Mutation analysis was performed in all tumors exhibiting loss of SDHB or SDHA staining by IHC. DNA was extracted from FFPE tissue blocks of macrodissected neoplastic tissue and, where available, from fresh frozen neoplastic tissue and whole blood both prospectively banked at the time of surgery (QIAamp DNA FFPE tissue kit and QIAamp DNA blood minikit; Qiagen, Melbourne, Vic., Australia). Mutation analysis of the entire coding sequence, including exon-intron boundaries, was performed for the 15 exons of SDHA (NCBI Ref Seq: NM_004168.2). Primer sequences were specifically designed to avoid amplification of the 3 pseudogenes (SDHAP1, SDHAP2, SDHAP3) as described previously,17 (link) and human control genomic DNA (Promega) was used to confirm that none of the pseudogenes were amplified. In some instances, 2 rounds of polymerase chain reaction were required to amplify the DNA from paraffin-embedded tissues. Mutations were confirmed by sequencing of 2 independent polymerase chain reactions. Mutation analysis of SDHB, SDHC, and SDHD was not performed as there were no tumors found to exhibit loss of SDHB alone by IHC.
This study was approved by the Northern Sydney Local Health District Human Research Ethics Committee.
This study was approved by the Northern Sydney Local Health District Human Research Ethics Committee.