Characterization of CADASIL among the Han Chinese in Taiwan: Distinct Genotypic and Phenotypic Profiles

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is originally featured with a strong clustering of mutations in NOTCH3 exons 3–6 and leukoencephalopathy with frequent anterior temporal pole involvement. The present study aims at characterizing the genotypic and phenotypic profiles of CADASIL in Taiwan. One hundred and twelve patients with CADASIL from 95 families of Chinese descents in Taiwan were identified by Sanger sequencing of exons 2 to 24 of NOTCH3. Twenty different mutations in NOTCH3 were uncovered, including 3 novel ones, and R544C in exon 11 was the most common mutation, accounting for 70.5% of the pedigrees. Haplotype analyses were conducted in 14 families harboring NOTCH3 R544C mutation and demonstrated a common haplotype linked to NOTCH3 R544C at loci D19S929 and D19S411. Comparing with CADASIL in most Caucasian populations, CADASIL in Taiwan has several distinct features, including less frequent anterior temporal involvement, older age at symptom onset, higher incidence of intracerebral hemorrhage, and rarer occurrence of migraine. Subgroup analyses revealed that the R544C mutation is associated with lower frequency of anterior temporal involvement, later age at onset and higher frequency of cognitive dysfunction. In conclusion, the present study broadens the spectrum of NOTCH3 mutations and provides additional insights for the clinical and molecular characteristics of CADASIL patients of Han-Chinese descents.     

CADASIL mutant NOTCH3(R90C) decreases the viability of HS683 oligodendrocytes via apoptosis

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary cerebral small vessel disease caused by mutations in NOTCH3. Prevailing models suggest that demyelination occurs secondary to vascular pathology. However, in zebrafish, NOTCH3 is also expressed in mature oligodendrocytes. Thus, we hypothesized that in addition to vascular defects, mutant NOTCH3 may alter glial function in individuals with CADASIL. The aim of this study was to characterize the direct effects of a mutant NOTCH3 protein in HS683 oligodendrocytes. HS683 oligodendrocytes transfected with wild-type NOTCH3, mutant NOTCH3(R90C), and empty control vector were used to study the impact of the NOTCH3(R90C) mutant on its protein hydrolytic processing, cell viability, apoptosis, autophagy, oxidative stress, and the related upstream events using immunoblotting, immunofluorescence, RT-PCR, and flow cytometry. We determined that HS683 oligodendrocytes transfected with mutant NOTCH3(R90C), which is the hotspot mutation site-associated with CADASIL, exhibited aberrant NOTCH3 proteolytic processing. Compared to cells overexpressing wild-type NOTCH3, cells overexpressing NOTCH3(R90C) were less viable and had a higher rate of apoptosis. Immunoblotting revealed that cells transfected with NOTCH3(R90C) had higher levels of intrinsic mitochondrial apoptosis, extrinsic death receptor path-related apoptosis, and autophagy compared with cells transfected with wild-type NOTCH3. This study suggests that in patients with CADASIL, early defects in glia influenced by NOTCH3(R90C) may directly contribute to white matter pathology in addition to secondary vascular defects. This study provides a potential therapeutic target for the future treatment of CADASIL.     

Two novel mutations of the NOTCH3 gene in Korean patients with CADASIL

Mutations in the NOTCH3 gene (NOTCH3) are responsible for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), an adult-onset hereditary angiopathy leading to ischemic episodes, vascular dementia and other neurologic deficits. All mutations of NOTCH3 described so far are strictly stereotyped, leading to the gain or loss of a cysteine residue in a given epidermal growth factor (EGF)-like repeat of NOTCH3. We report two novel mutations of NOTCH3, R587C and C988Y, each resulting in an odd number of cysteine residues in an EGF-like repeat of NOTCH3. We identified these mutations in two unrelated Korean families with CADASIL, who presented with magnetic resonance imaging (MRI) abnormalities typical of CADASIL. These findings confirm that mutations in NOTCH3 are associated with the pathogenesis of CADASIL across different ethnic backgrounds.     

Two novel mutations and a previously unreported intronic polymorphism in the NOTCH3 gene

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease of small vessel caused by mutations in the NOTCH3 gene (NCBI Gene ID: 4854) located on chromosome 19p13.1. NOTCH3 consists of 33 exons which encode a protein of 2321 amino acids. Exons 3 and 4 were found to be mutation hotspots, containing more than 65% of all CADASIL mutations. We performed direct sequencing on an ABI 3130 Genetic Analyser to screen for mutations and polymorphisms on 300 patients who were clinically suspected to have CADASIL. First, exons 3 and 4 were screened in NOTCH3 and if there were no variations found, then extended CADASIL testing (exons 2, 11, 18 and 19) was offered to patients. Here we report two novel non-synonymous mutations identified in the NOTCH3 gene. The first mutation, located in exon 4 was found in a 49-year-old female and causes an alanine to valine amino acid change at position 202 (605C>T). The second mutation, located in exon 11, was found in a 66-year-old female and causes a cysteine to arginine amino acid change at position 579 (1735T>C). We also report a 46-year-old male with a known polymorphism Thr101Thr (rs3815188) and an unreported polymorphism NM_000435.2:c.679+60G>A observed in intron 4 of the NOTCH3 gene. Although Ala202Ala (rs1043994) is a common polymorphism in the NOTCH3 gene, our reported novel mutation (Ala202Val) causes an amino acid change at the same locus. Our other reported mutation (Cys579Arg) correlates well with other known mutations in NOTCH3, as the majority of the CADASIL-associated mutations in NOTCH3 generally occur in the EGF-like (epidermal growth factor-like) repeat domain, causing a change in the number of cysteine residues. The intronic polymorphism NM_000435.2:c.679+60G>A lies close to the intron-exon boundary and may affect the splicing mechanism in the NOTCH3 gene.     

R54C Mutation of NOTCH3 Gene in the First Rungus Family with CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary stroke caused by mutations in NOTCH3 gene. We report the first case of CADASIL in an indigenous Rungus (Kadazan-Dusun) family in Kudat, Sabah, Malaysia confirmed by a R54C (c.160C>T, p.Arg54Cys) mutation in the NOTCH3. This mutation was previously reported in a Caucasian and two Korean cases of CADASIL. We recruited two generations of the affected Rungus family (n = 9) and found a missense mutation (c.160C>T) in exon 2 of NOTCH3 in three siblings. Two of the three siblings had severe white matter abnormalities in their brain MRI (Scheltens score 33 and 50 respectively), one of whom had a young stroke at the age of 38. The remaining sibling, however, did not show any clinical features of CADASIL and had only minimal changes in her brain MRI (Scheltens score 17). This further emphasized the phenotype variability among family members with the same mutation in CADASIL. This is the first reported family with CADASIL in Rungus subtribe of Kadazan-Dusun ethnicity with a known mutation at exon 2 of NOTCH3. The penetrance of this mutation was not complete during the course of this study.     

Prevalence of CADASIL and Fabry Disease in a Cohort of MRI Defined Younger Onset Lacunar Stroke

Background and Purpose

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by mutations in the NOTCH3 gene, is the most common monogenic disorder causing lacunar stroke and cerebral small vessel disease (SVD). Fabry disease (FD) due to mutations in the GLA gene has been suggested as an underdiagnosed cause of stroke, and one feature is SVD. Previous studies reported varying prevalence of CADASIL and FD in stroke, likely due to varying subtypes studied; no studies have looked at a large cohort of younger onset SVD. We determined the prevalence in a well-defined, MRI-verified cohort of apparently sporadic patients with lacunar infarct.

Methods

Caucasian patients with lacunar infarction, aged ≤70 years (mean age 56.7 (SD8.6)), were recruited from 72 specialist stroke centres throughout the UK as part of the Young Lacunar Stroke DNA Resource. Patients with a previously confirmed monogenic cause of stroke were excluded. All MRI’s and clinical histories were reviewed centrally. Screening was performed for NOTCH3 and GLA mutations.

Results

Of 994 subjects five had pathogenic NOTCH3 mutations (R169C, R207C, R587C, C1222G and C323S) all resulting in loss or gain of a cysteine in the NOTCH3 protein. All five patients had confluent leukoaraiosis (Fazekas grade ≥2). CADASIL prevalence overall was 0.5% (95% CI 0.2%-1.1%) and among cases with confluent leukoaraiosis 1.5% (95% CI 0.6%-3.3%). No classic pathogenic FD mutations were found; one patient had a missense mutation (R118C), associated with late-onset FD.

Conclusion

CADASIL cases are rare and only detected in SVD patients with confluent leukoaraiosis. No definite FD cases were detected.     

A Novel Cysteine-Sparing NOTCH3 Mutation in a Chinese Family with CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset cerebral small vessel disorder caused by the mutations of the neurogenic locus notch homolog protein 3 (NOTCH3) gene. The extracellular part of NOTCH3 is composed of 34 epidermal growth factor-like (EGF-like) repeat domains. Each EGF-like domain is rich of cysteine and glycine to produce three loops that are essential for high-affinity binding to its ligand. Nearly all reported CADASIL-associated mutations result in gain or loss of a cysteine residue within the EGF-like domains. Only a few cysteine-sparing NOTCH3 mutations have been documented in the patients with CADASIL to date. Here, we reported a Chinese CADASIL family with a cysteine-sparing NOTCH3 mutation. In this family, affected patients had dizziness, memory loss, gait instability, or hemiplegia. Brain magnetic resonance imaging (MRI) showed diffuse leukoencephalopathy with confluent signal abnormalities in the periventricular white matter, basal ganglia, and centrum semiovale bilaterally. By screening the entire coding region of NOTCH3, a novel missense mutation p.G149V (c.446G>T) was found. This mutation was not detected in 400 normal controls. Considering the critical position of glycine within the C-loop of EGF-like domain and its high conservation through evolution, p.G149V mutation could be a potential pathogenic cause for CADASIL.     

Autophagy-lysosomal defect in human CADASIL vascular smooth muscle cells

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial progressive degenerative disorder and is caused by mutations in NOTCH3 gene. Previous study reported that mutant NOTCH3 is more prone to form aggregates than wild-type NOTCH3 and the mutant aggregates are resistant to degradation. We hypothesized that aggregation or accumulation of NOTCH3 could be due to impaired lysosomal-autophagy machinery in VSMC.Here, we investigated the possible cause of accumulation/aggregation of NOTCH3 in CADASIL using cerebral VSMCs derived from control and CADASIL patients carrying NOTCH3^R133C mutation. Thioflavin-S-staining confirmed the increased accumulation of aggregated NOTCH3 in VSMC^R133C compared to VSMC^WT. Increased levels of the lysosomal marker, Lamp2, were detected in VSMC^R133C, which also showed co-localization with NOTCH3 using double-immunohistochemistry. Increased level of LC3-II/LC3-I ratio was observed in VSMC^R133C suggesting an accumulation of autophagosomes. This was coupled with the decreased co-localization of NOTCH3 with LC3, and Lamp2 and, further, increase of p62/SQSTM1 levels in VSMC^R133C compared to the VSMC^WT. In addition, Western blot analysis indicated phosphorylation of p-ERK, p-S6RP, and p-P70 S6K. Altogether, these results suggested a dysfunction in the autophagy-lysosomal pathway in VSMC^R133C.The present study provides an interesting avenue of the research investigating the molecular mechanism of CADASIL.     

Targeted next generation sequencing identifies novel NOTCH3 gene mutations in CADASIL diagnostics patients

Background

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic, hereditary, small vessel disease of the brain causing stroke and vascular dementia in adults. CADASIL has previously been shown to be caused by varying mutations in the NOTCH3 gene. The disorder is often misdiagnosed due to its significant clinical heterogeneic manifestation with familial hemiplegic migraine and several ataxia disorders as well as the location of the currently identified causative mutations. The aim of this study was to develop a new, comprehensive and efficient single assay strategy for complete molecular diagnosis of NOTCH3 mutations through the use of a custom next-generation sequencing (NGS) panel for improved routine clinical molecular diagnostic testing.

Results

Our custom NGS panel identified nine genetic variants in NOTCH3 (p.D139V, p.C183R, p.R332C, p.Y465C, p.C597W, p.R607H, p.E813E, p.C977G and p.Y1106C). Six mutations were stereotypical CADASIL mutations leading to an odd number of cysteine residues in one of the 34 NOTCH3 gene epidermal growth factor (EGF)-like repeats, including three new typical cysteine mutations identified in exon 11 (p.C597W; c.1791C>G); exon 18 (p.C977G; c.2929T>G) and exon 20 (p.Y1106C; c.3317A>G). Interestingly, a novel missense mutation in the CACNA1A gene was also identified in one CADASIL patient. All variants identified (novel and known) were further investigated using in silico bioinformatic analyses and confirmed through Sanger sequencing.

Conclusions

NGS provides an improved and effective methodology for the diagnosis of CADASIL. The NGS approach reduced time and cost for comprehensive genetic diagnosis, placing genetic diagnostic testing within reach of more patients.

     

Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL)is a rare hereditary cerebrovascular disease caused by a NOTCH3 mutation.However,the underlying cellular and molecular mechanisms remain unidentified.Here,we generated non-integrative induced pluripotent stem cells(iPSCs)from fibroblasts of a CADASIL patient harboring a heterozygous NOTCH3 mutation(c.3226C>T,p.R1076C).Vascular smooth muscle cells(VSMCs)differentiated from CADASIL-specific iPSCs showed gene expression changes associated with disease phenotypes,including activation of the NOTCH and NF-kB signaling pathway,cytoskeleton disorganization,and excessive cell proliferation.In comparison,these abnormalities were not observed in vascular endothelial cells(VECs)derived from the patients iPSCs.Importantly,the abnormal upregulation of NF-kB target genes in CADASIL VSMCs was diminished by a NOTCH pathway inhibitor,providing a potential therapeutic strategy for CADASIL.Overall,using this iPSCbased disease model,our study identified clues for studying the pathogenic mechanisms of CADASIL and developing treatment strategies for this disease.     

Role of Electron Microscopy in the Diagnosis of Cadasil Syndrome: A Study of 32 Patients

Background and Purpose

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by NOTCH3 gene mutations that result in vascular smooth muscle cell (VSMC) degeneration. Its distinctive feature by electron microscopy (EM) is granular osmiophilic material (GOM) detected in VSMC indentations and/or the extracellular space close to VSMCs. Reports of the sensitivity of EM in detecting GOM in biopsies from CADASIL patients are contradictory. We present data from 32 patients clinically suspected to have CADASIL and discuss the role of EM in its diagnosis in this retrospective study.

Methods

Skin, skeletal muscle, kidney and pericardial biopsies were examined by EM; the NOTCH3 gene was screened for mutations. Skin and muscle biopsies from 12 patients without neurological symptoms served as controls.

Results and Discussion

All GOM-positive patients exhibited NOTCH3 mutations and vice versa. This study i) confirms that EM is highly specific and sensitive for CADASIL diagnosis; ii) extends our knowledge of GOM distribution in tissues where it has never been described, e.g. pericardium; iii) documents a novel NOTCH3 mutation in exon 3; and iv) shows that EM analysis is critical to highlight the need for comprehensive NOTCH3 analysis. Our findings also confirm the genetic heterogeneity of CADASIL in a small Italian subpopulation and emphasize the difficulties in designing algorithms for molecular diagnosis.     

CADASIL-associated Notch3 mutations have differential effects both on ligand binding and ligand-induced Notch3 receptor signaling through RBP-Jk

Mutations in the NOTCH3 gene are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary angiopathy leading to strokes and dementia. Pathogenic mutations remove or insert cysteine residues within epidermal growth factor (EGF) repeats in the extracellular domain of the Notch3 receptor (N3ECD). Vascular smooth muscle cells (VSMC) are the predominant site of Notch3 expression in adults. In CADASIL patients, VSMC degenerate and N3ECD is deposited within the vasculature. However, the mechanisms underlying VSMC degeneration and N3ECD accumulation are still unknown. In this study, we investigated the consequences of three pathogenic Notch3 mutations on the biological activity of the receptor by analyzing ligand (Delta-/Jagged-)-induced signaling via RBP-Jk. Two mutations (R133C and C183R) that are located outside the putative ligand binding domain (LBD) of the receptor were found to result in normal Jagged1-induced signaling in A7r5 VSMC, whereas the third mutation (C455R located within the putative LBD) showed strongly reduced signaling activity. Ligand binding assays with soluble Delta1 and Jagged1 revealed that C455R interferes with ligand binding through disruption of the LBD which, as we show here, is located in EGF repeats 10/11 of Notch3. All mutant receptors including Notch3C455R were targeted to the cell surface but showed an elevated ratio between the unprocessed full-length 280-kDa receptor and S1-cleaved receptor fragments. Taken together, these data indicate that CADASIL-associated Notch3 mutations differ with respect to their consequences both on ligand binding and ligand-induced signaling through RBP-Jk, whereas they have similar effects on receptor maturation. Moreover, the data suggest that ligand-induced receptor shedding may not be required for N3ECD deposition in CADASIL.     

Pathogenesis of CADASIL: transgenic and knock-out mice to probe function and dysfunction of the mutated gene, Notch3, in the cerebrovasculature

Small vessel diseases (SVDs) of the brain are the leading cause of vascular cognitive impairment and a major contributor to stroke in the human adult, however, their pathogenesis is poorly understood. Dominant mutations in NOTCH3 cause CADASIL, one of the most prevalent inherited cerebral SVDs. The disease gene encodes a transmembrane receptor primarily expressed in smooth muscle cells of small arteries and pericytes of brain capillaries. Pathogenic mutations alter the number of cysteine residues in the extracellular domain of NOTCH3, leading to its abnormal accumulation in the vessels of patients. Mice lacking NOTCH3 have revealed a critical role for NOTCH3 in the elaboration of small arteries. Despite being incomplete disease models, transgenic mice expressing CADASIL-associated NOTCH3 mutations, have provided important insights into specific aspects of CADASIL pathogenesis, including the functional significance of disease-linked mutations and the earliest pathological events that initiate brain lesions. In this paper, I provide a critical overview of these studies. Moreover, I discuss future directions and further work that needs to be done.     

Renal involvement in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL): report of a case with a six-year follow-up

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a disorder of the cerebral small blood vessels caused by a mutation in the NOTCH3 gene, which encodes a large transmembrane receptor NOTCH3. It is associated with systemic arteriopathy involving small arteries, besides the brain, in skin, spleen, liver, muscle, aorta and in the kidney. The key pathological finding is the accumulation of granular osmiophilic material (GOM) on degenerating vascular smooth muscle cells. In the kidney GOMs have been described only in a very limited number of CADASIL patients. We describe a genetically confirmed CADASIL patient with mild renal dysfunction and GOMs in the interlobular and juxtaglomerular arteries and, for the first time, also within the glomerulus, whose nephrology conditions remained stable, whereas the neurological manifestations markedly worsened over a six-year follow-up period. The reasons for this discrepancy are probably related to differences in the structure and function of brain and kidney blood vessels.     

Apoptosis in CADASIL: An in vitro study of lymphocytes and fibroblasts from a cohort of Italian patients

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease affecting vascular smooth muscle cells of nearly all tissues. Clinical manifestations mainly concern the central nervous system with repeated TIA/stroke, migraine, psychiatric disturbances, and cognitive decline. Minor findings have been reported in muscle, nerve, and skin. CADASIL is due to NOTCH3 gene mutations. This gene has been identified as an up‐regulator of c‐FLIP, an inhibitor of Fas‐ligand‐induced apoptosis. The aim of this study was to assess the involvement of oxidative stress‐induced apoptosis in cells from 16 Italian CADASIL patients. Peripheral blood lymphocytes (PBLs) and fibroblasts from CADASIL patients were exposed to 2‐deoxy‐D ‐ribose (dRib), which induces apoptosis by oxidative stress. Apoptosis was analyzed by flow cytometry, agarose gel electrophoresis and fluorescence microscopy for caspase‐3 activation, phosphatidylserine exposure and mitochondrial membrane depolarization. PBLs and fibroblasts from CADASIL patients showed a significantly higher response to dRib‐induced apoptosis than those of controls. PBLs from CADASIL patients also showed a significantly higher percentage of apoptotic cells than PBLs from controls, even when cultured without dRib. The greater susceptibility of PBLs and fibroblasts from CADASIL patients to dRib‐induced apoptosis suggests that NOTCH3 mutations are an important apoptotic trigger. Since PBLs from patients showed higher levels of apoptosis even in the absence of an apoptotic stimulus, cells from CADASIL patients appear to be physiologically prone to apoptotic cell death. J. Cell. Physiol. 219: 494–502, 2009. © 2009 Wiley‐Liss, Inc.     

