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.