Maciej Tomaszewski

Gene Expression Profiling Reveals Renin mRNA Overexpression in Human Hypertensive Kidneys and a Role for MicroRNAs

The kidney has long been invoked in the etiology of essential hypertension. This could involve alterations in expression of specific genes and microRNAs (miRNAs). The aim of the present study was to identify, at the transcriptome-wide level, mRNAs and miRNAs that were differentially expressed between kidneys of 15 untreated hypertensive and 7 normotensive white male subjects of white European ancestry. By microarray technology we found 14 genes and 11 miRNAs that were differentially expressed in the medulla. We then selected and confirmed by real-time quantitative PCR expression differences for NR4A1, NR4A2, NR4A3, PER1, and SIK1 mRNAs and for the miRNAs hsa-miR-638 and hsa-let-7c. Luciferase reporter gene experiments in human kidney (HEK293) cells confirmed the predicted binding of hsa-let-7c to the 3′ untranslated region of NR4A2 mRNA. In the renal cortex we found differential expression of 46 genes and 13 miRNAs. We then confirmed expression differences for AIFM1, AMBP, APOE, CD36, EFNB1, NDUFAF1, PRDX5, REN, RENBP, SLC13A1, STX4, and TNNT2 mRNAs and for miRNAs hsa-miR-21, hsa-miR-126, hsa-miR-181a, hsa-miR-196a, hsa-miR-451, hsa-miR-638, and hsa-miR-663. Functional experiments in HEK293 cells demonstrated that hsa-miR-663 can bind to the REN and APOE 3′ untranslated regions and can regulate REN and APOE mRNA levels, whereas hsa-miR-181a regulated REN and AIFM1 mRNA. Our data demonstrated for the first time that miRNAs can regulate renin expression. The observed downregulation of 2 miRNAs in hypertension could explain the elevation in intrarenal renin mRNA. Renin, CD36, and other mRNAs, as well as miRNAs and associated pathways identified in the present study, provide novel insights into hypertension etiology.

Common Variants in Genes Underlying Monogenic Hypertension and Hypotension and Blood Pressure in the General Population

The genes responsible for several monogenic hypertensive and hypotensive disorders have been identified. Our aim was to evaluate whether common variants in these genes affect blood pressure in the general population. We studied 2037 adults from 520 nuclear families characterized for 24-hour ambulatory blood pressure and related cardiovascular traits. We genotyped 298 tagging and putative functional single nucleotide polymorphisms, achieving a median coverage of 82.4% across 11 candidate loci. Five polymorphisms in the KCNJ1 gene coding for the potassium channel, ROMK, showed associations with mean 24-hour systolic or diastolic blood pressure. The strongest association was with an intronic polymorphism, rs2846679, where the minor allele (frequency 16%) was associated with a −1.58 (95% CI −2.47 to −0.69) mm Hg change in mean 24-hour systolic blood pressure, after accounting for age, sex, and familial correlations (P=0.00048). Polymorphisms in the gene were also associated with clinic blood pressure and left ventricular mass as assessed by ECG Sokolow-Lyon voltage (P=0.0081 for rs675759). Associations with mean 24-hour systolic or diastolic blood pressure were also observed for variants in CASR, NR3C2, SCNN1B, and SCNN1G. The findings show that common variants in genes responsible for some Mendelian disorders of hypertension and hypotension affect blood pressure in the general population. Notably, variants in KCNJ1, which causes Bartter syndrome type 2, were strongly associated, potentially providing a novel target for intervention.

Genome-wide association analysis identifies novel blood pressure loci and offers biological insights into cardiovascular risk.

Elevated blood pressure is the leading heritable risk factor for cardiovascular disease worldwide. We report genetic association of blood pressure (systolic, diastolic, pulse pressure) among UK Biobank participants of European ancestry with independent replication in other cohorts, and robust validation of 107 independent loci. We also identify new independent variants at 11 previously reported blood pressure loci. In combination with results from a range of in silico functional analyses and wet bench experiments, our findings highlight new biological pathways for blood pressure regulation enriched for genes expressed in vascular tissues and identify potential therapeutic targets for hypertension. Results from genetic risk score models raise the possibility of a precision medicine approach through early lifestyle intervention to offset the impact of blood pressure–raising genetic variants on future cardiovascular disease risk.

