Divergent role of Mitochondrial Amidoxime Reducing Component 1 (MARC1) in human and mouse.

Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.

Rare coding variants in 35 genes associate with circulating lipid levels-A multi-ancestry analysis of 170,000 exomes.

Large-scale gene sequencing studies for complex traits have the potential to identify causal genes with therapeutic implications. We performed gene-based association testing of blood lipid levels with rare (minor allele frequency < 1%) predicted damaging coding variation by using sequence data from >170,000 individuals from multiple ancestries: 97,493 European, 30,025 South Asian, 16,507 African, 16,440 Hispanic/Latino, 10,420 East Asian, and 1,182 Samoan. We identified 35 genes associated with circulating lipid levels; some of these genes have not been previously associated with lipid levels when using rare coding variation from population-based samples. We prioritize 32 genes in array-based genome-wide association study (GWAS) loci based on aggregations of rare coding variants; three (EVI5, SH2B3, and PLIN1) had no prior association of rare coding variants with lipid levels. Most of our associated genes showed evidence of association among multiple ancestries. Finally, we observed an enrichment of gene-based associations for low-density lipoprotein cholesterol drug target genes and for genes closest to GWAS index single-nucleotide polymorphisms (SNPs). Our results demonstrate that gene-based associations can be beneficial for drug target development and provide evidence that the gene closest to the array-based GWAS index SNP is often the functional gene for blood lipid levels.

Germline Mutations in CIDEB and Protection against Liver Disease.

BACKGROUND: Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS: We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS: The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P = 4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P = 9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS: Rare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.).

Correction: A missense variant in Mitochondrial Amidoxime Reducing Component 1 gene and protection against liver disease.

[This corrects the article DOI: 10.1371/journal.pgen.1008629.].

A missense variant in Mitochondrial Amidoxime Reducing Component 1 gene and protection against liver disease.

Analyzing 12,361 all-cause cirrhosis cases and 790,095 controls from eight cohorts, we identify a common missense variant in the Mitochondrial Amidoxime Reducing Component 1 gene (MARC1 p.A165T) that associates with protection from all-cause cirrhosis (OR 0.91, p = 2.3*10-11). This same variant also associates with lower levels of hepatic fat on computed tomographic imaging and lower odds of physician-diagnosed fatty liver as well as lower blood levels of alanine transaminase (-0.025 SD, 3.7*10-43), alkaline phosphatase (-0.025 SD, 1.2*10-37), total cholesterol (-0.030 SD, p = 1.9*10-36) and LDL cholesterol (-0.027 SD, p = 5.1*10-30) levels. We identified a series of additional MARC1 alleles (low-frequency missense p.M187K and rare protein-truncating p.R200Ter) that also associated with lower cholesterol levels, liver enzyme levels and reduced risk of cirrhosis (0 cirrhosis cases for 238 R200Ter carriers versus 17,046 cases of cirrhosis among 759,027 non-carriers, p = 0.04) suggesting that deficiency of the MARC1 enzyme may lower blood cholesterol levels and protect against cirrhosis.

Genome-Wide Polygenic Score, Clinical Risk Factors, and Long-Term Trajectories of Coronary Artery Disease.

OBJECTIVE: To determine the relationship of a genome-wide polygenic score for coronary artery disease (GPSCAD) with lifetime trajectories of CAD risk, directly compare its predictive capacity to traditional risk factors, and assess its interplay with the Pooled Cohort Equations (PCE) clinical risk estimator. Approach and Results: We studied GPSCAD in 28 556 middle-aged participants of the Malmö Diet and Cancer Study, of whom 4122 (14.4%) developed CAD over a median follow-up of 21.3 years. A pronounced gradient in lifetime risk of CAD was observed-16% for those in the lowest GPSCAD decile to 48% in the highest. We evaluated the discriminative capacity of the GPSCAD-as assessed by change in the C-statistic from a baseline model including age and sex-among 5685 individuals with PCE risk estimates available. The increment for the GPSCAD (+0.045, P<0.001) was higher than for any of 11 traditional risk factors (range +0.007 to +0.032). Minimal correlation was observed between GPSCAD and 10-year risk defined by the PCE (r=0.03), and addition of GPSCAD improved the C-statistic of the PCE model by 0.026. A significant gradient in lifetime risk was observed for the GPSCAD, even among individuals within a given PCE clinical risk stratum. We replicated key findings-noting strikingly consistent results-in 325 003 participants of the UK Biobank. CONCLUSIONS: GPSCAD-a risk estimator available from birth-stratifies individuals into varying trajectories of clinical risk for CAD. Implementation of GPSCAD may enable identification of high-risk individuals early in life, decades in advance of manifest risk factors or disease.

