Related Experiment Video
Updated: Dec 30, 2025
Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides
Published on: May 7, 2020
Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL
Gido Gravesteijn1, Johannes G Dauwerse2, Maurice Overzier2
1Department of Clinical Genetics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
Insights
A NOTCH3 gene variant causing exon 9 skipping, mimicking therapeutic correction, was found in individuals with mild CADASIL symptoms. This suggests exon skipping may reduce NOTCH3 aggregation and disease severity, supporting therapeutic development.
Area of Science:
- Genetics and Neurology
- Molecular Medicine
- Vascular Biology
Background:
- Cerebral Autosomal-Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a genetic disorder affecting blood vessels in the brain.
- It is caused by mutations in the NOTCH3 gene, leading to protein aggregation and symptoms like stroke and dementia.
- Current treatments focus on managing symptoms, with limited options for addressing the underlying cause.
Purpose of the Study:
- To investigate a naturally occurring NOTCH3 exon 9 skipping event in individuals with a specific NOTCH3 variant.
- To evaluate the impact of this exon skipping on NOTCH3 aggregation and CADASIL phenotype.
- To explore the therapeutic potential of NOTCH3 exon skipping for CADASIL.
Main Methods:
- Genetic analysis (gene panel, RT-PCR, Sanger sequencing) to identify and confirm the NOTCH3 variant and exon skipping.
- Clinical assessment and neuroimaging (MRI) to evaluate the CADASIL phenotype.
- Biochemical and ultrastructural analysis (electron microscopy, immunohistochemistry) of skin biopsies to assess NOTCH3 aggregation.
- In vitro studies using cell models to test therapeutic strategies (antisense-mediated exon skipping, CRISPR/Cas9).
Main Results:
- A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant was identified, leading to significant exon 9 skipping in affected individuals.
- Individuals with the variant exhibited white matter hyperintensities but had a mild CADASIL phenotype with no lacunes, disability, or cognitive impairment above age 60.
- NOTCH3 aggregation was minimal in skin biopsies, and therapeutic strategies demonstrated successful exon 9 exclusion in cell models.
Conclusions:
- The study provides the first in-human evidence that NOTCH3 exon 9 skipping, mimicking therapeutic cysteine correction, is associated with reduced NOTCH3 aggregation.
- This exon skipping correlates with an attenuated CADASIL phenotype, suggesting a potential therapeutic benefit.
- The findings support further development of NOTCH3 cysteine correction strategies, including exon skipping, for CADASIL treatment.
Abstract:
CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL.
More Related Videos
05:48Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands
Published on: January 2, 2018
09:34Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Published on: April 4, 2018
Related Concept Videos
03:14Notch Signaling Pathway
The Notch gene came into the limelight in 1914 after the discovery that its mutation in Drosophila melanogaster leads to a serrated (or "notched") wing margin phenotype. It was not...
01:12Role Of Notch Signalling In Intestinal Stem Cell Renewal
Direct cell-to-cell contact is needed for the activation of Notch signaling. The signal is initiated when a notch ligand binds to a receptor on an adjacent cell, also...
01:23Cystic Fibrosis: Pathogenesis
CF is primarily caused by a genetic mutation in a chromosome 7 gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common gene mutation leading to CF is the ΔF508 mutation,...
02:18Alternative RNA Splicing
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
01:19Cadherins in Tissue Organization
Cell Sorting During Development
Cell sorting plays an...