title: A comprehensive next generation sequencing-based genetic testing strategy to improve diagnosis of inherited pheochromocytoma and paraganglioma.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  METHODS
  The study was performed in a diagnostic genetics laboratory.
  
  
  METHODS
  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.
  
  
  METHODS
  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.
authors.affiliation: Centre for Rare Diseases and Personalised Medicine, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TH, United Kingdom.
authors.forename: Eleanor
authors.initials: E
authors.lastname: Rattenberry
authors.forename: Lindsey
authors.initials: L
authors.lastname: Vialard
authors.forename: Anna
authors.initials: A
authors.lastname: Yeung
authors.forename: Hayley
authors.initials: H
authors.lastname: Bair
authors.forename: Kirsten
authors.initials: K
authors.lastname: McKay
authors.forename: Mariam
authors.initials: M
authors.lastname: Jafri
authors.forename: Natalie
authors.initials: N
authors.lastname: Canham
authors.forename: Trevor R
authors.initials: TR
authors.lastname: Cole
authors.forename: Judit
authors.initials: J
authors.lastname: Denes
authors.forename: Shirley V
authors.initials: SV
authors.lastname: Hodgson
authors.forename: Richard
authors.initials: R
authors.lastname: Irving
authors.forename: Louise
authors.initials: L
authors.lastname: Izatt
authors.forename: Márta
authors.initials: M
authors.lastname: Korbonits
authors.forename: Ajith V
authors.initials: AV
authors.lastname: Kumar
authors.forename: Fiona
authors.initials: F
authors.lastname: Lalloo
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Morrison
authors.forename: Emma R
authors.initials: ER
authors.lastname: Woodward
authors.forename: Fiona
authors.initials: F
authors.lastname: Macdonald
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Wallis
authors.forename: Eamonn R
authors.initials: ER
authors.lastname: Maher
mesh_terms: D000310:Adrenal Gland Neoplasms / Q000175:diagnosis* / Q000191:economics / Q000235:genetics / Q000378:metabolism; D015331:Cohort Studies; D017046:Cost Savings; D003365:Costs and Cost Analysis; D004252:DNA Mutational Analysis / Q000191:economics; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000191:economics / Q000379:methods; D018095:Germ-Line Mutation; D006258:Head and Neck Neoplasms / Q000175:diagnosis* / Q000191:economics / Q000235:genetics / Q000378:metabolism; D017048:Health Care Costs; D006801:Humans; D010235:Paraganglioma / Q000175:diagnosis* / Q000191:economics / Q000235:genetics / Q000378:metabolism; D010673:Pheochromocytoma / Q000175:diagnosis* / Q000191:economics / Q000235:genetics / Q000378:metabolism; D011446:Prospective Studies; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006113:United Kingdom; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D013385:Succinate Dehydrogenase; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; C497446:VHL protein, human