Paul S. de Vries

Adiposity as a cause of cardiovascular disease: a Mendelian randomization study

BACKGROUND Adiposity, as indicated by body mass index (BMI), has been associated with risk of cardiovascular diseases in epidemiological studies. We aimed to investigate if these associations are causal, using Mendelian randomization (MR) methods. METHODS The associations of BMI with cardiovascular outcomes [coronary heart disease (CHD), heart failure and ischaemic stroke], and associations of a genetic score (32 BMI single nucleotide polymorphisms) with BMI and cardiovascular outcomes were examined in up to 22,193 individuals with 3062 incident cardiovascular events from nine prospective follow-up studies within the ENGAGE consortium. We used random-effects meta-analysis in an MR framework to provide causal estimates of the effect of adiposity on cardiovascular outcomes. RESULTS There was a strong association between BMI and incident CHD (HR = 1.20 per SD-increase of BMI, 95% CI, 1.12-1.28, P = 1.9.10(-7)), heart failure (HR = 1.47, 95% CI, 1.35-1.60, P = 9.10(-19)) and ischaemic stroke (HR = 1.15, 95% CI, 1.06-1.24, P = 0.0008) in observational analyses. The genetic score was robustly associated with BMI (β = 0.030 SD-increase of BMI per additional allele, 95% CI, 0.028-0.033, P = 3.10(-107)). Analyses indicated a causal effect of adiposity on development of heart failure (HR = 1.93 per SD-increase of BMI, 95% CI, 1.12-3.30, P = 0.017) and ischaemic stroke (HR = 1.83, 95% CI, 1.05-3.20, P = 0.034). Additional cross-sectional analyses using both ENGAGE and CARDIoGRAMplusC4D data showed a causal effect of adiposity on CHD. CONCLUSIONS Using MR methods, we provide support for the hypothesis that adiposity causes CHD, heart failure and, previously not demonstrated, ischaemic stroke.

Incremental predictive value of 152 single nucleotide polymorphisms in the 10-year risk prediction of incident coronary heart disease: the Rotterdam Study

OBJECTIVE To examine the incremental predictive value of genetic risk scores of coronary heart disease (CHD) in the 10-year risk prediction of incident CHD. METHODS In 5899 subjects, we used 152 single nucleotide polymorphisms (SNPs) associated with coronary artery disease by the CARDIoGRAMplusC4D consortium to construct three weighted genetic risk scores: (i) GRS(gws) based on 49 genome-wide significant SNPs; (ii) GRS(fdr) based on 103 suggestively associated SNPs; and (iii) GRS(all) based on all 152 SNPs. We examined the changes in discrimination and reclassification of incident CHD when adding the genetic risk scores to models including traditional risk factors. We repeated the analysis for prevalent CHD. RESULTS The genetic risk scores were associated with incident CHD despite adjustment for traditional risk factors and family history: participants had a 13% higher rate of CHD per standard deviation increase in GRS(all). GRS(all )improved the C-statistic by 0.006 [95% confidence interval (CI): 0.000, 0.013] beyond age and sex, 0.003 (95% CI: -0.001, 0.008) beyond traditional risk factors and 0.003 (95% CI: -0.001, 0.007) beyond traditional risk factors and family history. The genetic risk scores did not improve reclassification. GRS(all) strongly improved both discrimination and reclassification of prevalent CHD, even beyond traditional risk factors and family history, with a C-statistic improvement of 0.009 (0.003, 0.015). CONCLUSIONS Although the genetic risk scores based on 152 SNPs were associated with incident CHD, they did not improve risk prediction. This discrepancy may be the result of SNP discovery for prevalent rather than incident CHD, since the SNPs do improve prediction for prevalent disease.

