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

Abstract

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. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. 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%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1.

Fig. 1. Drop in enrichment score of normal exon splice junctions for all NF1 patients is clearly visible in close proximity to their specific pathogenic splicing event.

Enrichment score of normal exon-exon splice junctions of the validation cohort (a) and the replication cohort (b). Shown are the ERS score for the normal exon splice junctions for the validation (a) and replication (b) cohort. Annotated in the figures are the drop in ERS due to the pathogenic splicing event. Normal reference exon splice junctions are expected to have a median ERS ~1. center line of the boxplot represents the median value (Q2), bounds of box are Q1 and Q3 respectively and whiskers Q1–1.5xIQR and Q3 + 1.5xIQR respectively. Outliers are depicted with color and shape matching the specific sample (see inner figure legend).

Fig. 2. For all patient derived samples with a pathogenic splicing event a concomitant drop of the normal exon junction in close proximity is visible.

Enrichment score of normal exon-exon splice junctions of the undetermined cohort (a), annotated in the figure are the drop in ERS due to the detected pathogenic splicing event. Normal reference exon splice junctions are expected to have a median ERS ~1. center line of the boxplot represents the median value (Q2), bounds of box are Q1 and Q3 respectively and whiskers Q1–1.5xIQR and Q3 + 1.5xIQR respectively. Outliers are depicted with color and shape matching the specific sample (see inner figure legend). Splicing of undetermined 1 and 8 explained (b/c). Shown are the median ERS and reads for all samples within the same run vs the actual ERS and reads of the sample indicated; undiagnosed 1 (b) and undiagnosed 8 (c).