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Related Concept Videos

RNA Splicing01:32

RNA Splicing

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Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
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Related Experiment Video

Updated: Jun 17, 2025

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
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Investigating Splice Defects in USH2A Using Targeted Long-Read Sequencing.

Shwetha Chandrasekhar1, Siying Lin1,2, Neringa Jurkute1,2,3

  • 1UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK.

Cells
|August 9, 2024
PubMed
Summary

This study uses RT-PCR and Oxford Nanopore Technology sequencing to analyze USH2A mRNA in nasal cells. It functionally characterizes splicing alterations from variants, improving molecular diagnosis for Usher Syndrome and retinitis pigmentosa.

Keywords:
Oxford nanopore sequencingUSH2Acryptic splice-altering variantdeep intronic variantinherited retinal diseaselong-read sequencingnasal epithelial cells

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Identification of Alternative Splicing and Polyadenylation in RNA-seq Data
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Area of Science:

  • Genetics and Genomics
  • Molecular Biology
  • Ophthalmology and Audiology

Background:

  • Biallelic variants in the USH2A gene cause retinitis pigmentosa (RP) and Usher Syndrome Type 2 (USH2), leading to vision impairment and hearing loss.
  • Despite advances in next-generation sequencing, a significant diagnostic gap persists for these conditions, potentially due to non-coding or uncertain significance variants.
  • Accurate molecular diagnosis is crucial for understanding disease mechanisms and developing targeted therapies for USH2A-associated disorders.

Purpose of the Study:

  • To demonstrate the clinical utility of reverse transcription-polymerase chain reaction (RT-PCR) coupled with Oxford Nanopore Technology (ONT) sequencing for analyzing USH2A mRNA transcripts.
  • To functionally characterize the splice-altering effects of candidate USH2A variants, including deep intronic and synonymous variants of uncertain significance.
  • To bridge the diagnostic gap in molecular testing for patients with RP and USH2 by identifying pathogenic splicing defects.

Main Methods:

  • Recruitment of five individuals with USH2 or non-syndromic RP and uncertain USH2A genotypes identified by whole genome sequencing.
  • Isolation of mRNA from nasal epithelial cells and subsequent RT-PCR amplification of USH2A transcripts.
  • Sequencing of amplified USH2A mRNA using Oxford Nanopore Technology (ONT) to detect and characterize splicing abnormalities, validated against in silico predictions (SpliceAI).

Main Results:

  • In silico splice-altering predictions for all candidate USH2A variants were confirmed by ONT sequencing.
  • Functional characterization revealed simple and complex mis-splicing patterns, including cryptic splicing, caused by deep intronic and uncertain significance variants.
  • A complex cryptic splicing pattern was observed for the c.3812-3_3837dup variant, resulting in predominant exon 18 skipping and a predicted stop gain, confirming its pathogenicity.

Conclusions:

  • RT-PCR-ONT sequencing of USH2A mRNA from nasal epithelial cells is a clinically valuable method for detecting and functionally characterizing splicing defects.
  • This approach successfully identified pathogenic simple and complex splicing alterations arising from previously uncharacterized deep intronic variants and variants of uncertain significance in USH2A.
  • The study significantly contributes to resolving the molecular basis of Usher Syndrome Type 2 and retinitis pigmentosa, addressing a critical gap in genetic diagnostics.