomas (WHO grade I)
feature a 5-year tumor recurrence rate of 5% as compared
to 50–80% for anaplastic meningiomas (grade III),
highlighting the importance of elucidating the molecular
mechanisms which contribute to tumor progression .
The most common genetic mutation in meningiomas
is NF2 inactivation, which is observed not only in
NF2associated tumors, but also in 47 to 72% of sporadic
meningiomas, and is thus considered an integral step for
meningioma tumor initiation [4–6]. Recent studies
utilizing high throughput whole-exome and whole-genome
sequencing have identified two distinct subtypes of
sporadic meningiomas: tumors with or without an
inactivated NF2 gene [7, 8]. Sporadic meningiomas with
disrupted NF2 tend to display greater genomic instability
(including several cases of chromothripsis) and higher
grades than non-NF2 meningiomas. Non-NF2 tumors
have been shown to contain recurrent oncogenic
mutations in AKT1, KLF4, TRAF7 and SMO, indicating the
alternate involvement of the PI3K-AKT and Hedgehog
NF2-associated meningiomas are rarer than their
sporadic counterparts and far fewer studies have investigated
the genetics underlying their initiation and progression.
Two case series evaluated meningiomas from NF2
patients only for the allelic imbalances most commonly
observed in sporadic meningiomas, and confirmed
frequent somatic inactivation of the NF2 gene, as well as
losses of chromosome arms 1p, 6q, 9p, 10q, 14q and 18q
[9, 10]. A more recent study used single nucleotide
polymorphism array analysis to report increased
chromosomal instability with increasing grade in NF2-associated
Here, we present an in-depth genomic study of grade I
and grade II meningiomas that resided in close proximity
in the brain of an NF2 patient. The tumors contained the
same NF2 germline mutation and similar somatic hits
affecting the normal remaining copy of the gene, yet differed
drastically in genomic architecture and growth rate. The
tumors were investigated using whole-exome sequencing
complemented with SKY and SNP-array copy-number
Materials and methods
A 35-year-old woman was enrolled in the Institutional
Review Board (IRB)-approved NF2 natural history study
(NIH#08-N-0044) at the National Institute of Neurologic
Disease and Stroke (NINDS). Prior MR imaging
confirmed the NF2 Manchester diagnostic criteria of
bilateral vestibular schwannoma in addition to numerous
other significant findings: intracranial schwannomas
involving cranial nerves V, VII, and VIII, intracranial
meningiomas, cervical ependymomas, schwannomas along
the cauda equina, and cervicothoracic meningiomas.
In preparation for surgery, the patient underwent
frameless stereotactic navigation imaging on a 1.5 Tesla
MRI scanner with and without gadolinium. 1-mm axial
images were obtained with sagittal and coronal
reconstruction. Image guidance registration was performed
intraoperatively using facial registration.
A single image-guided right frontal craniotomy was used
to resect an anterior grade II meningioma, in four
discrete sections, and a posterior grade I meningioma.
The two meningiomas were separated by an intervening
section of normal brain, and were resected through a
single image-guided right frontal craniotomy. The
anterior grade II meningioma was noted to be soft and was
removed in four anatomically discrete sections with
alternating steps of circumferential dissection and suction.
The posterior grade I meningioma was noted to be firm
and was removed en bloc.
Tumor specimens were fixed in 10% buffered formalin
immediately after removal, processed overnight, and
subsequently embedded in paraffin. Five μm-thick
sections were obtained from the paraffin blocks, and
stained using the standard hematoxylin and eosin
Frozen tumor tissue was processed with Proteinase K,
and DNA extraction was completed using the
phenol:chloroform procedure. Frozen tumor tissue was minced
with a scalpel, washed once in PBS, pH 7.4, and
incubated in a solution containing 100 mM TrisHCl, pH 8.0,
5 mM EDTA, 0.5% SDS and 200 μg/mL Proteinase K
(Invitrogen, Grand Island, NY) at 55 °C for 2–3 h or at
37 °C overnight. DNA was extracted by the
phenol:chloroform procedure and precipitated with ice cold
isopropanol. DNA pellets were air dried, re-suspended in
10 mM TrisHCl, pH 7.4 and 0.1 mM EDTA, aliquoted
and stored at −20 °C.
