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\begin{document}

\title{Functional assays combined with pre-mRNA splicing analysis improve
variant classification and diagnostics for individuals with
Neurofibromatosis type 1 and Legius syndrome.}



\author[1]{Mark Nellist}%
\author[2]{Hannie C.W. Douben}%
\author[2]{Marianne Hoogeveen-Westerveld}%
\author[1]{Jesse Louwen}%
\author[1]{Marian Kroos-de Haan}%
\author[2]{Mattijs Punt}%
\author[1]{Babeth van Ommeren}%
\author[1]{Leontine van Unen}%
\author[1]{P. Elfferich}%
\author[1]{Esmee Kasteleijn}%
\author[1]{Yolande Van Bever}%
\author[1]{Margreethe van Vliet}%
\author[2]{Rianne Oostenbrink}%
\author[1]{Jasper Saris}%
\author[1]{Anja Wagner}%
\author[2]{Yvette van Ierland}%
\author[1]{Tjakko J. van Ham}%
\author[2]{Rick Van Minkelen}%
\affil[1]{Erasmus MC Afdeling Klinische Genetica}%
\affil[2]{Erasmus MC}%


\vspace{-1em}



  
  \date{May 15, 2024}


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\selectlanguage{english}
\begin{abstract}
Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by
inactivating variants in \emph{NF1} and \emph{SPRED1}. \emph{NF1}
encodes neurofibromin (NF), a GTPase activating protein (GAP) for RAS,
that interacts with the \emph{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 \emph{NF1} and
\emph{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 \emph{NF1} and \emph{SPRED1} variant classification. To help
establish the pathogenicity of \emph{NF1} and \emph{SPRED1} variants
identified in individuals with NF1 or LS, we employed 4 assays: (i)
analysis of patient RNA by RT-PCR; (ii) \emph{in vitro} exon trap
analysis of \emph{NF1} pre-mRNA splicing; (iii) \emph{in vitro} analysis
of NF RAS GAP activity; and (iv) \emph{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 \emph{NF1} and \emph{SPRED1} variant classification and NF and LS
diagnostics.%
\end{abstract}%



\sloppy


\textbf{Functional assays combined with pre-mRNA splicing analysis
improve variant classification and diagnostics for individuals with
Neurofibromatosis type 1 and Legius syndrome.}

Hannie Douben\textsuperscript{1*}, Marianne
Hoogeveen-Westerveld\textsuperscript{1*}, Mark
Nellist\textsuperscript{1*}, Jesse Louwen\textsuperscript{1}, Marian
Kroos-de Haan\textsuperscript{1}, Mattijs Punt\textsuperscript{1,3},
Babeth van Ommeren\textsuperscript{1}, Leontine van
Unen\textsuperscript{1}, Peter Elfferich\textsuperscript{1}, Esmee
Kasteleijn\textsuperscript{1}, Yolande van Bever\textsuperscript{1},
Margreethe van Vliet\textsuperscript{1}, Rianne
Oostenbrink\textsuperscript{2,4}, Jasper J. Saris\textsuperscript{1},
Anja Wagner\textsuperscript{1}, Yvette van Ierland\textsuperscript{1,4},
Tjakko van Ham\textsuperscript{1}, Rick van
Minkelen\textsuperscript{1,4}

\textsuperscript{1.} Department of Clinical Genetics, Erasmus University
Medical Center, Rotterdam, The Netherlands

\textsuperscript{2.} Department of Pediatrics, Erasmus University
Medical Center, Rotterdam, The Netherlands

\textsuperscript{3.} current address: Department of Neuroscience,
Erasmus University Medical Center, Rotterdam, The Netherlands

\textsuperscript{4.} ENCORE Expertise Center for Neurodevelopmental
Disorders, Erasmus University Medical Center, Rotterdam, The Netherlands

* these authors contributed equally

Correspondence and requests for materials should be addressed to: Prof.
T. van Ham (t.vanham@erasmusmc.nl) or Dr. R. van Minkelen
(r.vanminkelen@erasmusmc.nl), Department of Clinical Genetics, Erasmus
University Medical Center, Wytemaweg 80, 3015CN Rotterdam, The
Netherlands.

\textbf{Abstract}

Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by
inactivating variants in \emph{NF1} and \emph{SPRED1} .
\emph{NF1}encodes neurofibromin (NF), a GTPase activating protein (GAP)
for RAS, that interacts with the \emph{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 \emph{NF1}
and\emph{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 \emph{NF1} and \emph{SPRED1} variant classification.

