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Sensitive Determination of BRAF Copy Number in Clinical Samples by Pyrosequencing

Setty, Prashanth PhD*; Gessi, Marco MD*; Waha, Anke PhD*; Hammes, Jennifer*; El-Maarri, Osman PhD; Pietsch, Torsten MD*; Waha, Andreas PhD*

doi: 10.1097/PDM.0b013e3182143817
Original Articles
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Pilocytic astrocytoma is the most frequently occurring brain tumor during childhood. It is classified as grade I by the World Health Organization and may rarely evolve into higher-grade tumors. Frequent genetic abnormalities documented in astrocytomas in children are gains on chromosomal arm 7q. Duplications at 7q34 lead to a fusion between genes KIAA1549 and BRAF resulting in constitutive activation of the BRAF kinase. The BRAF gene is located on chromosome 7q34 and a pseudogene has been identified on chromosome Xq13. We have developed a simple and sensitive pyrosequencing method for the determination of the BRAF copy number in clinical samples. The approach is based on the simultaneous amplification of a DNA fragment contained in exon 11 of BRAF and the respective pseudogene that is used as an internal control. Three different bases in the PCR product allow precise sequence assessment of products originating from the BRAF gene and the respective pseudogene and a calculation of gene copy numbers. After the calibration of the assay on 78 control DNA samples, 42 clinical PA samples were analyzed for variation in copy numbers by pyrosequencing and for fusion gene expression by reverse transcription-polymerase chain reaction. The results obtained from tumor DNA by the developed assay and the established reverse transcription-polymerase chain reaction assays show a high concordance. In summary, we have established a pyrosequencing-based assay allowing precise detection of BRAF copy numbers in DNA extracted from clinical samples.

*Department of Neuropathology

Institute of Experimental Hematology and Transfusion Medicine, University of Bonn, Germany

Supported by National Genome Research Network NGFNplus, Brain Tumor Net (grant 01GS08187, SP8, and SP9b), the Kinderkrebsstiftung (grant DKS 2006.03), German Ministry for Education and Research BMBF, Competence Network Pediatric Oncology and Hematology, Project Embryonal Tumors (grant 01GI0418), and BONFOR program of the Medical Faculty of the University of Bonn.

Reprints: Andreas Waha, PhD, Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud-Str 25, D-53105 Bonn, Germany (e-mail: awaha@uni-bonn.de).

© 2011 Lippincott Williams & Wilkins, Inc.