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<p>Cerebrovascular disease (CVD) is strongly linked to hypertension and generally occurs later in life than coronary artery disease (CAD). Three quarters of the patients with symptomatic CVD are above 65 years of age. The risk factors are the same for CVD and CAD, but the relative importance of the vascular risk factors differs greatly.</p><p>Genetic causes of stroke are relatively rare. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a hereditary disease which causes CVD in young adults and middle-aged people, with migraine, stroke, psychiatric illness and dementia as clinical manifestations.</p><p>The subject of this thesis is vascular function in stroke and CADASIL. Endothelium-dependent vasodilation (EDV) and arterial stiffness were investigated by different methods in stroke patients and CADASIL patients compared with healthy controls. Venous occlusion plethysmography with intra-arterial acetylcholine was used to evaluate EDV in the forearm resistance vessels. Flow-mediated vasodilation of the brachial artery was used to evaluate EDV in a conduit artery. Stroke patients displayed reduced EDV in resistance vessels compared with a healthy control group, but this reduction was not significant when, in a larger group of stroke patients, adjustments were made for blood pressure, antihypertensive treatment and other risk factors. Flow mediated vasodilation of the brachial artery was reduced in the stroke patients even after adjustment for risk factors. </p><p>Compared with controls, the CADASIL patients showed similar EDV in the conduit artery, but reduced EDV in resistance vessels.</p><p>Arterial compliance was evaluated by augmentation index from pulse wave analysis, by a ratio of cardiac stroke volume and pulse pressure, and by the distensibility of the carotid artery in relation to pulse pressure. Stroke patients and CADASIL patients did not display any significant increase in arterial stiffness when evaluated by these methods. </p>

Cerebrovascular disease (CVD) is strongly linked to hypertension and generally occurs later in life than coronary artery disease (CAD). Three quarters of the patients with symptomatic CVD are above 65 years of age. The risk factors are the same for CVD and CAD, but the relative importance of the vascular risk factors differs greatly. Genetic causes of stroke are relatively rare. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a hereditary disease which causes CVD in young adults and middle-aged people, with migraine, stroke, psychiatric illness and dementia as clinical manifestations. The subject of this thesis is vascular function in stroke and CADASIL. Endothelium-dependent vasodilation (EDV) and arterial stiffness were investigated by different methods in stroke patients and CADASIL patients compared with healthy controls. Venous occlusion plethysmography with intra-arterial acetylcholine was used to evaluate EDV in the forearm resistance vessels. Flow-mediated vasodilation of the brachial artery was used to evaluate EDV in a conduit artery. Stroke patients displayed reduced EDV in resistance vessels compared with a healthy control group, but this reduction was not significant when, in a larger group of stroke patients, adjustments were made for blood pressure, antihypertensive treatment and other risk factors. Flow mediated vasodilation of the brachial artery was reduced in the stroke patients even after adjustment for risk factors. Compared with controls, the CADASIL patients showed similar EDV in the conduit artery, but reduced EDV in resistance vessels. Arterial compliance was evaluated by augmentation index from pulse wave analysis, by a ratio of cardiac stroke volume and pulse pressure, and by the distensibility of the carotid artery in relation to pulse pressure. Stroke patients and CADASIL patients did not display any significant increase in arterial stiffness when evaluated by these methods.