To help establish the pathogenicity of \emph{NF1} and
\emph{SPRED1}variants identified in individuals with NF1 or LS, we
employed 4 assays: (i) analysis of patient RNA by RT-PCR; (ii) \emph{in
vitro} exon trap analysis of \emph{NF1} pre-mRNA splicing; (iii)
\emph{in vitro} analysis of NF RAS GAP activity; and (iv) \emph{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 \emph{NF1} and \emph{SPRED1} variant
classification and NF and LS diagnostics.

\textbf{Keywords}

Neurofibromatosis type 1, Legius syndrome, neurofibromin, SPRED1,
functional genetics

\textbf{Introduction}

Variant classification is key to diagnosing genetic disorders. Clinical
and genetic investigations, \emph{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-l\selectlanguage{ngerman}âit 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 \selectlanguage{english}~1/3500 and is caused by
inactivation of the \emph{NF1} tumour suppressor {[}Ratner and Miller,
2015{]}. The canonical 12 kb \emph{NF1} mRNA transcript, NM\_000267.3,
encodes a 2818 amino acid (320 kDa) GTPase activating protein (GAP)
called neurofibromin (NF). Loss or inactivation of \emph{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-l\selectlanguage{ngerman}âit macules, axillary and inguinal freckling,
lipomas, as well as macrocephaly, learning disabilities and
developmental delay {[}Brems et al., 2007{]}. LS has an incidence of
\selectlanguage{english}~1/75000 and is caused by inactivation of \emph{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; F\selectlanguage{ngerman}ührer 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 \emph{NF1} or \emph{SPRED1} for which pathogenicity is
uncertain in \selectlanguage{english}~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 \emph{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
in\emph{NF1} 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.

\textbf{Materials and Methods}

\emph{Editorial Policies and Ethical Considerations}

Informed consent was provided by all subjects, as required by the
institutional review board of the Erasmus Medical Center, and according
to standard diagnostic protocols.

\emph{Patient assessment and selection of variants for testing}

The Erasmus MC Department of Clinical Genetics NF1/LS cohort consists of
\textgreater{}4900 index cases suspected of NF1 or LS based on the
international clinical diagnostic criteria {[}Legius et al., 2021{]} for
whom DNA has been submitted for genetic testing of \emph{NF1}
and/or\emph{SPRED1} . Prior to functional testing, variants were
classified using the available clinical and genetic data. Nomenclature
for all the reported variants is according to HGVS guidelines {[}HGVS,
2016{]}. For assay validation, we included 8 variants that had been
classified as pathogenic and/or subjected to functional evaluation
previously: NF1 p.Leu90Pro {[}Xiao et al., 2018{]}, p.Met992del
{[}Koczkowska et al., 2020{]}, p.Asp1217Tyr {[}Hirata et al., 2016{]},
p.Arg1276Gly {[}Mattocks et al., 2004{]} and p.Lys1423Glu {[}Thomas et
al., 2012, Koczkowska et al., 2019{]}; and SPRED1 p.Val44Asp,
p.Thr102Met {[}Hirata et al., 2016{]} and p.Ser105Ala {[}Yan et al.,
2020{]}.

\emph{Constructs, antibodies and cell-lines}

\emph{NF1} minigene exon trap constructs and NF expression plasmids were
generated using standard cloning techniques {[}Sambrook et al., 1989{]},
Gibson assembly {[}Gibson et al., 2009{]} and/or site-directed
mutagenesis (see Supplementary Information for details). All constructs
were verified by sequencing of the complete insert. Nucleotide and amino
acid numbering are according to \emph{NF1} transcript NM\_000267.3 and
SPRED1 transcript NM\_152594.2, unless specified otherwise.

Antibodies were from Cell Signaling Technology (Danvers, U.S.A.)(rabbit
anti-HA; mouse anti-HA; 9B11 mouse anti-myc), Invitrogen (mouse anti-V5)
Sigma-Aldrich (St. Louis, U.S.A.) (mouse and rabbit anti-FLAG) and
Li-Cor Biosciences (Lincoln, U.S.A.)(goat anti-rabbit 680 nm and goat
anti-mouse 800 nm conjugates). Anti-FLAG affinity beads were from
Sigma-Aldrich, glutathione-sepharose was from GE Healthcare (Uppsala,
Sweden).

