A Method for Quantitative Analysis of Standard and High-Throughput qPCR Expression Data Based on Input Sample Quantity

Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR) have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR) and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells) and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA)) permits the normalization of results between different batches and between different instruments – regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1) the achievement of absolute quantification and (2) a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available

The AGTR1 gene A1166C polymorphism as a risk factor and outcome predictor of primary intracerebral and aneurysmal subarachnoid hemorrhages

Associations between the angiotensin II type 1 receptor (AGTR1) gene A1166C polymorphism and hypertension, aortic abdominal aneurysms (as a risk factor) as well as cardiovascular disorders (as a risk factor and an outcome predictor) have been demonstrated. We aimed to investigate the role of this polymorphism as risk factors and outcome predictors in primary intracerebral hemorrhage (PICH) and aneurysmal subarachnoid hemorrhage (aSAH). We have prospectively recruited 1078 Polish participants to the study: 261 PICH patients, 392 aSAH patients, and 425 unrelated control subjects. The A1166C AGTR1 gene polymorphism was studied using the tetra-primer ARMS-PCR method. Allele and genotype frequencies were compared with other ethnically different populations. The A1166C polymorphism was not associated with the risk of PICH or aSAH. Among the aSAH patients the AA genotype was associated with a good outcome, defined by a Glasgow Outcome Scale of 4 or 5 (p < 0.02). The distribution of A1166C genotypes in our cohort did not differ from other white or other populations of European descent. In conclusion, we found an association between the A1166C AGTR1 polymorphism and outcome of aSAH patients, but not with the risk of PICH or aSAH

The AGTR1 gene A1166C polymorphism as a risk factor and outcome predictor of primary intracerebral and aneurysmal subarachnoid hemorrhages

Associations between the angiotensin II type 1 receptor (AGTR1) gene A1166C polymorphism and hypertension, aortic abdominal aneurysms (as a risk factor) as well as cardiovascular disorders (as a risk factor and an outcome predictor) have been demonstrated. We aimed to investigate the role of this polymorphism as risk factors and outcome predictors in primary intracerebral hemorrhage (PICH) and aneurysmal subarachnoid hemorrhage (aSAH). We have prospectively recruited 1078 Polish participants to the study: 261 PICH patients, 392 aSAH patients, and 425 unrelated control subjects. The A1166C AGTR1 gene polymorphism was studied using the tetra-primer ARMS-PCR method. Allele and genotype frequencies were compared with other ethnically different populations. The A1166C polymorphism was not associated with the risk of PICH or aSAH. Among the aSAH patients the AA genotype was associated with a good outcome, defined by a Glasgow Outcome Scale of 4 or 5 (p&lt;0.02). The distribution of A1166C genotypes in our cohort did not differ from other white or other populations of European descent. In conclusion, we found an association between the A1166C AGTR1 polymorphism and outcome of aSAH patients, but not with the risk of PICH or aSAH

Polimorfizm –455G/A genu β-fibrynogenu a ryzyko udaru niedokrwiennego w populacji polskiej

Background and purpose: Ischaemic stroke is considered to be multifactorial and interactions between environmental and genetic factors play an important role. Although vascular risk factors are well known, the genetic ones are still undiscover - ed. In the present study, we assessed the significance of the β-fibrinogen –455G/A gene polymorphism and the risk of ischaemic stroke in a Polish population. Material and methods: 426 ischaemic stroke patients classified according to stroke aetiologies (small vessel disease, large vessel disease or cardioembolic stroke) and 234 controls were included in the study. The association of the β-fibrinogen genotypes with ischaemic stroke was tested using logistic regression analysis under dominant, recessive or additive models of inheritance. Results: The allele and genotype distributions of the β-fibri - nogen –455G/A gene polymorphism did not differ significantly between patients and controls (patients: G – 75%, GG – 56.6%, GA – 36.8%, AA – 6.6%; controls: G – 73.7%, GG – 57.3%, GA – 32.9%, AA – 9.8%; p > 0.05, χ2). In addition, logistic regression analysis adjusted for the known risk factors, i.e. hypertension, ischaemic heart disease, myocardial infarction, hypercholesterolaemia, diabetes mellitus and smoking, did not show a role of the studied polymorphism in ischaemic stroke. Conclusions: The β-fibrinogen –455G/A gene polymorphism is not a risk factor for ischaemic stroke in a Polish population

Polimorfizm –455G/A genu β-fibrynogenu a ryzyko udaru niedokrwiennego w populacji polskiej

