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

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene, often affecting tumor suppressor function.
• The specific splice-site mutation c.61-2A>G (rs1131691100) is rare and its pathogenic mechanism, particularly concerning splicing, is not well understood.
• Investigating splicing disorders requires a standardized framework, especially for exploring personalized genome editing therapies.

Purpose of the Study:

• To comprehensively characterize the pathogenic mechanism of the NF1 splice-site mutation c.61-2A>G using multi-omics analysis.
• To investigate the potential for therapeutic genome editing strategies for this specific NF1 mutation.
• To establish a framework for studying monogenic splicing disorders and developing precision therapies.

Main Methods:

• Integrated analysis of short- and long-read whole genome sequencing, whole transcriptome sequencing, and methylation profiling.
• Detailed examination of splicing alterations caused by the c.61-2A>G mutation.
• Assessment of CRISPR-Cas9 prime editing as a potential in vivo correction strategy.

Main Results:

• The c.61-2A>G mutation abolishes the canonical splice acceptor site, leading to activation of a cryptic site and a 16-nucleotide deletion in exon 2.
• This splicing alteration results in a frameshift, premature stop codon, and truncation of the NF1 protein's N-terminal region.
• Long-read sequencing identified a novel, methylated CpG dinucleotide created by the mutation, and CRISPR-Cas9 prime editing emerged as the sole viable in vivo correction approach.

Conclusions:

• This study provides the first in-depth multi-omics characterization of the NF1 c.61-2A>G mutation.
• The findings elucidate the molecular consequences of the splicing defect, including protein truncation and potential epigenetic modifications.
• A framework for precision therapeutic development in silico for monogenic splicing disorders is established, highlighting CRISPR-Cas9 prime editing's potential.