HEK 293T and COS-7 cells were maintained in Dulbecco's Modified Eagle
Medium (DMEM)(Lonza, Verviers, Belgium) containing 10\% fetal calf
serum, 50 U/ml penicillin and 50 \selectlanguage{greek}μ\selectlanguage{english}g/ml streptomycin in a humidified
37\textsuperscript{o}C, 5 - 10\% CO\textsubscript{2} incubator.

\emph{Assessment of the effects of NF1 variants on NF1 pre-mRNA splicing
in patient material}

Reverse transcriptase (RT) PCR was performed on 1 - 2 \selectlanguage{greek}µ\selectlanguage{english}g total RNA as
described in the Supplementary Information.

\emph{In vitro assessment of the effects of NF1 variants on NF1 pre-mRNA
splicing}

Exon trap experiments were performed as described previously
{[}Dufner-Almeida et al., 2019; Halim et al., 2017{]}. See Supplementary
Information for details.

\emph{In vitro assessment of RAS GAP activity}

To estimate RAS GAP activity, HA-H-RAS together with wild-type NF or
variant NF were expressed in COS-7 or HEK 293T cells. GTP-bound RAS was
subsequently isolated using glutathione-agarose beads coated with
recombinant GST-RAF-RBD {[}van Triest et al., 2001{]}. See Supplementary
Information for details.

\emph{In vitro assessment of the NF1-SPRED interaction}

To investigate the NF-SPRED interaction, FLAG-SPRED1 together with
wild-type or variant NF were co-expressed in HEK 293T cells. NF-SPRED1
complexes were immunoprecipitated using anti-FLAG affinity beads
(Sigma-Aldrich). See Supplementary Information for details.

\textbf{Results}

\emph{Assessment of the effects of NF1 variants on NF1 pre-mRNA
splicing}

We investigated the effects on \emph{NF1} pre-mRNA splicing of 34
variants that had been identified in individuals suspected of NF1
(Figure 1). In 13 cases, the results of the \emph{in vitro} exon trap
experiments were confirmed by analysis of RNA from a blood sample from
the corresponding individual; in 4 cases subject RNA was analyzed
directly, without performing exon trapping (Figure 1B). For the
remaining subjects, RNA was not available.

We detected one or more abnormal splice products, either \emph{in vitro}
, in subject RNA, or in both for 25 variants (74 \%). Exon skipping
(type I defect) {[}Anna and Monika, 2018{]} was observed for 18
variants; 2 variants resulted in incorporation of a pseudo-exon causing
premature truncation of the \emph{NF1} ORF (type II defect); utilization
of a non-canonical splice site, resulting in exon truncation (type III
defect) was observed for 4 variants; and 3 variants caused intron
retention (type IV defect). In 5 cases, abnormal splicing resulted in an
in-frame deletion (Supplementary Information, Table S1; Figure 1C). Of
these, the c.288+3 A\textgreater{}T, r.205\_288del, p.(Arg69\_Gly96del)
and c.2710A\textgreater{}T, r.2707\_2850del, p.(Cys904\_Val951del)
deletions were selected to determine whether the deletion affected NF
activity (see below). In 7 cases, a missense change completely prevented
canonical \emph{NF1} pre-mRNA splicing, making assessment of NF function
redundant. In 9 cases, we did not observe an effect of the variant
on\emph{NF1} pre-mRNA splicing, either \emph{in vitro} or in subject RNA
(Supplementary Information, Table S1). These cases included 5 missense
variants, of which 2 could be subjected to functional assessment (see
below). In total, analysis of \emph{NF1} pre-mRNA splicing assisted in
the classification of 25 variants as likely pathogenic (Supplementary
Information, Table S1).

\emph{In vitro assessment of the effects of NF1 variants on NF RAS GAP
activity}

The ability of the NF GAP-related domain (GRD; amino acids 1180 - 1504)
to inactivate RAS can be determined by measuring the amount of active,
GTP-bound RAS in the presence of the NF GRD. High levels of RAS-GTP
indicate that the activity of the NF GRD is impaired and can help
classify a variant as pathogenic {[}Thomas et al., 2012{]}. We
identified the \emph{NF1} c.3829G\textgreater{}C, p.(Gly1277Arg) and
c.3651T\textgreater{}A, p.(Asp1217Glu) variants in the NF GRD in 2
individuals with NF1. We introduced both variants and the
pathogenic\emph{NF1} c.3826C\textgreater{}G, p.(Arg1276Gly) variant into
a NF V5-p.1180\_1504 expression construct {[}Thomas et al. 20122{]} and
determined the RAS GAP activity of the variant proteins in transfected
cells using a pull-down assay for GTP-bound RAS (Figure 2). Neither the
p.Arg1276Gly variant nor the p.Gly1277Arg variant had RAS GAP activity,
as estimated from the levels of GTP-bound RAS (RAS GTP) in the pull-down
fraction (Figure 2C), supporting the likely pathogenicity of
the\emph{NF1} c.3829G\textgreater{}C, p.(Gly1277Arg) substitution.