Age- and Sex-Specific Causal Effects of Adiposity on Cardiovascular Risk Factors

Observational studies have reported different effects of adiposity on cardiovascular risk factors across age and sex. Since cardiovascular risk factors are enriched in obese individuals, it has not been easy to dissect the effects of adiposity from those of other risk factors. We used a Mendelian randomization approach, applying a set of 32 genetic markers to estimate the causal effect of adiposity on blood pressure, glycemic indices, circulating lipid levels, and markers of inflammation and liver disease in up to 67,553 individuals. All analyses were stratified by age (cutoff 55 years of age) and sex. The genetic score was associated with BMI in both nonstratified analysis (P = 2.8 × 10−107) and stratified analyses (all P < 3.3 × 10−30). We found evidence of a causal effect of adiposity on blood pressure, fasting levels of insulin, C-reactive protein, interleukin-6, HDL cholesterol, and triglycerides in a nonstratified analysis and in the <55-year stratum. Further, we found evidence of a smaller causal effect on total cholesterol (P for difference = 0.015) in the ≥55-year stratum than in the <55-year stratum, a finding that could be explained by biology, survival bias, or differential medication. In conclusion, this study extends previous knowledge of the effects of adiposity by providing sex- and age-specific causal estimates on cardiovascular risk factors.

Rare and low-frequency variants and their association with plasma levels of fibrinogen, FVII, FVIII, and vWF

Fibrinogen, coagulation factor VII (FVII), and factor VIII (FVIII) and its carrier von Willebrand factor (vWF) play key roles in hemostasis. Previously identified common variants explain only a small fraction of the trait heritabilities, and additional variations may be explained by associations with rarer variants with larger effects. The aim of this study was to identify low-frequency (minor allele frequency [MAF] ≥0.01 and <0.05) and rare (MAF <0.01) variants that influence plasma concentrations of these 4 hemostatic factors by meta-analyzing exome chip data from up to 76,000 participants of 4 ancestries. We identified 12 novel associations of low-frequency (n = 2) and rare (n = 10) variants across the fibrinogen, FVII, FVIII, and vWF traits that were independent of previously identified associations. Novel loci were found within previously reported genes and had effect sizes much larger than and independent of previously identified common variants. In addition, associations at KCNT1, HID1, and KATNB1 identified new candidate genes related to hemostasis for follow-up replication and functional genomic analysis. Newly identified low-frequency and rare-variant associations accounted for modest amounts of trait variance and therefore are unlikely to increase predicted trait heritability but provide new information for understanding individual variation in hemostasis pathways.

A Genetic Variant in the Seed Region of miR-4513 Shows Pleiotropic Effects on Lipid and Glucose Homeostasis, Blood Pressure, and Coronary Artery Disease

MicroRNAs (miRNA) play a crucial role in the regulation of diverse biological processes by post‐transcriptional modulation of gene expression. Genetic polymorphisms in miRNA‐related genes can potentially contribute to a wide range of phenotypes. The effect of such variants on cardiometabolic diseases has not yet been defined. We systematically investigated the association of genetic variants in the seed region of miRNAs with cardiometabolic phenotypes, using the thus far largest genome‐wide association studies on 17 cardiometabolic traits/diseases. We found that rs2168518:G>A, a seed region variant of miR‐4513, associates with fasting glucose, low‐density lipoprotein–cholesterol, total cholesterol, systolic and diastolic blood pressure, and risk of coronary artery disease. We experimentally showed that miR‐4513 expression is significantly reduced in the presence of the rs2168518 mutant allele. We sought to identify miR‐4513 target genes that may mediate these associations and revealed five genes (PCSK1, BNC2, MTMR3, ANK3, and GOSR2) through which these effects might be taking place. Using luciferase reporter assays, we validated GOSR2 as a target of miR‐4513 and further demonstrated that the miRNA‐mediated regulation of this gene is changed by rs2168518. Our findings indicate a pleiotropic effect of miR‐4513 on cardiometabolic phenotypes and may improve our understanding of the pathophysiology of cardiometabolic diseases.

