Discussion
To complement the molecular genetic test results from our diagnostic laboratory, we performed functional assessment of 105 NF1 andSPRED1 variants. We employed 4 assays: (i) analysis of subject RNA by RT-PCR; (ii) in vitro exon trap analysis of NF1pre-mRNA splicing; (iii) in vitro analysis of NF RAS GAP activity; and (iv) in vitro analysis of the NF-SPRED1 interaction. In 69 cases (66%) we obtained evidence to support pathogenicity (Supplementary Information, Tables S1, S2 and S3).
In contrast to laboratories that specialize in NF1 variant detection and classification using patient RNA, our diagnostic laboratory performs molecular screening primarily on DNA samples [van Minkelen et al., 2014]. The decision to focus on DNA variant identification was originally taken to simplify the workflow and allow the laboratory to apply a standard method to variant detection for a wide range of genetic disorders. Direct analysis of RNA was not considered practical for routine screening in our setting. The in vitro exon trap experiments therefore provided a useful screen for identifying variants likely to affect splicing, without having to re-sample patients. We did not observe major discrepancies between the exon trap and RT-PCR results that would have led to a different classification for any of the variants tested, consistent with other work from our laboratory [Douben et al., in revision; Dekker et al., submitted]. Information on the observed in vitro effects could be provided to the clinician and the relevant individual(s) prior to taking a sample for confirmation. Moreover, the exon trap approach was a simple method to help resolve allele-specific patterns of pre-mRNA splicing that were unclear from the subject RNA data. The exon trap experiments indicated whether a variant prevented canonical splicing of an exon completely, or had only a partial effect. In 4 cases there were some minor differences between the in vitro and in vivoRNA data (Supplementary Information, Table S1). However, we did not identify cases where a variant affected splicing in vitro but notin vivo , or vice versa . Analysis of pre-mRNA splicing was also a useful screen for the functional assessments as it was not always obvious whether a variant was likely to affect splicing and/or protein function. In some cases, RNA analysis revealed abnormal NF1splicing, making functional assessment redundant, whereas in other cases, RNA analysis indicated that functional assessment of an in-frame deletion was required to help establish pathogenicity.
Compared to the exon trapping and RT-PCR experiments, assessment of NF-SPRED1 function was labour-intensive, time-consuming and the results were sometimes more difficult to analyze and interpret. Nonetheless, we obtained insight into the effects of multiple NF1 andSPRED1 variants on NF-SPRED1 function (Supplementary Information, Tables S2 and S3). We focused on 3 characteristics: (i) NF RAS GAP activity, (ii) the NF-SPRED1 interaction and (iii) NF/SPRED1 expression/stability, and devised an empirical scheme to categorize the variants. It is possible that some variants that disrupted NF-SPRED1 function in our in vitro assays might still retain sufficient activity in vivo to prevent NF1 or LS. Furthermore, differences in estimated activity or expression might be due to variation in transfection efficiency, cell numbers, immunoblotting artefacts or other processing errors. Therefore the results had to be interpreted with caution and in the light of the clinical and genetic evidence. Despite these caveats, we considered a > 50% reduction in either RAS GAP activity or NF-SPRED1 binding as functional evidence to support pathogenicity. We did not consider a > 50% reduction in expression/stability as sufficient evidence for pathogenicity unless it was concordant with significant disruption of both the RAS GAP activity and NF-SPRED1 interaction (P < 0.05, Student’s paired t-test), even if the RAS GAP activity or NF-SPRED1 interaction was > 50% of the wild-type value (indicated in orange in Figures 3B and 4B and Supplementary Information, Table S2). Variants that did not show significant reductions in RAS GAP activity, or the interaction with SPRED1 remained of uncertain significance, unless other evidence was obtained to support or exclude pathogenicity.
We obtained evidence supporting pathogenicity for 43 NF1 and 2SPRED1 variants, including the known pathogenic variantsNF1 p.Leu90Pro, p.Met992del, p.Asp1217Tyr, p.Arg1276Gly and p.Lys1423Glu, and SPRED1 p.Val44Asp (Supporting Information, Tables S2 and S3). None of these variants were identified more than once in the gnomAD (v2.1) database [https://gnomad.broadinstitute.org/](accessed 7/3/2022), and none were classified as benign or likely benign in Clinvar [https://www.ncbi.nlm.nih.gov/clinvar/] (accessed 7/3/2022). In all cases, the variant was identified in one or more individuals suspected of NF1 or LS. The remaining variants did not show sufficient evidence for an effect on NF or SPRED1 function to support pathogenicity, even though several are described as likely pathogenic in Clinvar (Supplementary Information, Table S2). In one case we observed a discrepancy between the results of the RAS GAP assay with the NF V5-p.1180_1504-V5 GRD and the NF p.420ins10 protein. The NF1 p.Thr1199Ile variant impaired RAS GAP activity of the GRD but did not significantly affect RAS GAP activity of the NF p.420ins10 protein (compare Figures 2D and 3B). It is possible that the NF GRD and p.420ins10 proteins have distinct sensitivities to changes in secondary structure. The extra scaffolding around the active site of the GRD provided by the p.420ins10 protein might restrict structural changes and thereby maintain RAS GAP activity.
We could not exclude pathogenicity based on the results of the functional assessment. We only interrogated 3 aspects of NF-SPRED1 function: RAS GAP activity, the NF-SPRED1 interaction and expression/stability. We did not investigate other putative functions of NF or SPRED1 [D’Angelo et al., 2006; Welti et al., 2011; Fadhlullah et al., 2019]. Furthermore, we were unable to investigate NF1variants that mapped distal to the C-termini of the p.2069myc and p.420ins10myc proteins. It would obviously be desirable to be able test all variants in the context of full-length NF and efforts to efficiently derive NF1 variants in a full-length NF1 expression construct are on-going in our laboratory. Nonetheless, despite these limitations, our work provided valuable information for individuals with NF1 and LS regarding their disease status and genetic risks, confirming the utility of functional testing for NF1 and SPRED1variant classification.
In summary, we applied in vitro assays to investigate the effects of NF1 and SPRED1 variants on NF1 pre-mRNA splicing and NF-SPRED1 function. We tested 100 NF1 and 5 SPRED1variants and obtained evidence to support pathogenicity in 69 cases (66%)(Supplementary Information, Tables S1, S2 and S3). The results of our experiments have been submitted to the NF1 and SPRED1 Leiden Open Variation Databases (https://databases.lovd.nl/shared/genes/NF1; https://databases.lovd.nl/shared/genes/SPRED1). Our work demonstrates that functional testing helps identify likely pathogenic NF1 andSPRED1 variants. Implementation of these tests in our diagnostic laboratory, together with consideration of the clinical, population,in silico and segregation data, has resulted in improved molecular diagnostics for individuals with NF1 and LS and facilitated appropriate monitoring, treatment and prenatal diagnostic options for family planning.