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Background: Nitric oxide signaling plays a key role in the regulation of vascular tone and platelet activation. Here, we seek to understand the impact of a genetic predisposition to enhanced nitric oxide signaling on risk for cardiovascular diseases, thus informing the potential utility of pharmacological stimulation of the nitric oxide pathway as a therapeutic strategy. Methods: We analyzed the association of common and rare genetic variants in 2 genes that mediate nitric oxide signaling (Nitric Oxide Synthase 3 [ NOS3 ] and Guanylate Cyclase 1, Soluble, Alpha 3 [ GUCY1A3 ]) with a range of human phenotypes. We selected 2 common variants (rs3918226 in NOS3 and rs7692387 in GUCY1A3 ) known to associate with increased NOS3 and GUCY1A3 expression and reduced mean arterial pressure, combined them into a genetic score, and standardized this exposure to a 5 mm Hg reduction in mean arterial pressure. Using individual-level data from 335 464 participants in the UK Biobank and summary association results from 7 large-scale genome-wide association studies, we examined the effect of this nitric oxide signaling score on cardiometabolic and other diseases. We also examined whether rare loss-of-function mutations in NOS3 and GUCY1A3 were associated with coronary heart disease using gene sequencing data from the Myocardial Infarction Genetics Consortium (n=27 815). Results: A genetic predisposition to enhanced nitric oxide signaling was associated with reduced risks of coronary heart disease (odds ratio, 0.37; 95% confidence interval [CI], 0.31-0.45; P =5.5*10 –26 ], peripheral arterial disease (odds ratio 0.42; 95% CI, 0.26-0.68; P =0.0005), and stroke (odds ratio, 0.53; 95% CI, 0.37-0.76; P =0.0006). In a mediation analysis, the effect of the genetic score on decreased coronary heart disease risk extended beyond its effect on blood pressure. Conversely, rare variants that inactivate the NOS3 or GUCY1A3 genes were associated with a 23 mm Hg higher systolic blood pressure (95% CI, 12-34; P =5.6*10 –5 ) and a 3-fold higher risk of coronary heart disease (odds ratio, 3.03; 95% CI, 1.29-7.12; P =0.01). Conclusions: A genetic predisposition to enhanced nitric oxide signaling is associated with reduced risks of coronary heart disease, peripheral arterial disease, and stroke. Pharmacological stimulation of nitric oxide signaling may prove useful in the prevention or treatment of cardiovascular disease.

Background: Experimental disruption of both gene copies (“knockout”) in model organisms has proven useful to understand gene function. Identification of “human knockouts” (i.e., individuals in which null mutations disrupt both copies of a given gene) should therefore provide valuable insights to a gene function that could be of direct relevance to humans. Objective and Methods: We leverage naturally-occurring null mutations and consanguinity in the human population in order to: 1) identify humans knocked out for genes; and 2) characterize the phenotypic consequences of complete gene disruption. We performed whole-exome sequencing in 7,078 individuals living in Pakistan, a region of the world with high levels of consanguinity, and tested whether knockouts differed from wild-type participants across a range of >200 cardiometabolic traits. Results: Median length of genome-wide homozygosity among Pakistani participants was 6-7 times higher than participants of European (CEU, TSI), East Asian (CHB, JPT, CHD) and African ancestries (YRI, MKK), respectively. In Pakistanis, we were able to enumerate 47,656 mutations predicted to be null (nonsense, frameshift, or canonical splice-site); variants were further filtered based on MAF, transcript position, splice sites, and conservation to identify 36,850 “high-confidence” null mutations across 12,131 autosomal genes. Across all participants, 961 distinct genes were completely disrupted by homozygous null mutations. 1,306 participants (18.4%) had at least one gene knocked out. In a phenotypic screen, homozygosity for null mutations at APOC3 was associated with absent plasma apolipoprotein C-III levels; at PLAG27 , with absent enzymatic activity of lipoprotein-associated phospholipase A2 in the blood; at CYP2F1 , with higher plasma interleukin-8 concentrations; and at either A3GALT2 or NRG4 , with markedly reduced plasma insulin C-peptide concentrations. After physiologic challenge with oral fat, APOC3 knockouts, when compared to wild-type family members, displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Conclusion: These observations provide a roadmap to understand the consequences of complete disruption of a large fraction of genes in the human genome.

