Ahamad Hassan

Evaluating the Genetic Component of Ischemic Stroke Subtypes: A Family History Study

Background and Purpose— Twin and family history studies support a role for genetic factors in stroke risk. Because the etiology of ischemic stroke is heterogeneous, genetic factors may vary by etiologic subtype. We determined the familial aggregation of stroke risk in different stroke phenotypes and used the results to model estimated sample size requirements for case-control studies. Methods— One thousand consecutive white subjects with ischemic stroke and 800 white controls matched for age and sex were recruited. A first-degree family history of stroke and myocardial infarction was obtained by structured interview. Stroke subtype was determined with the use of modified Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. Results— A family history of stroke at ≤65 years was a significant risk factor for large-vessel disease (odds ratio [OR], 2.24; 95% CI, 1.49 to 3.36;P <0.001) and for small-vessel disease (OR, 1.93; 95% CI, 1.25 to 2.97;P =0.003). When only cases aged ≤65 years were considered, these ORs increased to 2.93 (95% CI, 1.68 to 5.13) (P <0.001) and 3.15 (95% CI, 1.81 to 5.50) (P <0.001), respectively. No significant associations were seen for cardioembolic stroke or stroke of undetermined etiology. Conclusions— A family history of vascular disease is an independent risk factor for both large-vessel atherosclerosis and small-vessel disease, especially in cases presenting before age 65 years. The estimated sample sizes for case-control studies illustrate how candidate gene studies for ischemic stroke might be made more effective by focusing on these specific phenotypes, in which the genetic component of the disease appears to be strongest.

Risk factor profile of cerebral small vessel disease and its subtypes

Background: The mechanisms of cerebral small vessel disease (SVD) are unclear. Both atherosclerosis and a non-atherosclerotic diffuse arteriopathy have been reported pathologically. Two pathological and radiological subtypes have been suggested: localised atherosclerotic disease in larger perforating arteries causing larger lacunar infarcts without leukoaraiosis, and diffuse disease in smaller arterioles causing multiple smaller lacunar infarcts with leukoaraiosis. If atherosclerosis were important in SVD as a whole or in one particular subtype, one would expect the risk factor profile to be similar to that of cerebral large vessel disease (LVD). Methods: Risk factor profiles were compared in Caucasian stroke patients with SVD (n = 414), LVD (n = 471) and 734 stroke-free Caucasian population controls. Patients with SVD were subdivided according to the presence or absence of confluent leukoaraiosis, into isolated lacunar infarction (ILI) and ischaemic leukoaraiosis (ILA). Results: Hypertension was commoner in SVD than LVD (odds ratio (OR) 3.43 (2.32 to 5.07); p<0.001) whereas hypercholesterolaemia (OR 0.34 (0.24 to 0.48); p<0.001), smoking (OR 0.63 (0.44 to 0.91); p = 0.012), myocardial infarction (OR 0.35 (0.20 to 0.59); p<0.001) and peripheral vascular disease (OR 0.32 (0.20 to 0.50); p<0.001) were commoner in LVD. Among SVD patients, age (OR 1.11 (1.09 to 1.14); p<0.001) and hypertension (OR 3.32 (1.56 to 7.07); p = 0.002) were associated with ILA and hypercholesterolaemia (OR 0.45 (0.28 to 0.74); p = 0.002), diabetes (OR 0.42 (0.21 to 0.84); p = 0.014) and myocardial infarction (OR 0.18 (0.06 to 0.52); p = 0.001) with ILI. Conclusion: SVD has a different risk factor profile from the typical atherosclerotic profile found in LVD, with hypertension being important. There are differences in the risk factor profile between the SVD subtypes; the association of ILI with hypercholesterolaemia, diabetes and myocardial infarction may be consistent with a more atherosclerotic aetiology.

Endothelial Nitric Oxide Gene Haplotypes and Risk of Cerebral Small-Vessel Disease

