Reduced Venous Oxygen Saturation Associates With Increased Dependence of Patients With Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy: A 7.0-T Magnetic Resonance Imaging Study.

Background and Purpose- Accumulating evidence has demonstrated hemodynamic abnormalities and cerebral hypoperfusion in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Increased venous susceptibility assessed by susceptibility weighted imaging and mapping has been shown to indicate a decrease in venous oxygen saturation. This study aimed to investigate whether altered venous oxygen saturation is related to clinical phenotypes of CADASIL patients. Methods- Using 7.0-T susceptibility weighted imaging and mapping, we compared venous susceptibility of cortical veins between 41 CADASIL patients and 43 age- and sex-matched healthy controls. The magnetic resonance imaging lesion load, mini-mental state examination score, Barthel Index, and modified Rankin Scale were examined in the patient group, and the correlations between venous susceptibility and clinical characteristics were analyzed. Results- Venous susceptibility increased with age (r=0.508, P=0.001) and was higher in CADASIL patients than in healthy controls (t=-4.673; P<0.001). We found a positive association between venous susceptibility and the age-related white matter change scores (r=0.364; P=0.019), number of lacunar infarctions (r=0.520; P<0.001), number of cerebral microbleeds (ρ=0.445; P=0.004), and small-vessel disease scores (ρ=0.465; P=0.002) in CADASIL patients. Moreover, increased venous susceptibility was associated with higher modified Rankin Scale scores in CADASIL patients after adjustment for age- and small-vessel disease scores (odds ratio=3.178; 95% CI, 1.101-9.179; P=0.033). Conclusions- Our findings indicate that extensive cerebral hypoperfusion may induce central nervous system impairment in CADASIL, and susceptibility weighted imaging and mapping could be used clinically to assess the condition of CADASIL patients.

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.

Generation and characterization of the human iPSC line IDISi001-A isolated from blood cells of a CADASIL patient carrying a NOTCH3 mutation.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of hereditary stroke disorder. It is caused by mutations in NOTCH3 that lead to progressive degeneration of the smooth muscle cells in blood vessels. There is currently no treatment for this disorder. We reprogrammed to pluripotency blood mononuclear cells isolated from a patient carrying a NOTCH3 mutation by using a commercially available non-integrating system. The success in the generation of this iPSC line (IDISi001-A) suggests that the NOTCH3 mutation did not limit cell reprogramming and offers an unprecedented opportunity for studying and modeling CADASIL pathology.

Circulating Biomarkers in Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy Patients.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited cerebral microangiopathy presenting with variable features, including migraine, psychiatric disorders, stroke, and cognitive decline and variable disability. On neuroimaging, CADASIL is characterized by leukoencephalopathy, multiple lacunar infarcts, and microbleeds. Previous studies suggest a possible role of endothelial impairment in the pathogenesis of the disease. METHODS: We assessed plasma levels of von Willebrand factor (vWF) and thrombomodulin (TM) and the blood levels of endothelial progenitor cells (EPCs) and circulating progenitor cells (CPCs) in 49 CADASIL patients and 49 age-matched controls and their association with clinical/functional and neuroimaging features. RESULTS: In multivariate analysis, CADASIL patients had significantly higher vWF and lower EPC levels. TM levels were similar in the 2 groups. CADASIL patients with a more severe clinical phenotype (history of stroke or dementia) presented lower CPC levels in comparison with patients with a milder phenotype. On correlation analysis, lower CPC levels were associated with worse performances on neuropsychological, motor and functional tests, and with higher lesion load on brain magnetic resonance imaging (degree of leukoencephalopathy and number of lacunar infarcts). CONCLUSIONS: This is the first CADASIL series in which multiple circulating biomarkers have been studied. Our findings support previous studies on the presence and the possible modulating effect of endothelial impairment in the disease. Furthermore, our research data suggest that blood CPCs may be markers of disease severity.

Blood biomarkers in a mouse model of CADASIL.

