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 NF1 c.3829G>C, p.(Gly1277Arg) and c.3651T>A, p.(Asp1217Glu) variants in the NF GRD in 2 individuals with NF1. We introduced both variants and the pathogenicNF1 c.3826C>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 theNF1 c.3829G>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 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 (P > 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 (P < 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 (P > 0.025; Student’s paired t-test with Bonferroni correction; black in Figure 2D).