Whole-exome sequencing (WES) of tumor and normal DNA
Capture of the coding portion (exome) of genomic DNA
and library preparation for next generation sequencing
was done using Roche NimbleGen (Madison, WI)
SeqCap EZ Exome + UTR library (64 Mb of coding
exons and miRNA regions plus 32 Mb
untranslated regions (UTR)) according to the
manufacturer’s instructions. As an input, 1 μg of tumor and
matching normal genomic DNA was used. Sequencing
was completed on the Illumina HiSeq 2500 system
(Illumina, San Diego, CA, USA). Among the six exomes
sequenced, the average breadth of coverage was 89%
(range 88–90%), and the average depth of coverage was
66X (range 54X-78X).
Raw sequencing data was further processed using an
analytical pipeline that included ELAND (Illumina, Inc.)
for initial alignment to the reference human genome
(GRCh37); Novoalign, v.2.08.02  for local
realignment; bam2mpg for genotype calling and
calculation of the quality score Most Probable Genotype
(MPG)  and ANNOVAR for functional annotation of
genetic variants [14, 15]. The resulting data was
formatted in VarSifter  format for further filtering.
Filtering consisted of removing all non-coding variants
and nucleotides whose genotypes were identical in both
the tumor and corresponding germline DNA, whose
quality score (Most Probable Genotype, MPG) was less
than 10 in either tumor or normal DNA, and whose
ratio of quality score to depth of coverage was below 0.5
in germline DNA and below 0.4 in tumor DNA. All
common variants (variants with minor allele frequency
above 0.03 in ClinSeq and 1000 Genomes databases)
were also removed. The resulting set was annotated with
PolyPhen, SIFT and CADD tools to identify pathogenic
Sanger validation of mutations identified by WES
PCR primers were designed using Primer3 (v. 0.4.0)
online software . PCR amplification was conducted
using a 20 μL reaction mixture containing 20–50 ng of
genomic DNA, 1x reaction buffer, 1.5 mM MgCl2, 4
dNTPs at 250 μM each, 10 pmole each of forward and
reverse primers, and 2 units of ThermoFisher Scientific
Taq DNA polymerase (Waltham, MA). PCR products
were analyzed on Agilent 2100 BioAnalyzer (Santa Clara,
CA) and sent for Sanger sequencing to ACGT, Inc.
(Wheeling, IL). Sequencing was done on ABI 3730 DNA
Analyzer, the data was processed with GeneMapper v.3.7
software (ThermoFisher Scientific), and the phred
quality score for sequenced nucleotides was visualized
using CodonCode Aligner v.6.0.2 (CodonCode Corp.,
Centerville, MA). Sequencing reaction tracks were
visualized using FinchTV v.1.5.0 (Geospiza, Inc., Seattle,
WA) and CodonCode Aligner software.
Sanger sequencing of NF2
Sanger sequencing of NF2 was conducted by Prevention
Genetics (Marshfield, WI), a CLIA-certified DNA testing
lab. PCR was used to amplify all NF2 coding exons, as
well as ~20 bp flanking intronic or other non-coding
sequence. Sequencing was performed separately in both
the forward and reverse directions and all differences
from the reference sequence were reported.
SNP genotyping was performed using HumanOmni
ExpressExome-8v1.2 Illumina BeadChip arrays (Illumina,
San Diego, CA) per the manufacturer’s instructions. The
arrays were read using the iScan platform (Illumina),
and visualized with GenomeStudio v.2011.1 software
(Illumina). The call rate for all the DNA samples was
>99%. Genomic coordinates are per hg19.
Copy-number variation analysis
Copy-number variation (CNV) analysis of all tumor
samples was performed using Nexus Copy Number
software v.6.1 (BioDiscovery, Inc., Hawthorne, CA). “Allelic
imbalance” refers to a locus with B-allele frequency
classes other than 0, 0.5 or 1. Allele-Specific Copy
number Analysis of Tumors (ASCAT) (v2.1) analysis of the
data was performed as described by Van Loo and
Spectral karyotyping (SKY)
Metaphase slide preparations were made from cultured
meningioma primary cell cultures established from the
grade II meningioma and hybridized with commercially
available SKY probe set (Applied Spectral Imaging Inc.,
Carlsbad, CA) according to the manufacturer’s
instructions. Mitotic arrest with colcemid (0.015 μg/mL, 2–4 h)
(GIBCO, Gaithersburg, MD) was followed by hypotonic
treatment (75 mM KCl, 20 min, 37 °C) and fixation in
methanol–acetic acid mixture (3:1).