Essential hypertension and beta(2)-adrenergic receptor gene linkage and association analysis

A region on human chromosome 5 (5q31.1-qter) contains several genes that encode important blood pressure regulators and thus is a good candidate for analysis of linkage and association with hypertension. We recruited 638 individuals from 212 Polish pedigrees with clustering of essential hypertension. These subjects were genotyped for 11 microsatellite markers that span this region to test for linkage to essential hypertension and systolic and diastolic blood pressures. The segment of this region of ≈7 cM delineated by D5S1480 and D5S500 markers was linked to blood pressures in multipoint analysis. In 2-point analysis, D5S1480—the marker in close proximity to β 2 -adrenergic receptor gene—reached the maximal linkage to essential hypertension and adjusted systolic and diastolic blood pressures, implicating this gene as a positional candidate for further association studies. Arg16Gly, Gln27Glu, and Thr164Ile—3 functional single nucleotide polymorphisms within the β 2 -adrenergic receptor gene—were tested for association with essential hypertension. None of these polymorphisms showed a significant association with essential hypertension, separately or in the haplotype analysis. This study provided evidence of linkage of 5q31.1-5qter region to essential hypertension in the European population. Moreover, it implicated the chromosomal segment in close proximity to D5S1480 and D5S500. The detailed analysis of 3 single nucleotide polymorphisms does not support the role of the β 2 -adrenergic receptor gene as a major causative gene for the detected linkage.A region on human chromosome 5 (5q31.1-qter) contains several genes that encode important blood pressure regulators and thus is a good candidate for analysis of linkage and association with hypertension. We recruited 638 individuals from 212 Polish pedigrees with clustering of essential hypertension. These subjects were genotyped for 11 microsatellite markers that span this region to test for linkage to essential hypertension and systolic and diastolic blood pressures. The segment of this region of ≈7 cM delineated by D5S1480 and D5S500 markers was linked to blood pressures in multipoint analysis. In 2-point analysis, D5S1480—the marker in close proximity to &bgr;2-adrenergic receptor gene—reached the maximal linkage to essential hypertension and adjusted systolic and diastolic blood pressures, implicating this gene as a positional candidate for further association studies. Arg16Gly, Gln27Glu, and Thr164Ile—3 functional single nucleotide polymorphisms within the &bgr;2-adrenergic receptor gene—were tested for association with essential hypertension. None of these polymorphisms showed a significant association with essential hypertension, separately or in the haplotype analysis. This study provided evidence of linkage of 5q31.1-5qter region to essential hypertension in the European population. Moreover, it implicated the chromosomal segment in close proximity to D5S1480 and D5S500. The detailed analysis of 3 single nucleotide polymorphisms does not support the role of the &bgr;2-adrenergic receptor gene as a major causative gene for the detected linkage.

Strikingly Low Circulating CRP Concentrations in Ultramarathon Runners Independent of Markers of Adiposity: How Low Can You Go?

Objective—This study was undertaken to evaluate to what extent C-reactive protein (CRP) can be reduced by exercise by examining its circulating concentrations in male ultramarathon runners and to determine if low leptin as a robust circulating marker of fat mass could account for low CRP in such men. Methods and Results—Sixty-seven male ultramarathon runners and 63 sedentary male controls of similar age and body mass index were recruited. CRP and leptin were measured by ELISA and radioimmunoassay, respectively. Median CRP concentration in lean (body mass index <25 kg/m2) marathon runners was less than half control median (0.4 [0.2 to 0.9] mg/L versus 0.9 [0.5 to 2.7] mg/L, P =0.0013) and, more strikingly, in nonlean runners was approximately 26% of control median (0.4 [0.3 to 0.8] mg/L versus 1.5 [0.9 to 2.5] mg/L, P =0.0002). Circulating leptin levels were also substantially lower in lean (45% less) and nonlean (63% less, both P <0.0001) ultramarathon runners. However, interleukin-6 levels were not different. Furthermore, leptin adjustment only minimally attenuated the case-control difference in CRP, suggesting that mechanisms other than fat mass reduction contribute to low concentrations of CRP in marathon runners. Conclusions—This study suggests that circulating CRP concentrations can be markedly suppressed, independently of total adiposity or indeed fat mass, by intense regular physical exercise.

