Analysis of CIC -associated CpG island methylation in oligoastrocytoma

© 2013 British Neuropathological Society

Analysis of CIC-associated CpG-island methylation in oligoastrocytoma1

Felix Sahm1,2, Ulrike Lass2, Christel Herold-Mende3, Andreas von Deimling1,2,

Christian Hartmann4, Wolf Mueller1, 5

1Department of Neuropathology, Ruprecht-Karls-Universität Heidelberg, D-69120

Heidelberg, Germany

2Clinical Cooperation Unit Neuropathology G380, German Cancer Research Center

(DKFZ), D-69120 Heidelberg, Germany

3Division of Neurosurgical Research, Department of Neurosurgery, Ruprecht-Karls-

Universität Heidelberg, D-69120 Heidelberg, Germany

4Department of Neuropathology, Institute of Pathology, Hannover Medical School, D-

30625 Hannover, Germany

5Department of Neuropathology, University Leipzig, D-04103 Leipzig, Germany

Corresponding Author

Prof. Dr. med. Wolf C Mueller, MD, PhD

Current address:

University Leipzig

Department of Neuropathology

Liebigstrasse 24 (Interim, Building G)

D-04103 Leipzig

Fon: +49 (0)341- 97- 15040

Fax: +49 (0) 341- 97- 15049

Email: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the

copyediting, typesetting, pagination and proofreading process, which may lead to differences between this

version and the Version of Record. Please cite this article as doi: 10.1111/nan.12045

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© 2013 British Neuropathological Society 2

Abstract

Aims: Combined deletion of the whole chromosomal arms 1p and 19q is a frequent

event in oligodendroglial tumours. Recent identification of recurrent mutations in CIC

on 19q and FUBP1 on 1p and their mutational patterns suggest a loss of function of

the respective proteins. Surprisingly, oligoastrocytomas harbouring identical genetic

characteristics regarding 1p/19q co-deletion and frequent IDH1/2 mutations have

been shown to carry CIC mutations in a significantly lower number of cases. The

present study investigates whether epigenetic modification may result in silencing of

CIC.

Methods: Since IDH1/2 mutation mediated DNA hypermethylation is a prominent

feature of these tumours, we analyzed a set of CIC wild-type oligoastrocytomas and

other diffuse gliomas with regard to 1p/19q status for presence of CIC-associated

CpG-island methylation by methylation-specific PCR.

Results: Both methylation specific PCR and subsequent bisulfite-sequencing of

selected cases revealed an unmethylated status in all samples.

Conclusion: Despite the hypermethylator- phenotype in IDH1/2 mutant tumours and

recent detection of gene silencing particularly on retained alleles in

oligodendendroglial tumours, hypermethylation of CIC-associated CpG-islands does

not provide an alternative mechanism of functional CIC protein abrogation.

Key words: oligoastrocytoma, CIC, 1p/19q, IDH1, CpG-island methylation, gCIMP

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Introduction

The prognostic and predictive impact of the common 1p/19q co-deletion in

oligodendroglial brain tumours has long been established [1] and very recently

gained further momentum by impressive reports on response to circumscribed

treatment regimens in co-deleted cases at the ASCO 2012 meeting [2, 3]. However,

the biological mechanisms mediating these effects are so far not understood. The

recent identification of recurrent mutations on the retained alleles of the far upstream

element (FUSE) binding protein 1- gene (FUBP1) and the homolog of the Drosophila

gene capicua (CIC) on 19q in oligodendroglioma and oligoastrocytoma promise

further insight [4-6]. While FUBP1 mutations were only found in a small fraction of

cases, or even no mutation in one study (5), CIC mutations were detected in up to

100% of oligodendroglioma [6] with isocitrate dehydrogenase gene 1/2 (IDH1/2)

mutation and combined deletions of 1p/19q. CIC is the human homologue of capicua

which has been extensively analyzed in Drosophila. Its activity is regulated through

regulation by mitogen- activated protein (MAP)- kinases and the currently available

data on its function in humans points towards a role in granular cell development [7].

Both the loss of one copy and the mutational characteristics of the remaining allele

including stop codons and frameshifts abrogating the DNA-binding function and Cro-

like domain expression support the hypothesis of a loss of function. Remarkably,

while the aforementioned vast majority of oligodendrogliomas with deletions of

1p/19q harboured CIC mutations in these studies, CIC mutations were detected in

only approximately 50% of oligoastrocytomas despite identical 1p/19q pattern and

IDH1/2 mutational status [6]. Thus, it is intriguing to speculate that expression and

function of CIC in the remaining cases might be affected by alternative mechanisms.