Role of Blood Lipids in the Development of Ischemic Stroke and its Subtypes: A Mendelian Randomization Study.

BACKGROUND AND PURPOSE: Statin therapy is associated with a lower risk of ischemic stroke supporting a causal role of low-density lipoprotein (LDL) cholesterol. However, more evidence is needed to answer the question whether LDL cholesterol plays a causal role in ischemic stroke subtypes. In addition, it is unknown whether high-density lipoprotein cholesterol and triglycerides have a causal relationship to ischemic stroke and its subtypes. Our aim was to investigate the causal role of LDL cholesterol, high-density lipoprotein cholesterol, and triglycerides in ischemic stroke and its subtypes through Mendelian randomization (MR). METHODS: Summary data on 185 genome-wide lipids-associated single nucleotide polymorphisms were obtained from the Global Lipids Genetics Consortium and the Stroke Genetics Network for their association with ischemic stroke (n=16 851 cases and 32 473 controls) and its subtypes, including large artery atherosclerosis (n=2410), small artery occlusion (n=3186), and cardioembolic (n=3427) stroke. Inverse-variance-weighted MR was used to obtain the causal estimates. Inverse-variance-weighted multivariable MR, MR-Egger, and sensitivity exclusion of pleiotropic single nucleotide polymorphisms after Steiger filtering and MR-Pleiotropy Residual Sum and Outlier test were used to adjust for pleiotropic bias. RESULTS: A 1-SD genetically elevated LDL cholesterol was associated with an increased risk of ischemic stroke (odds ratio: 1.12; 95% confidence interval: 1.04-1.20) and large artery atherosclerosis stroke (odds ratio: 1.28; 95% confidence interval: 1.10-1.49) but not with small artery occlusion or cardioembolic stroke in multivariable MR. A 1-SD genetically elevated high-density lipoprotein cholesterol was associated with a decreased risk of small artery occlusion stroke (odds ratio: 0.79; 95% confidence interval: 0.67-0.90) in multivariable MR. MR-Egger indicated no pleiotropic bias, and results did not markedly change after sensitivity exclusion of pleiotropic single nucleotide polymorphisms. Genetically elevated triglycerides did not associate with ischemic stroke or its subtypes. CONCLUSIONS: LDL cholesterol lowering is likely to prevent large artery atherosclerosis but may not prevent small artery occlusion nor cardioembolic strokes. High-density lipoprotein cholesterol elevation may lead to benefits in small artery disease prevention. Finally, triglyceride lowering may not yield benefits in ischemic stroke and its subtypes.

High Level of Fasting Plasma Proenkephalin-A Predicts Deterioration of Kidney Function and Incidence of CKD.

High levels of proenkephalin-A (pro-ENK) have been associated with decreased eGFR in an acute setting. Here, we examined whether pro-ENK levels predict CKD and decline of renal function in a prospective cohort of 2568 participants without CKD (eGFR>60 ml/min per 1.73 m2) at baseline. During a mean follow-up of 16.6 years, 31.7% of participants developed CKD. Participants with baseline pro-ENK levels in the highest tertile had significantly greater yearly mean decline of eGFR (Ptrend<0.001) and rise of cystatin C (Ptrend=0.01) and creatinine (Ptrend<0.001) levels. Furthermore, compared with participants in the lowest tertile, participants in the highest tertile of baseline pro-ENK concentration had increased CKD incidence (odds ratio, 1.51; 95% confidence interval, 1.18 to 1.94) when adjusted for multiple factors. Adding pro-ENK to a model of conventional risk factors in net reclassification improvement analysis resulted in reclassification of 14.14% of participants. Genome-wide association analysis in 4150 participants of the same cohort revealed the strongest association of pro-ENK levels with rs1012178 near the PENK gene, where the minor T-allele associated with a 0.057 pmol/L higher pro-ENK level per allele (P=4.67x10-21). Furthermore, the T-allele associated with a 19% increased risk of CKD per allele (P=0.03) and a significant decrease in the instrumental variable estimator for eGFR (P<0.01) in a Mendelian randomization analysis. In conclusion, circulating plasma pro-ENK level predicts incident CKD and may aid in identifying subjects in need of primary preventive regimens. Additionally, the Mendelian randomization analysis suggests a causal relationship between pro-ENK level and deterioration of kidney function over time.

