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.