A 25-year-old woman had multifocal, epithelioid gastric GIST (CARE checklist is available as Additional file 1). KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 12, 14, and 18) molecular analysis did not show evidence of mutations. The clinical/pathological features matched well with those describing dSDH GIST (female sex, young age, and multifocal and epithelioid gastric GIST). Moreover, in a gene expression analysis previously reported (GSE20710), we showed that the expression profile of this tumor (GIST_21) was similar to the profile of 3 patients (GIST_07, GIST_10, and GIST_24) who we subsequently identified as having mutated SDHA [3, 8]. To better characterize GIST_21, all SDHx subunits were sequenced by the Sanger method, but the analysis did not show any mutations.
To search for alterations that might not have been detected by Sanger sequencing, we performed paired-end whole exome sequencing of GIST_21 (see Additional file 2 for materials and methods), without finding any mutations in any SDHx coding sequences or flanking intronic regions. Exhaustive exome sequencing analysis revealed no pathogenic mutations in other GIST-related genes, such PDGFRA, BRAF, and neurofibromin 1, and only a nonsynonymous missense variant in exon 18 of KIT. This variant is described in the COSMIC database (COSM133780) in 1 case of malignant melanoma. Because the matched normal sample for this patient was not available, we were not able to determine if this KIT variant was somatic or germinal.
By gene expression analysis, we examined the expression levels of the 4 subunits of the SDH complex; as shown in Fig. 1a, we detected a remarkable down-modulation of SDHC mRNA in GIST_21 with respect to all other GIST samples, both SDHA-mutant and KIT/PDGFRA-mutant GIST. Moreover, Western blot immunoassay showed that GIST_21 had normal SDHA but markedly decreased SDHB protein expression (Fig. 1b) and would be predicted to have a loss of SDH complex activity. Additionally, SNP6.0 genotyping on GIST_21 showed a normal karyotype, with no detectable loss of heterozygosity or chromosome 1q deletion [9].
Hypermethylation of the SDHC promoter region was recently associated with Carney triad–related GIST and dSDH GIST without SDHx mutation [6, 7]. Thus, we designed a bisulfite methylation assay to look for potential epimutations in the 2 known CpG islands of the SDHC promoter regions, CpG17 and CpG27. This analysis was performed in 4 dSDH (including GIST_21 and 3 GIST without SDHx mutations) and in 8 KIT/PDGFRA-mutated GIST. Hypermethylation of the SDHC promoter was found only in GIST_21, and this alteration was detected in heterozygosis at all CpG sites (Fig. 1c). The methylated case showed a significant 0.63-fold decrease in SDHC mRNA expression level (P = 0.0001) in comparison with the 11 methylation-negative cases (Fig. 1d).