Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors

Abstract

Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof.

Keywords: DNA methylation; H19; IGF2; adrenocortical tumors; cancer; copy number analysis; imprinting; pheochromocytomas; tetraploidy.

Figure 1

Quantitative expression analysis of IGF2 and H19 in adrenocortical tumors. IGF2 and H19 expression values are normalized to the geometric mean of the three reference genes (SDHA, ATP5B, and CYC1). Data is presented on a logarithmic scale.

Figure 2

DNA methylation levels for the three DMRs of the IGF2/H19 locus for each subgroup of adrenocortical tumors. (A) Schematic presentation of the IGF2/H19 locus. IGF2 harbors 9 exons whereas H19 harbors five exons, which are indicated by the black and white squares. Transcription start sites are indicated with arrows. The IGF2/H19 locus has three differential methylated regions (DMRs) named DMR0, DMR2 and H19 DMR. The parental-specific (M=maternal allele, P=paternal allele) DNA methylation status is presented by black bars for each DMR (black=methylated, white=unmethylated). The H19 DMR makes up the imprinting control region (ICR) of the locus containing seven CTCF biding sites. For each DMR a number of CpG sites were analyzed (DMR0=3, DMR2=26, CTCF2=17, CTCF3=11, and for CTCF6=15) and the DNA methylation levels correlated highly between the three sites (ρ>0.75, P=2.2×10⁻¹⁶, Spearman's correlation). Each CpG site is presented as a filled circle. (B) The mean DNA methylation levels of single CpG sites are shown for each of the four adrenocortical tumor subtypes (Conn's adenoma, ACBTs, PCCs, and ACCs).

Figure 3

ASCAT profiles of adrenal tumors. (A) A Conn's adenoma sample (1) being diploid and with no somatic copy number changes at chr11p15.5 thus presenting a normal sample. (B) A tetraploid ACC sample (51), for which loss of a single allele resulted in triploidy of chr11p15. (C) An UPD-like genotype (57) being tetraploid with loss of two alleles of the same parental origin. (D) A diploid sample (39) with a copy number neutral variation presenting an UPD. (E) The PCC 33 represents samples being diploid and with loss of a single allele of chr11p15.

Figure 4

Doughnut diagrams integrating IGF2 gene expression data with somatic copy number changes and DNA methylation alterations of the IGF2/H19 locus. CAs and ACBTs were mainly characterized by normal IGF2 expression, absence of copy number changes, and normal DNA methylation levels of the H19 ICR. In contrast all PCCs showed overexpression of IGF2, where all samples with somatic copy number changes showed hypermethylation of the H19 ICR. Seventeen out of 20 ACCs presented with overexpression of IGF2 and this mainly correlated with somatic copy number changes of chr11p15.5 and hypermethylation of the H19 ICR. Somatic copy number changes of chr11p15.5 were not exclusively associated with high IGF2 expression levels as a ACC sample (57) with an UPD-like genotype had IGF2 expression levels comparable to the samples from patients with Conn's adenoma.