Italiano et al. BMC Cancer 2012, 12:408 http://www.biomedcentral.com/1471-2407/12/408 characterized by defects in cellular respiration and ac- tivation of pseudohypoxia signalling pathways [11]. The succinate dehydrogenase protein complex II (SDPC II) catalyzes the oxidation of succinate. SDHB is one of four protein subunits forming succinate dehydrogenase, the other three being SDHA, SDHC and SDHD. Loss of SDHB expression results in inhib- ition of the degradation of Hypoxia Inducing Factors (HIF), which in turn impairs apoptosis, and induces angiogenesis and glycolysis [12-15]. Loss of SDHB ex- pression was first identified in GIST occurring in the context of Carney triad and in a subset of pediatric and adult GISTs with similar characteristics [9]. Loss of SDHB is also seen in WT GIST occuring in the context of CSS with genlius mutation of SDHB or SDHC (II). However, such mutations were also found in about 10% of sporadic GIST lacking KIT or PDGFRA mutation [11]. The mechanisms involved in loss of SDHB expression in SDH-deficient GIST with- out an associated SDHB or SDHC mutations remain unclear. One possible explanation is loss of function mutations in the SDHA gene, which have been re- cently identified in four patients (one pediatric and 3 young adult) with sporadic GIST lacking KIT or PDGFRA mutations [16,17]. The aim of this study was to assess globally by next generation sequencing mutations in the SDH-pathway, as well as determine the mutational and expression status of SDHA in a series of syndromic and sporadic GIST without muta- tions in KIT or PDGFRA. Methods Patients Samples from 17 patients with gastric GIST selected on the basis of wild-type status for the KIT and PDGFRA genes were included for analysis. In 6 cases DNA from frozen tissue was available for next gener- ation sequencing; in the remaining cases DNA was available from paraffin embedded material. Thirteen cases were diagnosed in children (≤18 years) and four cases in young adults (defined as older than 18 but younger than age of 30; 3 females and 1 male). Among the 13 pediatric cases (11 females, 2 males), five were diagnosed in the context of Carney’s triad (CT). The mean age at diagnosis for pediatric patients and young adults was 12 (range 8–18) and 23 years old (range 21–26), respectively. In all cases, KIT (exons 9, 11, 13, and 17), PDGFRA (exon 12, 14, 18) and BRAF (exon 15) genotyping was performed as previously described [18]. The clinicopathologic and genotype findings of seven of the pediatric and two of the young adult GIST cases have been previously described [19]. The study was approved by the Insti- tutional Review Board (IRB-protocol 02–060). Page 2 of 7 Sequencing by Oligonucleotide Ligation and Detection (SOLiD) and variant detection Six cases, including five children, one of whom in the context of CT, and one young adult were analyzed by the SOLiDTM (Sequencing by Oligonucleotide Ligation and Detection) platform. This next generation sequen- cing technology interrogates two bases at a time by ligation chemistry and detection of one of four colors associated with those specific two bases. Whole exome capture was performed on 1–3 μg of high quality gen- omic DNA using the SureSelect Human All Exon Kit, which targets 38 MB of exonic sequences, according to the protocol provided by the manufacturer (Agilent, Santa Clara, CA, USA). Enriched DNA libraries are then sequenced on a SOLiD 3plus system (Applied Bio- systems, Carlsbad, CA, USA), generating 68 million reads (50 bp). 86% of the targeted region was sequenced at a 10x-coverage. The colorspace CSFASTA and QUAL files were first converted to double encoded FASTQ files which are then mapped to the target genome (hg19) using BWA with default options plus the colorspace mode option (−c). The output SAM files are tagged with read group ids merge across runs for the same library and then process with MarkDuplicates from Picard. Overlapping paired reads are resolved to remove redundant sequence. Then all the BAM for all samples are merged and process through the GATK toolkit to first Realign in/dels and the base Q scores are recalibrated with Recalibration tool. Paired samples (tumor/normal) are processed in pairs by muTect and we also run the entire cohort through the GATK Unified Genotyper to call both SNPs and in/dels. The variant output file (the VCF file) from the Unified Genotyper was then annotated using the SNPeff program with the UCSC RefSeq HG19 database to annotated the effect of the mutation. The raw output contained approximately 38,000 events. We then used a fairly stringent set of criteria to filter these calls. Only calls marked "PASS" be the Unified Genotype were retained all other events were filtered out. Further we removed events that had a non-reference allele fre- quency (NRAF) less than 10%. We also then removed any events that were annotated in dbSNP (v132) and removed those not annotated as HIGH IMPACT by SNPeff. This lead to a list of five events. For all 5 events we manually inspected the read pileups in IGV to look for possible artifacts. Of the 5 only one had no obvious defects. The other 4 had either strand bias issues and/or position bias (the variant reads tended to show the variants at the 3' end of the reads). Targeted exon resequencing The mutational status of SDHA (exon 2, 9 and 13) was assessed by direct Sanger sequencing of genomic DNA.