Functional assays combined with pre-mRNA splicing analysis improve
variant classification and diagnostics for individuals with
Neurofibromatosis type 1 and Legius syndrome.
Abstract
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). Establishing 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 variants on
pre-mRNA splicing and NF/SPRED1 function. The purpose of this work was
to improve NF1 and SPRED1 variant classification. To help
establish the pathogenicity of NF1 and SPRED1 variants
identified in individuals with NF1 or LS, we employed 4 assays: (i)
analysis of patient RNA by RT-PCR; (ii) in vitro exon trap
analysis of NF1 pre-mRNA splicing; (iii) in vitro analysis
of NF RAS GAP activity; and (iv) in vitro analysis of the
NF-SPRED1 interaction. In 69/105 (66%) cases we obtained evidence to
support variant pathogenicity according to American College of Medical
Genetics guidelines, demonstrating the utility of functional approaches
for NF1 and SPRED1 variant classification and NF and LS
diagnostics.