Heritability of Early Repolarization A Population-Based Study

Background— Early repolarization (ER), defined by J-point elevation in 12-lead ECG, was recently associated with increased risk for idiopathic ventricular fibrillation and cardiovascular mortality. The determinants of ER are unknown. We investigated its heritability in a large, family-based cohort.nnMethods and Results— The study sample comprised 1877 individuals from 505 white nuclear families representative of the British general population. Standard 12-lead ECGs were evaluated for the presence of ER, defined as J-point elevation of ≥0.1 mV in at least 2 adjacent inferior (II, III, and aVF) or anterolateral (I, aVL, and V4 through V6) leads. Narrow sense heritability estimates were computed adjusting for age, age2, and sex. The prevalence of ER was 7.7% (n=145) in the whole cohort, 5.9% (n=56) in parents, and 9.6% (n=89) in offspring. Heritability estimate for the presence of ER was calculated at h2=0.49 (standard error=0.14; P =2.7*10−4) and was higher when restricted to its presence in inferior leads (h2=0.61, standard error=0.18, P =4.3*10−4) or for the notching ER morphology (h2=0.81, standard error=0.19, P =2.4*10−5). Individuals with at least 1 affected parent had a 2.5-fold increased risk for presenting with ER on ECG (odds ratio, 2.54; 95% confidence interval, 1.33 to 4.84; P =0.005). Familial transmission was more frequent when the mother was affected (odds ratio, 3.84; 95% confidence interval, 1.41 to 10.43; P =0.008) than when the father was affected (odds ratio, 1.82; 95% confidence interval, 0.82 to 4.03; P =0.141), although this difference was not statistically significant ( P =0.18).nnConclusions— ER is a heritable phenotype. Offspring of ER-positive parents have a 2.5-fold increased risk of presenting with ER on their ECG.Background—Early repolarization (ER), defined by J-point elevation in 12-lead ECG, was recently associated with increased risk for idiopathic ventricular fibrillation and cardiovascular mortality. The determinants of ER are unknown. We investigated its heritability in a large, family-based cohort. Methods and Results—The study sample comprised 1877 individuals from 505 white nuclear families representative of the British general population. Standard 12-lead ECGs were evaluated for the presence of ER, defined as J-point elevation of ≥0.1 mV in at least 2 adjacent inferior (II, III, and aVF) or anterolateral (I, aVL, and V4 through V6) leads. Narrow sense heritability estimates were computed adjusting for age, age2, and sex. The prevalence of ER was 7.7% (n=145) in the whole cohort, 5.9% (n=56) in parents, and 9.6% (n=89) in offspring. Heritability estimate for the presence of ER was calculated at h2=0.49 (standard error=0.14; P=2.7*10−4) and was higher when restricted to its presence in inferior leads (h2=0.61, standard error=0.18, P=4.3*10−4) or for the notching ER morphology (h2=0.81, standard error=0.19, P=2.4*10−5). Individuals with at least 1 affected parent had a 2.5-fold increased risk for presenting with ER on ECG (odds ratio, 2.54; 95% confidence interval, 1.33 to 4.84; P=0.005). Familial transmission was more frequent when the mother was affected (odds ratio, 3.84; 95% confidence interval, 1.41 to 10.43; P=0.008) than when the father was affected (odds ratio, 1.82; 95% confidence interval, 0.82 to 4.03; P=0.141), although this difference was not statistically significant (P=0.18). Conclusions—ER is a heritable phenotype. Offspring of ER-positive parents have a 2.5-fold increased risk of presenting with ER on their ECG.

Association analyses based on false discovery rate implicate new loci for coronary artery disease

Genome-wide association studies (GWAS) in coronary artery disease (CAD) had identified 66 loci at genome-wide significance (P < 5 × 10−8) at the time of this analysis, but a much larger number of putative loci at a false discovery rate (FDR) of 5% (refs. 1,2,3,4). Here we leverage an interim release of UK Biobank (UKBB) data to evaluate the validity of the FDR approach. We tested a CAD phenotype inclusive of angina (SOFT; ncases = 10,801) as well as a stricter definition without angina (HARD; ncases = 6,482) and selected cases with the former phenotype to conduct a meta-analysis using the two most recent CAD GWAS. This approach identified 13 new loci at genome-wide significance, 12 of which were on our previous list of loci meeting the 5% FDR threshold, thus providing strong support that the remaining loci identified by FDR represent genuine signals. The 304 independent variants associated at 5% FDR in this study explain 21.2% of CAD heritability and identify 243 loci that implicate pathways in blood vessel morphogenesis as well as lipid metabolism, nitric oxide signaling and inflammation.