Expression levels of the wild-type and variant NF V5-p.1180\_1504
proteins were low. To facilitate detection, we modified the NF
V5-p.1180\_1504 construct by altering the sequence preceding the
initiation codon to correspond to the Kozak consensus sequence and by
introducing an extra C-terminal V5-epitope tag. We observed robust
expression of the resulting wild-type NF V5-p.1180\_1504-V5 protein and
derived 13 \emph{NF1} variants identified in individuals in our NF1
patient cohort in the NF V5-p.1180\_1504-V5 expression construct (Figure
2B). In 5 cases, the variant lacked RAS GAP activity: levels of
GTP-bound RAS (RAS-GTP) were not significantly different to those in the
absence of NF V5-p.1180\_1504-V5 (\emph{P} \textgreater{} 0.025;
Student's paired t-test with Bonferroni correction; shown in red in
Figure 2D). In 2 cases, RAS GAP activity was significantly reduced
compared to wild-type NF V5-p.1180\_1504-V5, but significantly increased
compared to the absence of NF V5-p.1180\_1504-V5 (\emph{P} \textless{}
0.025; Student's paired t-test with Bonferroni correction; orange in
Figure 2D), suggesting that these variants impaired RAS GAP activity but
did not inactivate the GRD completely. The remaining 6 variants retained
full RAS GAP activity (\emph{P} \textgreater{} 0.025; Student's paired
t-test with Bonferroni correction; black in Figure 2D).

\emph{In vitro assessment of the effects of NF1 variants on NF p.1\_2069
RAS GAP activity}

Many \emph{NF1} VUS identified in our cohort mapped outside the NF GRD.
Attempts to introduce nucleotide changes into a full-length
\emph{NF1}expression construct were unsuccessful. However, we were able
to introduce variants into 2 expression constructs that encoded the
N-terminal 2069 amino acids of NF (Figure 3A). The only difference
between these 2 constructs was the inclusion of sequences corresponding
to a neuron-specific \emph{NF1} transcript encoding a 10 amino acid
insertion {[}Geist and Gutmann, 1996{]}. We referred to the proteins
expressed from these constructs as NF p.2069myc, corresponding to
reference transcript NM\_000267.3, and NF p.420ins10myc, corresponding
to the transcript encoding the insertion. We did not detect significant
differences between the wild-type p.2069myc and p.420ins10myc proteins.
Both were expressed at similar levels and exhibited equivalent RAS GAP
activity.

We introduced 66 \emph{NF1} variants in these constructs, including 13
variants previously tested in the NF V5-p.1180\_1504 or NF
V5-p.1180\_1594-V5 constructs, and estimated the RAS GAP activity of all
the 66 variant proteins (Figure 3B; Supplementary Information, Table
S2). Some were as active as the corresponding wild-type NF protein, some
had severely attenuated RAS GAP activity and others had intermediate
levels of activity. This made it difficult to assign an exact cut-off
value to distinguish pathogenic, inactivating variants. Therefore, we
devised an empirical scheme to categorize the variants. We compared the
mean RAS GAP activities of the variants, and whether these were reduced
relative to the wild-type (\emph{P} \textless{} 0.05, Student's
paired\emph{t} -test). If the mean estimated RAS GAP activity was
\textless{} 50\% of the wild-type and if this reduction was
statistically significant, then we considered it evidence for disruption
of NF RAS GAP activity and supporting evidence for pathogenicity (ACMG
criteria PS3). This was the case for 25 variants (Figure 3B; variants
indicated in red: mean activity \textless{} 50\% of wild-type NF;
Student's paired\emph{t} -test \emph{P} \textless{} 0.05), 19 of which
mapped to the NF1 GRD. In 12 cases, the results with the NF
p.1180\_1504-V5 and V5-p.1180\_1504-V5 proteins were confirmed. We did
not detect significant impairment of RAS GAP activity by the NF
p.420ins10myc p.Thr1199Ile variant, in contrast to the reduction
associated with the NF V5-p.1180\_1504-V5 p.Thr1199Ile variant (compare
Figures 2D and 3B, and see Discussion).

