Denaturing high performance liquid chromatography detection of SDHB, SDHD, and VHL germline mutations in pheochromocytoma.
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.
Meyer-Rochow
Goswin Y
GY
Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Australia.
Smith
Janine M
JM
Richardson
Anne-Louise
AL
Marsh
Deborah J
DJ
Sidhu
Stan B
SB
Robinson
Bruce G
BG
Benn
Diana E
DE
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
C491545:SDHD protein, human; C108095:SDHB protein, human; D013385:Succinate Dehydrogenase; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human