The patient, over 12 months of enrollment, noted
progressively worsening right frontal headaches. The patient
had a family history of NF2 and was deaf from bilateral
vestibular schwannomas that were successfully treated in
the past with radiosurgery. Her neurological exam was
notable only for bilateral deafness. Over the 12-month
period, the patient was noted to have significant
radiographic progression of a right anterior frontal
meningioma, increasing to 335 times its original volume, while
other brain and spine tumors remained relatively stable
(Fig. 1). Due to the tumor’s symptomatic radiographic
progression, surgical resection was offered. Consent to
remove an adjacent, stable posterior frontal tumor was
also obtained in the setting that it was accessible for
resection without posing additional risk. The patient had
an unremarkable hospital course and post-operative
imaging confirmed gross total resection of both lesions. At
six weeks follow-up, the patient noted significant
improvement in her headaches.
Histopathological analysis of the tumors
Histological analysis of the anterior tumor revealed a
grade II meningioma with increased cellularity, nuclear
pleomorphism, cells with prominent nucleoli and areas
of patternless growth. Increased proliferation index as
evidenced by immunostain for MIB-1 antigen was also
observed. Pathology of the posterior specimen revealed a
grade I meningioma consisting of monomorphic cells
having ovoid to elongated nuclei, multiple cellular
whorls, scattered psammoma bodies and rare mitotic
figures (Fig. 2).
Germline and somatic mutations in the NF2 gene
Sequencing of exons and small flanking intronic regions
of the NF2 gene from peripheral white blood cell DNA
(germline) identified a constitutive mutation in the
intronic region, two nucleotides upstream of the 5′-end of
exon 13: c.1341-2A > C. This mutation likely disrupts
the acceptor site of intron 12, thus affecting RNA
splicing, and has been previously reported as pathogenic
(Human Gene Mutation Database, CS115647) by Ellis
and co-authors . It has not been previously
annotated in the ExAC (mean coverage 15x), 1000 Genomes,
or ESP datasets .
The mutation was identified in the patient’s peripheral
white blood cell DNA by a CLIA-certified genetic testing
lab (Prevention Genetics, Marshfield, WI, data not
shown). We performed secondary confirmation using
PCR amplification followed by Sanger sequencing of the
DNA fragment containing the mutant base. Both
Prevention Genetics (PG) and our analyses of germline
DNA revealed the presence of a mutant base C in the
sequence (Fig. 3). Examination of phred quality scores
for the mutant base and several surrounding nucleotides
Fig. 1 MRI and growth rate analysis of the grade I and grade II meningiomas. a MRI images of the patient’s tumors at the start of the study, and
6 and 12 month time points. The slowly and rapidly growing tumors are indicated with white and red arrows, respectively. Numbers 1 through 5
on the bottom image show the tumor samples taken for genomic analysis: 1- grade I (slowly growing) meningioma, and 2 through 5 - regions of
the grade II (rapidly growing) meningioma. b Growth rate volumetric analysis of the tumors shown in a, with the grade II tumor displaying
Fig. 2 Histological appearance of benign and atypical meningioma. a Histological analysis of the posterior specimen revealed a grade I
meningioma with typical whorl formations (bar 100 μm) and b psammoma bodies (bar 100 μm). c The anterior tumor revealed a grade II
meningioma with increased cellularity, nuclear pleomorphism, and prominent nucleoli as indicated by H&E (bar 50 μm) and d an increased
proliferation index by MIB-1 labeling (bar 50 μm)
revealed lower values in the patient’s germline DNA
compared to the normal control (Additional file 1). This
was in agreement with visual evaluation of the
chromatogram peaks: the presence of C signal in addition to
A signal at this nucleotide position made an
unambiguous call less certain, resulting in a lower quality score
(Fig. 3 and Additional file 1).
We noticed that the mutant signal (C) was weaker
than that of the reference allele (A) (Fig. 3, see
“Germline” panel). A similar A-to-C signal ratio was
observed in both the plus and minus strand DNA
sequences in both PG and our analyses (data in Fig. 3 is
shown for the plus strand only). A-to-C substitution in
the sequence 5′-GG[A]GGGCC-3′ converts it to a
GCrich 8 nucleotide-long stretch 5′-GG[C]GGGCC-3′.
Such sequences can be more difficult to analyze due to
the secondary DNA structure, which may explain the
decreased mutant base C signal.
