NOTCH3 Variant Position Affects the Phenotype at the Pluripotent Stem Cell Level in CADASIL

Area of Science:

• Neurogenetics
• Stem Cell Biology
• Molecular Medicine

Background:

• Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the leading genetic stroke cause.
• NOTCH3 gene variants, specifically cysteine-altering mutations in epidermal growth factor-like repeat (EGFr) domains, underlie CADASIL.
• While NOTCH3 variants in EGFr domains 1-6 are linked to severe disease and those in EGFr 7-34 to later onset, the direct impact of variant position on disease mechanisms remains unclear.

Purpose of the Study:

• To investigate whether the position of NOTCH3 pathogenic variants influences cellular phenotypes and protein profiles.
• To generate and analyze human-induced pluripotent stem cells (hiPSCs) from CADASIL patients with variants in different EGFr domains.

Main Methods:

• Generation of six hiPSC lines: two from patients with NOTCH3 EGFr 1-6 variants, two from patients with EGFr 7-34 variants, and two from healthy controls.
• Assessment of Notch3 aggregation and protein profiles within the established hiPSC lines.
• Evaluation of cell reprogramming efficiency and proteomic changes associated with variant position.

Main Results:

• NOTCH3 variants did not impede hiPSC reprogramming efficiency.
• hiPSC lines with NOTCH3 variants in EGFr domains 1-6 exhibited increased Notch3 protein accumulation compared to those with EGFr 7-34 variants.
• Proteomic analysis revealed alterations in cytoplasmic reorganization mechanisms in hiPSCs with EGFr 1-6 variants.

Conclusions:

• The position of NOTCH3 pathogenic variants directly influences cellular phenotypes in hiPSCs.
• Findings support the clinical observation linking NOTCH3 variant position to CADASIL disease severity.
• This study provides a cellular model for exploring genotype-phenotype correlations in CADASIL.