SDHA and SDHB mutations in KIT/PDGFRA WT gastrointestinal stromal tumors

Background: KIT/PDGFRA wild-type (WT) GISTs harbour mutations on SDHB and SDHC and, more recently, we described mutations on SDHA using massively parallel sequencing approach. We sequenced SDHA and SDHB genes in a larger series in order to validate the data. Methods: SDHA gene (1-15 exons) and SDHB gene (1-8 exons) (even not all exons in all samples) were sequenced on tumor (T) and/or peripheral blood (PB) of WT GIST patients by Sanger Sequencing method. DNA was extracted from tumor specimens by the QIAmp DNA Mini kit (Qiagen, Milan, Italy) and amplified with specific primer pairs designed to amplify exons but not SDHA pseudo-genes located on chromosomes 3 and 5. Then, PCR products were purified with the Qiaquick PCR purification kit (Qiagen, Milan, Italy) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems). Sanger sequencing was performed on ABI 3730 Genetic Analyzer (Applied Biosystems). Results: SDHA gene exons were sequenced on a total of 27 WT GIST patients, in particular on T, PB and both from 12, 6 and 9 patients respectively. SDHB gene exons were sequenced on a total of 18 out of 27 patients, in particular on T, PB and both from 7, 8 and 3 patients respectively. 8 SDHA mutations were found in 5 samples (18.5%). Besides those previously identified, 5 new SDHA mutations were found in other 3 samples: one sample harboured R171C and R589Q heterozygous missense mutation in exons 5 and 13 respectively. The other one harboured G419R and E564K heterozygous missense mutations in exons 9 and 13 respectively. The third sample harboured a delCAG immediately upstream of exon 5, in heterozygosis on PB and in homozygosis on T. A SDHB heterozygous mutation (301delT) in exon 4 was found on 1 PB sample. Conclusions: the presence of SDHA mutations has been confirmed in a subgroup of WT GIST patients. All subunits of SDH complex should be sequenced on WT GIST patients in order to explore the frequency and any linkage between each other and the pathogenetic and clinical significance.