Association of NOTCH3 Variant Risk Category With 2-Year Clinical and Radiologic Small Vessel Disease Progression in Patients With CADASIL.

BACKGROUND AND OBJECTIVES: Pathogenic variants in NOTCH3 are the main cause of hereditary cerebral small vessel disease (SVD). SVD-associated NOTCH3 variants have recently been categorized into high risk (HR), moderate risk (MR), or low risk (LR) for developing early-onset severe SVD. The most severe NOTCH3-associated SVD phenotype is also known as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). We aimed to investigate whether NOTCH3 variant risk category is associated with 2-year progression rate of SVD clinical and neuroimaging outcomes in CADASIL. METHODS: A single-center prospective 2-year follow-up study was performed of patients with CADASIL. Clinical outcomes were incident stroke, disability (modified Rankin Scale), and executive function (Trail Making Test B given A t-scores). Neuroimaging outcomes were mean skeletonized mean diffusivity (MSMD), normalized white matter hyperintensity volume (nWMHv), normalized lacune volume (nLV), and brain parenchymal fraction (BPF). Cox regression and mixed-effect models, adjusted for age, sex, and cardiovascular risk factors, were used to study 2-year changes in outcomes and differences in disease progression between patients with HR-NOTCH3 and MR-NOTCH3 variants. RESULTS: One hundred sixty-two patients with HR (n = 90), MR (n = 67), and LR (n = 5) NOTCH3 variants were included. For the entire cohort, there was 2-year mean progression for MSMD (ß = 0.20, 95% CI 0.17-0.23, p = 7.0 × 10-24), nLV (ß = 0.13, 95% CI 0.080-0.19, p = 2.1 × 10-6), nWMHv (ß = 0.092, 95% CI 0.075-0.11, p = 8.8 × 10-20), and BPF (ß = -0.22, 95% CI -0.26 to -0.19, p = 3.2 × 10-22), as well as an increase in disability (p = 0.002) and decline of executive function (ß = -0.15, 95% CI -0.30 to -3.4 × 10-5, p = 0.05). The HR-NOTCH3 group had a higher probability of 2-year incident stroke (hazard ratio 4.3, 95% CI 1.4-13.5, p = 0.011), and a higher increase in MSMD (ß = 0.074, 95% CI 0.013-0.14, p = 0.017) and nLV (ß = 0.14, 95% CI 0.034-0.24, p = 0.0089) than the MR-NOTCH3 group. Subgroup analyses showed significant 2-year progression of MSMD in young (n = 17, ß = 0.014, 95% CI 0.0093-0.019, p = 1.4 × 10-5) and premanifest (n = 24, ß = 0.012, 95% CI 0.0082-0.016, p = 1.1 × 10-6) individuals. DISCUSSION: In a trial-sensitive time span of 2 years, we found that patients with HR-NOTCH3 variants have a significantly faster progression of major clinical and neuroimaging outcomes, compared with patients with MR-NOTCH3 variants. This has important implications for clinical trial design and disease prediction and monitoring in the clinic. Moreover, we show that MSMD is a promising outcome measure for trials enrolling premanifest individuals.

Bi-allelic NIT1 variants cause a brain small vessel disease characterized by movement disorders, massively dilated perivascular spaces, and intracerebral hemorrhage.

PURPOSE: To describe a recessively inherited cerebral small vessel disease, caused by loss-of-function variants in Nitrilase1 (NIT1). METHODS: We performed exome sequencing, brain magnetic resonance imaging, neuropathology, electron microscopy, western blotting, and transcriptomic and metabolic analyses in 7 NIT1-small vessel disease patients from 5 unrelated pedigrees. RESULTS: The first identified patients were 3 siblings, compound heterozygous for the NIT1 c.727C>T; (p.Arg243Trp) variant and the NIT1 c.198_199del; p.(Ala68∗) variant. The 4 additional patients were single cases from 4 unrelated pedigrees and were all homozygous for the NIT1 c.727C>T; p.(Arg243Trp) variant. Patients presented in mid-adulthood with movement disorders. All patients had striking abnormalities on brain magnetic resonance imaging, with numerous and massively dilated basal ganglia perivascular spaces. Three patients had non-lobar intracerebral hemorrhage between age 45 and 60, which was fatal in 2 cases. Western blotting on patient fibroblasts showed absence of NIT1 protein, and metabolic analysis in urine confirmed loss of NIT1 enzymatic function. Brain autopsy revealed large electron-dense deposits in the vessel walls of small and medium sized cerebral arteries. CONCLUSION: NIT1-small vessel disease is a novel, autosomal recessively inherited cerebral small vessel disease characterized by a triad of movement disorders, massively dilated basal ganglia perivascular spaces, and intracerebral hemorrhage.

Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction.

Cysteine-altering missense variants (NOTCH3cys) in one of the 34 epidermal growth-factor-like repeat (EGFr) domains of the NOTCH3 protein are the cause of NOTCH3-associated small vessel disease (NOTCH3-SVD). NOTCH3-SVD is highly variable, ranging from cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) at the severe end of the spectrum to non-penetrance. The strongest known NOTCH3-SVD modifier is NOTCH3cys variant position: NOTCH3cys variants located in EGFr domains 1-6 are associated with a more severe phenotype than NOTCH3cys variants located in EGFr domains 7-34. The objective of this study was to further improve NOTCH3-SVD genotype-based risk prediction by using relative differences in NOTCH3cys variant frequencies between large CADASIL and population cohorts as a starting point. Scientific CADASIL literature, cohorts and population databases were queried for NOTCH3cys variants. For each EGFr domain, the relative difference in NOTCH3cys variant frequency (NVFOR) was calculated using genotypes of 2574 CADASIL patients and 1647 individuals from population databases. Based on NVFOR cut-off values, EGFr domains were classified as either low (LR-EGFr), medium (MR-EGFr) or high risk (HR-EGFr). The clinical relevance of this new three-tiered EGFr risk classification was cross-sectionally validated by comparing SVD imaging markers and clinical outcomes between EGFr risk categories using a genotype-phenotype data set of 434 CADASIL patients and 1003 NOTCH3cys positive community-dwelling individuals. CADASIL patients and community-dwelling individuals harboured 379 unique NOTCH3cys variants. Nine EGFr domains were classified as an HR-EGFr, which included EGFr domains 1-6, but additionally also EGFr domains 8, 11 and 26. Ten EGFr domains were classified as MR-EGFr and 11 as LR-EGFr. In the population genotype-phenotype data set, HR-EGFr individuals had the highest risk of stroke [odds ratio (OR) = 10.81, 95% confidence interval (CI): 5.46-21.37], followed by MR-EGFr individuals (OR = 1.81, 95% CI: 0.84-3.88) and LR-EGFr individuals (OR = 1 [reference]). MR-EGFr individuals had a significantly higher normalized white matter hyperintensity volume (nWMHv; P = 0.005) and peak width of skeletonized mean diffusivity (PSMD; P = 0.035) than LR-EGFr individuals. In the CADASIL genotype-phenotype data set, HR-EGFr domains 8, 11 and 26 patients had a significantly higher risk of stroke (P = 0.002), disability (P = 0.041), nWMHv (P = 1.8 × 10-8), PSMD (P = 2.6 × 10-8) and lacune volume (P = 0.006) than MR-EGFr patients. SVD imaging marker load and clinical outcomes were similar between HR-EGFr 1-6 patients and HR-EGFr 8, 11 and 26 patients. NVFOR was significantly associated with vascular NOTCH3 aggregation load (P = 0.006), but not with NOTCH3 signalling activity (P = 0.88). In conclusion, we identified three clinically distinct NOTCH3-SVD EGFr risk categories based on NFVOR cut-off values, and identified three additional HR-EGFr domains located outside of EGFr domains 1-6. This EGFr risk classification will provide an important key to individualized NOTCH3-SVD disease prediction.

Effect of NOTCH3 EGFr Group, Sex, and Cardiovascular Risk Factors on CADASIL Clinical and Neuroimaging Outcomes.

BACKGROUND: A retrospective study has shown that EGFr (epidermal growth factor-like repeat) group in the NOTCH3 gene is an important cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) disease modifier of age at first stroke and white matter hyperintensity (WMH) volume. No study has yet assessed the effect of other known CADASIL modifiers, that is, cardiovascular risk factors and sex, in the context of NOTCH3 EGFr group. In this study, we determined the relative disease-modifying effects of NOTCH3 EGFr group, sex and cardiovascular risk factor on disease severity in the first genotype-driven, large prospective CADASIL cohort study, using a comprehensive battery of CADASIL clinical outcomes and neuroimaging markers. METHODS: Patients with CADASIL participated in a single-center, prospective cohort study (DiViNAS [Disease Variability in NOTCH3 Associated Small Vessel Disease]) between 2017 and 2020. The study protocol included a clinical assessment, neuropsychological test battery and brain magnetic resonance imaging on a single research day. Multivariable linear, logistic and Cox regression models were used to cross-sectionally assess the effect of CADASIL modifiers on clinical severity (stroke, disability, processing speed) and neuroimaging markers (WMH volume, peak width of skeletonized mean diffusivity, lacune volume, brain volume, cerebral microbleed count). RESULTS: Two hundred patients with CADASIL participated, of which 103 harbored a NOTCH3 EGFr 1-6 variant and 97 an EGFr 7-34 variant. NOTCH3 EGFr 1-6 group was the most important modifier of age at first stroke (hazard ratio, 2.45 [95% CI, 1.39-4.31]; P=0.002), lacune volume (odds ratio, 4.31 [95% CI, 2.31-8.04]; P=4.0×10-6), WMH volume (B=0.81 [95% CI, 0.60-1.02]; P=1.1×10-12), and peak width of skeletonized mean diffusivity (B=0.65 [95% CI, 0.44-0.87]; P=1.6×10-8). EGFr 1-6 patients had a significantly higher WMH volume in the anterior temporal lobes and superior frontal gyri and a higher burden of enlarged perivascular spaces. After NOTCH3 EGFr group, male sex and hypertension were the next most important modifiers of clinical outcomes and neuroimaging markers. CONCLUSIONS: NOTCH3 EGFr group is the most important CADASIL disease modifier not only for age at first stroke and WMH volume but also strikingly so for a whole battery of clinically relevant disease measures such as lacune volume and peak width of skeletonized mean diffusivity. NOTCH3 EGFr group is followed in importance by sex, hypertension, diabetes, and smoking.

Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy Family Members With a Pathogenic NOTCH3 Variant Can Have a Normal Brain Magnetic Resonance Imaging and Skin Biopsy Beyond Age 50 Years.

BACKGROUND: To determine whether extremely mild small vessel disease (SVD) phenotypes can occur in NOTCH3 variant carriers from Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) pedigrees using clinical, genetic, neuroimaging, and skin biopsy findings. METHODS: Individuals from CADASIL pedigrees fulfilling criteria for extremely mild NOTCH3-associated SVD (mSVDNOTCH3) were selected from the cross-sectional Dutch CADASIL cohort (n=200), enrolled between 2017 and 2020. Brain magnetic resonance imaging were quantitatively assessed for SVD imaging markers. Immunohistochemistry and electron microscopy was used to quantitatively assess and compare NOTCH3 ectodomain (NOTCH3ECD) aggregation and granular osmiophilic material deposits in the skin vasculature of mSVDNOTCH3 cases and symptomatic CADASIL patients. RESULTS: Seven cases were identified that fulfilled the mSVDNOTCH3 criteria, with a mean age of 56.6 years (range, 50-72). All of these individuals harbored a NOTCH3 variant located in one of EGFr domains 7-34 and had a normal brain magnetic resonance imaging, except the oldest individual, aged 72, who had beginning confluence of WMH (Fazekas score 2) and 1 cerebral microbleed. mSVDNOTCH3 cases had very low levels of NOTCH3ECD aggregation in skin vasculature, which was significantly less than in symptomatic EGFr 7-34 CADASIL patients (P=0.01). Six mSVDNOTCH3 cases had absence of granular osmiophilic material deposits. CONCLUSIONS: Our findings demonstrate that extremely mild SVD phenotypes can occur in individuals from CADASIL pedigrees harboring NOTCH3 EGFr 7-34 variants with normal brain magnetic resonance imaging up to age 58 years. Our study has important implications for CADASIL diagnosis, disease prediction, and the counseling of individuals from EGFr 7-34 CADASIL pedigrees.

NOTCH3 variant position is associated with NOTCH3 aggregation load in CADASIL vasculature.

AIMS: CADASIL, the most prevalent hereditary cerebral small vessel disease, is caused by cysteine-altering NOTCH3 variants (NOTCH3cys ) 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 NOTCH3cys variant position is associated with NOTCH3 protein aggregation load. METHODS: We quantified vascular NOTCH3 aggregation in skin biopsies (n = 25) and brain tissue (n = 7) of CADASIL patients with a NOTCH3cys 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). RESULTS: Patients with NOTCH3cys 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 NOTCH3cys 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. CONCLUSIONS: 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 NOTCH3cys EGFr 7-34 and EGFr 1-6 variants.