PATH-61. Decoding Diagnostic Uncertainty for Tumor Predisposition Conditions: RNA Testing of Variants of Uncertain Significance in Neurofibromatosis

BACKGROUND The neurofibromatoses are classified as neurofibromatosis type 1 (NF1) or schwannomatosis (SWN). NF1 is caused by pathogenic variants in the NF1 gene; SWN is associated with variants in NF2, SMARCB1, LZTR1 or other yet undiscovered genes. When genetic testing for patients with suspected neurofibromatosis identifies variants of uncertain significance (VUS), evaluation with RNA analysis can clarify relevance and guide clinical management. Case Discussion • PATIENT 1 30-year-old female with severe multifocal neuropathic pain and multiple extracranial T2 hyperintense lesions on MRI. Genetic testing revealed a NF1 VUS (c.8050 + 93A>T) in blood. RNA analysis demonstrated aberrant mRNA splicing, leading to reclassification as pathogenic and confirming a diagnosis of NF1. • PATIENT 2 A 50-year-old female with intermittent lower extremity pain and a benign mixed schwannoma/neurofibroma on histology. A NF2 VUS (c.448-5T>C) in blood showed no impact on splicing by RNA analysis. The variant was reclassified as likely benign, ruling out germline NF2. • PATIENT 3 A 6-year-old with café-au-lait macules, axillary freckling, and negative NF1 germline testing was found to have a SPRED1 VUS (c.377-13T>A) in blood. RNA analysis demonstrated no impact on splicing. Thus this VUS was reclassified as likely benign and clinical attention was refocused on possible mosaic/segmental NF1. Discussion Although clinical criteria remain central to diagnosis, the functional characterization of VUS through RNA studies can refine diagnoses in patients with equivocal clinical presentations to guide surveillance or targeted drug therapies and inform family planning/testing of family members. CONCLUSION We report a previously uncharacterized NF1 variant now reclassified as pathogenic - impacting both treatment planning and genetic counseling - via RNA analysis. In contrast, two other variants were confirmed as benign, allowing patients to avoid inappropriate interventions. These cases underscore the value of RNA-based testing in the interpretation of VUS and the optimization of care in genetic tumor predisposition conditions.