In 16 cases, RAS GAP activity was significantly reduced compared to the
wild-type control (\emph{P} \textgreater{} 0.05; Student's paired
t-test), but was \textgreater{} 50\% of the wild-type value (Figure 3B;
variants indicated in orange). We considered this insufficient evidence
to support pathogenicity. The remaining variants did not show evidence
for impaired RAS GAP activity: mean activity was not significantly
different to wild-type NF (\emph{P} \textgreater{} 0.05; Student's
paired t-test; Figure 3B, variants indicated in black).

\emph{In vitro assessment of the effects of NF1 and SPRED1 variants on
the NF-SPRED1 interaction}

Pathogenic \emph{NF1} variants can disrupt the interaction between NF
and SPRED1 without affecting NF RAS GAP activity {[}8{]}. The wild-type
NF p.2069myc and p.420ins10myc proteins could be immunoprecipitated (IP)
together with co-expressed wild-type FLAG-SPRED1 (Figure 4A) and we used
this coimmunoprecipitation (coIP) assay to determine whether
59\emph{NF1} and 5 \emph{SPRED1} variants affected the NF-SPRED1
interaction. We compared the NF p.2069myc and p.420ins10myc wild-type
and variant signals in the IP fractions (Figure 4B) and categorized the
variants using the same criteria as for the RAS GAP assay: a significant
reduction (\emph{P} \textless{} 0.05, Student's paired t-test) of
\textgreater{} 50\% in the mean NF signal in the IP fraction was
evidence for an effect of a variant on the NF-SPRED1 interaction, and
supported pathogenicity.

The NF-SPRED1 interaction was reduced \textgreater{} 50\% for
26\emph{NF1} and 2 \emph{SPRED1} variants, including the
\emph{NF1}p.Asp1217Tyr and \emph{SPRED1} p.Val44Asp variants previously
shown to disrupt the NF-SPRED1 interaction {[}Hirata et al., 2016{]}
(Figures 4B, variants indicated in red). In 9 cases there was a
significant reduction in the NF signal in the IP fraction, but the mean
value was \textgreater{} 50\% of the wild-type and therefore not
sufficient evidence to support pathogenicity (Figure 4B, variants
indicated in orange). The remaining variants did not show significant
differences from the wild-type controls (\emph{P} \textgreater{} 0.05,
Student's paired t-test; Figures 4B, indicated in black).

\emph{In vitro assessment of the effects of NF1 and SPRED1 variants on
the expression and stability of NF and SPRED1}

The estimated RAS GAP activity and the estimated strength of the
NF-SPRED1 interaction might reflect differences in the expression and/or
stability of the variant proteins. To investigate effects on NF and
SPRED1 stability, we compared the wild-type and variant signals in the
cell lysates by immunoblotting. In 12 cases, the mean expression of a
variant was reduced to below 50\% of the wild-type (Figure 4C, indicated
in red). Of these, 6 also showed a significant reduction of
\textgreater{} 50\% in either RAS GAP activity, SPRED1 binding or both,
and in 2 cases, p.Leu90Arg and p.Arg69\_Gly96del, both RAS GAP activity
and NF-SPRED1 binding were significantly reduced, but by \textless{}
50\%. The remaining variants showed either significantly reduced
expression (\emph{P} \textless{} 0.05 Student's paired t-test), but
still \textgreater{} 50\% of wild-type (that is, \textless{} 50 \%
reduction; Figure 4C, indicated in orange), or did not affect NF or
SPRED1 expression or stability (\emph{P} \textgreater{} 0.05 Student's
paired t-test; Figure 4C, indicated in black).