Since virtually all CIC mutant cases carried IDH1/2 mutations which are associated

with a CpG-island hypermethylator phenotype (glioma CpG-island methylator

phenotype gCIMP) [8, 9], this epigenetic modification might result in loss of sufficient

protein expression in the remaining cases. Therefore, we screened a set of

oligodendroglial tumours for CpG-island methylation associated with the CIC gene

locus.

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Materials and Methods DNA from brain tumour samples of 56 patients without CIC- mutations and

established IDH1/2, 1p/19q- status were obtained from the archive of the Department

of Neuropathology, Heidelberg, including two astrocytoma WHO grade II, 7

anaplastic astrocytoma WHO grade III, 19 glioblastoma WHO grade IV, two

oligodendroglioma WHO grade II, one anaplastic oligodendroglioma WHO grade III,

10 oligoastrocytoma grade II and 17 anaplastic oligoastrocytoma WHO grade III

(Table 1). All samples were analysed in an anonymized manner as approved by the

local institutional review boards.

IDH1 exon 4 encompassing codon 132,IDH2 exon 4 encompassing codon 172 and

fragments spanning all 20 exons of CIC were bi- directionally sequenced as

previously described [6].

Loss of heterozygosity (LOH) of 1p and 19q was analyzed by employing at least

three polymorphic microsatellite markers for each chromosomal arm as previously

described [6]. MLPA analysis was performed using a commercially available kit

(Salsa MLPA, P088, MRC Holland, Amsterdam, the Netherlands) as described

previously [6].

Bisulfite sequencing (BS) and Methylation-specific PCR (MSP) were performed as

previously described [10, 11]. In detail, to evaluate the methylation status of the CIC-

promoter several fragments of the promoter were bisulfite sequenced. The promoter

region was divided into either adjacent or partly overlapping fragments. Initially,

bisulfite-modified DNA was amplified, electrophoretically separated and visualized by

ethidium bromide. Second, the PCR product was plasmid-incorporated using One

Shot Escherichia coli cells and the TOPO TA Cloning Kit (Invitrogen Life

Technologies Inc., Carlsbad, CA, USA). Cells were then plated and grown overnight

on prewarmed LB plates containing X-gal and 50 µg/ml ampicillin and 50 µg/ml

kanamycin. Colony PCR implementing plasmid- sequence- recognizing M13 primers

was performed on ten white colonies to validate the insert. Before sequencing, the

PCR products were purified (QIAquick PCR Purification Kit, Qiagen, Valencia, CA,

USA). Sequencing was performed on an ABI 3730XL DNA Sequencer (ABI, Foster

City, CA, USA) and analysed using Sequencher Software version 4.2. (Gene Codes

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Corporation, Ann Arbor, MI, USA). MSP primers were designed for CIC associated

CpG islands based on Reference Assembly NC_000019.9 Reference GRCh37.p9

and accession number NM_015125.3. This region was defined as CpG-island by the

human genome browser, position chr19:42784075-42785542 (genome.ucsc.edu).

Besides their CpG-pattern, these regions were predicted to serve as promoter [12].

Primer positions are depicted in Figure 1. Sequences of both bisulfite sequencing-

and MSP- primers are provided in supplementary Table 1.

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Results

In 28/56 CIC wild-type cases, an IDH1R132H mutation was identified, one anaplastic

astrocytoma WHO grade III harboured an IDH1R132C, and none an IDH2 mutation.

Combined losses of 1p/19q were observed in 18/56 cases, isolated loss of 19q in

16/56 cases and isolated loss of 1p in one case (Table 1). All samples were initially

analyzed by microsatellite markers and validated by MLPA [6].

Large parts of the CIC associated CpG-island were investigated by bisulfite

sequencing in selected tissues and all tumours were investigated for CpG-island

methylation by methylation specific PCR. DNA derived from non-tumourous normal

brain was bisulfite sequenced to exclude cell-type specific methylation of the CpG-

island and to identify potential methylation sites not associated with tumourigenesis.

