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.

Atypical Progeria Primarily Manifesting as Premature Cardiac Valvular Disease Segregates with LMNA-Gene Variants.

Mutations in the LMNA-gene can cause a variety of 'laminopathies'. These laminopathies are associated with a range of phenotypes, including disorders affecting the adipose tissue, peripheral nerves, the heart, such as dilated cardiomyopathy and conduction system abnormalities, and less commonly, progeroid disorders. This case series describes two families in which two novel LMNA-gene variants were identified, and who presented with an atypical progeroid phenotype with primarily premature aortic and mitral valve stenosis. Interestingly, these families exhibited no clear evidence of multisystem involvement, illustrating the complex role of lamins A/C.

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.

Reanalysis of whole-exome sequencing (WES) data of children with neurodevelopmental disorders in a standard patient care context.

This study aims to inform future genetic reanalysis management by evaluating the yield of whole-exome sequencing (WES) reanalysis in standard patient care in the Netherlands. Single-center data of 159 patients with a neurodevelopmental disorder (NDD), in which WES analysis and reanalysis were performed between January 1, 2014, and December 31, 2021, was retrospectively collected. Patients were included if they were under the age of 18 years at initial analysis and if this initial analysis did not result in a diagnosis. Demographic, phenotypic, and genotypic characteristics of patients were collected and analyzed. The primary outcomes of our study were (i) diagnostic yield at reanalysis, (ii) reasons for detecting a new possibly causal variant at reanalysis, (iii) unsolicited findings, and (iv) factors associated with positive result of reanalysis. In addition, we conducted a questionnaire study amongst the 7 genetic department in the Netherlands creating an overview of used techniques, yield, and organization of WES reanalysis. The single-center data show that in most cases, WES reanalysis was initiated by the clinical geneticist (65%) or treating physician (30%). The mean time between initial WES analysis and reanalysis was 3.7 years. A new (likely) pathogenic variant or VUS with a clear link to the phenotype was found in 20 initially negative cases, resulting in a diagnostic yield of 12.6%. In 75% of these patients, the diagnosis had clinical consequences, as for example, a screening plan for associated signs and symptoms could be devised. Most (32%) of the (likely) causal variants identified at WES reanalysis were discovered due to a newly described gene-disease association. In addition to the 12.6% diagnostic yield based on new diagnoses, reclassification of a variant of uncertain significance found at initial analysis led to a definite diagnosis in three patients. Diagnostic yield was higher in patients with dysmorphic features compared to patients without clear dysmorphic features (yield 27% vs. 6%; p = 0.001). CONCLUSIONS: Our results show that WES reanalysis in patients with NDD in standard patient care leads to a substantial increase in genetic diagnoses. In the majority of newly diagnosed patients, the diagnosis had clinical consequences. Knowledge about the clinical impact of WES reanalysis, clinical characteristics associated with higher yield, and the yield per year after a negative WES in larger clinical cohorts is warranted to inform guidelines for genetic reanalysis. These guidelines will be of great value for pediatricians, pediatric rehabilitation specialists, and pediatric neurologists in daily care of patients with NDD. WHAT IS KNOWN: • Whole exome sequencing can cost-effectively identify a genetic cause of intellectual disability in about 30-40% of patients. • WES reanalysis in a research setting can lead to a definitive diagnosis in 10-20% of previously exome negative cases. WHAT IS NEW: • WES reanalysis in standard patient care resulted in a diagnostic yield of 13% in previously exome negative children with NDD. • The presence of dysmorphic features is associated with an increased diagnostic yield of WES reanalysis.

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.

Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model.

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 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 TgN3R182C150 mice with aggregates composed of CADASIL-R133C mutated and wild-type EGF1-5 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.

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.

Further delineation of phenotypic spectrum of SCN2A-related disorder.

