A Pilot Genome-Scale Profiling of DNA Methylation in Sporadic Pituitary Macroadenomas: Association with Tumor Invasion and Histopathological Subtype

<div><p>Pituitary adenomas (PAs) are neoplasms that may cause a variety of neurological and endocrine effects. Although known causal contributors include heredity, hormonal influence and somatic mutations, the pathophysiologic mechanisms driving tumorigenesis and invasion of sporadic PAs remain unknown. We hypothesized that alterations in DNA methylation are associated with PA invasion and histopathology subtype, and that genome-scale methylation analysis may complement current classification methods for sporadic PAs. Twenty-four surgically-resected sporadic PAs with varying histopathological subtypes were assigned dichotomized Knosp invasion scores and examined using genome-wide DNA methylation profiling and RNA sequencing. PA samples clustered into subgroups according to functional status. Compared with hormonally-active PAs, nonfunctional PAs exhibited global DNA hypermethylation (mean beta-value 0.47 versus 0.42, <i>P</i> = 0.005); the most significant site of differential DNA methylation was within the promoter region of the potassium voltage-gated channel <i>KCNAB2</i> (FDR = 5.11×10⁻¹⁰). Pathway analysis of promoter-associated CpGs showed that nonfunctional PAs are potentially associated with the ion-channel activity signal pathway. DNA hypermethylation tended to be negatively correlated with gene expression. DNA methylation analysis may be used to identify candidate genes involved in PA function and may potentially complement current standard immunostaining classification in sporadic PAs. DNA hypermethylation of <i>KCNAB2</i> and downstream ion-channel activity signal pathways may contribute to the endocrine-inactive status of nonfunctional PAs.</p></div

Top 30 significant genes with differential DNA methylation between NFAs and FAs.

a<p>Probe_CpG: the Illumina HM450 probe ID.</p>b<p>SD: standard deviation.</p>c<p>NFA-FA: the mean beta value subtractive difference between NFA tumors and FA tumors.</p>d<p>FDR: False Discovery Rate.</p>e<p>TSS200: 200 bp within transcription start site.</p>f<p>TSS1500: 1500 bp within the transcription start site.</p><p>* Promoter Associated.</p><p>Link to the whole information of the Probe_CpG: <a href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL13534" target="_blank">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL13534</a>.</p

Gene expression.

<p>A, Linear regression analysis showed negative trend of DNA methylation and gene expression. B, Three overlapped significant genes of DNA methylation and gene expression when compared NFAs to FAs. C, gene <i>ODAM</i> expression in one FA subject of d0079 which secreted hormones of GH and TSH, <i>GAPDH</i> gene expression was used as control.</p

Patient and tumor characteristics.

<p>M: Male, F: Female; Age: Year unit; PRL: Prolactin; TR × CC: Transverse (mm) × Antero-posterior (mm). The average size of all PAs was 23.55 TR mm × 19.36 CC mm; NA: Not applicable. Invasion: “0” represents noninvasive PAs, “1” represents invasive PAs; Ave: Average; Knosp: Knosp grade of PAs.</p

Hierarchical Clustering of DNA methylation in 24 PA cases.

<p>The HM450 probes (7,674) with the highest 2% of standard deviation across the set of 383,718 global HM450 probes were used.</p

Global CpG DNA methylation profiles in PAs.

<p>A, Comparison of mean DNA methylation levels across 383,718 HM450 probes in noninvasive (Knosp grade 0, 1) and invasive (Knosp grade 2, 3, 4) PAs. B, Mean DNA methylation levels in PA cases across the five different Knosp grades (0–4) among the set of 383,718 HM450 probes. C, Comparison of mean DNA methylation levels across 383,718 HM450 probes in FAs and NFAs. P-value was calculated via simple t-test. D, Comparison of mean DNA methylation levels across 383,718 HM450 probes in only NFAs. P-value was calculated via simple t-test. All of the above DNA methylation beta values are shown as mean ± SEM in different groups. E and F, DNA methylation levels in gene regions (TSS1500, TSS200, 5′UTR, 1stExon, gene body and 3′UTR) for NFAs (panel E) and FAs (Panel F). TSS200: 200 bp within transcription start site; TSS1500: 1500 bp within transcription start site; UTR: untranslated region; 1stExon: the first exon of gene.</p