Comprehensive Analysis of the NF1 gene Using Long-Read Sequencing Improved Neurofibromatosis type 1 Molecular Diagnosis

Clinical diagnosing Neurofibromatosis type 1 (NF1) in pediatrics are facing challenges because of limited presence of age-dependent phenotypes, and the limited detection rate by current approaches for the complexity of the NF1 gene. Here we developed a comprehensive analysis of NF1 (CANF1) combining 14 long-range locus-specific PCR, 25 gap primers and long-read sequencing (LRS) for sequence analysis of the NF1 gene. In this blind retrospective study, the clinical utility of CANF1 was evaluated in 191 samples (181 pediatric probands, 10 NF1 parents) by comparing to the control methods, mainly next generation sequencing (NGS). The results exhibited 176 probands (176/181 = 97.2%) having concordant results, and the other 5 probands (2.8%) with improved findings including: one was established a new diagnosis (c.5812 + 332A > G in deep intron) and four were improved with precise CNV breakpoints. In 127 pediatric NF1 probands with limited clinical manifestations, this assay received a detection rate of 92.9%, which is higher than NGS. In conclusion, this study constructed a comprehensive analysis of NF1 employing LRS, which can reliably identify various type variants of the NF1 gene in one assay. This CANF1 assay can help in screening NF1 with more precise molecular diagnosis than conventional methods, particularly for individuals with unfulfilling NF1 diagnosis solely by clinical phenotypes.