Cystatin C based estimation of chronic kidney disease and amyotrophic lateral sclerosis in the ALS registry Swabia: associated risk and prognostic value.

Kidney function as part of metabolic changes could be associated with amyotrophic lateral-sclerosis (ALS). We investigated the associations between estimated chronic kidney disease (CKD), based on the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) cystatin C equation, and the risk at onset and prognostic value of CKD for ALS. Between October 2010 and June 2014, 362 ALS cases (59.4% men, mean age 65.7 years) and 681 controls (59.5% men, means age 66.3 years) were included in a population-based case-control study based on the ALS registry Swabia in Southern Germany. All ALS cases were followed-up (median 89.7 months), 317 died. Serum samples were measured for cystatin C to estimate the glomerular filtration rate (eGFR) according to the CKD-EPI equation. Information on covariates were assessed by an interview-based standardized questionnaire. Conditional logistic regression models were applied to calculate odds ratios (OR) for risk of ALS associated with eGFR/CKD stages. Time-to-death associated with renal parameters at baseline was assessed in ALS cases only. ALS cases were characterized by lower body mass index, slightly lower smoking prevalence, more intense occupational work and lower education than controls. Median serum cystatin-C based eGFR concentrations were lower in ALS cases than in controls (54.0 vs. 59.5 mL/min pro 1.73 m2). The prevalence of CKD stage ≥ 3 was slightly higher in ALS cases than in controls (14.1 vs. 11.0%). In the adjusted models, CKD stage 2 (OR 1.82, 95% CI 1.32, 2.52) and stage 3 (OR 2.34, 95% CI 1.38, 3.96) were associated with increased ALS risk. In this cohort of ALS cases, eGFR and CKD stage ≥ 3 (HR 0.94; 95% CI 0.64, 1.38) were not associated with prognosis. In this case-control study, higher CKD stages were associated with increased ALS risk, while in the prospective cohort of ALS cases, no indication of an association of CysC-based CKD on mortality was seen. In addition, our work strengthens the importance to evaluate renal function using a marker independent of muscle mass in ALS patients.

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.

Predictors of Clinical Worsening in Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy: Prospective Cohort Study.

BACKGROUND AND PURPOSE: Predictors of clinical worsening in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy remain unknown. This study aims to identify demographic, clinical, and magnetic resonance imaging predictors of incident strokes, incident dementia, clinical deterioration, and death in patients with this genetically proven disease. METHODS: Two hundred ninety subjects (mean age, 50.6±11.4 years) were assessed at baseline and followed up for 36 months. Incident clinical events were recorded, and clinical scores included the Mini Mental State Examination, Mattis Dementia Rating Scale, modified Rankin Scale, and Barthel index. The number of lacunes and microbleeds, the volume of white-matter hyperintensities, and brain parenchymal fraction were assessed on baseline magnetic resonance imaging. Data were analyzed by ANCOVA, multivariable logistic regression, and Cox proportional hazard models. RESULTS: Incident stroke occurred in 55 of 278 patients (19.8%). Moderate or severe disability developed in 19 of 210 (9%) nondisabled individuals, incident dementia in 49 of 231 (20%) nondemented subjects, and 4.8% of patients died. Active smoking, the number of lacunes, and brain parenchymal fraction independently predicted incident stroke during follow-up. Gait disturbance, dementia, and brain parenchymal fraction predicted progression toward moderate or severe disability. Active smoking, disability, and brain parenchymal fraction predicted incident dementia. Age was the only significant predictor of death. CONCLUSIONS: Clinical assessment and brain magnetic resonance imaging aid in predicting incident clinical events and clinical deterioration in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. There is a bidirectional relationship between dementia and moderate or severe disability in predicting each other's onset. Active smoking is a modifiable risk factor associated with clinical progression in Notch3 mutation carriers.

Cysteine-sparing CADASIL mutations in NOTCH3 show proaggregatory properties in vitro.

