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

Area of Science:

• Genetics and Molecular Biology
• Cancer Genomics
• Cell Signaling

Background:

• Neurofibromatosis type 1 is caused by heterozygous loss-of-function variants in the NF1 gene, which encodes neurofibromin, a RAS-GTPase-activating protein.
• Missense variants in NF1 outside the GAP-related domain (GRD) are challenging to classify due to limited functional data.
• The NF1 variant c.6394T>C (p.Ser2132Pro) is currently classified as a variant of uncertain significance in ClinVar.

Purpose of the Study:

• To investigate the functional consequences of the NF1 variant c.6394T>C (p.Ser2132Pro).
• To determine if functional evidence supports reclassifying this variant as likely pathogenic under the ACMG/AMP framework.

Main Methods:

• In silico analysis including population databases, evolutionary conservation, and predictive tools.
• Structural mapping onto the neurofibromin cryo-EM model.
• Functional studies in HEK293T and A375 cells using prime editing to introduce the variant at the endogenous NF1 locus, assessing neurofibromin levels, RAS-GTP dynamics, and MAPK pathway activity.

Main Results:

• The p.Ser2132Pro variant was absent in population databases, affected a conserved residue, and received deleterious in silico predictions.
• Endogenous expression of p.Ser2132Pro significantly reduced neurofibromin protein abundance (88-95%) with minimal impact on NF1 transcript levels.
• The variant impaired RAS-GTP signal termination, increased steady-state RAS-GTP, and elevated ERK phosphorylation under specific treatment conditions.

Conclusions:

• The NF1 c.6394T>C (p.Ser2132Pro) variant leads to a cellular loss-of-function primarily through reduced neurofibromin protein abundance and impaired RAS regulation.
• The combined functional and in silico evidence supports a Likely Pathogenic classification for NF1 c.6394T>C (p.Ser2132Pro) under the ACMG/AMP guidelines.