Denaturing High Performance Liquid Chromatography Detection of SDHB, SDHD, and VHL Germline Mutations in Pheochromocytoma

Abstract

Pheochromocytomas are neuroendocrine tumors of chromaffin cell origin which arise from the adrenal medulla and less commonly the extra-adrenal sympathetic paraganglia. Pheochromocytomas are component tumors of the familial syndromes multiple endocrine neoplasia Type 2, von Hippel Lindau disease, Neurofibromatosis Type 1, and the pheochromocytoma/paraganglioma syndromes caused by mutations in the RET, VHL, NF1, SDHB, and SDHD genes, respectively. The aim of this study was to evaluate denaturing high performance liquid chromatography (dHPLC) as a screening tool for the detection of germline mutations within VHL, SDHB, and SDHD in pheochromocytoma patients. Polymerase chain reaction of all exons of VHL, SDHB, and SDHD genes was performed on leukocyte DNA extracted from stored blood samples of 74 unrelated patients treated for pheochromocytoma. After dHPLC analysis, all samples demonstrating variance were selected for sequencing. Of the 74 patients, 12 mutations and 16 polymorphisms were identified by dHPLC and confirmed on sequencing. More specifically, a total of 5 mutations and 15 polymorphisms were detected in SDHB and 7 mutations and 1 polymorphism were identified in VHL. No SDHD mutations or polymorphisms were identified. By sequencing only dHPLC variants, the total amount of DNA sequencing required was reduced by approximately 88%. dHPLC is an effective screening tool for the detection of germline mutations in SDHB, SDHD, and VHL and has application for diagnostic germline mutation analysis in pheochromocytoma patients.