The VHL gene is epigenetically inactivated in pheochromocytomas and abdominal paragangliomas

Abstract

Pheochromocytoma (PCC) and abdominal paraganglioma (PGL) are neuroendocrine tumors that present with clinical symptoms related to increased catecholamine levels. About a third of the cases are associated with constitutional mutations in pre-disposing genes, of which some may also be somatically mutated in sporadic cases. However, little is known about inactivating epigenetic events through promoter methylation in these very genes. Using bisulphite pyrosequencing we assessed the methylation density of 11 PCC/PGL disease genes in 96 tumors (83 PCCs and 13 PGLs) and 34 normal adrenal references. Gene expression levels were determined by quantitative RT-PCR. Both tumors and normal adrenal samples exhibited low methylation index (MetI) in the EGLN1 (PDH2), MAX, MEN1, NF1, SDHB, SDHC, SDHD, SDHAF2 (SDH5), and TMEM127 promoters, not exceeding 10% in any of the samples investigated. Aberrant RET promoter methylation was observed in two cases only. For the VHL gene we found increased MetI in tumors as compared with normal adrenals (57% vs. 27%; P < 0.001), in malignant vs. benign tumors (63% vs. 55%; P < 0.05), and in PGL vs. PCC (66% vs. 55%; P < 0.0005). Decreased expression of the VHL gene was observed in all tumors compared with normal adrenals (P < 0.001). VHL MetI and gene expressions were inversely correlated (R = −0.359, P < 0.0001). Our results show that the VHL gene promoter has increased methylation compared with normal adrenals (MetI > 50%) in approximately 75% of PCCs and PGLs investigated, highlighting the role of VHL in the development of these tumors.

Keywords: :

• Pheochromocytoma
• paraganglioma
• VHL
• DNA promoter methylation
• gene expression
• pyrosequencing
• qRT-PCR

10.4161/epi.26686

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

The study was financially supported by Karolinska Institutet, StratCan (Strategic Research Program in Cancer at Karolinska Institutet), the Swedish Research Council, The Cancer Society in Stockholm, Gustav V Jubilee Foundation, the Swedish Cancer Society, as well as the Regional Agreement on Medical Training and Clinical Research (ALF) between Stockholm County Council and Karolinska Institutet.

Acknowledgments

The authors wish to thank Ms Susanne Bergström for valuable Think-Cell guidance and Ms Lisa Ånfalk at the Department of Oncology-Pathology, Karolinska University Hospital Solna, for excellent sample collection and handling.

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/epigenetics/article/26686