Background and Purpose— Genetic influences are important in multifactorial cerebral small-vessel disease (SVD) and may act via endothelial dysfunction. Nitric oxide (NO) synthesized by endothelial nitric oxide synthase (eNOS) is a key mediator of endothelial function. We determined the role of 3 potentially functional eNOS polymorphisms (T-786C, intron 4ab, G894T) located toward the 5′ flanking end of the gene as risk factors for SVD and different SVD subtypes: isolated lacunar infarction (n=137) and ischemic leukoaraiosis (n=160). Methods— Three hundred patients with SVD and 600 community controls were studied. Genotypes were determined through polymerase chain reaction with or without restriction fragment digestion. Nitrate (NOx) levels were determined in a subgroup by use of a Griess method. Polymorphisms were tested individually and in combination with haplotype analysis. Results— The intron 4a variant was protective against SVD. This effect was confined to isolated lacunar infarction (odds ratio, 0.55; 95% confidence interval, 0.35 to 0.86; P =0.01). Haplotypes encountered were significantly different in this subtype compared with controls (P =0.001), with the -786C promoter/intron 4a combination particularly underrepresented. NOx levels were associated with the T-786C locus (P =0.03) but only in the presence of the intron 4a allele (P =0.07 for interaction). Conclusions— The intron 4ab insertion/deletion genotype was associated with isolated lacunar infarction. Haplotype and functional studies suggested that the protective effect of the 4a variant could be mediated through changes in eNOS promoter activity and increased NO levels. The specific association with isolated symptomatic lacunar infarction and not ischemic leukoaraiosis may reflect different etiopathogeneses of the 2 subtypes. Lack of NO could predispose to localized microatheroma in proximal arterioles rather than diffuse arteriosclerosis affecting distal perforating vessels.

Yield of Screening for CADASIL Mutations in Lacunar Stroke and Leukoaraiosis

Background and Purpose— Cerebral autosomal dominant arteriopathy subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic disorder typified by early onset lacunar strokes, subcortical dementia, psychiatric disturbances, and migraine. Mutations in the Notch3 gene are responsible. Atypical phenotypes have been recognized, and the disease is probably underdiagnosed in the wider stroke population. Therefore, we determined the yield of screening for Notch3 mutations in lacunar stroke with or without leukoaraiosis. Methods— Two hundred eighteen consecutive patients were studied. All had brain and carotid imaging. Polymerase chain reaction-single-stranded conformational polymorphism analysis was used to screen exons 3, 4, 5, and 6 of the Notch3 gene for mutations and polymorphisms. Results— A single mutation in exon 4 (C697T) was identified in a young patient, giving an overall carrier frequency of 0.05% (95% CI, 0.0 to 2.0). For patients with onset of lacunar stroke at ≤65 years and leukoaraiosis, the yield was 2.0% (95% CI, 0.4 to 10.9). Conclusions— Notch3 mutations are rare in patients with typical strokes due to cerebral small-vessel disease. In the absence of classic features suggestive of CADASIL, screening for Notch3 mutations has a low yield.

Planning genetic studies in human stroke: Sample size estimates based on family history data

BackgroundIdentification of stroke risk genes in humans has relied on case-control methods to determine the association between candidate genes and disease. Alternative approaches include linkage analysis using affected sibling pairs, transmission disequilibrium testing (TDT), and sibling TDT (S-TDT). Despite theoretical benefits, the feasibility of these methods in stroke remains unknown. MethodsFamily history was determined in 727 patients with ischemic stroke and 623 control subjects. These data were used to estimate the number of stroke patients required for the different study designs. ResultsA family history of any stroke occurring at ≤65 years was an independent risk factor for ischemic stroke at all ages (OR 1.47, 95% CI 1.02 to 2.12, p = 0.04) and a stronger risk factor for young (≤65 years) ischemic stroke (OR 2.25, 95% CI 1.43 to 3.55, p < 0.0001). For early-onset ischemic stroke, the sibling risk ratio was estimated to be 3.08. Assuming three major stroke loci, collection of 953 affected sibling pairs (both ≤65 years) would be needed for a linkage study, and 115,472 ischemic stroke patients would have to be screened to achieve this sample size from the authors’ population. The predicted sample sizes for association studies to detect a gene conferring an OR of 2.0 were case-control methodology (414), TDT (414), and S-TDT (617), which would require screening of 820, 31,680, and 3,062 cases. ConclusionAlternative genetic approaches are feasible, but TDT and linkage studies using the affected sib-pair methodology may require large multicenter collaborations. S-TDT approaches appear more practical. These estimates will aid in planning of such studies.

