Association of NOTCH3 Variant Position With Stroke Onset and Other Clinical Features Among Patients With CADASIL

Abstract

Background and objectives: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by a cysteine-altering variant in 1 of the 34 epidermal growth factor-like repeat (EGFR) domains of the NOTCH3 protein. CADASIL has a variable phenotypic presentation, and NOTCH3 variants in EGFRs 1-6 have been found correlated with greater disease severity. We examined clinical and radiologic features and performed bioinformatic annotation of variants in a large CADASIL cohort to further understand these associations.

Methods: We examined the association of NOTCH3 variant position on stroke onset and other clinical features among patients with CADASIL from the United Kingdom. We also explored how in silico predicted protein aggregation differed by variant position and the extent to which this affected stroke risk.

Results: We identified 76 different cysteine-altering NOTCH3 variants in our cohort of 485 patients (mean age: 50.1 years; % male: 57.5). After controlling for cardiovascular risk factors, variants in EGFRs 1-6 were associated with earlier onset of stroke (hazard ratio [HR]: 2.05, 95% CI: 1.43-2.94) and encephalopathy (HR: 2.70, 95% CI: 1.15-6.37), than variants in EGFRs 7-34. Although the risk of stroke was higher in the patients with predicted protein aggregation (HR: 1.50, 95% CI: 1.05-2.14), this association was no longer significant after controlling for variant site. Further analysis suggested that lower stroke risk was observed for variants in EGFRs 10-17 compared with variants in the other EGFR domains.

Discussion: NOTCH3 variant position is a predictor of stroke and encephalopathy in CADASIL independent of cardiovascular risk factors. Lower stroke risk was found for variants in EGFRs 10-17. Molecular factors that influence CADASIL disease severity remain to be determined.

Figure 1. Lolliplot Showing the Distribution of Distinct Variants Present in the Patient Cohort Across the Reverse Strand of the NOTCH3 Gene

Lighter circles represent gain-of-cysteine variants; darker circles represent loss-of-cysteine variants. Lighter rectangular boxes represent the exons that encode EGFRs 7–34. Darker rectangular boxes represent the exons that encode EGFRs 1–6. EGFR = epidermal growth factor-like repeat.

Figure 2. Kaplan-Meier Plot Showing the Difference in Age at Onset of the First Stroke for Patients With EGFR Domain 1–6 Variants and Those With EGFR Domain 7–34 Variants

HR was calculated through Cox regression with adjustment for sex and cardiovascular risk factors. EGFR = epidermal growth factor-like repeat; HR = hazard ratio.

Figure 3. Kaplan-Meier Plot Showing the Difference in Age at Onset of the First Encephalopathy for Patients With EGFR Domain 1–6 Variants and Those With EGFR Domain 7–34 Variants

HR was calculated through Cox regression with adjustment for sex and cardiovascular risk factors. EGFR = epidermal growth factor-like repeat; HR = hazard ratio.

Figure 4. Forest Plot Showing the Risk of Stroke Associated With Variants in Different EGFR Location Compared With EGFRs 3–4

Prevalence of stroke cases was analyzed with the presence of variants in different EGFR location through Cox regression, with the EGFRs 3–4 variant group being used as the reference and with adjustment for sex and cardiovascular risk factors (N = 485). EGFR = epidermal growth factor-like repeat; HR = hazard ratio.

Figure 5. Mediation Analyses

(A) Effect of the presence of gain-of-cysteine variant on the relationship between EGFRs 1–6 variant and stroke. (B) Effect of the presence of proaggregation variant on the relationship between EGFRs 1–6 variant and stroke. (C) Effect of the presence of gain-of-cysteine variant on the relationship between EGFRs 1–6 variant and proaggregation variant. OR was calculated through logistic regression with adjustment for age and sex. HR was calculated through Cox regression with adjustment for sex and EGFRs 1–6 variants. Regression models were bootstrapped 10,000 times to examine whether a mediation effect was statistically significant. EGFR = epidermal growth factor-like repeat; HR = hazard ratio; OR = odds ratio.