Mutations in NOTCH 3 are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a neurological disorder characterized by stroke, and vascular cognitive impairment and dementia. Loss of vascular smooth muscle cells (VSMC) and accumulation of granular osmiophilic material (GOM) deposits are hallmarks of CADASIL. There are no therapies for CADASIL and experimental endpoints to examine the preclinical efficacy of potential drugs are lacking. This study aims to use a mouse carrying the C455R mutation in Notch 3 to identify biomarkers associated with CADASIL. Mass spectrometry and antibody arrays were used to explore the aorta and blood proteomes of CADASIL mice, ELISA assays were utilized for biomarker validation, a ligand-dependent assay was applied to examine the relationship between Notch signaling and biomarker expression, and retinal histology was performed for quantification of VSMC loss in arteries. Two-hundred day-old mice with the C455R CADASIL mutation in Notch 3 mice display robust VSMC loss in retinal arteries and had increased plasma levels of collagen18α1/endostatin (col18α1) and high-temperature requirement A serine peptidase 1 (HTRA1) and reduced levels of Notch 3 extracellular domain (N3ECD), compared to control wild type mice. Measurements of plasma endostatin, HTRA1 and N3ECD, along with VSMC quantification in retinal arteries, may serve as surrogate endpoints for assessing efficacy in preclinical therapeutic studies of CADASIL using mice.

CADASIL and autoimmunity: coexistence in a family with the R169C mutation at exon 4 of the NOTCH3 gene.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease caused by mutations of the NOTCH3 gene, which result in degeneration of vascular smooth muscle cells, arteriolar stenosis, and impaired cerebral blood flow. For clinicians this is the commonest hereditary adult-onset condition causing stroke and vascular dementia at middle age. Atypical phenotypes have been recognized, and the disease is probably underdiagnosed in the wider stroke population. Coexistence of autoimmunity is atypical and has been described only in occasional patients. METHODS: Three members of a Greek family from the island of Lesvos of North East Greece were evaluated. The patients come from a four-generation family in which there were at least seven members with clinical data suggestive of CADASIL. We describe here the clinical, imaging and biochemical findings in this family with R169C mutation at exon 4 and presenting additional clinical and biochemical findings suggestive of autoimmune disorder. DNA was extracted from whole blood using standard procedures for sequencing. RESULTS: Three affected members of this family carried the R169C. In a phenotypic analysis of affected individuals from four generations with CADASIL, the disease was characterized by migraine attacks, recurrent subcortical infarcts, and cognitive decline with typical anterior temporal lobe white matter lesions. At least 3 mutation carriers from two generations had increased antinuclear antibody (ANA) titers and various combinations of rash, joint pains, photosensitivity, and renal involvement. CONCLUSION: This is a rare description of the coexistence of autoimmunity in CADASIL patients with possible worsening clinical effects. The study extends the spectrum of atypical presentation of CADASIL. The coexistence of autoimmunity does not necessarily exclude CADASIL, but may cause an additional diagnostic and therapeutic challenge. This autoimmune disorder may have increased the severity of the disease and, additionally, may be related to the pathogenetic mechanisms of CADASIL. It is possible that the NOTCH3 mutation alone is not enough to trigger autoimmunity since, in the case of our family, the R169C mutation has already been described in other families with no evidence of coexistent autoimmunity. Other genetic or environmental factors or interactions and/or common pathways between the vascular and immune systems are probably co-operating. Further, prospective studies are needed to clarify the prevalence and types of autoimmune disorders present in CADASIL families.

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.