Epigenome-wide association study (EWAS) on lipids: the Rotterdam Study

BackgroundDNA methylation is a key epigenetic mechanism that is suggested to be associated with blood lipid levels. We aimed to identify CpG sites at which DNA methylation levels are associated with blood levels of triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and total cholesterol in 725 participants of the Rotterdam Study, a population-based cohort study. Subsequently, we sought replication in a non-overlapping set of 760 participants.ResultsGenome-wide methylation levels were measured in whole blood using the Illumina Methylation 450 array. Associations between lipid levels and DNA methylation beta values were examined using linear mixed-effect models. All models were adjusted for sex, age, smoking, white blood cell proportions, array number, and position on array. A Bonferroni-corrected p value lower than 1.08 × 10−7 was considered statistically significant. Five CpG sites annotated to genes including DHCR24, CPT1A, ABCG1, and SREBF1 were identified and replicated. Four CpG sites were associated with triglycerides, including CpG sites annotated to CPT1A (cg00574958 and cg17058475), ABCG1 (cg06500161), and SREBF1 (cg11024682). Two CpG sites were associated with HDL-C, including ABCG1 (cg06500161) and DHCR24 (cg17901584). No significant associations were observed with LDL-C or total cholesterol.ConclusionsWe report an association of HDL-C levels with methylation of a CpG site near DHCR24, a protein-coding gene involved in cholesterol biosynthesis, which has previously been reported to be associated with other metabolic traits. Furthermore, we confirmed previously reported associations of methylation of CpG sites within CPT1A, ABCG1, and SREBF1 and lipids. These results provide insight in the mechanisms that are involved in lipid metabolism.

Pleiotropy among common genetic loci identified for cardiometabolic disorders and C-reactive protein

Pleiotropic genetic variants have independent effects on different phenotypes. C-reactive protein (CRP) is associated with several cardiometabolic phenotypes. Shared genetic backgrounds may partially underlie these associations. We conducted a genome-wide analysis to identify the shared genetic background of inflammation and cardiometabolic phenotypes using published genome-wide association studies (GWAS). We also evaluated whether the pleiotropic effects of such loci were biological or mediated in nature. First, we examined whether 283 common variants identified for 10 cardiometabolic phenotypes in GWAS are associated with CRP level. Second, we tested whether 18 variants identified for serum CRP are associated with 10 cardiometabolic phenotypes. We used a Bonferroni corrected p-value of 1.1×10-04 (0.05/463) as a threshold of significance. We evaluated the independent pleiotropic effect on both phenotypes using individual level data from the Women Genome Health Study. Evaluating the genetic overlap between inflammation and cardiometabolic phenotypes, we found 13 pleiotropic regions. Additional analyses showed that 6 regions (APOC1, HNF1A, IL6R, PPP1R3B, HNF4A and IL1F10) appeared to have a pleiotropic effect on CRP independent of the effects on the cardiometabolic phenotypes. These included loci where individuals carrying the risk allele for CRP encounter higher lipid levels and risk of type 2 diabetes. In addition, 5 regions (GCKR, PABPC4, BCL7B, FTO and TMEM18) had an effect on CRP largely mediated through the cardiometabolic phenotypes. In conclusion, our results show genetic pleiotropy among inflammation and cardiometabolic phenotypes. In addition to reverse causation, our data suggests that pleiotropic genetic variants partially underlie the association between CRP and cardiometabolic phenotypes.

Association of Rare Loss-Of-Function Alleles in HAL, Serum Histidine: Levels and Incident Coronary Heart Disease