Background: Fibroblast growth factor 21 (FGF-21) is an adipokine produced predominantly in the liver and is a metabolic regulator of glucose and lipid metabolism. Circulating FGF-21 levels have been shown to be independently associated with a host of cardio-metabolic traits, including: BMI and adiposity, metabolic syndrome, diabetes, cholesterol and triglycerides, and cardiovascular disease outcomes. Despite these epidemiological observations, causal relationships between FGF-21 and cardio-metabolic outcomes remain uncertain. Objectives: To identify the cross-sectional correlates of FGF-21 levels and to explore the causal relationship between FGF-21 and cardio-metabolic traits using a Mendelian randomization based approach. Methods & Results: FGF-21 levels were measured by ELISA in 9,572 participants from the Pakistan Risk of Myocardial Infarction Study (PROMIS), a multi-ethnic case-control study aimed at identifying genetic and non-genetic determinants of cardio-metabolic diseases. Log serum FGF-21 levels were weekly but significantly correlated with measures of obesity (BMI r=0.1, p < 0.0001) and serum lipids (total cholesterol r=0.08, p < 0.0001; LDL-C r=0.06, p < 0.0001; log triglycerides r=0.15, p < 0.0001); there was, however, no significant association with biochemical indicators of glycemic control. A Genome Wide Association Study (GWAS) was conducted to identify genetic determinants of FGF-21 levels; genetic variants were further assessed for specific associations with FGF21 levels in order to to identify instruments suitable for Mendelian Randomization studies. Variants that specifically increased FGF21 levels were not found to be associated with either Type-2 diabetes (80,000 cases and 230,000 controls) or coronary heart disease (63,746 cases and 130,681 controls). Conclusions: Although FGF-21 is weekly correlated with a number of cardiovascular risk factors; however its relationship to cardio-metabolic outcomes is less likely to be causal.

Inflammation has been proposed as a contributor to different stages in the pathogenesis of coronary heart disease (CHD), including the lifelong process of atherogenesis, the acute atherothrombotic event that causes ischemic necrosis in acute myocardial infarction, and the myocardial damage after ischemia.1,2 C-reactive protein (CRP), an acute-phase plasma protein synthesized by the liver, is the most extensively studied systemic marker of inflammation.3 Since the original report of an association between modest increases in baseline circulating CRP and subsequent cardiovascular outcomes in patients with unstable angina at the initial examination,4,5 CRP has been the focus of intense investigation. Measurement of CRP has been advocated as a means of improving cardiovascular risk prediction.6 Because CRP binds to low-density lipoprotein (LDL)7 and is present in atherosclerotic plaques,8 there is also considerable interest in whether CRP may play a direct causal role in CHD (and, by implication, could be an important therapeutic target for disease prevention). As discussed below, the evidence relating to this hypothesis has been derived from several different routes of enquiry, including observational epidemiology, human genetic studies, experimental and animal models, and randomized clinical trials of statins. Collectively, several recent findings, such as those in this issue of Circulation from Koike et al9 on human CRP transgenic rabbits, have reduced the likelihood that CRP itself is a major causal mediator in CHD. In this editorial, we briefly summarize the strengths and limitations of the available data, with an emphasis on the recent major findings. Article see p 2088 In regard to observational epidemiology, the first population-based prospective study of CRP concentration and CHD risk appeared in 1996.10 By 2009, >50 additional such studies had been reported.11–14 The rapid appearance of epidemiological studies on CRP has been facilitated greatly by the relative stability …

Background— Many epidemiological studies have reported on associations between serum triglyceride concentrations and the risk of coronary heart disease, but this association has not been reliably quantified. In the present study, we report 2 separate nested case-control comparisons in 2 different prospective, population-based cohorts, plus an updated meta-analysis of 27 additional prospective studies in general Western populations. Methods and Results— Measurements were made in a total of 3582 incident cases of fatal and nonfatal coronary heart disease and 6175 controls selected from among the 44 237 men and women screened in the Reykjavik and the European Prospective Investigation of Cancer (EPIC)-Norfolk studies. Repeat measurements were obtained an average of 4 years apart in 1933 participants in the EPIC-Norfolk Study and an average of 12 years apart in 379 participants in the Reykjavik study. The long-term stability of log-triglyceride values (within-person correlation coefficients of 0.64 [95% CI, 0.60 to 0.68] over 4 years and 0.63 [95% CI, 0.57 to 0.70] over 12 years) was similar to those of blood pressure and total serum cholesterol. After adjustment for baseline values of several established risk factors, the strength of the association was substantially attenuated, and the adjusted odds ratio for coronary heart disease was 1.76 (95% CI, 1.39 to 2.21) in the Reykjavik study and 1.57 (95% CI, 1.10 to 2.24) in the EPIC-Norfolk study in a comparison of individuals in the top third with those in the bottom third of usual log-triglyceride values. Similar overall findings (adjusted odds ratio, 1.72; 95% CI, 1.56 to 1.90) were observed in an updated meta-analysis involving a total of 10 158 incident coronary heart disease cases from 262 525 participants in 29 studies. Conclusions— Available prospective studies in Western populations consistently indicate moderate and highly significant associations between triglyceride values and coronary heart disease risk. Because these associations depend considerably on levels of established risk factors, however, further studies are needed to help assess the nature of any independent associations.

Background —It is unknown whether modest increases of fibrin D-dimer, a circulating marker of fibrin turnover, are relevant to coronary heart disease (CHD) in the general population. Methods and Results —We measured serum concentrations of D-dimer antigen in the stored baseline blood samples of 630 CHD cases and 1269 controls “nested” in a prospective cohort of 5661 men who were monitored for 16 years, and we conducted a meta-analysis of previous relevant studies to place our findings in context. In a comparison of men in the top third compared with those in the bottom third of baseline fibrin D-dimer values (tertile cutoffs, >94 versus P Conclusion —Although there may be an association between circulating D-dimer values and CHD, further studies are needed to determine the extent to which this is causal.