Genetic Architecture of Ambulatory Blood Pressure in the General Population. Insights From Cardiovascular Gene-Centric Array

Genetic determinants of blood pressure are poorly defined. We undertook a large-scale, gene-centric analysis to identify loci and pathways associated with ambulatory systolic and diastolic blood pressure. We measured 24-hour ambulatory blood pressure in 2020 individuals from 520 white European nuclear families (the Genetic Regulation of Arterial Pressure of Humans in the Community Study) and genotyped their DNA using the Illumina HumanCVD BeadChip array, which contains ≈50 000 single nucleotide polymorphisms in >2000 cardiovascular candidate loci. We found a strong association between rs13306560 polymorphism in the promoter region of MTHFR and CLCN6 and mean 24-hour diastolic blood pressure; each minor allele copy of rs13306560 was associated with 2.6 mm Hg lower mean 24-hour diastolic blood pressure (P=1.2×10−8). rs13306560 was also associated with clinic diastolic blood pressure in a combined analysis of 8129 subjects from the Genetic Regulation of Arterial Pressure of Humans in the Community Study, the CoLaus Study, and the Silesian Cardiovascular Study (P=5.4×10−6). Additional analysis of associations between variants in gene ontology-defined pathways and mean 24-hour blood pressure in the Genetic Regulation of Arterial Pressure of Humans in the Community Study showed that cell survival control signaling cascades could play a role in blood pressure regulation. There was also a significant overrepresentation of rare variants (minor allele frequency: <0.05) among polymorphisms showing at least nominal association with mean 24-hour blood pressure indicating that a considerable proportion of its heritability may be explained by uncommon alleles. Through a large-scale gene-centric analysis of ambulatory blood pressure, we identified an association of a novel variant at the MTHFR/CLNC6 locus with diastolic blood pressure and provided new insights into the genetic architecture of blood pressure.

Association of the Human Y Chromosome with Cholesterol Levels in the General Population

Objective—Males are at higher risk of cardiovascular diseases than females. The aim of the study was to test whether the potential of the Y chromosome to affect cardiovascular risk could be attributed to its influence on lipids. Methods and Results—1288 Polish men (1157 subjects from young healthy cohort and 131 individuals from middle-aged hypertensive population) were phenotyped for determinants of cardiovascular risk including BMI, blood pressures, lipids, and testosterone. Each subject was genotyped for the Hin dIII(+/−) polymorphism within the nonrecombining region of the Y chromosome. Men with the Hin dIII(−) variant exhibited significantly higher total cholesterol (TC) and low-density lipoprotein cholesterol (LDL) levels than subjects with the Hin dIII(+) genotype in both populations. The differences between the genotypes were 0.15 mmol/L (P =0.0107) and 0.45 mmol/L (P =0.0377) in TC and 0.15 mmol/L (P =0.0059) and 0.41 mmol/L (P =0.0432) in LDL among young apparently healthy men and middle-aged hypertensive men, respectively. The Hin dIII(+) was associated with a significant increase in blood pressure of the middle-aged men. Testosterone serum concentrations correlated positively with HDL-cholesterol levels, and this association was independent of the Y chromosome. Conclusions—The results indicate that a locus/loci on the Y chromosome may influence LDL levels, independent of testosterone levels.

A Common Variant in Low-Density Lipoprotein Receptor–Related Protein 6 Gene (LRP6) Is Associated With LDL-Cholesterol

Objective—A rare mutation in low-density lipoprotein receptor-related protein 6 gene (LRP6) was identified as the primary molecular defect underlying monogenic form of coronary artery disease. We hypothesized that common variants in LRP6 could predispose subjects to elevated LDL-cholesterol (LDL-C). Methods and Results—Twelve common (minor allele frequency ≥0.1) single nucleotide polymorphisms in LRP6 were genotyped in 703 individuals from 213 Polish pedigrees (Silesian Cardiovascular Study families). The family-based analysis revealed that the minor allele of rs10845493 clustered with elevated LDL-C in offspring more frequently than expected by chance (P=0.0053). The quantitative analysis restricted to subjects free of lipid-lowering treatment confirmed the association between rs10845493 and age-, sex-, and BMI-adjusted circulating levels of LDL-C in families as well as 2 additional populations − 218 unrelated subjects from Silesian Cardiovascular Study replication panel and 1138 individuals from Young Men Cardiovascular Association cohort (P=0.0268, P=0.0476, and P=0.0472, respectively). In the inverse variance weighted meta-analysis of the 3 populations each extra minor allele copy of rs10845493 was associated with 0.14 mmol/L increase in age-, sex-, and BMI-adjusted LDL-C (SE=0.05, P=0.0038). Conclusions—Common polymorphism in the gene underlying monogenic form of coronary artery disease impacts on risk of LDL-C elevation.