Influence of SNP rs4773144 genotype on gene transcription.

<p>Primary cultures of vascular SMCs (A) and ECs (B) of the A/G genotype for SNP rs4773144 from different individuals were subjected to chromatin immunoprecipitation using an antibody against Pol II, followed by an allelic imbalance analysis of SNP rs4773144, with the use of the TaqMan method to determine the ratio of Ct value of the A allele (detected by a VIC fluorescein-labeled probe) versus the Ct value of the G allele (detected by a FAM fluorescent dye-labeled probe). Upper panels show amplification curves of the A allele (red) and G allele (green). Column charts in the lower panels show the relative fold difference in G to A ratio of Ct values between input DNA and anti-Pol II antibody precipitated chromatin DNA. Columns and error bars represent mean and SEM values.</p

Association between SNP rs4773144 and MI incidence/prevalence in CHD patients.

<p>Association between SNP rs4773144 and MI incidence/prevalence in CHD patients.</p

Influence of SNP rs4773144 genotype on apoptosis.

<p><b>(A)</b> Primary cultures of SMCs from different individuals were genotyped for rs4773144 and subjected to apoptosis assay. Shown in the graph are relative differences in cell apoptotic rate in different genotype groups. Columns and error bars represent mean and SEM values; <i>p</i>-values shown are for an additive genetic model. <b>(B)</b> Primary cultures of ECs of different genotypes for rs4773144 were subjected to immunoblot analysis of the anti-apoptotic protein BCL2. Shown on the left are representative immunoblot images and on the right a graphic presentation of BCL2 band intensities standardized against band intensities of the loading control HSC70. Columns and error bars represent mean and SEM values; <i>p</i>-values shown are for an additive genetic model.</p

Effect of SNP rs4773144 on transcription modulating activity of a DNA sequence encompassing the SNP and on nuclear protein interaction with the DNA sequence.

<p><b>(A)</b> Vascular SMCs were transfected with a firefly luciferase reporter gene plasmid (pGL3-promoter vector) with an insert corresponding to a DNA sequence encompassing the rs4773144 site of the A or G allele, or with the empty plasmid (control), together with a plasmid (pRL-TK) containing a <i>renilla</i> luciferase gene to serve as a transfection efficiency reference. At 48 hour after transfection, activities of firefly luciferase and <i>renilla</i> luciferase were measured. Columns and error bars represent mean and SEM values of the ratio of firefly luciferase activity to <i>renilla</i> luciferase activity. <b>(B)</b> A representative image of electrophoretic mobility shift assays. Nuclear protein extracts from vascular ECs were incubated with biotin-labeled probes corresponding to the A allele (lanes 1–7) or the G allele (lanes 8–14) of SNP rs4773144 in the absence or presence of competitors in 20-fold or 50-fold molar excess, as indicated underneath the image.</p

Influence of SNP rs4773144 genotype on <i>COL4A2</i> and <i>COL4A1</i> expression levels.

<p>Primary cultures of vascular SMCs and ECs from different individuals were genotyped for rs4773144 and subjected to quantitative reverse transcriptase–polymerase chain reaction analysis of <i>COL4A2</i> and <i>COL4A1</i>. Showed in the graphs are relative fold differences in <i>COL4A2</i> (<b>A</b> and <b>B</b>) and <i>COL4A1</i> (<b>C</b> and <b>D</b>) RNA levels in SMCs (<b>A</b> and <b>C</b>) and ECs (<b>B</b> and <b>D</b>), respectively. Columns and error bars represent mean and SEM values; <i>p</i>-values shown are for an additive genetic model.</p

Association of SNP rs4773144 with collagen IV level in atherosclerotic plaques and plaque cap thickness.

<p>Atherosclerotic coronary arteries from different individuals were genotyped for SNP rs4773144 and subjected to histopathological analyses using Image-Pro software. <b>(A)</b> A representative image of immunostaining, blue color indicates COL4A2 staining, and brown smooth muscle alpha-actin. ×400 original magnification. <b>(B)</b> Percentages of COL4A2 immunostain positive areas in atherosclerotic plaques in different genotype groups. <b>(C)</b> Atherosclerotic plaque cap thickness in different genotype groups. <b>(D)</b> Atherosclerotic plaque cap/intima ratios in different genotype groups. Columns and error bars in <b>B</b>-<b>D</b> are mean and SEM values; <i>p</i>-values shown are for an additive genetic model.</p

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Effects of calcium, magnesium, and potassium concentrations on ventricular repolarization in unselected individuals.

Background: Subclinical changes on the electrocardiogram are risk factors for cardiovascular mortality. Recognition and knowledge of electrolyte associations in cardiac electrophysiology are based on only in vitro models and observations in patients with severe medical conditions.Objectives: This study sought to investigate associations between serum electrolyte concentrations and changes in cardiac electrophysiology in the general population.Methods: Summary results collected from 153,014 individuals (54.4% women; mean age 55.1 ± 12.1 years) from 33 studies (of 5 ancestries) were meta-analyzed. Linear regression analyses examining associations between electrolyte concentrations (mmol/l of calcium, potassium, sodium, and magnesium), and electrocardiographic intervals (RR, QT, QRS, JT, and PR intervals) were performed. The study adjusted for potential confounders and also stratified by ancestry, sex, and use of antihypertensive drugs.Results: Lower calcium was associated with longer QT intervals (-11.5 ms; 99.75% confidence interval [CI]: -13.7 to -9.3) and JT duration, with sex-specific effects. In contrast, higher magnesium was associated with longer QT intervals (7.2 ms; 99.75% CI: 1.3 to 13.1) and JT. Lower potassium was associated with longer QT intervals (-2.8 ms; 99.75% CI: -3.5 to -2.0), JT, QRS, and PR durations, but all potassium associations were driven by use of antihypertensive drugs. No physiologically relevant associations were observed for sodium or RR intervals.Conclusions: The study identified physiologically relevant associations between electrolytes and electrocardiographic intervals in a large-scale analysis combining cohorts from different settings. The results provide insights for further cardiac electrophysiology research and could potentially influence clinical practice, especially the association between calcium and QT duration, by which calcium levels at the bottom 2% of the population distribution led to clinically relevant QT prolongation by >5 ms.</p

Consistently higher effect sizes for women for all seven loci.

<p>Shown are beta-estimates and 95% confidence intervals for the seven identified SNPs (also stating the phenotype for which the SNP was selected for).</p

Seven SNPs show sex difference.

a<p>Trait and sex for which the SNP was selected;</p>b<p>Gene labels state the nearest gene or the gene as published previously; details on all genes near the association signal can be found in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003500#pgen.1003500.s002" target="_blank">Figure S2</a>;</p>c<p>One-sided P-Values.</p>d<p>larger sample size due to one additional study that did not have hip circumference, and therefore could not contribute to WHRadjBMI.</p>e<p>smaller sample size as this SNP was not on Metabochip.</p><p>Shown are the seven SNPs with significant (at 5% false discovery rate) sex difference in the follow-up data. These seven SNPs exhibit genome-wide significant association in women (joint discovery and follow-up <i>P_women</i><5×10−8) and only two of these show nominally significant association in men (joint <i>P_men</i><0.05). The three loci MAP3K1, HSD17B4, and PPARG are shown here for the first time for their anthropometric trait association as well as for sex-difference.</p