\emph{Categorization of NF1 and SPRED1 variants, based on the results of
the functional assessment}

To use the results of the functional experiments to help classify the
variants, we applied empirical rules to define 2 groups. The first group
consisted of variants that clearly disrupted NF or SPRED1 function in
the \emph{in vitro} functional assays. We defined clear disruption as a
significant reduction (\emph{P} \textless{} 0.05, Student's paired
t-test) of \textgreater{} 50\% in either RAS GAP activity or in
NF-SPRED1 binding. In addition, if the mean expression of a variant was
significantly reduced by \textgreater{} 50\%, and both RAS GAP activity
and SPRED1 were significantly reduced but the mean reduction was
\textless{} 50\%, then we concluded that the effect on NF expression
could be biologically relevant, and therefore that there was evidence to
support pathogenicity (Supplementary Information, Tables S2 and S3). The
second group did not show clear evidence for disruption of NF1 or SPRED1
function. We considered a reduction of \textgreater{} 50\% in expression
as insufficient evidence to support pathogenicity if we detected normal
levels of RAS GAP activity and SPRED1 binding.

\textbf{Discussion}

To complement the molecular genetic test results from our diagnostic
laboratory, we performed functional assessment of 105 \emph{NF1}
and\emph{SPRED1} variants. We employed 4 assays: (i) analysis of subject
RNA by RT-PCR; (ii) \emph{in vitro} exon trap analysis of
\emph{NF1}pre-mRNA splicing; (iii) \emph{in vitro} analysis of NF RAS
GAP activity; and (iv) \emph{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 \emph{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 \emph{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 \emph{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 \emph{in vitro} and \emph{in vivo}RNA
data (Supplementary Information, Table S1). However, we did not identify
cases where a variant affected splicing \emph{in vitro} but not\emph{in
vivo} , or \emph{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
\emph{NF1}splicing, 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 \emph{NF1}
and\emph{SPRED1} 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 \emph{in vitro} assays might still retain sufficient
activity \emph{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 \textgreater{} 50\% reduction in either
RAS GAP activity or NF-SPRED1 binding as functional evidence to support
pathogenicity. We did not consider a \textgreater{} 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 (\emph{P} \textless{} 0.05, Student's paired
t-test), even if the RAS GAP activity or NF-SPRED1 interaction was
\textgreater{} 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 \emph{NF1} and
2\emph{SPRED1} variants, including the known pathogenic
variants\emph{NF1} p.Leu90Pro, p.Met992del, p.Asp1217Tyr, p.Arg1276Gly
and p.Lys1423Glu, and \emph{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
\emph{NF1}variants 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 \emph{NF1} variants in a full-length \emph{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 \emph{NF1} and
\emph{SPRED1}variant classification.

In summary, we applied \emph{in vitro} assays to investigate the effects
of \emph{NF1} and \emph{SPRED1} variants on \emph{NF1} pre-mRNA splicing
and NF-SPRED1 function. We tested 100 \emph{NF1} and 5
\emph{SPRED1}variants 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 \emph{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 \emph{NF1}
and\emph{SPRED1} variants. Implementation of these tests in our
diagnostic laboratory, together with consideration of the clinical,
population,\emph{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.

\textbf{Data availability}

Data and materials are available from the corresponding authors upon
reasonable request.

\textbf{Acknowledgements}

We thank Dr. Thomas and Prof. Upadhyaya (Institute of Medical Genetics,
Cardiff, United Kingdom), Prof. Yoshimura (Keio University, Japan),
Prof. Scheffzek (Medical University of Innsbruck, Austria), Prof.
Elgersma (Erasmus Medical Center, The Netherlands) and Prof. Zwartkruis
(University of Utrecht, The Netherlands) for providing plasmid
constructs. Jacqueline Boonman, Paul van den Berg, Astrid Dorhout, Femke
Hendriks and Willemieke Nobel and Sandra van der Sluijs - de Heer are
thanked for technical assistance and data curation. We thank Dr. van
Dooren and Dr. van de Laar (Erasmus Medical Center, The Netherlands),
Dr. Lesnik and Dr. Moghadasi (Leiden University Medical Center, The
Netherlands), Dr. Sinnema (Maastricht University Medical Center, The
Netherlands), Dr. Corsten (University Medical Center Groningen, The
Netherlands), Dr. Plomp (Amsterdam University Medical Center, The
Netherlands), Dr. Zekveld-Vroon (University Medical Center Utrecht, The
Netherlands) and Dr. Spruijt (Radboud University Medical Center, The
Netherlands) for providing patient samples and information.

This research is supported (not financially) by the European Reference
Network on Genetic Tumour Risk Syndromes (ERN GENTURIS)---Project ID No
739547. ERN GENTURIS is partly co-funded by the European Union within
the framework of the Third Health Programme ``ERN-2016---Framework
Partnership Agreement 2017--2021''.

