Currás-Freixes, MariaPiñeiro-Yañez, ElenaMontero-Conde, CristinaApellániz-Ruiz, MaríaCalsina, BrunaMancikova, VeronikaRemacha, LauraRichter, SusanErcolino, ToninoRogowski-Lehmann, NatalieDeutschbein, TimoCalatayud, MaríaGuadalix, SonsolesÁlvarez-Escolá, CristinaLamas, CristinaAller, JavierSastre-Marcos, JuliaLázaro, ConxiGalofré, Juan CPatiño-García, AnaMeoro-Avilés, AmparoBalmaña-Gelpi, JudithDe Miguel-Novoa, PazBalbín, MilagrosMatías-Guiu, XavierLetón, RocíoInglada-Pérez, LucíaTorres-Pérez, RafaelRoldán-Romero, Juan MRodríguez-Antona, CristinaFliedner, Stephanie M JOpocher, GiuseppePacak, KarelKorpershoek, Estherde Krijger, Ronald RVroonen, LaurentMannelli, MassimoFassnacht, MartinBeuschlein, FelixEisenhofer, GraemeCascón, AlbertoAl-Shahrour, FatimaRobledo Batanero, Mercedes2025-01-142025-01-142017-07J Mol Diagn . 2017 Jul;19(4):575-588.https://hdl.handle.net/20.500.12105/26019Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.Supported by the Fondo de Investigaciones Sanitarias project PI14/00240, European Regional Development Fund, Spanish Group of Neuroendocrine Tumors, the European Union Seventh Framework Program FP7/2007-2013 grant 259735, the Paradifference foundation, and Interdisciplinary Center for Clinical Research of the University of Wurzburg grant Z2/57 (T.D.). M.C. is a predoctoral fellow supported by the Severo Ochoa Excellence Program project SEV-2011-0191, C.M. is supported by a postdoctoral fellowship from the Spanish Association Against Cancer Foundation, L.I.-P. is. supported by Biomedical Research Networking Center on Rare Diseases (CIBERER), M.A.-R. is a predoctoral fellow of la Caixa/Spanish National Cancer Research Centre international Ph.D. program, J.M.R.-R. is a predoctoral fellow of la Caixa/Doctorates at Spanish Universities and research centers, and L.R. is a predoctoral fellow supported by the Fondo de Investigaciones Sanitarias project PI12/00236.engSUCCINATE-DEHYDROGENASEGERMLINE MUTATIONSGENE-MUTATIONSNF1 GENEMALIGNANT PHEOCHROMOCYTOMASPRENATAL-DIAGNOSISNECK PARAGANGLIOMARET PROTOONCOGENEHIF2A MUTATIONSHIGH-FREQUENCYresearch article28552549194575-588J Mol Diagnosticsopen access