Introduction
Variant classification is key to diagnosing genetic disorders. Clinical
and genetic investigations, in silico predictions, population
data, and analysis of pre-mRNA splicing and protein structure and
function all help establish variant pathogenicity.
Neurofibromatosis type 1 (NF1; MIM# 162200) is an autosomal dominant
disorder characterized by cafe-au-lâit macules, Lisch nodules, axillary
freckling, cutaneous neurofibromas and a wide range of patient specific
symptoms [Peltonen et al., 2012; Jett and Friedman, 2010; Ly and
Blakeley, 2019]. NF1 has an incidence of ˜1/3500 and is caused by
inactivation of the NF1 tumour suppressor [Ratner and Miller,
2015]. The canonical 12 kb NF1 mRNA transcript, NM_000267.3,
encodes a 2818 amino acid (320 kDa) GTPase activating protein (GAP)
called neurofibromin (NF). Loss or inactivation of NF1 results in
increased RAS signaling and the development of lesions characteristic
for NF1 [Cichowski and Jacks, 2001].
Legius syndrome (LS; MIM# 611431) is an autosomal dominant disorder
characterized by cafe-au-lâit macules, axillary and inguinal freckling,
lipomas, as well as macrocephaly, learning disabilities and
developmental delay [Brems et al., 2007]. LS has an incidence of
˜1/75000 and is caused by inactivation of SPRED1 that encodes the
Sprouty-related protein with an EVH (Ena/Vasp homology) domain 1
(SPRED1). SPRED1 recruits NF to the plasma membrane where it stimulates
GTP hydrolysis by membrane-bound RAS [Stowe et al., 2012]. The
functional relationship between NF and SPRED1 helps explain the
phenotypic overlap between NF1 and LS. Indeed, while some amino acid
substitutions impair NF RAS GAP activity to cause NF1, other changes
that do not affect RAS GAP activity cause NF1 by disrupting the
interaction with SPRED1 [Dunzendorfer-Matt et al., 2016]. Similarly,
changes to SPRED1 disrupt the interaction with NF and cause LS [Hirata
et al., 2016; Führer et al., 2019; Yan et al., 2020].
Molecular genetic analysis can establish a diagnosis of NF1 or LS: the
identification of an inactivating change, such as a frameshift or
nonsense variant provides strong evidence to support pathogenicity.
However, variants that could affect pre-mRNA splicing or introduce
damaging changes into the NF or SPRED1 proteins are more difficult to
classify. In our center, DNA-based molecular screening identifies a
variant in NF1 or SPRED1 for which pathogenicity is
uncertain in ˜20% of cases [van Minkelen et al., 2014]. The
American College of Medical Genetics and Genomics (ACMG) has provided
guidelines for the interpretation of genetic variants [Richards et
al., 2015]. Strong evidence for classifying a variant as pathogenic
can be obtained by performing functional experiments (ACMG criterium
PS3) [Hirata et al., 2016; Yan et al., 2020; Zatkova et al., 2004; Ars
et al., 2003; Messiaen et al., 2000; Thomas et al., 2012]. We
initiated functional assessment of pre-mRNA splicing and NF-SPRED1
function for variants of uncertain clinical significance (VUS)
identified in our cohort of individuals with NF1 or LS, and implemented
these tests in our diagnostic laboratory. Establishing variant
pathogenicity would provide individuals with certainty regarding their
affection status and follow-up, and would facilitate prenatal
diagnostics.
We investigated the effects of 34 variants on NF1 pre-mRNA
splicing and 66 variants on NF and SPRED1 function. The combination of
RNA and protein studies enabled us to fully investigate the effects of
the different variants. For some variants, mRNA splicing analysis was
required to identify the correct protein variant to test in the
functional assays. In other cases, the identification of defects inNF1 pre-mRNA splicing made functional testing of putative
missense changes redundant. The results of the RNA and functional
experiments, together with clinical and genetic data, were used to
classify the variants, following ACMG guidelines.