Effects of different regional cerebral blood flow on white matter hyperintensity in CADASIL patients

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an early-onset inherited small vessel disease. Decreased cerebral blood flow (CBF) may contribute to white matter hyperintensity (WMH) severity in CADASIL, but more evidence is needed to support this hypothesis. This study comprised six patients with CADASIL who harbored mutations in the coding sequence of NOTCH3 and twelve age-matched neurologically healthy controls. We collected clinical and imaging data from patients with CADASIL and divided the brain into four regions: WMH, normal-appearing white matter (NAWM), gray matter (GM), and global brain. We analyzed the relationship between CBF of each region and the WMH volume. Compared with the control group, CBF was significantly decreased in all four regions in the CADASIL group. Lower CBF in these regions was correlated with higher WMH volume in CADASIL. CBF in the NAWM, GM and global regions was positively correlated with that in WMH region. However, after correction tests, only CBF in the WMH region but not in NAWM, GM and global regions was associated with WMH volume. Our findings suggest that CBF in the WMH region is an influencing factor of the WMH severity in CADASIL.     

Hypomorphic NOTCH3 Alleles Do Not Cause CADASIL in Humans

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by stereotyped missense mutations in NOTCH3. Whether these mutations lead to the CADASIL phenotype via a neomorphic effect, or rather by a hypomorphic effect, is subject of debate. Here, we report two novel NOTCH3 mutations, both leading to a premature stop codon with predicted loss of NOTCH3 function. The first mutation, c.307C>T, p.Arg103*, was detected in two brothers aged 50 and 55 years, with a brain MRI and skin biopsy incompatible with CADASIL. The other mutation was found in a 40‐year‐old CADASIL patient compound heterozygous for a pathogenic NOTCH3 mutation (c.2129A>G, p.Tyr710Cys) and an intragenic frameshift deletion. The deletion was inherited from his father, who did not have the skin biopsy abnormalities seen in CADASIL patients. These individuals with rare NOTCH3 mutations indicate that hypomorphic NOTCH3 alleles do not cause CADASIL.     

Two novel mutations in NOTCH3 gene causes cerebral autosomal dominant arteriopathy with subcritical infarct and leucoencephalopathy in two Chinese families

Objective: To investigate the genetic pathogenic causes of cerebral autosomal dominant arteriopathy with subcritical infarct and leucoencephalopathy (CADASIL) in two Chinese families, to provide the molecular basis for genetic counseling and antenatal diagnosis. Methods: The genetic mutation of gene NOTCH3 of propositus and family members was analyzed in these two CADASIL families by polymerase chain reaction and DNA sequencing technology directly. At the same time, the NOTCH3 gene mutation point of 100 healthy collators was detected, to explicit the pathogenic mutation by function prediction with Polyphen-2 and SIFT. Results: Both propositus of the two families and patients with symptom were all accorded with the clinical features of CADASIL. It was shown by DNA sequencing that the 19^th exon [c. 3043 T > A (p.Cys1015Ser)] in gene NOTCH3 of propositus, 2 patients (II3, III7), and a presymptomatic patient (IV1) in Family I all had heterozygosity missense mutation; and the 3^rd exon [c.316T > G, p. (Cys106Gly)] in gene NOTCH3 of the propositus, a patient (IV3) and two presymptomatic patients (IV5, 6) in Family II all had heterozygosity missense mutation; and no mutations were detected in the 100 healthy collators. It was indicated by analyzing the function prediction that the mutation of [c. 3043 T > A (p.Cys1015Ser)] and [c.316T > G, p. (Cys106Gly)] may both influence encoding protein in NOTCH3. By analysis of the conservatism of mutation point in each species, these two basic groups were highly conserved. Conclusion: The heterozygosity missense mutation of 19^th exon [c. 3043 T > A (p.Cys1015Ser)] and the 3^rd exon [c.316T > G, p. (Cys106Gly)] in NOTCH3 gene are the new pathogenic mutations of CADASIL, and enriches the mutation spectrum of NOTCH3 gene.     

