Pathogenic Neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq

Purpose Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. A targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. Methods RNA was extracted from lymphocytes, followed by targeted NF1 RNAseq. An in-house developed tool (QURNAS) was used to calculate the enrichment score (ERS) for each splicing event. Results This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). Conclusion Targeted NF1 RNAseq can be successfully used to detect pathogenic RNA splicing variants, complementary to DNA based diagnostics.