Identification of protein variants in gastrointestinal stromal tumor KIT/PDGFRA wild type (WT GISTs) with RNA massively parallel sequencing and computational analysis

Massively parallel RNA sequencing, followed by data analysis, allows to discover novel single nucleotide variants and to identify new potential target genes in rare diseases, such as WT-GISTs. By this, mutations at the level of the A subunit of the succinate dehydrogenase (complex II) of the mitochondrial inner membrane were characterized and validated with Sanger sequencing as tumoral common markers of two young adults (1). After SNP calling and validation, the effect of the variation at the protein level is either a truncation of the protein chain or a mutation that is identified as disease-related with a predictor, SNPs & GO (2), suited to identify disease-associated residue substitution on the basis of the protein sequence and its function. Further investigation of the effects of the mutation on the protein stability indicates that the disease associate mutations hamper folding stability by decreasing the number of hydrogen bonds and of other stabilizing interactions in the FAD binding domain of SDHA. This approach highlights also private variants in the two young adults. Among the private disease-related variants we identified in one patient a K1775E substitution in the myosin heavy chain 9 (MYH9) and a R206H substitution in the triosephosphate isomerase 1 (TPI1). The second patient carried a V815M mutation in a oxoglutarate dehydrogenase-like protein (OGDHL). Interestingly all the mutations that are labeled disease-associated with SNPs & GO are also promoting protein destabilization according to a predictor suited to evaluate protein destabilization upon mutation starting from the protein sequence (I-Mutant2, 3). The results are also confirmed with protein structure computational analysis. We performed Sanger sequencing on both tumor and peripheral blood samples and found that private mutations were germline heterozygous.