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.