Abstract The mitochondrial oxidative phosphorylation system (OXPHOS) generates energy but also deleterious reactive oxygen species (ROS). Changes in the cytoskeleton, composed mainly of microfilaments, microtubules and intermediate filaments, have been observed in OXPHOS deficiency. The 3243A>G point mutation in mitochondrial DNA (mtDNA) leads to mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), which is the most common mitochondrial disease. Interestingly, mitochondrial aberrations have been demonstrated in patients with a mutation in NOTCH3, the genetic cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Randomization of vimentin intermediate filament direction and length together with slower population growth was observed in myoblasts with 3243A>G, with no difference in the amount of apoptotic cell death. Upon complex IV inhibition (with or without the microtubule-depolymerizing compound nocodazole) or a lack of mtDNA (ρ0) in osteosarcoma cells the vimentin network collapsed perinuclearly, forming thick bundles, whereas complex I inhibition led to thinner vimentin network bundles. Furthermore, the amount of vimentin was increased in ρ0 cells. Mitochondria accumulated around the nucleus upon complex IV inhibition and in ρ0 cells. Analysis of the total proteome revealed that specific OXPHOS deficiencies led to changes in the expression of cytoskeletal proteins and proteins involved in apoptosis, OXPHOS, glycolysis and oxidative stress response. Muscle histochemical and genetic analysis showed ragged red fibres and cytochrome c oxidase-negative fibres to be associated with 5650G>A in a patient with R133C in NOTCH3 and 5650G>A in MTTA. Immunolabelling of cells with R133C and 5650G>A revealed a sparse tubulin network with asters and less abundant mitochondria by comparison with control cell lines. Comparison of nucleotide diversity between CADASIL pedigrees and controls showed increased mtDNA sequence variation in the CADASIL patients. Also maternal relatives in two CADASIL pedigrees differed from each other in their mtDNA. These findings suggest that defects in OXPHOS lead to selective changes in the vimentin network, which may have a role in the pathophysiology of mitochondrial diseases. They also suggest a relationship between NOTCH3 and mtDNA, and establish the pathogenicity of 5650G>A. The overall results emphasize that a deficiency in the energy converting system together with oxidative stress can lead to cytoskeletal changes.

CADASIL is a neurodegenerative autosomal dominant hereditary disease with late onset. Main symptoms are migraines with aura, cerebral ischemic events, cognitive impairment and dementia. The disease is caused by a mutation in the NOTCH3 gene. The major mutation type changes the number of cysteine residues in the EGF-like repeats of the Notch3 protein. In Czech Republic, currently used methods for molecular genetic analysis of the CADASIL disease are Sanger sequencing and MLPA. But there are patients with CADASIL-like symptoms who were not confirmed by these methods. Therefore, the aim of this thesis was to implement transcript analysis by Sanger sequencing of cDNA PCR products and quantitative real-time PCR (qPCR) to analyze gross deletions and duplications to clarify the molecular genetic basis of the disease. By transcript analysis, the existence of the transcript variant X1 was experimentally confirmed in control samples. Moreover, the results from transcript analysis showed that non-typical missense mutation c.1725G>A (p.T575=) which does not directly change the number of cysteine residues, can cause the CADASIL disease via missplicing and subsequent causing deletion including cysteine residues. The other tested variants did not show any changes in the transcript level. The qPCR method did not...

Background: Rare genetic mutations cause familial early-onset stroke disorders, known as “Mendelian strokes”. The broader relevance of rare mutations in unrelated young stroke patients is uncertain. We hypothesize that rare mutations in known and novel genes are important risk factors for stroke. Methods: Exome sequencing was used to characterize rare disruptive protein-altering mutations in 185 young cases and 185 matched controls from INTERSTROKE, a large and globally representative stroke study. The major objectives were: 1) to precisely define the role of known Mendelian stroke genes and 2) to discover novel gene and pathway associations. Results: A focused assessment of known Mendelian stroke genes revealed a significant contribution from NOTCH3, the causal gene for Cerebral Autosomal Dominant Arteriopathies with Subcortical Infarcts and Leucoencephalopathies (CADASIL). CADASIL mutations were identified in six cases and no controls (P=0.03). The clinical presentation of CADASIL mutation carriers deviated from known symptomatology, consisting of small-vessel ischemic strokes (SVIS) accompanied by secondary features including migraine and depression. A novel role for non-CADASIL NOTCH3 mutations in ICH was also elucidated (OR=2.86; 95% CI, 1.13 to 7.93, P=0.02). Such mutations were present in 22% of ICH cases and 8% of matching controls. An agnostic evaluation of all genes did not reveal any genome-wide significant associations. However, NOTCH3 was among the top ICH genes out of 13,706 tested, and many others were also biologically relevant, notably, AARS2 and NBEAL2. A protective association was identified for the renin angiotensin system (P=8.1x10-4), whereas type II diabetes mellitus was associated with increased risk (P=1.9x10-2). Conclusion: Rare mutations influence risk of early-onset stroke. CADASIL mutations play an important role in unrelated stroke patients. Beyond CADASIL, a novel role was uncovered for other NOTCH3 mutations as common and significant risk factors for ICH. Novel biologically relevant genes and pathways may also affect stroke susceptibility. / Thesis / Master of Science in Medical Sciences (MSMS)