Identification of single nucleotide variants in gastrointestinal stromal tumor KIT/PDGFRA wild-type (WT GISTs) by massively parallel sequencing.

Abstract

10046 Background: Recently SDH variations were identified in KIT and PDGFRA wild type GISTs. However the causative genomic alterations of these GISTs still remain unknown. Massively parallel sequencing allows identifying novel putative variants. Methods: Whole transcriptome paired-end RNA sequencing was performed by Illumina GAIIx system using a 75 bases paired-end strategy on tumor samples of two young adults patients (P1 and P2) affected by gastric WT-GIST (age 28 and 30 years). Sequences were aligned with BWA against exon+junction references. SNVmix2 was used for SNP calling, identifying by this 2045 and 1780 coding non-synonymous novel single nucleotide variants (SNVs) in P1 and P2, respectively. After checking misalignments by SAMTools, the variants were filtered to increasing SNPcall confidence: SNVs with quality read score > 30 (error probability of 0.1%), total coverage > 40, and ratio between coverage of alterate base and total coverage >0.3 were labeled high confidence. Single point mutations were translated at the protein level and their likelihood of being disease-associated was computed with SNP&GO and confirmed with Sanger sequencing. Results: In both patients common mutations in SDHA and different private SNV were highlighted. Among the private disease-related SNVs we identified mutations in MYH9 (myosin heavy chain 9) and TPI1 (triosephosphate isomerase 1) in patient P1, and a mutation in OGDHL (oxoglutarate dehydrogenase-like) in P2. MYH9 is involved in cell motility and cytokinesis, while OGDHL is an enzyme of the Krebs cycle, as the SDH complex, and TPI1 is involved in glycolysis. Ongoing validation by Sanger sequencing on DNA from tumor and peripheral blood (PB) already confirmed the presence of heterozygous MYH9 K1775E in P1 and OGDHL V815M in P2, both in tumor and PB, showing that these mutations are germinal. Conclusions: Massively parallel RNA sequencing, followed by data analysis, allows to discover novel single nucleotide variants and to identify new potential target genes in WT-GIST.