SCN2A-related disorders include intellectual disability, autism spectrum disorder, seizures, episodic ataxia, and schizophrenia. In this study, the phenotype-genotype association in SCN2A-related disorders was further delineated by collecting detailed clinical and molecular characteristics. Using previously proposed genotype-phenotype hypotheses based on variant function and position, the potential of phenotype prediction from the variants found was examined. Patients were identified through the Deciphering Developmental Disorders study and gene matching strategies. Phenotypic information and variant interpretation evidence were collated. Seventeen previously unreported patients and five patients who had been previously reported (but with minimal phenotypic and segregation data) were included (10 males, 12 females; median age 10.5 years). All patients had developmental delays and the majority had intellectual disabilities. Seizures were reported in 15 of 22 (68.2%), four of 22 (18.2%) had autism spectrum disorder and no patients were reported with episodic ataxia. The majority of variants were de novo. One family had presumed gonadal mosaicism. The correlation of the use of sodium channel-blocking antiepileptic drugs with phenotype or genotype was variable. These data suggest that variant type and position alone can provide some predictive information about the phenotype in a proportion of cases, but more precise assessment of variant function is needed for meaningful phenotype prediction.

Cysteine-Altering NOTCH3 Variants Are a Risk Factor for Stroke in the Elderly Population.

BACKGROUND 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.

Broad phenotype of cysteine-altering NOTCH3 variants in UK Biobank: CADASIL to nonpenetrance.

OBJECTIVE: To determine the small vessel disease spectrum associated with cysteine-altering NOTCH3 variants 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-altering NOTCH3 variants. 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-altering NOTCH3 variant (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 with NOTCH3 variants 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-altering NOTCH3 variant have a higher small vessel disease MRI burden than controls, almost half have no MRI abnormalities up to age 70 years. This shows that NOTCH3 cysteine altering variants are associated with an extremely broad phenotypic spectrum, ranging from CADASIL to nonpenetrance.

Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients.

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 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.

Progression and Classification of Granular Osmiophilic Material (GOM) Deposits in Functionally Characterized Human NOTCH3 Transgenic Mice.

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 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 NOTCH3Arg182Cys 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.

Serum Neurofilament light correlates with CADASIL disease severity and survival.

Objective: To validate whether serum Neurofilament Light-chain (NfL) levels correlate with disease severity in CADASIL, and to determine whether serum NfL predicts disease progression and survival. Methods: Fourty-one (pre-) manifest individuals with CADASIL causing NOTCH3 mutations and 22 healthy controls were recruited from CADASIL families. At baseline, MRI-lesion load and clinical severity was determined and serum was stored. Disease progression was measured in 30/41 patients at 7-year follow-up, and survival of all individuals was determined at 17-year follow-up. Serum NfL levels were quantified using an ultra-sensitive molecule array. Generalized estimated equation regression (GEE) was used to analyze association between serum NfL, MRI-lesion load, disease severity, and disease progression. With GEE-based Cox regression, survival was analyzed. Results: At baseline, serum NfL levels correlated with MRI-lesion load [lacune count (s = 0.64, P = 0.002), brain atrophy (r = -0.50, P = 0.001), and microbleed count (s = 0.48, P = 0.044)], cognition [CAMCOG (s = -0.45, P = 0.010), MMSE (r = -0.61, P = 0.003), GIT (r = -0.61, P < 0.001), TMT-A (r = 0.70, P < 0.001)) and disability (mRS (r = 0.70, P = 0.002)]. Baseline serum NfL predicted 7-year changes in disability (B = 0.34, P < 0.001) and cognition (CAMCOG B = -4.94, P = 0.032), as well as 17-year survival. Higher NfL levels were associated with increased mortality (HR=1.8 per twofold increase in NfL levels, P = 0.006). Interpretation: Serum NfL levels correlate with disease severity, disease progression and 17-year survival in CADASIL patients. Serum NfL is a promising biomarker to monitor and predict disease course in CADASIL, as well as potentially assessing therapeutic response in future clinical trials.

The effect of NOTCH3 pathogenic variant position on CADASIL disease severity: NOTCH3 EGFr 1-6 pathogenic variant are associated with a more severe phenotype and lower survival compared with EGFr 7-34 pathogenic variant.

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 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.

Correction: Putting genome-wide sequencing in neonates into perspective.

The original version of this Article contained an error in the spelling of the author Pleuntje J. van der Sluijs, which was incorrectly given as Eline (P. J.) van der Sluijs. This has now been corrected in both the PDF and HTML versions of the Article.

Correction: The effect of NOTCH3 pathogenic variant position on CADASIL disease severity: NOTCH3 EGFr 1-6 pathogenic variant are associated with a more severe phenotype and lower survival compared with EGFr 7-34 pathogenic variant.

This Article was originally published under Nature Research's License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly.