In vitro assessment of the effects of NF1 and SPRED1 variants on the NF-SPRED1 interaction
Pathogenic 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 59NF1 and 5 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 (P < 0.05, Student’s paired t-test) of > 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 > 50% for 26NF1 and 2 SPRED1 variants, including the NF1p.Asp1217Tyr and 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 > 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 (P > 0.05, Student’s paired t-test; Figures 4B, indicated in black).