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

Roelof Koster ,Constance T R M Stumpel ,Han G Brunner ,M J Louwers,C.n Van Oosterhoud ,Maaike Vreeburg ,Aimée Paulussen ,Van Der Ppam Paul Schoot ,Floor A.m Duijkers ,Martijn P G Broen ,Marjolijn C.j Jongmans ,Michel Van Geel ,Demis Tserpelis ,Kasper Derks ,Rita D Brandão ,Astrid S Plomp ,Radek Szklarczyk ,C E P Van Roozendaal,Mala Misra-Isrie ,Margje Sinnema ,Arthur Van Den Wijngaard ,Saskia A.j Lesnik Oberstein ,Liesbeth Spruijt ,Kathleen Claes ,Marinus J Blok

doi:10.1038/s41525-021-00258-w

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.

Highlights

Neurofibromatosis type 1 (NF1; MIM 162200) is one of the most common autosomal dominant tumor-predisposition disorders affecting ~1 in 3000 individuals[1,2]
The only missed previously reported normal rare event is from a transcript ‘ins 9a/9br’ that is supposed to be specific for neuronal tissue, and not detectable in blood[25,26,27]
DNA and RNA diagnostics is the presence of a mosaic postzygotic NF1 pathogenic variant, in specific affected tissue, but absent in Alternative splicing is an important mechanism wherein different blood

Summary

INTRODUCTION

Neurofibromatosis type 1 (NF1; MIM 162200) is one of the most common autosomal dominant tumor-predisposition disorders affecting ~1 in 3000 individuals[1,2]. A variant that shows an effect with a well-established functional study; especially a robust, reproducible and validated study in a clinical diagnostic laboratory will get a pathogenic strong 3 (PS3) score. As such a potential splice variant that shows a loss-of-function effect could get a PS3 score and in some cases a PVS1 score. The functional study most often employed for splice variant validation is conventional Reverse Transcription (RT)-PCR, which has its drawbacks It is laborious, time consuming and prone to preferential amplification of transcripts depending on primer choice. A diagnostic procedure was designed that uses an in-house developed tool QURNAs (Quantitative enrichment of aberrant splicing events in targeted RNAseq) to facilitate the detection and quantification of normal and/or pathogenic RNA splicing events in targeted RNA sequencing data

RESULTS

DISCUSSION

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