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
Oliveira, D.V.; Coupland, K.G.; Shao, W.C.; Jin, S.B.; Gaudio, F. del; Wang, S.L.; ... ; Karlstrom, H. 2022
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial small vessel disease; no preventive or curative... Show moreCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial small vessel disease; no preventive or curative therapy is available. CADASIL is caused by mutations in the NOTCH3 gene, resulting in a mutated NOTCH3 receptor, with aggregation of the NOTCH3 extracellular domain (ECD) around vascular smooth muscle cells. In this study, we have developed a novel active immunization therapy specifically targeting CADASIL-like aggregated NOTCH3 ECD. Immunizing CADASIL TgN3R182C(150) mice with aggregates composed of CADASIL-R133C mutated and wild-type EGF(1-5) repeats for a total of 4 months resulted in a marked reduction (38-48%) in NOTCH3 deposition around brain capillaries, increased microglia activation and lowered serum levels of NOTCH3 ECD. Active immunization did not impact body weight, general behavior, the number and integrity of vascular smooth muscle cells in the retina, neuronal survival, or inflammation or the renal system, suggesting that the therapy is tolerable. This is the first therapeutic study reporting a successful reduction of NOTCH3 accumulation in a CADASIL mouse model supporting further development towards clinical application for the benefit of CADASIL patients. 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
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
The research described in this thesis was aimed at identifying and understanding biological mechanisms and molecular pathways involved in the pathophysiology of stroke and migraine, including the... Show moreThe research described in this thesis was aimed at identifying and understanding biological mechanisms and molecular pathways involved in the pathophysiology of stroke and migraine, including the detrimental connection between them. The thesis consists of two parts. Part 1 describes multiple experimental stroke research projects in mice in which we set out to: (I) improve the methodology of stroke research, and (II) unravel the stroke-migraine connection using diff erent research strategies, methods, and transgenic mouse models. The mouse models express human pathogenic mutations found in CADASIL, RVCL-S and FHM1 and represent the clinical spectrum of monogenic disorders linking ischemic stroke and migraine. Part 2 includes multiple clinical projects in which we set out to study a large cohort of ischemic stroke patients with and without migraine in search for means to investigate stroke characteristics and vascular pathology. Show less
CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most prevalent hereditary small vessel disease. CADASIL patients typically develop... Show moreCADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most prevalent hereditary small vessel disease. CADASIL patients typically develop recurrent strokes from mid-adult age onwards, leading to cognitive impairment and ultimately vascular dementia. As there is currently no therapy that can delay or prevent CADASIL, the CADASIL research group of the Leiden University Medical Center is developing a therapeutic approach for CADASIL, called “NOTCH3 cysteine correction”.The aim of this PhD-project was to advance CADASIL therapy development.The work in this thesis provides the first in human evidence that the therapeutic approach of NOTCH3 cysteine correction leads to reduced protein aggregation, by describing a family with naturally occurring NOTCH3 cysteine correction. Furthermore, this thesis includes the results of the longest follow-up study to date of individuals with CADASIL, as well as and the identification of Neurofilament Light-chain (NfL) as blood biomarker in CADASIL. In a pre-clinical CADASIL disease model, potential pre-clinical biomarkers were explored and this resulted in the development of a GOM deposit classification system. Show less
Boer, I. de; MaassenVanDenBrink, A.; Terwindt, G.M. 2020
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease characterised by recurrent ischemic stroke, cognitive... Show moreCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease characterised by recurrent ischemic stroke, cognitive decline progressing to dementia, psychiatric disturbances and apathy. More than half of mutation carriers suffer from migraine, most often migraine with aura. Recently, a CADASIL patient was treated with a monoclonal antibody targeting the calcitonin gene-related peptide (CGRP) receptor. Monoclonal antibodies targeting the CGRP system have been demonstrated to be safe, well tolerated, and effective in reducing migraine attacks. There is, however, abundant evidence that CGRP is important in maintaining cardiovascular homeostasis under (patho)physiological conditions. CGRP may act as a vasodilatory safeguard during cerebral and cardiac ischemia and blockage of the system could, therefore, potentially worsen ischemic events. Therefore, we caution against treating patients with small vessel diseases, such as the monogenic disorder CADASIL, with these drugs until relevant safety data and long term follow up results are available. Alternative preventive migraine treatments in CADASIL may be acetazolamide, sodium valproate, lamotrigine, topiramate, verapamil, or flunarizine. 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
Bersano, A.; Kraemer, M.; Burlina, A.; Mancuso, M.; Finsterer, J.; Sacco, S.; ... ; Markus, H.S. 2020
Despite intensive investigations, about 30% of stroke cases remains of undetermined origin. After exclusion of common causes of stroke, there is a number of rare heritable and non-heritable... Show moreDespite intensive investigations, about 30% of stroke cases remains of undetermined origin. After exclusion of common causes of stroke, there is a number of rare heritable and non-heritable conditions, which often remain misdiagnosed, that should be additionally considered in the diagnosis of cryptogenic stroke. The identification of these diseases requires a complex work up including detailed clinical evaluation for the detection of systemic symptoms and signs, an adequate neuroimaging assessment and a careful family history collection. The task becomes more complicated by phenotype heterogeneity since stroke could be the primary or unique manifestation of a syndrome or represent just a manifestation (sometimes minor) of a multisystem disorder. The aim of this review paper is to provide clinicians with an update on clinical and neuroradiological features and a set of practical suggestions for the diagnostic work up and management of these uncommon causes of stroke. The identification of these stroke causes is important to avoid inappropriate and expensive diagnostic tests, to establish appropriate management measures, including presymptomatic testing, genetic counseling, and, if available, therapy. Therefore, physicians should become familiar with these diseases to provide future risk assessment and family counseling. 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
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
Donato, I. di; Bianchi, S.; Stefano, N. de; Dichgans, M.; Dotti, M.T.; Duering, M.; ... ; Federico, A. 2017
The aim of this thesis was to work towards pre-clinical proof-of-concept for NOTCH3 cysteine corrective exon skipping as a rational therapeutic approach for CADASIL. To address all aspects required... Show moreThe aim of this thesis was to work towards pre-clinical proof-of-concept for NOTCH3 cysteine corrective exon skipping as a rational therapeutic approach for CADASIL. To address all aspects required for therapeutic development, the work performed for this thesis included not only in vitro testing of NOTCH3 exon skipping in CADASIL patient derived vascular smooth muscle cells and studies into the function of the cysteine corrected proteins, but also the generation of a relevant humanized in vivo model, pre-clinical biomarker development, and studies defining prevalence, spectrum and characteristics of NOTCH3 mutations worldwide. Show less
