Denaturing High Performance Liquid Chromatography Detection of SDHB, SDHD, and VHL Germline Mutations in Pheochromocytoma

Meyer-Rochow, Goswin Y.; Smith, Janine M.; Richardson, Anne-Louise; Marsh, Deborah J.; Sidhu, Stan B.; Robinson, Bruce G.; Benn, Diana E.

doi:10.1016/j.jss.2008.07.043

« Previous Next »Journal of Surgical Research
Volume 157, Issue 1 , Pages 55-62, November 2009

Denaturing High Performance Liquid Chromatography Detection of SDHB, SDHD, and VHL Germline Mutations in Pheochromocytoma

Goswin Y. Meyer-Rochow, F.R.A.C.S.
- Cancer Genetics, Hormones, and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Sydney, Australia
- University of Sydney Department of Endocrine Surgery, Royal North Shore Hospital, St Leonards, Australia
Janine M. Smith, M.B.Ch.B.
- Department of Clinical Genetics, The Children's Hospital at Westmead and University of Sydney, Westmead, NSW, Australia
Anne-Louise Richardson, B.Sc.
- Cancer Genetics, Hormones, and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Sydney, Australia
Deborah J. Marsh, Ph.D.
- Functional Genomics Laboratory, Hormones and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital, and University of Sydney, Sydney, Australia
Stan B. Sidhu, Ph.D.
- Cancer Genetics, Hormones, and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Sydney, Australia
- University of Sydney Department of Endocrine Surgery, Royal North Shore Hospital, St Leonards, Australia
Bruce G. Robinson, M.D.
- Cancer Genetics, Hormones, and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Sydney, Australia
Diana E. Benn, Ph.D.
- Cancer Genetics, Hormones, and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, Sydney, Australia
- To whom correspondence and reprint requests should be addressed at Cancer Genetics Laboratory, Hormones, and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital St. Leonards, Sydney, NSW 2065, Australia.

Received 8 May 2008 published online 05 September 2008.

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.

Key Words: dHPLC, pheochromocytoma, germline mutations, VHL, SDHB, SDHB