BACKGROUND AND PURPOSE: Mutations in NOTCH3 cause cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common monogenic cause of stroke and vascular dementia. Misfolding and aggregation of NOTCH3 proteins triggered by cysteine-affecting mutations are considered to be the key disease mechanisms. However, the significance of cysteine-sparing mutations is still debated. METHODS: We studied a family with inherited small vessel disease by standardized medical history, clinical examination, MRI, ultrastructural analysis of skin biopsies, and Sanger sequencing of all NOTCH3 exons. In addition, we performed in vitro characterization of NOTCH3 variants using recombinant protein fragments and a single-particle aggregation assay. RESULTS: We identified a novel cysteine-sparing NOTCH3 mutation (D80G) in 4 family members, which was absent in a healthy sibling. All mutation carriers exhibited a CADASIL typical brain imaging and clinical phenotype, whereas skin biopsy showed inconsistent results. In vitro aggregation behavior of the D80G mutant was similar compared with cysteine-affecting mutations. This was reproduced with cysteine-sparing mutations from previously reported families having a phenotype consistent with CADASIL. CONCLUSIONS: Our findings support the view that cysteine-sparing mutations, such as D80G, might cause CADASIL with a phenotype largely indistinguishable from cysteine mutations. The in vitro aggregation analysis of atypical NOTCH3 mutations offers novel insights into pathomechanisms and might represent a tool for estimating their clinical significance.

Subclinical hyperthyroidism is a risk factor for poor functional outcome after ischemic stroke.

BACKGROUND AND PURPOSE: Subclinical hyperthyroidism is associated with adverse cardiovascular events, including stroke and atrial fibrillation. However, its impact on functional outcome after stroke remains unexplored. METHODS: A total of 165 consecutively recruited patients admitted for ischemic stroke were included in this observational prospective study. Blood samples were taken in the morning within 3 days after symptom onset, and patients were divided into the following 3 groups: subclinical hyperthyroidism (0.1< thyroid-stimulating hormone ≤ 0.44 µU/mL), subclinical hypothyroidism (2.5 ≤ thyroid-stimulating hormone <20 µU/mL), and euthyroid state (0.44< thyroid-stimulating hormone <2.5 µU/mL). Patients with overt thyroid dysfunction were excluded. Follow-up took place 3 months after stroke. Primary outcome was functional disability (modified Rankin Scale), and secondary outcome was level of dependency (Barthel Index). Ordinal logistic regression analysis was used to adjust for possible confounders. Variables previously reported to be affected by thyroid function, such as atrial fibrillation, total cholesterol, or body mass index, were included in an additional model. RESULTS: Nineteen patients (11.5%) had subclinical hyperthyroidism, and 23 patients (13.9%) had subclinical hypothyroidism. Patients with subclinical hyperthyroidism had a substantially increased risk of functional disability 3 months after stroke compared with subjects with euthyroid state (odds ratio, 2.63; 95% confidence interval, 1.02-6.82, adjusted for age, sex, smoking status, and time of blood sampling). The association remained significant, when including the baseline NIHSS, TIA, serum CRP, atrial fibrillation, body mass index, and total cholesterol as additional variables (odds ratio, 3.95; 95% confidence interval, 1.25-12.47), and was confirmed by the secondary outcome (Barthel Index: odds ratio, 9.12; 95% confidence interval, 2.08-39.89). CONCLUSIONS: Subclinical hyperthyroidism is a risk factor for poor outcome 3 months after ischemic stroke.

Preceding intravenous thrombolysis facilitates endovascular mechanical recanalization in large intracranial artery occlusion.

BACKGROUND AND AIMS: Acute occlusions of the large intracranial arteries are relatively resistant to intravenous thrombolysis. Therefore, multimodal approaches combining intravenous thrombolysis with endovascular mechanical recanalization are increasingly being applied. In this setting, intravenous thrombolysis may facilitate subsequent mechanical thrombectomy. To test this hypothesis, we analyzed the influence of intravenous thrombolysis on net intervention time in subsequent endovascular mechanical recanalization. METHODS: In this retrospective single-center analysis, we compared net intervention time with and without preceding intravenous thrombolysis in patients treated by endovascular mechanical recanalization between 01/2003 and 06/2010. The net intervention time was defined as the interval between the onset of endovascular thrombus manipulation and successful vessel recanalization. RESULTS: We identified 65 eligible patients, 35 of whom were treated by intravenous thrombolysis before mechanical therapy. Recanalization was achieved in 26 patients with (74%) and 23 patients without preceding intravenous thrombolysis (77%). In the case of successful recanalization, the net intervention time was significantly shorter in patients with preceding intravenous thrombolysis (24·8 ± 22·8 vs. 44·2 ± 40·5 min; P<0·05). This difference remained significant after restricting the analysis to the patients treated by the Penumbra Stroke System(©) (n=32). After three-months, patients with preceding intravenous thrombolysis were more likely to be functionally independent (modified Rankin Scale≤2) than those without (P<0·05). CONCLUSIONS: Our findings suggest that preceding intravenous thrombolysis may reduce the intervention time in patients treated by endovascular mechanical recanalization. However, due to the retrospective design of our study, these findings have to be interpreted with caution and need confirmation in a larger patient population.