Copy number analysis of the tumors revealed that the
grade I meningioma contained loss of entire
chromosome 22, and all fragments of the grade II meningioma
harbored loss of chromosome 22q (Fig. 4). Thus, loss of
heterozygosity (LOH) was the likely mechanism of
somatic NF2 inactivation. PCR amplification followed by
Sanger-sequencing of the region surrounding
c.13412A > C substitution in all tumor samples revealed mostly
homozygous mutant genotype (C/C), confirming loss of
the remaining wild-type copy of the NF2 gene. One can
observe a weak reference allele A signal in the tumor
sequencing chromatograms, due to the presence of
20–30% of non-tumor stromal cells that still contain the
reference allele (Fig. 3, bottom four panels, “Grade I’,
“Grade II-1”-“Grade II-4”, green peaks). These
observations were also confirmed by lower phred scores of the
mutant nucleotide in all tumor samples compared to
normal DNA control (Additional file 1).
Copy-number SNP-array analysis
We investigated copy-number variation (CNV) in the
grade I and grade II meningiomas on Illumina
SNParrays, followed by Nexus CNV software analysis (Fig. 5).
Besides LOH of entire chromosome 22, the grade I
tumor contained only a single 22 kilobase deletion on
chromosome 8p23.2, in an intronic region of CSMD1. In
contrast, we observed multiple gains and losses, ranging
from a few kilobases to 100 megabases in eleven
different chromosomes in the grade II tumor (Table 1 and
Additional file 2). The deleted and amplified regions in
the grade II tumor harbor more than 3000 genes
(Additional file 3), 54 of which are known cancer genes
(Additional file 4).
Spectral karyotyping (SKY) of grade II meningioma cells
SNP-array analysis, while providing data on
copynumber variation in the genome, does not permit
detection of structural chromosomal rearrangements such as
inversions and translocations. To address this, we used
primary cell cultures established from the grade II
meningioma for SKY analysis. Viable cultures from the
grade II-2, grade II-3 and grade II-4 tumor fragments
Fig. 3 Sanger analysis of grade I and grade II tumors. Sequencing tracks of the genomic region surrounding the splice site mutation (Exon 13;
c.1341-2A > C) upstream of exon 13 in the NF2 gene in normal control DNA, and germline and tumor DNA of the NF2 patient. “Grade II-1”
through “Grade II-4” labels denote the four fragments of the grade II meningioma analyzed in this study. Arrows point at the mutant nucleotide,
which was heterozygous in germline and all tumor fragments. The relative font size for the alleles in each sample reflects the difference in signal
strength for A and C nucleotides at the site of the mutation
contained cells of normal ploidy, and both unaffected
cells (20–40%) and cells with multiple chromosomal
translocations (Table 2). Within each culture,
approximately half of the translocations were recurrent. In one
culture (grade II-2), we observed highly abnormal cells
with chromosomes that appeared “shattered” or broken
into multiple fragments.
Whole-exome sequencing (WES) of tumors and Sanger
verification of mutations
After WES data processing and filtering, we identified
two potentially damaging somatic mutations in the grade I
meningioma, and nine somatic mutations in the four
fragments of the grade II meningioma (Additional file 5). Of
the nine mutations in the grade II meningioma, two were
found in all four fragments.
Of the two mutations in the grade I meningioma, one
(EPHB3) mutation was verified by Sanger sequencing,
and of the nine mutations in the grade II tumor, two
(CAPN5 and ADAMTSL3) were verified by Sanger
sequencing. Importantly, the mutations in CAPN5 and
ADAMTSL3 were detected by WES in all four fragments
of grade II tumor and were verified by Sanger in all four
fragments as well, suggesting that these mutations
were likely present in the cell undergoing fast clonal
To our knowledge, this is the first whole-exome
sequencing study of NF2-associated grade I and grade II
meningiomas. Besides chromosome 22 loss, the genome of
the grade I meningioma closely resembled that of a
Fig. 4 Somatic inactivation of NF2 via chromosome 22 deletion in grade I and grade II meningiomas. SNP-array analysis of grade I (top panel)
and grade II (middle panel) meningiomas and normal (germline) DNA (bottom panel). Each panel consists of two plots: B-allele frequency (top)
and intensity (bottom). Cytoband map of chromosome 22 is shown on the bottom of the figure. The arrows show the start of the deletion region
in the grade II meningioma. Data for only one of the four fragments of grade II tumor is shown, since the data for the remaining three is essentially
normal diploid cell, while the genome of the grade II
tumor contained several chromosomal rearrangements
previously observed in meningiomas, including losses in
1p, 2p, 2q, 3p, 3q, 6q, 12p, 14q, 18q, Xp, gain in 1q
[21–25], and multiple translocations. Our observations
confirm previous findings that inactivation of NF2 is
likely to be the primary step in NF2-associated
meningioma formation . In addition, we show that
both benign and atypical tumors had a low somatic
mutation burden. Although limited to a single patient,
this data permits speculation that tumor progression
to a higher grade likely occurs through multiple
chromosomal gains, losses and translocations and to a
lesser extent from the accumulation of point
mutations and small indels.