Proteome analysis of cultivated vascular smooth muscle cells from a CADASIL patient

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementing disease caused by mutations in the NOTCH3 gene, most which are missense mutations leading to an uneven number of cysteine residues in epidermal growth factor-like repeats in the extracellular domain of Notch3 receptor (N3ECD). CADASIL is characterized by degeneration of vascular smooth muscle cells (VSMC) and accumulation of N3ECD on the VSMCs of small and middle-sized arteries. Recent studies have demonstrated that impairment of Notch3 signaling is not the primary cause of the disease. In the present study we used proteomic analysis to characterize the protein expression pattern of a unique material of genetically genuine cultured human CADASIL VSMCs. We identified 11 differentially expressed proteins, which are involved in protein degradation and folding, contraction of VSMCs, and cellular stress. Our findings indicate that misfolding of Notch3 may cause endoplasmic reticulum stress and activation of unfolded protein response, leading to increased reactive oxygen species and inhibition of cell proliferation. In addition, upregulation of contractile proteins suggests an alteration in the signaling system of VSMC contraction. The accumulation of N3ECD on the cell surface possibly upregulates the angiotensin II regulatory feedback loop and thereby enhances the readiness of the cells to respond to angiotensin II stimulation.     

NOTCH3基因c.520T>G突变致CADASIL一家系报告及文献复习

目的:研究探讨一CADASIL家系的临床特征及基因突变情况。方法:收集同一家系中3例CADASIL患者的临床资料,并对3例患者及先证者之兄进行全外显子测序(Whole Exome Sequencing, WES)。结果:该家系中3例患者临床表现多样,女性患者均有头痛病史,先证者及先证者之姐中年起病,先证者临床表现缺乏特异性,主要表现为头昏,认知功能检查正常,心理评估示轻度焦虑抑郁状态。先证者之姐主要表现为假性球麻痹及锥体束受损,认知功能检查示重度痴呆。先证者之女自4岁起诊断为癫痫-失神发作,认知功能检查示轻度认知功能障碍。影像学显示该家系3例患者均有脑白质病变,且随着年龄增大呈进行性发展,WES显示3例患者均存在NOTCH3基因第4外显子区域杂合突变:c.520T>G,导致氨基酸改变p.Cys174Gly。结论:NOTCH3基因c.520T>G所致该家系的临床表现具有多样性,且该家系中下一代起病较早,临床表现可与父代具有较大异质性,影像学表现可在青少年时期出现,并呈现进行加重的趋势。WES显示该家系中NOTCH3基因突变为第4外显子的杂合突变,该位点突变致CADASIL为国内首次报道。     

Is the Oxidant/Antioxidant Status Altered in CADASIL Patients?

The altered aggregation of proteins in non-native conformation is associated with endoplasmic reticulum derangements, mitochondrial dysfunction and excessive production of reactive oxygen species. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary systemic vasculopathy, caused by NOTCH3 mutations within the receptor extracellular domain, that lead to abnormal accumulation of the mutated protein in the vascular wall. NOTCH3 misfolding could cause free radicals increase also in CADASIL. Aim of the study was to verify whether CADASIL patients have increased oxidative stress compared to unrelated healthy controls. We enrolled 15 CADASIL patients and 16 gender- and age-matched healthy controls with comparable cardiovascular risk factor. Blood and plasma reduced and total aminothiols (homocysteine, cysteine, glutathione, cysteinylglycine) were measured by HPLC and plasma 3-nitrotyrosine by ELISA. Only plasma reduced cysteine (Pr-Cys) and blood reduced glutathione (Br-GSH) concentrations differed between groups: in CADASIL patients Br-GSH levels were higher (p = 0.019) and Pr-Cys lower (p = 0.010) than in controls. No correlation was found between Br-GSH and Pr-Cys either in CADASIL patients (rho 0.25, P=0.36) or in controls (rho -0.15, P=0.44). Conversely, 3-nitrotyrosine values were similar in CADASIL and healthy subjects (p = 0.82). The high levels of antioxidant molecules and low levels of oxidant mediators found in our CADASIL population might either be expression of an effective protective action against free radical formation at an early stage of clinical symptoms or they could suggest that oxidative stress is not directly involved in the pathogenesis of CADASIL.