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RNA Splicing01:32

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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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Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay
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Pathogenic neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq.

R Koster1, R D Brandão1,2, D Tserpelis1

  • 1Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands.

NPJ Genomic Medicine
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Summary

Targeted RNA sequencing effectively detects splicing errors in the NF1 gene, improving diagnosis for Neurofibromatosis type 1 (NF1) patients. This method identifies pathogenic variants missed by standard genetic tests, aiding in molecular confirmation.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Medical Diagnostics

Background:

  • Neurofibromatosis type 1 (NF1) is a genetic disorder caused by NF1 gene mutations.
  • A significant portion of NF1-causing variants affect RNA splicing, often evading conventional DNA sequencing and in silico analysis.
  • Accurate identification of splicing variants is crucial for NF1 diagnosis and patient management.

Purpose of the Study:

  • To develop and validate a targeted RNA sequencing (RNAseq) approach for detecting pathogenic RNA splicing variants in NF1.
  • To assess the efficacy of this RNAseq method in identifying known and novel splicing alterations.
  • To improve molecular diagnostic yield in NF1 patients, particularly those with unexplained symptoms.

Main Methods:

  • RNA was extracted from patient lymphocytes.
  • Targeted RNA sequencing was performed on the extracted RNA.
  • An in-house tool, QURNAs, was utilized to calculate the enrichment score (ERS) for splicing events.
  • The method was validated on patient cohorts with known pathogenic splice-variants in NF1.

Main Results:

  • The targeted RNAseq approach successfully detected all reference transcript exon splice junctions, previously described, and novel non-reference splicing events across tested cohorts.
  • All expected pathogenic splice-variants within the NF1 gene were identified.
  • In a cohort of 11 NF1 patients, the method confirmed splicing effects in 3 individuals with known variants and identified pathogenic deep-intronic splice variants in 2 of 8 previously undiagnosed patients (25% diagnostic yield).

Conclusions:

  • Targeted RNA sequencing is a robust and effective method for identifying pathogenic RNA splicing variants in NF1.
  • This approach enhances the molecular diagnostic capabilities for NF1, especially for cases involving complex splicing defects.
  • The findings support the integration of targeted RNAseq into routine diagnostics for NF1 to improve diagnostic accuracy and identify causative variants.