Spectrum of NOTCH3 mutations in Korean patients with clinically suspicious cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the NOTCH3 gene on chromosome 19. Previous studies showed that NOTCH3 contains mutational hotspots that can vary among individuals of different ethnic backgrounds. In this study, we investigated the spectrum of NOTCH3 mutations in Korean patients with CADASIL. We retrospectively analyzed 156 patients who underwent NOTCH3 gene testing for molecular diagnosis of CADASIL using Sanger sequencing with a tiered approach. First, we screened previously reported mutational hotspots (exons 2-6, 8, 11, 18, 19, and 22). If no mutation was detected and samples were available, we extended our analysis to additional exons (7, 9, 10, 14, 15, 20, 21, 23, and 25). In 45 of 156 patients (28.8%), 29 mutations and 16 novel variants of unknown significance (VUS) were identified. The p.R544C mutation in exon 11 of NOTCH3 was the most frequently observed mutation (n = 8), followed by p.R75P in exon 3 (n = 7), p.R332C in exon 6 (n = 3), p.R54C in exon 2 (n = 2), and p.R90C in exon 3 (n = 2). Among the VUS, p.R1175W in exon 22, p.S414C in exon 8, and p.N1207S in exon 22 were found in 5, 3, and 2 patients, respectively. Other mutations and VUS were observed in 1 patient each. Although this was not a prospective, nationwide cohort study, the results above suggested that the spectrum of NOTCH3 mutations might be different in Koreans than in individuals of Caucasian ethnicity. Therefore, further analysis of Koreans with CADASIL might be necessary to implement a Korean-specific mutation screening paradigm.     

A novel mutation (C67Y)in the NOTCH3 gene in a Korean CADASIL patient

We report a 52-yr-old Korean woman with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) whose diagnosis was confirmed by skin biopsy and the presence of a novel mutation in the NOTCH3 gene. The patient's clinical features were rather unusual in that 1) clinical presentations were only two episodes of stroke and mild dementia unaccompanied by mood disturbances or migraine, and 2) there was no family history. Brain MRI showed T2 hyperintensities in both temporal pole areas in line with the recent suggestion by O'Sullivan et al. that the abnormality could be a radiologic marker of CADASIL. FDG-PET also showed a hypometabolism in the temporal pole areas with an abnormal finding on MRI in addition to the hypometabolism in cortical and subcortical regions. We could learn from this case that CADASIL may be included in the differential diagnoses in patients with vascular dementia associated with a small vessel disease, even in the absence of a family history, especially when there are no known stroke risk factors and when the MRI shows T2 hyperintensity in the temporal pole regions.     

Genetic analysis of adult leukoencephalopathy patients using a custom‐designed gene panel

Leukoencephalopathies encompass all clinical syndromes that predominantly affect brain white matter. Genetic diagnosis informs clinical management of these patients, but a large part of the genetic contribution to adult leukoencephalopathy remains unresolved. To examine this genetic contribution, we analyzed genomic DNA from 60 Japanese patients with adult leukoencephalopathy of unknown cause by next generation sequencing using a custom‐designed gene panel. We selected 55 leukoencephalopathy‐related genes for the gene panel. We identified pathogenic mutations in 8 of the 60 adult leukoencephalopathy patients (13.3%): NOTCH3 mutations were detected in 5 patients, and EIF2B2, CSF1R, and POLR3A mutations were found independently in 1 patient each. These results indicate that cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 mutations is the most frequent adult leukoencephalopathy in our cohort. Moreover, brain imaging analysis indicates that CADASIL patients who do not present typical phenotypes may be underdiagnosed if not examined genetically.     

CADASIL in Arabs: clinical and genetic findings

Background  

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is increasingly recognized as an inherited arterial disease leading to a step-wise decline and eventually to dementia. CADASIL is caused by mutations in NOTCH3 epidermal growth factor-like repeat that maps to chromosome 19. CADASIL cases have been identified in most countries of Western and Central Europe, the Americas, Japan, Australia, the Caribbean, South America, Tanzania, Turkey, South Africa and Southeast Asia, but not in Arabs.     

