{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:59:48Z","timestamp":1785405588773,"version":"3.56.0"},"reference-count":56,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T00:00:00Z","timestamp":1784592000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32470667"],"award-info":[{"award-number":["32470667"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32470667"],"award-info":[{"award-number":["32470667"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"National High Level Hospital Clinical Research Funding","award":["2025-PUMCH-C-008"],"award-info":[{"award-number":["2025-PUMCH-C-008"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>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&gt;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\u201395% 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&gt;C (p.Ser2132Pro).<\/jats:p>","DOI":"10.3390\/genes17070838","type":"journal-article","created":{"date-parts":[[2026,7,21]],"date-time":"2026-07-21T16:16:39Z","timestamp":1784650599000},"page":"838","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Prime Editing-Based Functional Characterization Supports a Likely Pathogenic Interpretation of NF1 c.6394T&gt;C (p.Ser2132Pro)"],"prefix":"10.3390","volume":"17","author":[{"given":"Jiayu","family":"Wu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Complex, Severe, and Rare Diseases, Center for Bioinformatics, National Infrastructures for Translational Medicine, Institute of Clinical Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangyu","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Lab of Molecular Oncology, National Cancer Center\/National Clinical Research Center for Cancer\/Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Song","family":"Liu","sequence":"additional","affiliation":[{"name":"Center for Bioinformatics, National Infrastructures for Translational Medicine, Institute of Clinical Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenyu","family":"Ma","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Complex, Severe, and Rare Diseases, Center for Bioinformatics, National Infrastructures for Translational Medicine, Institute of Clinical Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoyue","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Complex, Severe, and Rare Diseases, Center for Bioinformatics, National Infrastructures for Translational Medicine, Institute of Clinical Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1038\/s41586-019-1879-7","article-title":"A brief history of human disease genetics","volume":"577","author":"Claussnitzer","year":"2020","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"D835","DOI":"10.1093\/nar\/gkz972","article-title":"ClinVar: Improvements to accessing data","volume":"48","author":"Landrum","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"D1313","DOI":"10.1093\/nar\/gkae1090","article-title":"ClinVar: Updates to support classifications of both germline and somatic variants","volume":"53","author":"Landrum","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_4","unstructured":"The Impact of Genomic Variation on Function (IGVF) Consortium (2024). 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