DISCUSSION
cDNA model system. Since discovery of the NF1 gene in
1990 research efforts have been hindered by the lack of a full-length
coding cDNA. This is partially due to the size of the gene and toxicity
of the human cDNA construct. We are aware of three potentially availableNF1 cDNAs: mouse Nf1 (Wallis et al., 2018) (isoform 2 with
2839 amino acids), codon optimized human NF1 (Bonneau, Lenherr,
Pena, Hart, & Scheffzek, 2009) (isoform 1 with 2818 aa), and a humanNF1 with mini-intron 35-36 (Cui & Morrison, 2019) (isoforms 1
and 2). As the mNf1 cDNA is highly homologous to endogenous humanNF1 , we have developed and validated the mouse Nf1 cDNA
expression system that allows us to examine the biochemical effects of
any Nf1 genetic variant. We have been able to perform
dose-response studies to titrate in varying amounts of mNf1 cDNA
and are able to detect a clear dose-response in terms of levels of
neurofibromin and repression of GTP-Ras levels and pERK/ERK ratios,
giving us confidence in both the cDNA and the functional assays.
Assay Performance: We see similar dynamic ranges between the
GTP-Ras and pERK/ERK experiments, with WT cDNA able to repress both
GTP-Ras and pERK/ERK ratios by about half that seen with EV control. The
Morrison lab reported that inactive variants (R1276P) served as better
controls than empty vectors (Cui & Morrison, 2019). While our R1276Q
and R1276X both displayed similar GTP-Ras levels as our EV, both showed
insignificantly higher pERK/ERK ratios. Indeed, we see significant
variability in the pERK/ERK ratios, leading to large error bars.
Regardless, each individual cDNA performs similarly between the two
assays and across multiple experiments, indicating that the results are
reliable. Each variant’s ability to affect Ras signaling relative to WT
cDNA is consistent between assays; however, we have noted exceptions.
delM992 and C379R both have significantly different GTP-Ras levels,
whereas pERK/ERK ratios fail to reach significance; and R1748X has a
significantly different pERK/ERK ratio, but GTP-Ras doesn’t reach
significance.
Using our previously published cDNA expression system (Wallis et al.,
2018) we can evaluate functional significance of variants in individuals
with NF1. These data indicate that each variant has a slightly different
functional profile in terms of both protein stability and the ability to
inhibit Ras signaling. We incorporated non-pathogenic variants in our
assays: E1327G, Q1336R, and P2782L. These three cDNAs performed
similarly to the WT cDNA in terms of neurofibromin levels and the
ability to inhibit Ras signaling, adding confidence to our functional
profile characterizations. Some pathogenic variants result in
instability of neurofibromin but still retain GRD function. The best
examples of this are with the G848R and L1957P variants, which retain
the ability to repress Ras activity (Figure 5) in both of our assays,
yet are unstable and produce less than 50% of the neurofibromin that is
observed with WT cDNA protein (Figure 4). Some variants result in stable
protein but have lost GRD function. For example, K1423E and S1997R both
produce neurofibromin levels similar to WT cDNA, yet display
significantly elevated Ras activity compared with WT cDNA. We also
observed that certain variants demonstrated both unstable protein and
loss of function; L1490P and D1623G exhibited lowered neurofibromin
levels, with inability to repress Ras signaling. Nonsense variants
resulting in truncated proteins have variable stability. Nonsense
variants with truncations after the GRD may maintain GRD function in
these overexpression assays. While both R1947X and R2550X show increased
Ras signaling, it is not statistically different from WT cDNA.