伴有皮层下梗死和白质脑病的常染色体显性遗传性脑动脉病一家系的临床及基因研究

目的 探讨伴有皮层下梗死和白质脑病的常染色体显性遗传性脑动脉病(cerebral autosomaldominant arteriopathy with subcortical infarcts and leukoencephalopathy,CADASIL)患者的临床特征及基因诊断.方法 对1个CADASIL先证者及其家族的发病情况、遗传方式、临床表现、影像学特征、分子遗传学及治疗等方面进行研究.结果 患者均有记忆力减退、乏力、脑卒中样发作等特点,没有高血压、动脉硬化证据,有家族聚集性,头颅CT、MRI示多发性梗死灶、脑白质变性,临床符合CADASIL的诊断.存活者中基因测序显示NOTCH3基因第3、4外显子突变,可确诊为CADASIL.该家系4代中有3代10人呈临床或亚临床发病,符合常染色体显性遗传.结论 该家系的临床及分子遗传学特征符合CADASIL诊断.     

Activating NOTCH3 mutation in a patient with small-vessel-disease of the brain

The most common causative diagnosis of hereditary small-vessel-disease of the brain, CADASIL, is due to highly stereotyped mutations in the NOTCH3 receptor. NOTCH3 has 33 exons but all CADASIL mutations occur within the Epidermal Growth Factor-like Repeats encoded by exons 2-24, lead to an odd number of cysteine residues and are associated with GOM deposits and abnormal NOTCH3 protein accumulation. The majority of CADASIL mutations appear to retain normal level of signaling activity, while very few mutations show reduced activity. Herein we identified a novel heterozygous missense mutation (c.4544T>C) in exon 25 of NOTCH3 in a patient with cerebral small-vessel-disease but lacking GOM deposits and NOTCH3 accumulation. The mutation should result in a p.L1515P substitution in the evolutionarily highly conserved juxtamembranous region of NOTCH3, which constitutes the heterodimerization domain. The p.L1515P mutant exhibits increased canonical NOTCH3 signaling, although in a ligand-independent fashion. Biochemical analysis suggests that the mutation renders NOTCH3 hyperactive through destabilization of the heterodimer. Therefore, our study suggests that the p.L1515P mutation falls in a novel mechanistic class of NOTCH3 mutations and that NOTCH3 activating mutations should be further considered for molecular analysis of patients with cerebral small-vessel-disease.     

High recurrence of the R1006C NOTCH3 mutation in central Italian patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a heritable small-vessel disease caused by mutations in NOTCH3 gene and clinically characterized by recurrent ischemic strokes, migraine with aura, psychiatric symptoms, cognitive decline and dementia. Direct sequencing of NOTCH3 gene in 90 Italian patients of sixty-three unrelated families identified four heterozygous mutations (R141C and C144F in exon 4, G528C in exon 10 and R1006C in exon 19) in fifteen probands and sixteen relatives. We detected seventeen heterozygous/homozygous polymorphisms, four of them novel. Here we report the high recurrence of R1006C mutation in ten families all originate from a restricted area of central Italy, the town of Ascoli Piceno and same neighbour villages. We also developed a PCR–Restriction Fragment Length Polymorphism (RFLP) assay to analyze the R1006C mutation. Our findings might suggest, for this mutation, the presence of a common ancestor.     

CADASIL and CARASIL

CADASIL and CARASIL are hereditary small vessel diseases leading to vascular dementia. CADASIL commonly begins with migraine followed by minor strokes in mid‐adulthood. Dominantly inherited CADASIL is caused by mutations (n > 230) in NOTCH3 gene, which encodes Notch3 receptor expressed in vascular smooth muscle cells (VSMC). Notch3 extracellular domain (N3ECD) accumulates in arterial walls followed by VSMC degeneration and subsequent fibrosis and stenosis of arterioles, predominantly in cerebral white matter, where characteristic ischemic MRI changes and lacunar infarcts emerge. The likely pathogenesis of CADASIL is toxic gain of function related to mutation‐induced unpaired cysteine in N3ECD. Definite diagnosis is made by molecular genetics but is also possible by electron microscopic demonstration of pathognomonic granular osmiophilic material at VSMCs or by positive immunohistochemistry for N3ECD in dermal arteries. In rare, recessively inherited CARASIL the clinical picture and white matter changes are similar as in CADASIL, but cognitive decline begins earlier. In addition, gait disturbance, low back pain and alopecia are characteristic features. CARASIL is caused by mutations (presently n = 10) in high‐temperature requirement. A serine peptidase 1 (HTRA1) gene, which result in reduced function of HTRA1 as repressor of transforming growth factor‐β (TGF β) ‐signaling. Cerebral arteries show loss of VSMCs and marked hyalinosis, but not stenosis.     

Whole-exome sequencing of Finnish patients with vascular cognitive impairment

Cerebral small vessel disease (CSVD) is the most important cause of vascular cognitive impairment (VCI). Most CSVD cases are sporadic but familial monogenic forms of the disorder have also been described. Despite the variants identified, many CSVD cases remain unexplained genetically. We used whole-exome sequencing in an attempt to identify novel gene variants underlying CSVD. A cohort of 35 Finnish patients with suspected CSVD was analyzed. Patients were screened negative for the most common variants affecting function in NOTCH3 in Finland (p.Arg133Cys and p.Arg182Cys). Whole-exome sequencing was performed to search for a genetic cause of CSVD. Our study resulted in the detection of possibly pathogenic variants or variants of unknown significance in genes known to associate with CSVD in six patients, accounting for 17% of cases. Those genes included NOTCH3, HTRA1, COL4A1, and COL4A2. We also identified variants with predicted pathogenic effect in genes associated with other neurological or stroke-related conditions in seven patients, accounting for 20% of cases. This study supports pathogenic roles of variants in COL4A1, COL4A2, and HTRA1 in CSVD and VCI. Our results also suggest that vascular pathogenic mechanisms are linked to neurodegenerative conditions and provide novel insights into the molecular basis of VCI.Subject terms: Stroke, Sequencing, Genetics research, Dementia     

CADASIL mutations impair Notch3 glycosylation by Fringe

Mutations in the NOTCH3 gene trigger adult-onset stroke and vascular dementia in patients with CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). All CADASIL mutations described to date affect the epidermal growth factor-like (EGF-like) repeats located in the extracellular domain of the Notch3 receptor. These domains are also the target of sequential complex O-linked glycosylation mediated by protein O-fucosyltransferase 1 and Fringe. We investigated whether O-fucosylation or Fringe-mediated elongation of O-fucose on Notch3 is impaired by CADASIL mutations. Biochemical studies of a Notch3 fragment containing the first five EGF-like repeats of Notch3, including the mutational hot spot, showed that CADASIL mutations do not affect the addition of O-fucose but do impair carbohydrate chain elongation by Fringe. CADASIL changes also induced aberrant homodimerization of mutant Notch3 fragments and heterodimerization of mutant Notch3 with Lunatic Fringe itself. Together, these data suggest that Fringe plays a role in CADASIL pathophysiology.     

Overlapping but distinct roles for NOTCH receptors in human cardiovascular disease

The NOTCH signalling pathway is an essential pathway, involved in many cellular processes, including cell fate decision, cell proliferation, and cell death and important in the development of most organs. Mutations in genes encoding components of the NOTCH signalling pathway lead to a spectrum of congenital disorders. Over the past decades, mutations in human NOTCH signalling genes have been identified in several diseases with cardiovascular involvement. NOTCH1 mutations have been described in bicuspid aortic valve disease, left-sided congenital heart disease, and Adams-Oliver syndrome. NOTCH2 mutations lead to the development of Alagille syndrome, while mutations in NOTCH3 cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. To date, mutations in NOTCH4 have not been associated with cardiovascular disease. This review focuses on the mutations described in NOTCH1, NOTCH2, and NOTCH3 and their associated cardiovascular phenotypes.     

The archetypal R90C CADASIL-NOTCH3 mutation retains NOTCH3 function in vivo

Cerebral Autosomal Dominant Arteriopathy with Subcortical infarcts and Leukoencephalopathy (CADASIL) is the most prominent known cause of inherited stroke and vascular dementia in human adult. The disease gene, NOTCH3, encodes a transmembrane receptor primarily expressed in arterial smooth muscle cells (SMC). Pathogenic mutations lead to an odd number of cysteine residues within the NOTCH3 extracellular domain (NOTCH3(ECD)), and are associated with progressive accumulation of NOTCH3(ECD) at the SMC plasma membrane. The murine homolog, Notch3, is dispensable for viability but required post-natally for the elaboration and maintenance of arteries. How CADASIL-associated mutations impact NOTCH3 function remains a fundamental, yet unresolved issue. Particularly, whether NOTCH3(ECD) accumulation may titrate the ligand and inhibit the normal pathway is unknown. Herein, using genetic analyses in the mouse, we assessed the functional significance of an archetypal CADASIL-associated mutation (R90C), in vivo, in brain arteries. We show that transgenic mouse lines expressing either the wild-type human NOTCH3 or the mutant R90C human NOTCH3, at comparable and physiological levels, can rescue the arterial defects of Notch3-/- mice to similar degrees. In vivo assessment of NOTCH3/RBP-Jk activity provides evidence that the mutant NOTCH3 protein exhibits normal level of activity in brain arteries. Remarkably, the mutant NOTCH3 protein remains functional and does not exhibit dominant negative interfering activity, even when NOTCH3(ECD) accumulates. Collectively, these data suggest a model that invokes novel pathogenic roles for the mutant NOTCH3 protein rather than compromised NOTCH3 function as the primary determinant of the CADASIL arteriopathy.     

Exome sequencing reveals an unexpected genetic cause of disease: NOTCH3 mutation in a Turkish family with Alzheimer's disease

Alzheimer's disease (AD) is a genetically complex disorder for which the definite diagnosis is only accomplished postmortem. Mutations in 3 genes (APP, PSEN1, and PSEN2) are known to cause AD, but a large number of familial cases do not harbor mutations in these genes and several unidentified genes that contain disease-causing mutations are thought to exist. We performed whole exome sequencing in a Turkish patient clinically diagnosed with Alzheimer's disease from a consanguineous family with a complex history of neurological and immunological disorders and identified a mutation in NOTCH3 (p.R1231C), previously described as causing cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Complete screening of NOTCH3 in a cohort of 95 early onset AD cases and 95 controls did not reveal any additional pathogenic mutations. Although the complex history of disease in this family precluded us to establish segregation of the mutation found with disease, our results show that exome sequencing is a rapid, cost-effective and comprehensive tool to detect genetic mutations, allowing for the identification of unexpected genetic causes of clinical phenotypes. As etiological based therapeutics become more common, this method will be key in diagnosing and treating disease.     

Transgenic mice expressing mutant Notch3 develop vascular alterations characteristic of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an increasingly recognized adult-onset autosomal dominant vascular dementia, caused by highly stereotyped mutations in the Notch3 receptor. CADASIL is a widespread angiopathy characterized by a degeneration of vascular smooth muscle cells (VSMCs) and the abnormal accumulation of electron-dense granular material called GOM and Notch3 protein, because of an impaired clearance. Evidence that VSMCs are the primary target of the pathogenic process is supported by the restricted expression of Notch3 in these cells but mechanisms of their degeneration remain essentially unknown. We generated transgenic mice in which the SM22alpha promoter drove, in VSMCs, the expression of a full-length human Notch3 carrying the Arg90Cys mutation, a CADASIL archetypal mutation. Transgenic mice showed no evidence of prominent brain parenchyma damage but demonstrated the two hallmarks of the CADASIL angiopathy, GOM deposits and Notch3 accumulation, within both the cerebral and peripheral arteries. Of interest, arteries of the tail were more severely affected with prominent signs of VSMC degeneration. Time-course analysis of vessel changes revealed that disruption of normal VSMC anchorage to adjacent extracellular matrix and cells, VSMC cytoskeleton changes as well as starting signs of VSMC degeneration, which were detected around 10 months of age, preceded Notch3 and GOM accumulation appearance, which were observed only by 14 to 16 months of age. In conclusion, we have generated transgenic mice that recapitulate the characteristic vascular lesions observed in CADASIL. Our results indicate that Notch3 or GOM accumulation are unlikely to be the prerequisites for the induction of VSMC degeneration and suggest that degeneration of VSMCs may rather be triggered by the disruption of their normal anchorage, based on the important role of adhesion for cell survival.     

Neoplastic lesions in CADASIL syndrome: report of an autopsied Japanese case

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is one of the most common heritable causes of stroke and dementia in adults. The gene involved in the pathogenesis of CADASIL is Notch3; in which mutations affect the number of cysteine residues in its extracellular domain, causing its accumulation in small arteries and arterioles of the affected individuals. Besides the usual neurological and vascular findings that have been well-documented in CADASIL patients, this paper additionally reports multiple neoplastic lesions that were observed in an autopsy case of CADASIL patient; that could be related to Notch3 mutation. The patient was a 62 years old male, presented with a past history of neurological manifestations, including gait disturbance and frequent convulsive attacks. He was diagnosed as CADASIL syndrome with Notch3 Arg133Cys mutation. He eventually developed hemiplegia and died of systemic convulsions. Autopsy examination revealed-besides the vascular and neurological lesions characteristic of CADASIL- multiple neoplastic lesions in the body; carcinoid tumorlet and diffuse idiopathic pulmonary neuro-endocrine cell hyperplasia (DIPNECH) in the lungs, renal cell carcinoma (RCC), prostatic adenocarcinoma (ADC) and adenomatoid tumor of the epididymis. This report describes a spectrum of neoplastic lesions that were found in a case of CADASIL patient that could be related to Notch3 gene mutations.     

Hereditary cerebral small vessel disease: Assessment of a HTRA1 variant using protein stability predictors and 3D modelling

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an autosomal recessive vascular disorder caused by biallellic variants in HTRA1. Recently, it has been reported that several heterozygous mutations in HTRA1 are responsible for a milder late-onset cerebral small vessel disease (CSVD) with an autosomal dominant pattern of inheritance. The majority of them are missense that affects the Htr1A protease activity due to a dominant-negative effect caused by defective trimerization or monomer activation. The molecular mechanism related to the structural destabilization of the protein supports the practical utility of integrating computational stability predictors to prioritize candidate variants in this gene. In this work, we report a family with several members diagnosed with subcortical ischemic events and progressive cognitive impairment caused by the novel c.820C > G, p.(Arg274Gly) heterozygous variant in HTRA1 segregating in an autosomal dominant manner and propose its molecular mechanism by a three-dimensional model of the protein's structure.     

Detection of the founder effect in Finnish CADASIL families

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited cerebrovascular disease characterized by brain infarcts, cognitive decline and dementia. The disease is caused by at least 91 missense mutations, four deletions and one splice site mutation in the NOTCH3 gene, which maps to 19p13.1. In 18 out of the 21 Finnish CADASIL families so far identified, the causative mutation is an arginine to cysteine substitution in position 133 (R133C). Most of the families carrying this mutation originate from the western coast of Finland, thus suggesting a founder effect. No previous reports of a founder effect in CADASIL have been published. We haplotyped 60 patients from these 18 families for 10 microsatellite markers in order to determine whether the families descend from a common ancestor. We found a similar haplotype linked to the mutation in all 18 pedigrees, which indicates a single common ancestor for all the Finnish R133C families. The age analysis of the founder mutation places the introduction of the mutation in the late 1600s or early 1700s.