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 ... Show moreCysteine-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. Show less
Hack, R.J.; Cerfontaine, M.N.; Gravesteijn, G.; Tap, S.; Hafkemeijer, A.; Grond, J. van der; ... ; Oberstein, S.A.J.L. 2022
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... Show moreBACKGROUND: 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=4.0x 10(-6)), WMH volume (B=0.81 [95% CI, 0.60-1.02]; P=1.1x 10(-12)), and peak width of skeletonized mean diffusivity (B=0.65 [95% CI, 0.44-0.87]; P=1.6x 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. Show less
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... Show moreBackground: 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 (mSVD(NOTCH3)) 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 (NOTCH3(ECD)) aggregation and granular osmiophilic material deposits in the skin vasculature of mSVD(NOTCH3) cases and symptomatic CADASIL patients. Results: Seven cases were identified that fulfilled the mSVD(NOTCH3) 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. mSVD(NOTCH3) cases had very low levels of NOTCH3(ECD) aggregation in skin vasculature, which was significantly less than in symptomatic EGFr 7-34 CADASIL patients (P=0.01). Six mSVD(NOTCH3) 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. Show less
Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics... Show moreNeurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1. Show less
Crosstalk between endothelial cells (ECs) and pericytes or vascular smooth muscle cells (VSMCs) is essential for the proper functioning of blood vessels. This balance is disrupted in several... Show moreCrosstalk between endothelial cells (ECs) and pericytes or vascular smooth muscle cells (VSMCs) is essential for the proper functioning of blood vessels. This balance is disrupted in several vascular diseases but there are few experimental models which recapitulate this vascular cell dialogue in humans. Here, we developed a robust multi-cell type 3D vessel-on-chip (VoC) model based entirely on human induced pluripotent stem cells (hiPSCs). Within a fibrin hydrogel microenvironment, the hiPSC-derived vascular cells self-organized to form stable microvascular networks reproducibly, in which the vessels were lumenized and functional, responding as expected to vasoactive stimulation. Vascular organization and intracellular Ca2+ release kinetics in VSMCs could be quantified using automated image analysis based on open-source software CellProfiler and ImageJ on widefield or confocal images, setting the stage for use of the platform to study vascular (patho)physiology and therapy. Show less
Guey, S.; Oberstein, S.A.J.L.; Tournier-Lasserve, E.; Chabriat, H. 2021
Cerebral small vessel diseases represent a frequent cause of stroke and cognitive or motor disability in adults. A small proportion of cerebral small vessel diseases is attributable to monogenic... Show moreCerebral small vessel diseases represent a frequent cause of stroke and cognitive or motor disability in adults. A small proportion of cerebral small vessel diseases is attributable to monogenic conditions. Since the characterization in the late 1990s of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, several other monogenic conditions leading to adult-onset ischemic or hemorrhagic stroke have been described. In this practical guide, we summarize the key features that should elicit the differential diagnosis of a hereditary cerebral small vessel diseases in adult stroke patients, describe the main clinical and imaging characteristics of the major hereditary cerebral small vessel diseases that can manifest as stroke, and provide general recommendations for the clinical management of affected patients and their relatives. Show less
Aims 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... Show moreAims 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. Methods 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). Results Patients with NOTCH3(cys) EGFr 7-34 variants had lower NOTCH3 scores (P = 1.3 center dot 10(-5)) and lower GOM counts (P = 8.2 center dot 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. 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 NOTCH3(cys) EGFr 7-34 and EGFr 1-6 variants. Show less
Background and Purpose:Cysteine altering NOTCH3 variants, which have previously been exclusively associated with the rare hereditary small vessel disease cerebral autosomal dominant arteriopathy... Show moreBackground and Purpose:Cysteine altering NOTCH3 variants, which have previously been exclusively associated with the rare hereditary small vessel disease cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, have a population frequency of 1:300 worldwide. Using a large population database, and taking genotype as a starting point, we aimed to determine whether individuals harboring a NOTCH3 cysteine altering variant have a higher load of small vessel disease markers on brain magnetic resonance imaging than controls, as well as a higher risk of stroke and cognitive impairment.Methods:A cross-sectional study using integrated clinical, neuroimaging, and whole-exome sequencing data of 92 456 participants from the Geisinger DiscovEHR initiative cohort. The case group consisted of individuals harboring a NOTCH3 cysteine altering variant (n=118). The control group consisted of randomly selected age- and sex-matched individuals who did not have any nonsynonymous variants in NOTCH3 (n=184). Medical records including brain magnetic resonance imagings were evaluated for clinical and neuroimaging findings associated with small vessel disease. Group comparisons were done using Fisher exact test and ordinal logistic regression models. Risk of stroke was assessed using Cox regression.Results:Of the 118 cases, 39.0% were men, mean age 58.1 +/- 16.9 years; 12.6% had a history of stroke, compared with 4.9% of controls. The risk of stroke was significantly increased after age 65 years (hazard ratio, 6.0 [95% CI, 1.4-26.3]). Dementia, mild cognitive impairment, migraine with aura and depression were equally prevalent in cases and controls. Twenty-nine cases (25%) and 45 controls (24%) had an available brain magnetic resonance imaging. After age 65 years, cases had a higher white matter lesion burden and more lacunes. A severe small vessel disease phenotype compatible with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy was rarely seen.Conclusions:Cysteine altering NOTCH3 variants are an important contributor to the risk of stroke, lacunes, and white matter hyperintensities in the elderly population. Show less
