DNA hypermethylation and aberrant expression of the EMP3 gene at 19q13.3 in Human Gliomas

Abstract

Allelic losses on 19q are found in the majority of oligodendroglial tumors and approximately one-third of diffuse astrocytomas. However, the tumor suppressor genes (TSG) on 19q are still elusive. Using cDNA microarray expression profiling, EMP3 at 19q13.3 was among those genes showing the most pronounced expression differences. In line with this, other authors reported EMP3 as being epigenetically silenced in neuroblastomas and astrocytomas. To further investigate EMP3 as a TSG candidate on 19q13.3, we performed molecular analysis of this gene in 162 human gliomas. Mutation analysis did not reveal EMP3 alteration in 132 gliomas. In oligodendroglial tumors, we found that aberrant methylation in the 5'-region of EMP3 was significantly associated with reduced mRNA expression and LOH 19q. In astrocytomas, EMP3 hypermethylation was also paralleled by reduced expression but was independent of the 19q status. EMP3 hypermethylation was detected in more than 80% of diffuse, anaplastic astrocytomas and secondary glioblastomas. Primary glioblastomas, however, mostly lacked EMP3 hypermethylation and frequently overexpressed EMP3. Our data corroborate that oligodendroglial and astrocytic gliomas often show EMP3 hypermethylation and aberrant expression. Furthermore, our findings suggest that primary and secondary glioblastomas are not only characterized by distinct genetic profiles but also differ in their epigenetic aberrations.

Figure 1

Schematic representation of the results obtained in 162 gliomas and three non‐neoplastic brain tissue samples (NB1‐3) concerning allelic deletions on 19q, EMP3 mRNA expression and EMP3 hypermethylation. Methyl‐ ation at each of the 16 investigated CpG sites in the EMP3 promoter region was determined as described in Materials and Methods. The location of the transcription start site is indicated on the top of the figure. The results are represented in a 4‐tiered semiquantitative grey‐scale pattern: white square, not methylated (0); light gray, weakly methylated (1); gray, moderately methylated (2); black, strongly methylated (3). EMP3 mRNA expression was determined by real‐time reverse transcription‐polymerase chain reaction analysis and normalized to the mRNA expression of ARF1. The EMP3 expression levels shown are calculated relative to non‐neoplastic brain tissue. LOH = loss of heterozygosity; RET = retention of heterozygosity; n.d. = not determined.

Figure 2

EMP3 hypermethylation and mRNA expression analysis in oligodendrogliomas and oligoastrocytomas of WHO grade II and III. A. Sequencing of parts of the EMP3 5′‐CpG‐rich region after sodium bisulfite modification revealed methylation of CpG sites in tumor O22 (arrowheads in upper lane) but not in tumor AO22 and in a non‐neoplastic brain tissue sample (NB2) (middle and lower lane) (shown is the reverse sequence from chr. 19 nucleotides 53520611 to 53520671, UCSC genome browser, Mar 2006 (hg18) assembly, http://genome.ucsc.edu/). B. Dot plot diagrams of EMP3 expression levels in oligodendroglial tumors with (n = 34) and without (n = 7) EMP3 hypermethylation (left side; meth., EMP3 hypermethylated; non‐meth., EMP3 not hypermethylated), as well as with (n = 27) and without (n = 13) allelic losses on 19q (right side; LOH = loss of heterozygosity; RET = retention of heterozygosity). Note significantly lower mean expression levels (indicated by horizontal bars) in tumors with EMP3 hypermethylation (P = 0.01; Student’s t‐test) and tumors with 19q losses (P = 0.01).

Figure 3

Univariable analyses of the association between EMP3 hypermethylation (A,B) or allelic losses on 1p and 19q (C,D) and overall survival in patients with oligodendroglial tumors. A,B. Kaplan−Meier survival curve estimates in relation to the EMP3 methylation status obtained for 46 patients (A), including 21 patients with WHO grade II and 25 patients with WHO grade III tumors, as well as for the subgroup of 25 patients with WHO grade III tumors (B). Note association (log rank tests) of EMP3 hypermethylation with overall survival (OS) in the entire group of oligodendroglial patients (A), but not in patients with anaplastic oligodendroglial tumors (B). C,D. Kaplan−Meier survival curves of the same patient cohort (except for one patient with a WHO grade II tumor) stratified according to the 1p/19q allelic status. Note 1p and 19q losses are significantly associated with longer OS in the entire group of patients (C) and in the subgroup of 25 patients with WHO grade III tumors (D). meth. = EMP3 hypermethylated; non‐meth. = EMP3 not hypermethylated LOH = loss of heterozygosity; RET = retention of heterozygosity.