title: Denaturing high performance liquid chromatography detection of SDHB, SDHD, and VHL germline mutations in pheochromocytoma.
abstract: BACKGROUND
  Pheochromocytomas are neuroendocrine tumors of chromaffin cell origin which arise from the adrenal medulla and less commonly the extra-adrenal sympathetic paraganglia. Pheochromocytomas are component tumors of the familial syndromes multiple endocrine neoplasia Type 2, von Hippel Lindau disease, Neurofibromatosis Type 1, and the pheochromocytoma/paraganglioma syndromes caused by mutations in the RET, VHL, NF1, SDHB, and SDHD genes, respectively. The aim of this study was to evaluate denaturing high performance liquid chromatography (dHPLC) as a screening tool for the detection of germline mutations within VHL, SDHB, and SDHD in pheochromocytoma patients.
  
  
  METHODS
  Polymerase chain reaction of all exons of VHL, SDHB, and SDHD genes was performed on leukocyte DNA extracted from stored blood samples of 74 unrelated patients treated for pheochromocytoma. After dHPLC analysis, all samples demonstrating variance were selected for sequencing.
  
  
  RESULTS
  Of the 74 patients, 12 mutations and 16 polymorphisms were identified by dHPLC and confirmed on sequencing. More specifically, a total of 5 mutations and 15 polymorphisms were detected in SDHB and 7 mutations and 1 polymorphism were identified in VHL. No SDHD mutations or polymorphisms were identified. By sequencing only dHPLC variants, the total amount of DNA sequencing required was reduced by approximately 88%.
  
  
  CONCLUSIONS
  dHPLC is an effective screening tool for the detection of germline mutations in SDHB, SDHD, and VHL and has application for diagnostic germline mutation analysis in pheochromocytoma patients.
authors.affiliation: Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Australia.
authors.forename: Goswin Y
authors.initials: GY
authors.lastname: Meyer-Rochow
authors.forename: Janine M
authors.initials: JM
authors.lastname: Smith
authors.forename: Anne-Louise
authors.initials: AL
authors.lastname: Richardson
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Marsh
authors.forename: Stan B
authors.initials: SB
authors.lastname: Sidhu
authors.forename: Bruce G
authors.initials: BG
authors.lastname: Robinson
authors.forename: Diana E
authors.initials: DE
authors.lastname: Benn
mesh_terms: D000293:Adolescent; D000310:Adrenal Gland Neoplasms / Q000175:diagnosis / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards; D005260:Female; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D018095:Germ-Line Mutation; D006801:Humans; D008297:Male; D008875:Middle Aged; D009691:Nucleic Acid Denaturation; D010673:Pheochromocytoma / Q000175:diagnosis / Q000235:genetics*; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D013385:Succinate Dehydrogenase / Q000235:genetics*; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics*; D055815:Young Adult
chemical_list: C491545:SDHD protein, human; C108095:SDHB protein, human; D013385:Succinate Dehydrogenase; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human