Prime Editing-Based Functional Characterization Supports a Likely Pathogenic Interpretation of NF1 c.6394T>C...

Background/Objectives: NF1 encodes neurofibromin, a RAS-GTPase-activating protein (GAP), and heterozygous loss-of-function variants cause neurofibromatosis type 1. Missense variants outside the GAP-related domain (GRD) are difficult to classify because supporting functional evidence is limited. NF1 c.6394T>C (p.Ser2132Pro) is currently listed in ClinVar as a variant of uncertain significance. We examined its functional consequences and evaluated whether the resulting evidence supports a likely pathogenic interpretation under the ACMG/AMP framework. Methods: We evaluated p.Ser2132Pro using population databases, evolutionary conservation, calibrated in silico predictors, and structural mapping onto the full-length cryo-EM model of neurofibromin. The variant was then introduced at the endogenous NF1 locus in HEK293T and A375 cells by prime editing, and we measured neurofibromin abundance, transcript levels, RAS-GTP dynamics, and MAPK pathway reactivation after PLX4032 treatment. Evidence was integrated under the ACMG/AMP framework. Results: p.Ser2132Pro was absent from population databases, affected a highly conserved residue buried within the C-terminal HEAT domain, and received concordant deleterious predictions from calibrated in silico tools. At the endogenous locus, p.Ser2132Pro reduced neurofibromin abundance by 88-95% in both cell models while NF1 transcript levels were only modestly reduced, impaired RAS-GTP signal termination, doubled steady-state RAS-GTP in A375 cells, and produced 5-fold higher ERK phosphorylation than the non-targeting control under PLX4032 treatment. Conclusions: These findings support a cellular loss-of-function effect of p.Ser2132Pro characterized primarily by reduced neurofibromin abundance with impaired neurofibromin-dependent RAS regulation. Under a conservative ACMG/AMP interpretation, the combined evidence supports a Likely Pathogenic interpretation of NF1 c.6394T>C (p.Ser2132Pro).