Background— Histidine is a semiessential amino acid with antioxidant and anti-inflammatory properties. Few data are available on the associations between genetic variants, histidine levels, and incident coronary heart disease (CHD) in a population-based sample. Methods and Results— By conducting whole exome sequencing on 1152 African Americans in the Atherosclerosis Risk in Communities (ARIC) study and focusing on loss-of-function (LoF) variants, we identified 3 novel rare LoF variants in HAL , a gene that encodes histidine ammonia-lyase in the first step of histidine catabolism. These LoF variants had large effects on blood histidine levels ( β =0.26; P =1.2×10−13). The positive association with histidine levels was replicated by genotyping an independent sample of 718 ARIC African Americans (minor allele frequency=1%; P =1.2×10−4). In addition, high blood histidine levels were associated with reduced risk of developing incident CHD with an average of 21.5 years of follow-up among African Americans (hazard ratio=0.18; P =1.9×10−4). This finding was validated in an independent sample of European Americans from the Framingham Heart Study (FHS) Offspring Cohort. However, LoF variants in HAL were not directly significantly associated with incident CHD after meta-analyzing results from the CHARGE Consortium. Conclusions— Three LoF mutations in HAL were associated with increased histidine levels, which in turn were shown to be inversely related to the risk of CHD among both African Americans and European Americans. Future investigations on the association between HAL gene variation and CHD are warranted.Background—Histidine is a semiessential amino acid with antioxidant and anti-inflammatory properties. Few data are available on the associations between genetic variants, histidine levels, and incident coronary heart disease (CHD) in a population-based sample. Methods and Results—By conducting whole exome sequencing on 1152 African Americans in the Atherosclerosis Risk in Communities (ARIC) study and focusing on loss-of-function (LoF) variants, we identified 3 novel rare LoF variants in HAL, a gene that encodes histidine ammonia-lyase in the first step of histidine catabolism. These LoF variants had large effects on blood histidine levels (&bgr;=0.26; P=1.2×10−13). The positive association with histidine levels was replicated by genotyping an independent sample of 718 ARIC African Americans (minor allele frequency=1%; P=1.2×10−4). In addition, high blood histidine levels were associated with reduced risk of developing incident CHD with an average of 21.5 years of follow-up among African Americans (hazard ratio=0.18; P=1.9×10−4). This finding was validated in an independent sample of European Americans from the Framingham Heart Study (FHS) Offspring Cohort. However, LoF variants in HAL were not directly significantly associated with incident CHD after meta-analyzing results from the CHARGE Consortium. Conclusions—Three LoF mutations in HAL were associated with increased histidine levels, which in turn were shown to be inversely related to the risk of CHD among both African Americans and European Americans. Future investigations on the association between HAL gene variation and CHD are warranted.

Genetic variants in the ADAMTS13 and SUPT3H genes are associated with ADAMTS13 activity

A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) cleaves von Willebrand factor, reducing its prothrombotic activity. The genetic determinants of ADAMTS13 activity remain unclear. We performed a genome-wide association study of ADAMTS13 activity in the Rotterdam Study, a population-based cohort study. We used imputed genotypes of common variants in a discovery sample of 3443 individuals and replication sample of 2025 individuals. We examined rare exonic variant associations in ADAMTS13 in 1609 individuals using an exome array. rs41314453 in ADAMTS13 was associated with ADAMTS13 activity in both our discovery (β, -20.2%; P = 1.3 × 10(-33)) and replication sample (P = 3.3 × 10(-34)), and explained 3.6% to 6.5% of the variance. In the combined analysis of our discovery and replication samples, there were 2 further independent associations at the ADAMTS13 locus: rs3118667 (β, 3.0; P = 9.6 × 10(-21)) and rs139911703 (β, -11.6; P = 3.6 × 10(-8)). In addition, rs10456544 in SUPT3H was associated with a 4.2 increase in ADAMTS13 activity (P = 1.13.6 × 10(-8)). Finally, we found 3 independent associations with rare coding variants in ADAMTS13: rs148312697 (β, -32.2%; P = 3.7 × 10(-6)), rs142572218 (β, -46.0%; P = 3.9 × 10(-5)), and rs36222275 (β, -13.9%; P = 2.9 × 10(-3)). In conclusion, we identified rs41314453 as the main genetic determinant of ADAMTS13 activity, and we present preliminary findings for further associations at the ADAMTS13 and SUPT3H loci.

CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non–foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1&agr; and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1&agr; via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1&agr;/VEGF-A pathway.