Long-read sequencing for NF1 gene analysis: enhancing diagnostic accuracy for Neurofibromatosis type 1

The Clinical diagnosis of Neurofibromatosis type 1 (NF1) in pediatric patients is challenged by incomplete manifestation of age-dependent phenotypes, and molecular genetic testing is usually required to confirm the diagnosis. Early differential diagnosis is particularly crucial for children presenting solely with multiple Cafe-au-lait spots (CALMs). Here we developed a comprehensive analysis of the NF1 gene (CANF1) based on long-range PCR and long-read sequencing (LRS) for genetic testing of NF1. This blinded retrospective study evaluated the clinical utility of CANF1 in 191 samples from 189 individuals (180 probands, 9 NF1 family members) by comparing it to next-generation sequencing (NGS), primarily exome sequencing (ES), as control methods. The results demonstrated concordant findings in 97.4% (186/191) of samples and 97.2% (175/180) of probands, and discordant results in 2.6% (5/191) of samples and 2.8% (5/180) of probands, including one newly established diagnosis due to a patient harboring the pathogenic deep intronic variant c.5812 + 332A > G. Among 126 pediatric probands with NF1, this assay achieved a diagnostic yield of 92.1%, outperforming ES with cost-competitive advantages. In conclusion, this study established an NF1 genetic assay employing LRS, demonstrating reliable detection for various variant types of the NF1 gene. The CANF1 assay provides an alternative screening approach for precise and cost-effective NF1 diagnosis, particularly valuable for pediatric cases not fulfilling NF1 clinical diagnostic criteria but presenting with a characteristic NF1 feature such as CALMs.