Brain microvascular accumulation and distribution of the NOTCH3 ectodomain and granular osmiophilic material in CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of familial brain arteriopathy, is associated with deposition of granular osmiophilic material (GOM). We used immunohistochemistry and immunogold electron microscopy (EM) to examine the distribution of GOM and NOTCH3 ectodomain (N3ECD) protein in microvasculature of brain gray matter and white matter in patients with CADASIL, non-CADASIL hereditary small-vessel disease and sporadic age-related degenerative disease, and comparable-age controls. We observed intense immunostaining patterns with 2 different anti-N3ECD antibodies in CADASIL but not in young and older controls or other small-vessel disease patients. In addition, CADASIL samples exhibited immunoreactivity in arterial walls and in most capillaries. Electron microscopy revealed profound and widespread extracellular distribution of 0.2- to 2-µm GOM deposits associated with meningeal vessels and perforating arteries and arterioles. Granular osmiophilic material was adjacent to capillaries even within white matter. Immunogold EM with antibody A1-1 to N3ECD revealed abundant particles in GOM within microvessels, vascular smooth muscle cell membranes, and perivascular cells. Granular osmiophilic material did not exhibit immunogold labeling for smooth muscle α-actin or collagen IV. These results showed the specificity of the antibodies and confirm the predominant localization of N3ECD within GOM deposits. The extensive distribution of N3ECD-GOM complexes within meninges, arteries, arterioles, and particularly capillaries in the brains of CADASIL patients suggests that NOTCH3 fragments are major components of GOM deposits, which may be eliminated via perivascular routes.

Multi-organ investigation in 16 CADASIL families from central Italy sharing the same R1006C mutation.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) may involve many target organs with relevant variability among affected individuals. We performed a multi-organ assessment tapping nervous system, skeletal muscle and cardiovascular system in thirty-nine individuals belonging to 16 families from Central Italy sharing the same R1006C CADASIL mutation. Stroke prevalence was larger in female patients (66.7%) than in males (23.8%); high levels of CKemia were quite frequent (21.6%) and were related to a myopathy without mitochondrial alterations; several individuals had atrial septal aneurysm (10.3%). No specific relationships between common cardiovascular risk factors and clinical manifestations were found. The present systematic study thus identified several gender-related, myopathic and cardiovascular peculiarities of R1006C mutation. This kind of comprehensive approach is necessary to define clinical course, prognosis and treatment options for a multi-organ disease such as CADASIL.

Clinical and genetic features in a family with CADASIL and high lipoprotein (a) values.

We present a family with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and elevated lipoprotein(a) [Lp(a)] levels. In addition to neurological examinations, ultrasound of extra- and intracranial arteries, laboratory tests, and cerebral magnetic resonance imaging (MRI), a whole genome screening with mutation analyses was performed. Rather untypical for CADASIL, stenoses of large intracranial arteries were detected in the index patient. All affected subjects lacked a history of migraine, mood disturbances, and cognitive decline despite extensive white matter lesions in two individuals. Furthermore, evidence of early cerebral microangiopathy was demonstrated in three children (age 9, 11 and 13). We were able to explain the mechanism of elevated Lp(a) on the basis of the kringle IV type 2 repetition size. A mutation S118C located in exon 4 of Notch3 was responsible for CADASIL. Elevated Lp(a) might have contributed to the cerebrovascular phenotype in this family.

Plasma levels of asymmetric dimethylarginine in cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy.

BACKGROUND: Asymmetric dimethylarginine (ADMA) is a marker of endothelial dysfunction and a new independent risk factor for adverse cerebrovascular events in small vessel disease. Conversely, L-arginine (LARG) may have a protective role. METHODS: To assess ADMA, LARG levels and LARG/ADMA ratio in 16 patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and normal controls, and to look for possible correlations with white matter changes. Plasma levels of ADMA and LARG were assayed by high-performance liquid chromatography in all subjects. The overall T(1) and T(2) lesion load was obtained from brain MRI of patients with CADASIL. RESULTS: ADMA plasma concentrations (1.5 +/- 2.0 microM) were significantly higher (p < 0.05) in CADASIL patients than in controls (0.35 +/- 0.075 microM). Analyzing only CADASIL subjects, an inverse borderline-significant correlation was found between LARG/ADMA (190 +/- 20) and T(2)-weighted lesion volumes (57.9 +/- 46.5; r = -0.578, p = 0.024). CONCLUSION: Our results may indicate the possible coexistence of endothelial dysfunction in CADASIL patients, broadening the range of potentially pathogenetic mechanisms in this disease and providing insights for future therapeutic strategies.