Background —Studies are needed to test claims that potentially virulent strains of Helicobacter pylori are more strongly related to coronary heart disease (CHD) than are other strains. Methods and Results —We measured serum IgG antibodies to mixed H pylori antigens and separately to the virulence-associated H pylori antigen CagA (cytotoxin-associated gene product A) in 505 CHD cases and in 1025 age-matched controls “nested” in a prospective study of 7735 British men (mean duration of follow-up in controls, 16 years). Of the 505 cases, 401 (79%) were seropositive for H pylori antibodies compared with 740 (72%) of the 1025 controls, yielding an odds ratio for CHD of 1.55 (95% CI 1.19 to 2.03), which fell to 1.30 (95% CI 0.88 to 1.90) after adjustments were made for standard vascular risk factors and indicators of socioeconomic status. Of the CHD cases, 240 (48%) were seropositive for IgG antibodies to CagA compared with 450 (44%) of the controls. When CagA-seropositive individuals were compared with H pylori –seronegative individuals, the odds ratio for CHD was 1.42 (95% CI 1.06 to 1.91), which fell to 1.10 (95% CI 0.71 to 1.71) after adjustments. In an analysis restricted to the 1141 (75%) H pylori –seropositive participants, the odds ratio for CHD was 1.0 (95% CI 0.78 to 1.29) in CagA–seropositive men. No strong associations were observed between H pylori seropositivity and blood lipids, blood pressure, markers of systemic inflammation, or plasma homocysteine. Conclusions — H pylori infection is not strongly related to the incidence of CHD in late middle-aged men, and CagA-positive strains appear to be no more strongly related to the disease than other strains. However, further studies are required to confirm or refute the existence of any moderate associations, particularly at younger ages.

Background— Measurement of B-type natriuretic peptide (BNP) concentration or its precursor (N-terminal fragment [NT-proBNP]) is recommended in patients with symptoms of left ventricular dysfunction and in other settings, but the relevance of these peptides to cardiovascular disease (CVD) in general populations or in patients with stable vascular disease is uncertain. Methods and Results— Data were collated from 40 long-term prospective studies involving a total of 87 474 participants and 10 625 incident CVD outcomes. In a comparison of individuals in the top third with those in the bottom third of baseline values of natriuretic peptides, the combined risk ratio (RR), adjusted for several conventional risk factors, was 2.82 (95% confidence interval [CI], 2.40 to 3.33) for CVD. Analysis of the 6 studies with at least 250 CVD outcomes (which should be less prone to selective reporting than are smaller studies) yielded an adjusted RR of 1.94 (95% CI, 1.57 to 2.39). RRs were broadly similar with BNP or NT-proBNP (RR, 2.89 [95% CI, 1.91 to 4.38] and 2.82 [95% CI, 2.35 to 3.38], respectively) and by different baseline vascular risk (RR, 2.68 [95% CI, 2.07 to 3.47] in approximately general populations; RR, 3.35 [95% CI, 2.38 to 4.72] in people with elevated vascular risk factors; RR, 2.60 [95% CI, 1.99 to 3.38] in patients with stable CVD). Assay of BNP or NT-proBNP in addition to measurement of conventional CVD risk factors yielded generally modest improvements in risk discrimination. Conclusions— Available prospective studies indicate strong associations between circulating concentration of natriuretic peptides and CVD risk under a range of different circumstances. Further investigation is warranted, particularly in large general population studies, to clarify any predictive utility of these markers and to better control for publication bias.

Background —Studies of iron status and coronary heart disease (CHD) have yielded conflicting results. In a systematic review (“meta-analysis”), we quantitatively assessed epidemiological associations reported in prospective studies. Methods and Results —Studies were identified by computer-assisted searches of the published literature, scanning of relevant reference lists, hand searching of relevant journals, and discussions with relevant authors. The following was abstracted: size and type of cohort, mean age, mean duration of follow-up, assay methods, degree of adjustment for confounders, and relationship of CHD risk to the baseline assay results. Twelve studies were identified, involving a total of 7800 CHD cases, with several reporting on >1 marker of iron status. For serum ferritin, with 570 CHD cases in 5 studies, comparison of individuals with baseline values ≥200 versus Conclusions —Published prospective studies do not provide good evidence to support the existence of strong epidemiological associations between iron status and CHD.

We thank Dr Pischon and colleagues for their interest in our work.1 We have reported the largest single prospective population-based study of adiponectin levels and coronary heart disease thus far, reinforced by a meta-analysis of 6 previous studies totaling 1313 incident cases.1 Overall, the adjusted risk ratio for coronary heart disease was 0.84 (95% CI: 0.70 to 1.01) in a comparison of individuals in the top third with those in the bottom third of baseline adiponectin concentrations, and there was no evidence of heterogeneity among the 7 reports (χ …