Also, a representative case with CIC mutation and a wild-type case were analyzed by

bisulfite sequencing in an effort to visualize potential differences in methylation

patterns outside the CpG-dinucleotides investigated by methylation specific PCR.

Bisulfite sequencing excluded cell-type specific methylation of the CpG-island in

normal brain (Figure 1). Normal brain derived DNA was practically devoid of

methylated CpG-sites. Methylation patterns distinguishing tumours with and without

CIC mutations were detected neither by bisulfite-sequencing nor by MSP. All

investigated tumours harbored a non-methylated CIC associated CpG-island.

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Discussion

CIC mutations are found in the majority of oligodendroglioma with combined

deletions of 1p/19q [4, 5], but only approximately 50% of oligoastrocytoma with

identical characteristics [6]. Thus, we analyzed a set of brain tumours including 27

oligoastrocytoma with CIC wild-type status for CIC associated CpG-island

methylation. In fact, a second study underlining the discrepancy between

oligodendroglioma and oligoastrocytoma regarding CIC mutation frequency and its

importance for classification has been presented recently [13]. Of note, these

tumours are known to present with a hypermethylator phenotype mediated by IDH1/2

mutation [9] and silencing of genes on the retained allele has been proven to result in

reduced or even absent expression: For example, downregulation of the epithelial

membrane protein 3- gene (EMP3) [14] on 19q or the transcriptional coactivator 4-

(CITED4) and the peroxiredoxin 1- gene (PRDX1) on 1p [15, 16] by hypermethylation

has been shown in oligodendroglial tumour cells. Moreover, a CpG- island

methylation dependent lack of protein expression has recently been identified on the

retained 1p with the cation proton antiporter 1- (NHE-1) gene being silenced resulting

in reduced pH regulation capabilities [17]. However, the CpG-islands predicted as a

likely promoter of CIC did neither show a methylation in CIC wild-type

oligoastrocytoma nor in CIC mutant oligoastrocytoma or CIC wild type astrocytic

tumours which served as control (Figure 1, Table 1). In line with these results semi-

quantitative RT- PCR of CIC- cDNA and immunohistochemistry of CIC in non-

tumourous normal human brain and selected CIC- wild- type gliomas with and

without LOH 19q confirmed unaltered CIC- expression patterns (supplementary

Figure 1). Thus, the discrepancy between CIC mutation frequencies in these entities

cannot be resolved by such epigenetic alterations. Even if alternative epigenetic or

post-transcriptional modifications might determine loss of functional CIC in the

remaining cases, it would still constitute an apparent contrast between

oligodendrogliomas and oligoastrocytomas. Possibly, this effect might be a result of

tissue sampling of the “hybrid”-tumour oligoastrocytoma. Very recently, further

evidence for the bi-clonal development of these tumours was provided by analyzing

IDH1/2 mutation, 1p/19q status and morphology in a set of primary and recurrent

oligoastrocytomas, indicating that the oligodendroglial and astrocytic fractions both

present with IDH1/2 mutation while only the oligodendroglial compartment harbours

combined deletion of 1p/19q [18]. In line with the favorable prognostic effect of LOH

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1p/19q, recurring tumours showed a higher astrocytic proportion. Thus, CIC

mutations might be restricted to the oligodendroglial fraction whose proportion in

DNA extracted samples might vary. However, we assured that microsatellite

analyses, multiplex ligation-dependent probe amplification (MLPA), IDH1/2- and CIC-

sequencing as well as methylation analysis were performed from the same DNA

sample, limiting the risk of this sample effect.

Since CIC mutation occur virtually in the whole coding sequence of CIC [4-6, 13],

high quality DNA from fresh or frozen tissue should be analyzed to ultimately address

this question. However, suitable samples are hardly available in sufficient amounts

for unequivocal dissection of both compartments.

Conclusively, this data clarifies that the hypermethylation phenotype of IDH1/2

mutant oligodendroglial tumours does not affect CIC associated CpG-islands and

does not account for CIC- protein abrogation in CIC- wild-type cases with 1p/19q co-

deletion.

Contribution Author(s)

Study concepts: WM, FS

Study design: WM, FS, UL

Data acquisition: WM, FS, UL, CH

Quality control of data and algorithms: WM, FS, UL, CH, AvD

Data analysis and interpretation: WM, FS, UL, CH, CHM, AvD

Statistical analysis: n/a

Manuscript preparation: WM, FS

Manuscript editing: WM, FS

Manuscript review: all authors.

