Whole USH2A Gene Sequencing Identifies Several New Deep Intronic Mutations

Area of Science:

• Genetics
• Molecular Biology
• Ophthalmology

Background:

• Deep intronic mutations causing pseudoexon insertions are often missed.
• USH2A gene mutations are a primary cause of Usher syndrome type II (USH2).
• Transcript analysis for USH2A mutations is challenging, leaving some USH2 patients with unidentified mutations.

Purpose of the Study:

• To develop and validate a DNA next-generation sequencing (NGS) approach for identifying deep intronic variants in USH2A.
• To assess the impact of these variants on gene splicing.
• To offer an alternative strategy for detecting deep intronic mutations when RNA analysis is not feasible.

Main Methods:

• Developed and validated a DNA NGS approach for deep intronic variant detection in USH2A.
• Utilized minigene assays to confirm the splicing consequences of identified mutations.
• Tested an antisense morpholino oligonucleotide in vitro to evaluate its potential for splicing restoration.

Main Results:

• Identified three novel deep intronic mutations in USH2A.
• Confirmed that these mutations lead to pseudoexon insertions and affect splicing.
• Demonstrated high inhibition rates with an antisense morpholino oligonucleotide for a specific mutation, indicating therapeutic potential.

Conclusions:

• The developed DNA NGS strategy is effective for identifying deep intronic mutations in USH2A, even when RNA analysis is difficult.
• This bioinformatics pipeline is gene-size independent, suggesting broad applicability for other disease-linked genes.
• The findings support a promising strategy for diagnosing USH2 and highlight potential avenues for molecular therapy.