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 in vitro functional assays. We defined clear disruption as a significant reduction (P < 0.05, Student’s paired t-test) of > 50% in either RAS GAP activity or in NF-SPRED1 binding. In addition, if the mean expression of a variant was significantly reduced by > 50%, and both RAS GAP activity and SPRED1 were significantly reduced but the mean reduction was < 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 > 50% in expression as insufficient evidence to support pathogenicity if we detected normal levels of RAS GAP activity and SPRED1 binding.