Functional Assays Combined with Pre-mRNA-Splicing Analysis Improve Variant Classification and Diagnostics for...

Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by inactivating variants in NF1 and SPRED1. NF1 encodes neurofibromin (NF), a GTPase-activating protein (GAP) for RAS that interacts with the SPRED1 product, Sprouty-related protein with an EVH (Ena/Vasp homology) domain 1 (SPRED1). Obtaining a clinical and molecular diagnosis of NF1 or LS can be challenging due to the phenotypic diversity, the size and complexity of the NF1 and SPRED1 loci, and uncertainty over the effects of some NF1 and SPRED1 variants on pre-mRNA splicing and/or protein expression and function. To improve NF1 and SPRED1 variant classification and establish pathogenicity for NF1 and SPRED1 variants identified in individuals with NF1 or LS, we analyzed patient RNA by RT-PCR and performed in vitro exon trap experiments and estimated NF and SPRED1 protein expression, RAS GAP activity, and interaction. We obtained evidence to support pathogenicity according to American College of Medical Genetics guidelines for 73/114 variants tested, demonstrating the utility of functional approaches for NF1 and SPRED1 variant classification and NF and LS diagnostics.