\textbf{Author contributions}

Conceptualization: MN, JJS, TvH, RvM; experimentation: HD, MH-W, MN, JL,
MK-dH, MP, BvO, LvU, PE, EK; clinical investigation: YvB, MvV, RO, AW,
YvI; supervision: MH-W, MN, AW, TvH, RvM; writing, review and editing:
MN, AW, YvI, TvH, RvM.

\textbf{Conflict of Interest Statement}

All authors declare that there are no conflicts of interest.

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\textbf{Figure legends}

\textbf{Figure 1.} Analysis of \emph{NF1} pre-mRNA splicing.

(A) Schematic overview of \emph{NF1} exon trap constructs derived as
part of this study, showing the approximate location of the tested
variants. Exonic sequences are shown as boxes, intronic sequences as
horizontal lines; intronic sequence representing a pseudo-exon is
indicated by the shaded box. SS indicates a variant also subjected to
functional assessment (see Supplementary Table S2). Variant nomenclature
is according to reference transcript NM\_000267.3, except P: reference
transcript NM\_001042492.2.

(B) Approximate location of the \emph{NF1} variants investigated with
RT-PCR and Sanger sequencing in patient RNA in relation to the NF1
reference transcript (NM\_000267.3).

(C) Exon trap and RT-PCR analysis of the
NM\_000267.3(NF1):c.2710T\textgreater{}A variant. Agarose gel
electrophoresis of the NF1 exon 21 wild-type (WT) and
c.2710T\textgreater{}A exon trap RT-PCR products (left) and RT-PCR
products from the index patient and 3 control individuals (center). A
indicates the canonical exon 21 product; B indicates the product
obtained due to the cryptic splice site at c.2706 created by the
c.2710T\textgreater{}A substitution. Electropherogram of Sanger sequence
analysis of the RT-PCR products, confirming the abnormal r.2707\_2850del
transcript.

(D) Exon trap and RT-PCR analysis of the
NM\_000267.3(NF1):c.3871-3T\textgreater{}G and c.3974+5G\textgreater{}C
variants. Agarose gel electrophoresis of the NF1 exon 29 wild-type (WT)
and c.3871-3T\textgreater{}G, c.3974+5G\textgreater{}C and
c.3871-3T\textgreater{}G + c.3974+5G\textgreater{}C exon trap RT-PCR
products (indicated with the red box). Note the complete loss of the
wild-type product from the constructs containing both substitutions.

\textbf{Figure 2.} \emph{In vitro} functional assessment of RAS GAP
activity of the NF GRD.

(A) Schematic overview of the pull-down assay for NF RAS GAP activity.
Variants were first introduced into the wild-type NF expression
construct by site-directed mutagenesis (SDM), co-transfected into
mammalian cells in culture, together with a RAS reporter expression
construct and, after 5 minutes stimulation with EGF, the cells were
lysed and GTP-bound RAS subjected to GST-RAF-RBD pull-down with
glutathione-agarose beads. Lysate and pull-down fractions were
subsequently analyzed by immunoblotting.

(B) Schematic overview of NF (above), showing the different proposed
functional domains (CSRD, orange; TBD, green; SPRED1 interaction,
violet; SEC14-PH, cyan; HEAT, pink; SBD, blue) and the NF GRD expression
constructs used in this study (below). Amino acid changes are given
according to reference transcript NM\_000267.3. Variants derived in the
NF V5-p.1180\_1504 expression construct {[}Thomas et al., 2012{]} are
indicated in cyan; other variants were derived from the wild-type NF
p.V5-1180\_1504-V5 expression construct.

(C) Relative RAS GAP activity of the NF p.V5-1180\_1504 variants. NF
V5-p.1180\_1504 variants were co-expressed with HA-H-RAS and RAS GAP
activity was estimated in a pull-down assay using recombinant
GST-RAF-RBD to specifically bind RAS-GTP. The signals for HA-H-RAS in
the pull-down fractions were determined in 3 independent experiments.
The mean estimated RAS GAP activity is shown relative to the wild-type
(V5-GRD); = 1.0). Error bars represent the standard error of the mean.
Variants showing significantly reduced RAS GAP activity are indicated in
red (see main text for details).

(D) Relative RAS GAP activity of the NF p.V5-1180\_1504 variants.
Pull-down assay was performed as in (C); RAS GAP activity was estimated
relative to the wild-type control (V5-GRD-V5). Variants showing no
evidence for RAS GAP activity are indicated in red; variants with
reduced RAS GAP activity are shown in orange; active variants are
indicated in black (see main text for details). Quantification data of
at least 3 independent experiments is shown.

