# Neurofibromin 1 (NF1) Splicing Mutation c.61-2A>G: From Aberrant mRNA Processing to Therapeutic Implications In Silico

## TL;DR

This study explores a rare NF1 mutation that causes abnormal protein production and identifies a potential gene-editing treatment.

## Contribution

The paper introduces a new framework for studying splicing disorders and identifies CRISPR-Cas9 prime editing as a viable treatment for the NF1 c.61-2A>G mutation.

## Key findings

- The NF1 c.61-2A>G mutation activates a cryptic splice site, causing a frameshift and protein truncation.
- The mutation creates a novel methylated CpG site, revealed through long-read sequencing.
- CRISPR-Cas9 prime editing is the only viable in vivo correction strategy for this mutation.

## Abstract

The neurofibromin 1 (NF1) splice-site mutation c.61-2A>G (rs1131691100) is a rare, pathogenic, autosomal dominant variant that disrupts NF1 tumor-suppressor function, causing neurofibromatosis type 1 (NF1). Its pathogenic mechanism is poorly understood, and the potential for personalized therapeutic genome editing remains unknown due to the absence of a standard framework for investigating splicing disorders. Here, we performed a comprehensive multi-omics analysis of a de novo c.61-2A>G case from South Korea, integrating short- and long-read whole genome sequencing, whole transcriptome sequencing, and methylation profiling. We confirm that c.61-2A>G abolishes the canonical splice acceptor site, activating a cryptic splice acceptor 16 nucleotides downstream in exon 2. This splicing shift generates a 16-nucleotide deletion, causing a frameshift and premature stop codon that truncates the protein’s N-terminal region. Long-read sequencing further reveals that the mutation creates a novel CpG dinucleotide, which is methylated in the majority of reads. Finally, we assessed therapeutic correction strategies, revealing that CRISPR-Cas9 prime editing is the only viable approach for in vivo correction. This study provides the first comprehensive multi-omics characterization of the NF1 c.61-2A>G mutation and establishes a minimal framework for precision therapeutic development in silico in monogenic splicing disorders.