CADASIL mutations enhance spontaneous multimerization of NOTCH3.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic cause of stroke and vascular dementia. Disease-causing mutations invariably affect cysteine residues within epidermal growth factor-like repeat domains in the extracellular domain of the NOTCH3 receptor (N3(ECD)). The biochemical and histopathological hallmark of CADASIL is the accumulation of N3(ECD) at the cell surface of vascular smooth muscle cells which degenerate over the course of the disease. The molecular mechanisms leading to N3(ECD) accumulation remain unknown. Here we show that both wild-type and CADASIL-mutated N3(ECD) spontaneously form oligomers and higher order multimers in vitro and that multimerization is mediated by disulfide bonds. Using single-molecule analysis techniques ('scanning for intensely fluorescent targets'), we demonstrate that CADASIL-associated mutations significantly enhance multimerization compared with wild-type. Taken together, our results for the first time provide experimental evidence for N3 self-association and strongly argue for a neomorphic effect of CADASIL mutations in disease pathogenesis.

Spectrum of mutations in biopsy-proven CADASIL: implications for diagnostic strategies.

BACKGROUND: Mutations in the NOTCH3 gene are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is an important cause of stroke in young adults. Mutations are typically located within epidermal growth factor-like repeat domains in the extracellular part of the Notch3 receptor. Identification of the mutation is critical for genetic counseling and testing of relatives at risk. OBJECTIVES: To identify the spectrum of NOTCH3 mutations in CADASIL and to discuss the implications for diagnostic strategies. DESIGN: Screening for NOTCH3 mutations was performed in 125 unrelated German CADASIL patients with biopsy-proven disease by direct sequencing of exons coding for epidermal growth factor-like repeats. Results were compared with those of previously published studies. RESULTS: We detected 54 distinct mutations (117 missense mutations and 3 in-frame deletions) in 120 (96.0%) of the 125 patients. Of the mutations, 58.3% were located in exon 4 and 85.8% in exons 2 through 6. In 5 patients (4.0%), no mutation was identified. CONCLUSIONS: Almost 90% of mutations could be detected within a few exons (exons 2-6). Thus, genetic testing should initially be focused on these exons, with some variation depending on the population in whom it is being performed. Yet, genetic testing for CADASIL is associated with a nameable proportion of false-negative results. Cases with a high index of clinical suspicion should be investigated by skin biopsy if genetic testing is negative.

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.

Long-term prognosis and causes of death in CADASIL: a retrospective study in 411 patients.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary angiopathy caused by mutations in the NOTCH3 gene. The clinical course is highly variable. Little is known about the long-term prognosis and the causes of death in CADASIL patients. Likewise, the impact of gender and NOTCH3 genotype on disease progression remains largely unexplored. We identified 411 subjects (196 men, 215 women) with a definite diagnosis of CADASIL. Age at onset for stroke, immobilization and death as well as the causes of death and clinical status at onset of the cause of death were determined systematically. Weibull regression models were used to calculate times to event, with gender and NOTCH3 genotype as covariates. At the time of the study, 73 patients had died. The median age at onset for stroke was 50.7 years [95% confidence interval (CI) = 48.2-53.1 years] in men and 52.5 years (95% CI = 50.0-54.9 years) in women (P = n.s.). The median ages at onset for inability to walk without assistance [men 58.9 years (95% CI = 56.6-61.3 years); women 62.1 years (59.7-64.4 years)], bedriddenness [men 62.1 years (59.6-64.7 years), women 66.5 years (63.9-69.1 years); and death [men 64.6 years (61.7-67.6 years); women 70.7 years (67.6-73.9 years)] were significantly lower in men than in women (all P < or = 0.01). The median survival time of men was significantly shorter than expected from German life tables (64.6 versus 69.3 years, P = 0.01). In contrast, the median survival time of women was not significantly reduced (70.7 versus 72.2 years). The C117F mutation was associated with a lower age at death and the C174Y mutation with a lower age at onset for stroke, immobilization and death (adjusted P values <0.05). At onset of the cause of death, 78% of the subjects were completely dependent. Sixty-three per cent were confined to bed. Pneumonia was the most frequent cause of death (38%), followed by sudden unexpected death (26%) and asphyxia (12%). We conclude that male sex is a risk factor for early immobilization and death in CADASIL. Our findings suggest possible genotype-phenotype correlations with regard to disease progression. The data presented may serve as source material for counselling CADASIL patients and for designing future interventional trials.