A comprehensive next generation sequencing-based genetic testing strategy to improve diagnosis of inherited pheochromocytoma and paraganglioma

CONTEXT: Pheochromocytomas and paragangliomas are notable for a high frequency of inherited cases, many of which present as apparently sporadic tumors.

OBJECTIVE: The objective of this study was to establish a comprehensive next generation sequencing (NGS)-based strategy for the diagnosis of patients with pheochromocytoma and paraganglioma by testing simultaneously for mutations in MAX, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL.

DESIGN: After the methodology for the assay was designed and established, it was validated on DNA samples with known genotype and then patients were studied prospectively.

SETTING: The study was performed in a diagnostic genetics laboratory.

PATIENTS: DNA samples from 205 individuals affected with adrenal or extraadrenal pheochromocytoma/head and neck paraganglioma (PPGL/HNPGL) were analyzed. A proof-of-principle study was performed using 85 samples known to contain a variant in 1 or more of the genes to be tested, followed by prospective analysis of an additional 120 samples.

MAIN OUTCOME MEASURES: We assessed the ability to use an NGS-based method to perform comprehensive analysis of genes implicated in inherited PPGL/HNPGL.

RESULTS: The proof-of-principle study showed that the NGS assay and analysis gave a sensitivity of 98.7%. A pathogenic mutation was identified in 16.6% of the prospective analysis cohort of 120 patients.

CONCLUSIONS: A comprehensive NGS-based strategy for the analysis of genes associated with predisposition to PPGL and HNPGL was established, validated, and introduced into diagnostic service. The new assay provides simultaneous analysis of 9 genes and allows more rapid and cost-effective mutation detection than the previously used conventional Sanger sequencing-based methodology.