title: Next-generation sequencing in the clinical genetic screening of patients with pheochromocytoma and paraganglioma.
abstract: BACKGROUND
  Recent findings have shown that up to 60% of pheochromocytomas (PCCs) and paragangliomas (PGLs) are caused by germline or somatic mutations in one of the 11 hitherto known susceptibility genes: SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, HIF2A (EPAS1), RET, NF1, TMEM127 and MAX. This list of genes is constantly growing and the 11 genes together consist of 144 exons. A genetic screening test is extensively time consuming and expensive. Hence, we introduce next-generation sequencing (NGS) as a time-efficient and cost-effective alternative.
  
  
  METHODS
  Tumour lesions from three patients with apparently sporadic PCC were subjected to whole exome sequencing utilizing Agilent Sureselect target enrichment system and Illumina Hi seq platform. Bioinformatics analysis was performed in-house using commercially available software. Variants in PCC and PGL susceptibility genes were identified.
  
  
  RESULTS
  We have identified 16 unique genetic variants in PCC susceptibility loci in three different PCC, spending less than a 30-min hands-on, in-house time. Two patients had one unique variant each that was classified as probably and possibly pathogenic: NF1 Arg304Ter and RET Tyr791Phe. The RET variant was verified by Sanger sequencing.
  
  
  CONCLUSIONS
  NGS can serve as a fast and cost-effective method in the clinical genetic screening of PCC. The bioinformatics analysis may be performed without expert skills. We identified process optimization, characterization of unknown variants and determination of additive effects of multiple variants as key issues to be addressed by future studies.
authors.affiliation: Department of Surgical Sciences Uppsala University S-751 85, Uppsala Sweden.
authors.forename: Joakim
authors.initials: J
authors.lastname: Crona
authors.forename: Alberto Delgado
authors.initials: AD
authors.lastname: Verdugo
authors.forename: Dan
authors.initials: D
authors.lastname: Granberg
authors.forename: Staffan
authors.initials: S
authors.lastname: Welin
authors.forename: Peter
authors.initials: P
authors.lastname: Stålberg
authors.forename: Per
authors.initials: P
authors.lastname: Hellman
authors.forename: Peyman
authors.initials: P
authors.lastname: Björklund
keywords: exome sequencing; paraganglioma; pheochromocytoma; whole genome sequencing