Bridging the Diagnostic Gap in Neurofibromatosis Type 1: When to Consider NF1 cDNA Sequencing.

Molecular confirmation of neurofibromatosis type 1 (NF1) is essential for accurate diagnosis, reproductive counseling, and lifelong surveillance, yet genomic DNA (gDNA)-based next-generation sequencing (NGS) panels may miss deep intronic or complex structural variants. We report five children meeting the 2021 NF1 clinical diagnostic criteria who had negative DNA-based NGS testing but were subsequently diagnosed using NF1 cDNA-based testing (NF1 cDNA testing), which identified three deep intronic variants, one 5'UTR variant, and one exonic insertion, including three novel variants. Four of the five variants produced abnormal-to-normal transcript ratios of approximately 1:1, consistent with complete loss of normal transcript from the affected allele. These variants, detected by NF1 cDNA testing, are predicted to result in the same clinical phenotype and natural history as NF1 exonic variants, underscoring the value of NF1 cDNA testing as a second-tier test when clinical suspicion remains high despite negative gDNA-based tests.