Chromosomal translocations leading to the disruption
of tumor suppressors or activation of proto-oncogenes
are common in many neoplasms [27, 28]. Limited
evidence suggests that chromosomal translocations may
also be present in meningiomas  and systematic
studies addressing this mechanism of tumorigenesis in
meningiomas are emerging. We observed numerous
Fig. 5 Genomic distribution of CNVs in grade I and four fragments of grade II meningiomas. Chromosomal deletions and duplications in
individual samples (lower panel) and as aggregation of all samples (upper panel) are shown as red and blue bars, respectively. Regions of allelic
imbalance are shown in purple. Individual chromosomes, 1 through 22 and X, are shown as alternating light blue and white columns. The height
of the red and blue bars in the upper panel reflects the number of samples the CNV is detected in. The percent scale is shown on the left of the
chromosomal translocations (both balanced and
unbalanced) as well as one case of highly irregular, shattered
chromosomes. Interestingly, similar to the observation
made by Brastianos et al. , close examination of
SNParray plots of chromosome 1 in the tumor revealed
deletion of the 5′-half of the NEGR1 gene (not shown).
These findings suggest that structural aberrations might
be more frequent than previously believed in NF2-driven
familial and sporadic meningiomas, and could represent
one of the mechanisms of genetic instability and routes
of tumor progression to higher grades.
By analyzing the genomic architecture and somatic
mutations in multiple fragments of the grade II tumor,
we gained insight into the clonal evolution of this fast
growing neoplasm. We observed not only a remarkably
uniform pattern of chromosomal gains and losses, but
also the consistent presence of the only two potentially
pathogenic mutations, in ADAMTSL3 and CAPN5, in all
four fragments. These findings indicate that the
aberrations were likely present in the initial cell undergoing
fast clonal expansion, and that any of these aberrations/
mutations could impact tumor progression and
accelerate growth rate.
Table 1 Grade II meningioma chromosomal aberrations
identified by SNP-array analysis
Table 2 Chromosomal translocations identified by SKY analysis
in grade II meningioma fragments 2, 3 and 4
46, XX, t(8;8), t(11;12)
Percent of normal metaphases and metaphases containing chromosomal
translocations (abnormal) is shown in the top row for each meningioma
grade II primary tissue culture analyzed (Grade II-2, II-3 and II-4, note that
culture from fragment II-1 could not be established). Abnormal metaphases
were further divided into recurrent or non-recurrent. Fragment 2 of the tumor
(Grade II-2) also contained 20% of cells with highly fragmented,
Germline mutations in CAPN5 (Calpain 5), which
encodes a calcium-dependent endopeptidase, have been
associated with neovascular inflammatory
vitreoretinopathy . Though the role of the protein in neoplastic
transformation is unclear, a recent study reported
association of CAPN5 with promyelocytic leukemia nuclear
bodies, which are involved in transcriptional regulation,
cell differentiation, apoptosis, and cell senescence .
The protein encoded by ADAMTSL3 (A Disintegrin And
Metalloproteinase with TromboSpondin Like 3) is
involved with extracellular matrix function and to cell–
matrix interactions, and is frequently mutated and
under-expressed in colorectal cancer . The gene
belongs to a large family of proteins associated with
microfibrils in the extracellular matrix, thus mediating
sequestration of the TGFB superfamily of proteins and
affecting wide array of cellular functions such as
adhesion, migration, proliferation and angiogenesis [33, 34].
The majority of meningiomas are benign and
asymptomatic tumors that require little or no treatment [35, 36].
However, a subset of tumors becomes more clinically
aggressive as they evolve toward atypical and anaplastic
stages, causing increased morbidity and mortality.
Remarkably, the tumors we investigated had the same NF2
germline mutation, the same genetic background, similar
chromosome 22 LOH and were residing within a few
millimeters from one another in the patient’s brain, yet
one remained as a slowly growing asymptomatic grade I
meningioma and the other evolved into a fast growing
grade II tumor. This observation underscores the
importance of stochastic factors in meningioma progression,
which are still poorly understood.
