Development of a novel RNA sequencing approach that identifies aberrant splicing in cancer predisposing genes

Background: High-throughput sequencing efforts in molecular tumor diagnostics detect increased numbers of novel genetic variants, including the ones that are predicted to affect splicing. Current approach for predicting whether a specific variant may result in aberrant RNA transcripts often consists of in-silico prediction using bioinformatics prediction tools, which is sometimes followed by RT-PCR analysis of the RNA extracted from blood, or functional splicing reporter assays. Although high-quality patient RNA analysis is usually preferred, such RNA is not always available, or the analysis is hampered due to degradation of aberrant transcripts through nonsense-mediated decay. To overcome this issue, we investigated the possibility of analyzing RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues, with the underlying hypothesis that formalin fixation may inhibit RNA degradation, enabling the detection of aberrant RNA products. Methods: RNA analysis was performed for 13 canonical splice site variants predicted or known to affect splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Of 11 variants, total nucleic acid was obtained from FFPE tumor tissues. For two variants, no FFPE material was available, but EBV-transformed B-cells carrying a splice variant were cultured. Primers were designed to amplify the exon-exon boundary predicted to be affected. Housekeeping gene expression was assessed to determine presence of RNA. Results: Total nucleic acid was successfully isolated for 10 variants from eight FFPE tumor tissues and two EBV-transformed B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known splice variant, present in the B-cell line, the known aberrant splice product could only be detected after formalin fixation of the cells, suggesting that formalin fixation possibly inhibited RNA degradation. Conclusion: Aberrant splicing was successfully identified in all known splice variants and three of four variants predicted to affect splicing, of which the splice effect was previously unknown. Using a RNA sequencing approach, somatic splice variants could be easily and rapidly analyzed, enabling retrospective analysis of variants predicted to result in splicing when only FFPE material is available in clinical settings. Citation Format: Anne M. Jansen, Heleen M. van der Klift, Jaap D. van Eendenburg, Carli M. Tops, Juul T. Wijnen, Frederik J. Hes, Ajay Goel, Hans Morreau, Tom van Wezel. Development of a novel RNA sequencing approach that identifies aberrant splicing in cancer predisposing genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 649.