Background and purpose Ischaemic stroke is considered to be multifactorial and interactions between environmental and genetic factors play an important role. Although vascular risk factors are well known, the genetic ones are still undiscovered. In the present study, we assessed the significance of the β-fibrinogen –455G/A gene polymorphism and the risk of ischaemic stroke in a Polish population. Material and methods 426 ischaemic stroke patients classified according to stroke aetiologies (small vessel disease, large vessel disease or cardioembolic stroke) and 234 controls were included in the study. The association of the β-fibrinogen genotypes with ischaemic stroke was tested using logistic regression analysis under dominant, recessive or additive models of inheritance. Results The allele and genotype distributions of the β-fibrinogen –455G/A gene polymorphism did not differ significantly between patients and controls (patients: G – 75%, GG – 56.6%, GA – 36.8%, AA – 6.6%; controls: G – 73.7%, GG – 57.3%, GA – 32.9%, AA – 9.8%; p &gt; 0.05, χ2). In addition, logistic regression analysis adjusted for the known risk factors, i.e. hypertension, ischaemic heart disease, myocardial infarction, hypercholesterolaemia, diabetes mellitus and smoking, did not show a role of the studied polymorphism in ischaemic stroke. Conclusions The β-fibrinogen –455G/A gene polymorphism is not a risk factor for ischaemic stroke in a Polish population.Wstęp i cel pracy Etiologia udaru niedokrwiennego mózgu jest wieloczynnikowa. Istotną rolę odgrywają w niej interakcje pomiędzy czynnikami środowiskowymi i genetycznymi. Naczyniowe czynniki ryzyka udaru mózgu są dość dobrze poznane, natomiast rola czynników genetycznych pozostaje wciąż niejasna. W prezentowanym badaniu oceniano znaczenie polimorfizmu –455G/A genu β-fibrynogenu w kontekście ryzyka wystąpienia udaru niedokrwiennego mózgu w populacji polskiej. Materiał i metody Do badania włączono 426 chorych na udar niedokrwienny mózgu sklasyfikowanych zgodnie z etiologią udaru (choroba małych naczyń, choroba dużych naczyń lub udar sercowozatorowy) oraz 234 osoby z grupy kontrolnej. Związek pomiędzy badanym polimorfizmem a udarem niedokrwiennym mózgu został zbadany przy użyciu regresji logistycznej w dominującym, recesywnym i addytywnym modelu dziedziczenia. Wyniki Nie stwierdzono istotnej różnicy w rozkładzie alleli i genotypów polimorfizmu –455G/A genu β-fibrynogenu pomiędzy pacjentami a osobami z grupy kontrolnej (pacjenci: G – 75%, GG – 56,6%, GA – 36,8%, AA – 6,6%; grupa kontrolna: G – 73,7%, GG – 57,3%, GA – 32,9%, AA – 9,8 %; p &gt; 0,05, test χ2). Ponadto w modelu regresji logistycznej uwzględniającym wpływ znanych czynników ryzyka, takich jak: nadciśnienie tętnicze, choroba niedokrwienna serca, zawał mięśnia sercowego, hipercholesterolemia, cukrzyca i palenie tytoniu, nie wykazano roli badanego polimorfizmu w udarze niedokrwiennym mózgu. Wnioski Polimorfizm –455G/A genu β-fibrynogenu nie jest czynnikiem ryzyka udaru niedokrwiennego mózgu w populacji polskiej

Vascular cognitive impairment linked to brain endothelium inflammation in early stages of heart failure in mice

Background Although advanced heart failure ( HF ) is a clinically documented risk factor for vascular cognitive impairment, the occurrence and pathomechanisms of vascular cognitive impairment in early stages of HF are equivocal. Here, we characterize vascular cognitive impairment in the early stages of HF development and assess whether cerebral hypoperfusion or prothrombotic conditions are involved. Methods and Results Tgαq*44 mice with slowly developing isolated HF triggered by cardiomyocyte‐specific overexpression of G‐αq*44 protein were studied before the end‐stage HF , at the ages of 3, 6, and 10 months: before left ventricle dysfunction; at the stage of early left ventricle diastolic dysfunction (with preserved ejection fraction); and left ventricle diastolic/systolic dysfunction, respectively. In 6‐ to 10‐month‐old but not in 3‐month‐old Tgαq*44 mice, behavioral and cognitive impairment was identified with compromised blood‐brain barrier permeability, most significantly in brain cortex, that was associated with myelin sheet loss and changes in astrocytes and microglia. Brain endothelial cells displayed increased E‐selectin immunoreactivity, which was accompanied by increased amyloid‐β 1‐42 accumulation in piriform cortex and increased cortical oxidative stress (8‐ OH dG immunoreactivity). Resting cerebral blood flow measured by magnetic resonance imaging in vivo was preserved, but ex vivo NO ‐dependent cortical arteriole flow regulation was impaired. Platelet hyperreactivity was present in 3‐ to 10‐month‐old Tgαq*44 mice, but it was not associated with increased platelet‐dependent thrombogenicity. Conclusions We report for the first time that vascular cognitive impairment is already present in the early stage of HF development, even before left ventricle systolic dysfunction. The underlying pathomechanism, independent of brain hypoperfusion, involves preceding platelet hyperreactivity and brain endothelium inflammatory activation. </jats:sec

Atrial fibrillation genetic risk differentiates cardioembolic stroke from other stroke subtypes

AbstractObjectiveWe sought to assess whether genetic risk factors for atrial fibrillation can explain cardioembolic stroke risk.MethodsWe evaluated genetic correlations between a prior genetic study of AF and AF in the presence of cardioembolic stroke using genome-wide genotypes from the Stroke Genetics Network (N = 3,190 AF cases, 3,000 cardioembolic stroke cases, and 28,026 referents). We tested whether a previously-validated AF polygenic risk score (PRS) associated with cardioembolic and other stroke subtypes after accounting for AF clinical risk factors.ResultsWe observed strong correlation between previously reported genetic risk for AF, AF in the presence of stroke, and cardioembolic stroke (Pearson’s r=0.77 and 0.76, respectively, across SNPs with p &lt; 4.4 × 10−4 in the prior AF meta-analysis). An AF PRS, adjusted for clinical AF risk factors, was associated with cardioembolic stroke (odds ratio (OR) per standard deviation (sd) = 1.40, p = 1.45×10−48), explaining ∼20% of the heritable component of cardioembolic stroke risk. The AF PRS was also associated with stroke of undetermined cause (OR per sd = 1.07, p = 0.004), but no other primary stroke subtypes (all p &gt; 0.1).ConclusionsGenetic risk for AF is associated with cardioembolic stroke, independent of clinical risk factors. Studies are warranted to determine whether AF genetic risk can serve as a biomarker for strokes caused by AF.</jats:sec