We performed an in-depth genomic study of
NF2associated benign and atypical meningiomas. Both
tumors had inactivated second copies of NF2 and a low
burden of somatic mutations. However, unlike the
benign tumor, the atypical meningioma presented with
widespread genomic aberrations, implying that
chromosomal instability may be a key driving force in tumor
progression. In addition, we identified two candidate
driver genes, CAPN5 and ADAMTSL3, which could
contribute to the elevated growth rate of the grade II
meningioma. Future efforts should be focused on
understanding the mechanistic links between NF2 deficiency
and genomic instability.
Additional file 1: Phred scores for Sanger sequencing of the
heterozygous mutant (red font) and surrounding homozygous wt
nucleotides. Normal control sample is shown on the top (green fill).
Phred scores for both forward and reverse sequencing reactions are
shown. Note that a heterozygous nucleotide would usually affect
(decrease) phred scores of a few adjacent wt homozygous nucleotides.
(PDF 44 kb)
Additional file 2: Losses, gains and regions with allelic imbalance in the
grade I meningioma and all four fragments of the grade II meningioma.
(PDF 173 kb)
Additional file 4: Cancer genes affected by losses or gains in the grade
II meningioma. (PDF 62 kb)
Additional file 5: Mutations selected for Sanger verification. (PDF 115 kb)
ADAMTSL3: A Disintegrin And Metalloproteinase with TromboSpondin Like 3;
AKT1: V-akt murine thymoma viral oncogene homolog 1; BRD8: Bromodomain
containing 8; CAPN5: Calpain 5; CNV: Copy-number variation; EPHB3: EPH
(ephrin) receptor B3; KLF4: Kruppel-like factor 4 (gut); LOH: Loss of
heterozygosity; NF2: Neurofibromatosis type 2; PI3K:
Phosphatidylinositol-4,5bisphosphate 3-kinase; SKY: Spectral karyotyping; SMO: Smoothened, frizzled
class receptor; SNP: Single nucleotide polymorphism; TGFB: Transforming
growth factor beta; TRAF7: TNF receptor associated factor 7; WES: Whole-exome
This study was supported by the Intramural Research Programs of the
National Institute of Neurologic Disease and Stroke (NINDS), the Division of
Cancer Epidemiology and Genetics of the National Cancer Institute (NCI),
and the National Human Genome Research Institute (NHGRI).
This study was supported by funding from the Intramural Research Program
of the National Institute of Neurologic Disease and Stroke (NINDS), the
Division of Cancer Epidemiology and Genetics of the National Cancer
Institute (NCI), and the National Human Genome Research Institute (NHGRI).
The roles of each funding body were as follows: NINDS for study design,
collection of data, and writing of the manuscript; NCI for study design,
collection, analysis, and interpretation of data, and writing of the manuscript;
and NHGRI for collection, analysis, and interpretation of data.
RD carried out MRI volumetric image analysis, DNA samples preparation,
genomic data analysis, assisted with clinical sample collection and co-drafted
the manuscript (with AP); AP analyzed genomic data, participated in the study
design (genomics and molecular biology) and co-drafted the manuscript
(with RD); ASD analyzed SKY data and prepared the data for publication; EDP
carried out the SKY experiments; NAE carried out histological sample
preparation; AR-C performed pathological evaluation of tumors; NFH carried
out WES data preparation; SCC carried out SNP-array experiments; JCM
supervised WES sequencing and WES data preparation; ARA assisted with
clinical sample collection; WES was carried out at NISC CSP; JDH supervised all
clinical aspects of the study; DRS participated in the study design and critically
evaluated the manuscript; AVG provided clinical care to the study’s patient,
conceived of the study, carried out the surgery and tumor tissue collection, and
critically evaluated the manuscript. All authors have read and approved the
Consent for publication
Written consent was obtained for publication of patient-related data in
accordance with the NIH#08-N-0044 protocol for patient enrollment and
informed consent, which is approved by the National Institute of Neurologic
Disease and Stroke Institutional Review Board. A copy of the consent is
available for review.
Ethics approval and consent to participate
Ethics approval was obtained in accordance with the NIH#08-N-0044
protocol which is approved by the National Institute of Neurologic Disease
and Stroke Institutional Review Board. Written informed consent was
obtained from the patient for study of her tissue in accordance with the
NIH#08-N-0044 protocol for patient enrollment and informed consent, which
is approved by the National Institute of Neurologic Disease and Stroke
Institutional Review Board. A copy of the consent is available for review.
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