In vitro assessment of the effects of NF1 variants on NF p.1_2069
RAS GAP activity
Many NF1 VUS identified in our cohort mapped outside the NF GRD.
Attempts to introduce nucleotide changes into a full-length NF1expression 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 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 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 (P < 0.05, Student’s pairedt -test). If the mean estimated RAS GAP activity was <
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 < 50% of wild-type NF; Student’s pairedt -test P < 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 (P > 0.05; Student’s paired
t-test), but was > 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 (P > 0.05; Student’s
paired t-test; Figure 3B, variants indicated in black).