CADASIL, the most prevalent hereditary cerebral small vessel disease, is caused by cysteine‐altering NOTCH3 variants (NOTCH3^cys) leading to vascular NOTCH3 protein aggregation. It has recently been shown that variants located in one of NOTCH3 protein epidermal growth‐factor like repeat (EGFr) domains 1–6, are associated with a more severe phenotype than variants located in one of the EGFr domains 7–34. The underlying mechanism for this genotype–phenotype correlation is unknown. The aim of this study was to analyse whether NOTCH3^cys variant position is associated with NOTCH3 protein aggregation load.

We quantified vascular NOTCH3 aggregation in skin biopsies (n = 25) and brain tissue (n = 7) of CADASIL patients with a NOTCH3^cys EGFr 1–6 variant or a EGFr 7–34 variant, using NOTCH3 immunohistochemistry (NOTCH3 score) and ultrastructural analysis of granular osmiophilic material (GOM count). Disease severity was assessed by neuroimaging (lacune count and white matter hyperintensity volume) and disability (modified Rankin scale).

Patients with NOTCH3^cys EGFr 7–34 variants had lower NOTCH3 scores (P = 1.3·10⁻⁵) and lower GOM counts (P = 8.2·10⁻⁵) than patients with NOTCH3^cys EGFr 1–6 variants in skin vessels. A similar trend was observed in brain vasculature. In the EGFr 7–34 group, NOTCH3 aggregation levels were associated with lacune count (P = 0.03) and white matter hyperintensity volume (P = 0.02), but not with disability.

CADASIL patients with an EGFr 7–34 variant have significantly less vascular NOTCH3 aggregation than patients with an EGFr 1–6 variant. This may be one of the factors underlying the difference in disease severity between NOTCH3^cys EGFr 7–34 and EGFr 1–6 variants.