Serum NOTCH3 Extracellular Domain in Patients With CADASIL

Abstract

Background and objectives: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary small vessel disease caused by variants in the NOTCH3 gene. The extracellular domain of NOTCH3 (N3ECD) has been proposed as a potential serum biomarker for CADASIL. However, its diagnostic utility remains uncertain. The aim of this study was to evaluate serum N3ECD levels in patients with CADASIL and assess their potential as a diagnostic or prognostic biomarker.

Methods: Serum N3ECD levels were measured using ELISA in 2 independent CADASIL cohorts: a Korean cohort (n = 78) and a Japanese cohort (n = 64). Each group included patients with genetically confirmed CADASIL. In addition, the Korean cohort included age-matched and sex-matched healthy controls and patients with sporadic ischemic stroke. Statistical analyses assessed group differences and correlations with clinical and radiologic features.

Results: Serum N3ECD levels did not differ significantly among patients with CADASIL, healthy controls, and patients with ischemic stroke in the Korean cohort (p = 0.149). However, in the Korean cohort, N3ECD levels showed a positive correlation with age (r = 0.329, p = 0.001), particularly in individuals carrying the p.Arg544Cys variant. Similarly, in the Japanese cohort, age-related increases in N3ECD levels were observed in p.Arg75Pro variant carriers (r = 0.661, p = 0.014). No significant associations were found between N3ECD levels and MRI markers or clinical outcomes such as stroke occurrence or cognitive impairment.

Discussion: These findings suggest that serum N3ECD alone has limited value as a standalone diagnostic marker for CADASIL. However, the age-dependent increase in N3ECD levels observed in specific variant carriers may reflect underlying pathophysiologic changes and could serve as a clue to disease progression. Further studies are warranted to clarify the mechanistic role of N3ECD in CADASIL and to evaluate its potential as a marker for monitoring disease progression or therapeutic response. Continued biomarker discovery efforts are needed to improve diagnosis and management of CADASIL.

Figure 1. Quantification of N3ECD Protein Levels in the Blood Serum of Healthy Controls, Stroke Patients, and Those With CADASIL

(A) Serum N3ECD levels were measured in patients with CADASIL (n = 78) and compared with those in healthy controls (n = 78) and stroke patients (n = 78). (B) N3ECD levels were compared between patients with cysteine-altering missense variants and those with noncysteine variants. One of 3 independent experiments is shown. (C) Comparison of serum N3ECD levels between patients harboring variants in EGFr domains 1–6 and domains 7–34. (D) Correlation analysis between risk classification of EGFr domains based on NVFOR cutoff values and N3ECD levels. Each symbol represents values from a single participant; the line in the middle of the violin shows the median value of the data set. ns, not significant; *, p < 0.05; paired one-way ANOVA(A); Student t test (B–D). CADASIL = cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; N3ECD = NOTCH3 extracellular domain.

Figure 2. Associations Between Serum N3ECD Levels and Disability/Cognitive Scores in Patients With CADASIL (n = 78)

As shown in panels (A–G), the analysis revealed no statistically significant associations between serum N3ECD levels and the measured variables: stroke (A), dementia (B), headache (C), and Clinical Dementia Rating (CDR) (D); Korean-Mini Mental State Examination (K-MMSE) score (E); SNSB total score (F); and neurofilament light chain (NFL) (G). Each symbol represents data from an individual participant. The central line in each violin plot represents the median value of the data set. Statistical significance is denoted as ns (not significant). Statistical tests performed include Student t test (A C), one-way ANOVA (D), Spearman correlation (E), and Pearson correlation (F and G). CADASIL = cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; N3ECD = NOTCH3 extracellular domain.

Figure 3. Associations Between Serum N3ECD Levels and MRI Markers in Patients With CADASIL (n = 59)

As shown in panels (A–D), the analysis revealed no statistically significant associations between serum N3ECD levels and MRI markers, including cerebral microbleeds (CMBs) (A), lacunes (B), brain parenchymal fraction (BPF) (C), and normalized white matter hyperintensity (nWMH) volume (D). Statistical analyses were conducted using Pearson correlation (A and B) and Spearman correlation (C and D). CADASIL = cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; N3ECD = NOTCH3 extracellular domain.

Figure 4. Validation of Serum N3ECD Levels in the Japanese CADASIL Cohort

(A) Serum N3ECD levels were measured in patients with CADASIL (n = 64) and healthy controls (n = 14) from an independent Japanese cohort. (B) Serum N3ECD levels were compared between patients with CADASIL with the p.Arg75Pro variant and those with other genotypes. (C) Comparison of serum N3ECD levels between patients with variants located in EGFr domains 1–6 and those with variants in domains 7–34. (D) Correlation analysis between high-risk or medium-risk EGFr domains and N3ECD levels. As shown in panels (E–G), the analysis revealed no statistically significant associations between serum N3ECD levels and the measured disability/cognitive scores. (E) Stroke, (F) dementia, and (G) migraines. Statistical tests performed include Student t test. CADASIL = cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; N3ECD = NOTCH3 extracellular domain.