A Comprehensive Next Generation Sequencing–Based Genetic Testing Strategy To Improve Diagnosis of Inherited Pheochromocytoma and Paraganglioma

Abstract

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. (J Clin Endocrinol Metab 98: E1248 -E1256, 2013) P heochromocytomas and paragangliomas are rare, mostly benign, tumors that are notable for a high frequency of inherited cases (1-5). Both pheochromocytomas (which arise from the adrenal medulla) and paragangliomas (also known as extraadrenal pheochromocytomas) (together referred to as PPGLs) usually present with symptoms caused by the cardiovascular effects of excess catecholamine secretion. Head and neck paragangliomas (HNPGLs) are generally nonfunctional and are derived from parasympathetic ganglia. Before 2000, about 10% of PPGLs were thought to be inherited (6) and mostly associated with defined clinical syndromes: von

Consensus Statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas

“…All participants have adopted, and reported on, NGS-based technologies in their research and/or clinical practice [14][15][16][17][18][19][20][21][22][23][24][25][26] . Discussions took place via conference calls, e-mail communications and file exchanges and one plenary session (at the 14th ENS@T Scientific Meeting on November 20th, 2015, Munich, Germany).…”

Genetic test in multiple endocrine neoplasia type 1 syndrome: An evolving story

“…In the presence of a MEN1 mutation lifelong specific clinical surveillance is suggested, as specifically reported in Table 1. These guidelines are reported according to the most recent "clinical practice guidelines for Multiple Endocrine Neoplasia type 1" drafted by the leading worldwide specialists in this field [16] . Moreover, a positive test can also determine the type of surgical intervention for MEN1-associated primary hyperparathyroidism, leading to the decision for a total parathyroidectomy to prevent future recurrences of parathyroid adenomas, instead of the selective ablation of only the adenomatous gland/glands in subjects with a negative MEN1 genetic test.…”

“…In 2013 Rattenberry et al [16] tested a targeted NGS platform, including 9 causative genes, for the genetic diagnosis of patients with pheochromocytoma and paraganglioma, allowing this NGS-based test to be established, validated and introduced into diagnostic practice. Very recently Welander et al [17] enlarged the targeted NGS platform for the mutation analysis of pheochromocytoma and paraganglioma, including 14 different susceptibility genes, proving to be a cost-and time-reducing effective genetic diagnostic method for these tumours.…”

Új módszertani lehetőségek és ezek alkalmazása a hormonális rendszer daganatainak genetikai kivizsgálásában

“…Rattenberry és mtsai amplikonszekvenálást végeztek GS Junior készülékkel. Ösz-szesen 9 gén egyidejű vizsgálatára terveztek amplifi ká-ciót [26]. Welander és mtsai 14 gén vizsgálatát végezték el Illumina MiSeq platformon [27].…”

“…A GS Junior-mérés során a 164 beazonosított eltérés közül 46 álpozitív volt, ami elsősorban a homopolimer hibájára volt visszavezethető. A szerzők ugyanakkor hangsúlyozták, hogy az elemzésben alkalmazott szűrésekkel ezek száma jelentő-sen csökkenthető [26]. A MiSeq készülékkel a módszer specifi citása >99% volt, egyértelműen jelezve a módszer alkalmazhatóságát [27].…”