Rutten, J.W.; Hack, R.J.; Duering, M.; Gravesteijn, G.; Dauwerse, J.G.; Overzier, M.; ... ; Oberstein, S.A.J.L. 2020
Objective To determine the small vessel disease spectrum associated with cysteine-alteringNOTCH3variants in community-dwelling individuals by analyzing the clinical and neuroimaging features of UK... Show moreObjective To determine the small vessel disease spectrum associated with cysteine-alteringNOTCH3variants in community-dwelling individuals by analyzing the clinical and neuroimaging features of UK Biobank participants harboring such variants. Methods The exome and genome sequencing datasets of the UK Biobank (n = 50,000) and cohorts of cognitively healthy elderly (n = 751) were queried for cysteine-alteringNOTCH3variants. Brain MRIs of individuals harboring such variants were scored according to Standards for Reporting Vascular Changes on Neuroimaging criteria, and clinical information was extracted with ICD-10 codes. Clinical and neuroimaging data were compared to age- and sex-matched UK Biobank controls and clinically diagnosed patients from the Dutch cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) registry. Results We identified 108 individuals harboring a cysteine-alteringNOTCH3variant (2.2 of 1,000), of whom 75% have a variant that has previously been reported in CADASIL pedigrees. Almost all variants were located in 1 of the NOTCH3 protein epidermal growth factor-like repeat domains 7 to 34. White matter hyperintensity lesion load was higher in individuals withNOTCH3variants than in controls (p= 0.006) but lower than in patients with CADASIL with the same variants (p< 0.001). Almost half of the 24 individuals with brain MRI had a Fazekas score of 0 or 1 up to age 70 years. There was no increased risk of stroke. Conclusions Although community-dwelling individuals harboring a cysteine-alteringNOTCH3variant have a higher small vessel disease MRI burden than controls, almost half have no MRI abnormalities up to age 70 years. This shows thatNOTCH3cysteine altering variants are associated with an extremely broad phenotypic spectrum, ranging from CADASIL to nonpenetrance. Show less
CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering... Show moreCADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL. Show less
Gravesteijn, G.; Munting, L.P.; Overzier, M.; Mulder, A.A.; Hegeman, I.; Derieppe, M.; ... ; Oberstein, S.A.J.L. 2019
CADASIL is a NOTCH3-associated cerebral small vessel disease. A pathological ultrastructural disease hallmark is the presence of NOTCH3-protein containing deposits called granular osmiophilic... Show moreCADASIL is a NOTCH3-associated cerebral small vessel disease. A pathological ultrastructural disease hallmark is the presence of NOTCH3-protein containing deposits called granular osmiophilic material (GOM), in small arteries. How these GOM deposits develop over time and what their role is in disease progression is largely unknown. Here, we studied the progression of GOM deposits in humanized transgenic NOTCH3(Arg182Cys) mice, compared them to GOM deposits in patient material, and determined whether GOM deposits in mice are associated with a functional CADASIL phenotype. We found that GOM deposits are not static, but rather progress in ageing mice, both in terms of size and aspect. We devised a GOM classification system, reflecting size, morphology and electron density. Six-month-old mice showed mostly early stage GOM, whereas older mice and patient vessels showed predominantly advanced stage GOM, but also early stage GOM. Mutant mice did not develop the most severe GOM stage seen in patient material. This absence of end-stage GOM in mice was associated with an overall lack of histological vascular pathology, which may explain why the mice did not reveal functional deficits in cerebral blood flow, cognition and motor function. Taken together, our data indicate that GOM progress over time, and that new GOM deposits are continuously being formed. The GOM staging system we introduce here allows for uniform GOM deposit classification in future mouse and human studies, which may lead to more insight into a potential association between GOM stage and CADASIL disease severity, and the role of GOM in disease progression. Show less
Rutten, J.W.; Akker, E.B. van den; Oberstein, S.A.J.L. 2019
Purpose: CADASIL is a small-vessel disease caused by a cysteine-altering pathogenic variant in one of the 34 epidermal growth factor-like repeat (EGFr) domains of the NOTCH3 protein. We recently... Show morePurpose: CADASIL is a small-vessel disease caused by a cysteine-altering pathogenic variant in one of the 34 epidermal growth factor-like repeat (EGFr) domains of the NOTCH3 protein. We recently found that pathogenic variant in EGFr domains 7-34 have an unexpectedly high frequency in the general population (1:300). We hypothesized that EGFr 7-34 pathogenic variant more frequently cause a much milder phenotype, thereby explaining an important part of CADASIL disease variability.Methods: Age at first stroke, survival and white matter hyperintensity volume were compared between 664 CADASIL patients with either a NOTCH3 EGFr 1-6 pathogenic variant or an EGFr 7-34 pathogenic variant. The frequencies of NOTCH3 EGFr 1-6 and EGFr 7-34 pathogenic variant were compared between individuals in the genome Aggregation Database and CADASIL patients.Results: CADASIL patients with an EGFr 1-6 pathogenic variant have a 12-year earlier onset of stroke than those with an EGFr 7-34 pathogenic variant, lower survival, and higher white matter hyperintensity volumes. Among diagnosed CADASIL patients, 70% have an EGFr 1-6 pathogenic variant, whereas EGFr 7-34 pathogenic variant strongly predominate in the population.Conclusion: NOTCH3 pathogenic variant position is the most important determinant of CADASIL disease severity, with EGFr 7-34 pathogenic variant predisposing to a later onset of stroke and longer survival. Show less
Gravesteijn, G.; Rutten, J.W.; Verberk, I.M.W.; Bohringer, S.; Liem, M.K.; Grond, J. van der; ... ; Oberstein, S.A.J.L. 2019