Blood pressure and haemoglobin A1c are associated with microhaemorrhage in CADASIL: a two-centre cohort study.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a hereditary arteriopathy caused by mutations of the Notch3 gene. The risk factors for cerebral microhaemorrhages (CM), their relationship to other MRI lesions in the disease and their potential clinical impact have not been previously defined. Our purpose was to examine the frequency, number and location of microhaemorrhages in a multicentre cohort study, defining predisposing factors and associated radiographic markers in CADASIL patients. We collected clinical data from 147 consecutive patients enrolled in an ongoing prospective cohort study. Degree of neurological disability and cognitive impairment were assessed by standardized scales. T(1)-weighted, FLAIR and T2*-weighted gradient-echo (GE) MRI sequences were performed. Volume and location of lacunar infarcts and white matter hyperintensity (WMH) were assessed. Number and location of CM were recorded. CM were present in 35% patients, most commonly occurring in the thalamus, brainstem and basal ganglia. The location of CM qualitatively differed from areas of lacunar infarction and WMH. There was a significant association between the presence of CM and a history of hypertension (P = 0.005), systolic blood pressure (SBP) (P = 0.014), haemoglobin A1c (HbA1c) (P = 0.004) and the volume of lacunar infarcts (P = 0.010) and WMHs (P = 0.046). The number of CM was independently associated with SBP (P = 0.005), the diagnosis of hypertension (P = 0.0004), volume of WMH (P = 0.0005) and lacunar infarcts (P = 0.004). In contrast, no association was found between blood pressure or HbA1c and the load of WMH or lacunar infarcts. The presence of CM was independently associated with increased modified Rankin scores. CM are independently associated with blood pressure and HbA1c as well as with lacunar infarct and WMH volume in CADASIL. Both the vascular risk factors and regional distribution of CM appear distinct from those associated with other MRI markers, suggesting a distinct pathological process. These lesions have a potential clinical impact in CADASIL. These findings further suggest that modulation of blood pressure and glucose levels might influence the course of the disease.

[Clinical features in 4 Chinese families with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)].

OBJECTIVE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is an inherited cerebral arteriolar disease in adulthood, which is caused by NOTCH3 gene mutation. The main symptoms were migraine, cerebral stroke, later with mood disorders and dementia in Caucasian patients. Recently, the disease was also recognized in Asian patients, in whom the migraine is rarely reported. In order to give the clinical features of Chinese patients, we described the clinical symptoms in 4 CADASIL families. METHODS: CADASIL was diagnosed by the investigation of ultra-structure changes of arteriole in sural nerve and NOTCH3 gene mutation in the 4 index cases. Detailed clinical and routine laboratory examinations were performed in these 4 patients, including electrocardiography, nerve conduction velocity, serum glycogen, and serum homocysteine. Additionally, we also collected the clinical data of the other 83 family members through interviews and the available medical records. RESULTS: Of the 83 persons, 29 were classified as clinical suspected patients, who presented one or more of the disease-related neurological symptoms, such as cerebral ischemic events and the cognitive impairment. All of them showed no common risk factors for stroke, such as diabetic mellitus, hypertension, and heart disease. The clinical suspected patients distributed in every consecutive generations and involved both sexes, which was according to the autosomal dominant inherited pattern. The onset age of the disease ranged from 28 to 70-year-old and mainly between the 4th and the 5th decades. The main symptoms were recurrent episodic vertigo, with or without hemiplegia. At the same time or a little bit later, the cognitive impairment was developed in some patients. Compared with the typical presentations of the disease in European patients, none of our 29 patients showed migraine,one index case showed mild sensory disturbance in extremities. Elevated serum homocysteine level and abnormal of nerve conduction study in two index cases (3 and 4) were noticed. CONCLUSION: The onset age of the disease of our patients is similar to that of Caucasian patients. The main symptoms were stroke and dementia. Involvement of post circulation system was the main clinical feature for ischemic events in our patients. Dementia could be found in the early stage of disease. Migraine should not be regarded as a common clinical feature in our patients. The involvement of the peripheral nerves expanded the disease expression outside the central nervous system.