Asymmetric Dimethylarginine in Cerebral Small Vessel Disease

Background and Purpose— Endothelial dysfunction may play a causal role in cerebral small vessel disease (SVD). Asymmetric dimethylarginine (ADMA), a circulating endogenous inhibitor of nitric oxide, has been implicated in endothelial dysfunction, particularly in hyperhomocystinemia, a known risk factor for SVD. We determined if ADMA was elevated in SVD, correlated with disease severity, and interacted with homocysteine. Methods— ADMA and homocysteine levels were determined in 47 consecutive symptomatic SVD patients and 38 controls. SVD was graded by leukoariosis severity and number of lacunar infarcts. Results— Mean (and SD) ADMA was higher in SVD patients compared with controls (0.814 [0.145] versus 0.747 [0.184] &mgr;mol/L; P=0.014) after controlling for age, gender, vascular risk factors, and creatinine clearance. Additionally controlling for homocysteine had only a small effect on this relationship (P=0.055). Mean homocysteine was higher in SVD cases compared with controls (15.14 [5.59] versus 12.49 [4.15] &mgr;mol/L; P=0.035). Leukoariosis grade correlated positively with ADMA (P=0.026) and homocysteine (P=0.003). Lacunar grade correlated with homocysteine (P=0.017), but not ADMA. Conclusions— ADMA is independently associated with SVD and correlates with leukoariosis severity.

Polymorphisms in Genes of the Endothelin System and Cerebral Small-Vessel Disease

Background and Purpose— Endothelial dysfunction has been implicated in the pathogenesis of cerebral small-vessel disease (SVD). Endothelin (ET), released by the endothelium, plays a crucial role in vasoconstriction in the cerebral circulation and could contribute to the pathogenesis of cerebral SVD. Circulating ET levels may not reflect vascular production of endothelin-1 (ET-1), most of which is abluminal. Studying genetic associations, particularly of functional polymorphisms that alter activity of the ET system, is an attractive method of determining whether ET plays a role in SVD pathogenesis. We determined whether genetic variants in components of the ET system are a risk factor for cerebral SVD. Methods— Three hundred SVD patients and 600 community controls were genotyped. Polymorphisms in the ET-1 gene (K198N), the ET receptor type A (ETA), (−231G>A and +1222C>T), and the ET type B (ETB) receptor (G57S and L277L) were genotyped. Polymorphisms were studied both individually and as haplotypes. With brain imaging, cases were subtyped into those with lacunar infarct without leukoaraiosis and those with leukoaraiosis. Results— No significant differences were observed between SVD cases and controls for any individual single-nucleotide polymorphism or the ETA haplotype. There were no differences between cases with isolated lacunar infarct or with lacunar infarct and leukoaraiosis. Conclusions— This study, in a well-phenotyped population, does not support a role for genetic variation in the ET system as a risk factor for cerebral SVD.

Therapeutic efficacy of chloroquine and sulfadoxine/pyrimethamine against Plasmodium falciparum infection in Somalia

OBJECTIVE To assess the efficacy of chloroquine and sulfadoxine/pyrimethamine in the treatment of uncomplicated Plasmodium falciparum infections in Somalia. METHODS Patients with clinical malaria in Merca, an area of high transmission of the disease, were treated with the standard regimens of chloroquine (25 mg/kg) or sulfadoxine/pyrimethamine (25 mg sulfadoxine and 1.25 mg pyrimethamine per kg). Similar patients in Gabiley, an area of low transmission, received the standard regimen of chloroquine. The clinical and parasitological responses were monitored for 14 days. FINDINGS Chloroquine treatment resulted in clinical failure in 33% (n = 60) and 51% (n = 49) of the patients in Merca and Gabiley respectively. There were corresponding parasitological failures of 77% RII/RIII and 35% RII/RIII. Patients who experienced clinical failure had significantly higher initial parasitaemia than those in whom there was an adequate clinical response, both in Merca (t = 2.2; P t = 2.8; P n = 50) of the patients achieved an adequate clinical response despite a parasitological failure rate of 76% RII/RIII. CONCLUSION Chloroquine should no longer be considered adequate for treating clinical falciparum malaria in vulnerable groups in the areas studied. Doubts about the therapeutic life of sulfadoxine/pyrimethamine in relation to malaria are raised by the high levels of resistance in the Merca area and underline the need to identify suitable alternatives.

Atrial natriuretic peptide gene G664A polymorphism and the risk of ischemic cerebrovascular disease

The atrial natriuretic peptide (ANP) gene may underlie stroke susceptibility and sensitivity to cerebral ischemia in an animal model of stroke. The authors investigate its role in humans by genotyping a polymorphism (G664A) in 436 patients with ischemic cerebrovascular disease and 295 community control subjects. The frequency of this variant was similar in both groups and across the different stroke subtypes. The ANP gene G664A polymorphism is therefore unlikely to be an important risk factor for ischemic stroke in this population.

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.