(E) Representative immunoblot showing the GST-RAF-RBD pull-down
fractions.

(F) Cell lysate fractions corresponding to the samples shown in (E).

\textbf{Figure 3.} \emph{In vitro} functional assessment of NF RAS GAP
activity.

(A) Schematic overview of neurofibromin (NF) (top) and the expressed
truncated NF proteins used for the functional experiments (below). The
C-terminal region absent from the expressed NF p.1\_2069-myc and
p.420ins10-myc proteins is indicated and the approximate positions of
the different variants are shown, including the 10 amino acid insertion
(420ins10) that differentiates between the two expressed wild-type
proteins. Variants derived in the NF p.1\_2069ins10-myc construct are
indicated in cyan; variants derived in the NF p.1\_2069-myc construct
are shown in black. To determine the RAS GAP activity of the variants,
the scheme shown in Figure 2A was used. Briefly, NF p.1\_2069-myc and NF
p.1\_2069ins10-myc variants were co-expressed with HA-H-RAS. RAS GAP
activity was estimated in a pull-down assay using recombinant
GST-RAF-RBD in at least 4 independent experiments, relative to the
wild-type NF p.1\_2069-myc or NF p.1\_2069ins10-myc (indicated in cyan)
proteins.

(B) Quantification of \emph{NF1} variant RAS GAP activity. Variants
showing evidence of disruption of RAS GAP activity (\textgreater{} 50\%
reduction in activity; \emph{P} \textless{} 0.05) are indicated in red;
variants with reduced RAS GAP activity are shown in orange (\textless{}
50\% reduction; \emph{P} \textless{} 0.05); active variants (no evidence
for reduced RAS GAP activity)(\emph{P} \textgreater{} 0.05) are
indicated in black (see main text for details). IT/RC: NF1
p.Ile1799Thr/Arg1809Cys (double \emph{cis} variant).

(C) Representative immunoblot showing the lysate (above) and GST-RAF-RBD
pull-down (below) fractions.

\textbf{Figure 4.} Functional assessment of the effects of \emph{NF1}and
\emph{SPRED1} variants on NF-SPRED1 interaction and NF expression.

(A) Schematic overview of the \emph{in vitro} functional assessment of
the NF1-SPRED1 interaction. Variants were introduced into the wild-type
(WT) expression construct by site-directed mutagenesis (SDM) and the NF
p.1\_2069-myc or p.1\_2069ins10-myc and FLAG-SPRED1 expression
constructs co-transfected into mammalian cells. NF-SPRED1 complexes were
isolated by immunoprecipitation (IP) using anti-FLAG agarose beads.
Lysate and IP fractions were subsequently analyzed by immunoblotting.
Signals for the variants relative to the WT proteins (NF p.1\_2069-myc,
NF p.1\_2069ins10-myc or FLAG-SPRED1) were determined in at least 3
independent experiments.

(B) Quantification of the IP signals for the \emph{NF1} (left)
and\emph{SPRED1} (right) variants. Variants showing a reduced
interaction (\textgreater{} 50\% reduction in signal in the coIP
fraction; \emph{P}\textless{} 0.05) are indicated in red; variants
showing \textless{} 50\% reduction (\emph{P} \textless{} 0.05) are shown
in orange; variants showing a comparable interaction to wild-type
NF-SPRED1 (\emph{P}\textgreater{} 0.05) are indicated in black (see main
text for details). IT/RC: NF1 p.Ile1799Thr/Arg1809Cys (double \emph{cis}
variant).

(C) Quantification of the signals for the expressed \emph{NF1} (left)
and \emph{SPRED1} (right) variants. Variants showing \textgreater{} 50\%
reduction (\emph{P} \textless{} 0.05) are indicated in red; variants
showing \textless{} 50\% reduction (\emph{P} \textless{} 0.05) are shown
in orange; variants expressed at levels comparable to the wild-type
control (\emph{P} \textgreater{} 0.05) are indicated in black (see main
text for details). IT/RC: NF1 p.Ile1799Thr/Arg1809Cys (double,\emph{cis}
variant).

(D) Representative immunoblot showing the anti-FLAG IP (above) and
lysate (below) fractions for \emph{NF1} variants.\selectlanguage{english}
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