WM: Wolf Mueller

FS: Felix Sahm

UL: Ulrike Lass

CHM: Christel Herold- Mende

AvD: Andreas von Deimling

CH: Christian Hartmann

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Table 1

Diagnosis

IDH1/2

1p

19q

MSP CIC

Diagnosis

IDH1/2

1p

19q

MSP CIC

O II wt ret ret U A II mut ret ret U

O II mut ret ret U A II wt ret DEL U

OA II mut ret ret U A III mut DEL DEL U

OA II mut ret ret U A III mut ni DEL U

OA II mut ret ret U A III mut ni DEL U

OA II mut ret ret U A III mut DEL DEL U

OA II mut DEL DEL U A III mut ni ret U

OA II mut DEL DEL U A III wt ret DEL U

OA II mut DEL DEL U A III wt ret DEL U

OA II mut ret ret U GBM wt DEL DEL U

OA II wt DEL DEL U GBM wt ret DEL U

OA II mut ret ret U GBM wt ret DEL U

OA III nd DEL DEL U GBM wt ret DEL U

OA III nd DEL DEL U GBM wt DEL DEL U

OA III mut DEL DEL U GBM wt ret ret U

OA III mut DEL DEL U GBM wt ret DEL U

OA III* mut ret ret U GBM wt ret DEL U

OA III mut ret ret U GBM wt ret DEL U

OA III mut ret ret U GBM mut ret DEL U

OA III mut DEL DEL U GBM nd ret ret U

OA III wt ret ret U GBM wt DEL DEL U

OA III mut DEL DEL U GBM wt ret DEL U

OA III mut DEL DEL U GBM wt DEL DEL U

OA III wt ret ret U GBM wt ret DEL U

OA III wt ret ret U GBM wt DEL DEL U

OA III mut ret ret U GBM wt DEL ret U

OA III mut ret ret U GBM wt ret DEL U

OA III mut ret ret U GBM mut ret DEL U

Legend to Table 1:

Characteristics of samples without CIC- mutations analyzed for methylation status.

OII - oligodendroglioma WHO grade II, OIII - anaplastic oligodendroglioma WHO

grade III, OAII - oligoastrocytoma WHO grade II, OAIII - anaplastic oligoatrocytoma

WHO grade III, AII - astrocytoma grade II, AIII - anaplastic astrocytoma WHO grade

III, GBM - glioblastoma WHO grade IV, gcGBM - giant cell glioblastoma WHO grade

IV, DEL – deletion, ret – both parental alleles retained, wt - wild-type, mut – mutant,

nd – not determinable, ni - not informative, * IDH1R132C mutation

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Legend to Figure 1:

Top: Position of CpG-islands and primers for methylation-specific PCR and Bisulfite-

sequencing in selected gliomas relative to ATG-site.

Bottom: Bisulfite-sequencing results of 10 individual clones in a representative CIC

mutant and wild-type case and brain control tissue. Each circle represents one

individual CpG- dinucleotide within the promoter sequence. Empty circles represent

unmethylated CpG dinucleotides, black circles highlight methylated CpGs. The

glioma samples with and without CIC- mutation reveal a similar methylation pattern of

only single, scattered and randomly distributed methylated CpGs as compared to

normal human brain.

Legend to supplementary Figure 1:

CIC cDNA- and protein- expression in normal human brain and glioma samples

without CIC- mutation either with or without LOH19q.

Top 2A: Semi- quantitative RT- PCR of CIC- cDNA reveals similar expression levels

for normal human brain compared to CIC- wild- type gliomas with (ID41368) and

without (ID41364) LOH19q.

Bottom 2B: CIC- Immunohistochemistry confirms RT- PCR results of similar CIC

expression in normal human brain compared to CIC- wild- type gliomas with

(ID41368) and without (ID41364) LOH19q. (Magnification x200, scale bar= 50µm.) In

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Brain

C p G i s l a n d

Bisulfite Seq Frag. 1



-3314 -4663 GCGC

-4702 -4560

-4534 -4353

-3400 -3238

-4221/11 -4157/44

Fragment 1 Fragment 2 Fragment 3

Figure 1

MSP primer

ATG

ATG

ATG

ATG

ATG

ID23204 CIC mut

ID23076 CIC wt

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Documents

Analysis of CIC -associated CpG island methylation in oligoastrocytoma