Association of low-frequency and rare coding-sequence variants with blood lipids and coronary heart disease in 56,000 whites and blacks.

Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the "Exome Array" to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121*], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited.

Loss-of-function mutations in APOC3, triglycerides, and coronary disease.

BACKGROUND: Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. METHODS: We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. RESULTS: An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G→A and IVS3+1G→T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P<1×10(-20)), and circulating levels of APOC3 in carriers were 46% lower than levels in noncarriers (P=8×10(-10)). The risk of coronary heart disease among 498 carriers of any rare APOC3 mutation was 40% lower than the risk among 110,472 noncarriers (odds ratio, 0.60; 95% confidence interval, 0.47 to 0.75; P=4×10(-6)). CONCLUSIONS: Rare mutations that disrupt APOC3 function were associated with lower levels of plasma triglycerides and APOC3. Carriers of these mutations were found to have a reduced risk of coronary heart disease. (Funded by the National Heart, Lung, and Blood Institute and others.).

The MBOAT7-TMC4 Variant rs641738 Increases Risk of Nonalcoholic Fatty Liver Disease in Individuals of European Descent.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver damage and is characterized by steatosis. Genetic factors increase risk for progressive NAFLD. A genome-wide association study showed that the rs641738 C>T variant in the locus that contains the membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7, also called LPIAT1) and transmembrane channel-like 4 gene (TMC4) increased the risk for cirrhosis in alcohol abusers. We investigated whether the MBOAT7-TMC4 is a susceptibility locus for the development and progression of NAFLD. METHODS: We genotyped rs641738 in DNA collected from 3854 participants from the Dallas Heart Study (a multi-ethnic population-based probability sample of Dallas County residents) and 1149 European individuals from the Liver Biopsy Cross-Sectional Cohort. Clinical and anthropometric data were collected, and biochemical and lipidomics were measured in plasma samples from participants. A total of 2736 participants from the Dallas Heart Study also underwent proton magnetic resonance spectroscopy to measure hepatic triglyceride content. In the Liver Biopsy Cross-Sectional Cohort, a total of 1149 individuals underwent liver biopsy to diagnose liver disease and disease severity. RESULTS: The genotype rs641738 at the MBOAT7-TMC4 locus associated with increased hepatic fat content in the 2 cohorts, and with more severe liver damage and increased risk of fibrosis compared with subjects without the variant. MBOAT7, but not TMC4, was found to be highly expressed in the liver. The MBOAT7 rs641738 T allele was associated with lower protein expression in the liver and changes in plasma phosphatidylinositol species consistent with decreased MBOAT7 function. CONCLUSIONS: We provide evidence for an association between the MBOAT7 rs641738 variant and the development and severity of NAFLD in individuals of European descent. This association seems to be mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling.

TCF7L2 type 2 diabetes risk variant, lifestyle factors, and incidence of prostate cancer.

BACKGROUND: Variation in transcription factor 7-like 2 (TCF7L2), the strongest genetic risk factor for type 2 diabetes (T2D), may play a role in prostate cancer (PCa) depending on lifestyle factors. The aims of this study were to determine if TCF7L2 rs7903146 is associated with risk of PCa and if the association is modified by lifestyle factors independently of T2D status. METHODS: We prospectively followed 8,558 men in the Malmö Diet and Cancer Study from baseline 1991-1996 until end of 2009. Cox regression models were used to assess the association between rs7903146 T2D-risk allele (T) and PCa. Effect modification by incident T2D status, fasting glucose levels, dietary, and lifestyle risk factors were tested. RESULTS: During follow-up 855 incident PCa cases were registered. We observed a non-significant tendency for the TCF7L2 variant to associate with higher risk of PCa, which was unaffected by adjustment for incident T2D (HR = 1.24; 95% CI: 0.96, 1.60; P = 0.079) but more pronounced among subjects who developed T2D (HR = 1.91, 95% CI: 0.88, 4.14; P = 0.064). In a sub-sample of hyperglycemic men we observed an increased risk of PCa among T-allele carriers (HR = 2.72, 95% CI: 1.22, 6.04; P = 0.014; P(interaction) = 0.056). T-allele carriers with higher number of lifestyle risk factors had an increased risk of PCa (P(interaction) = 0.006). CONCLUSIONS: We found no independent association between TCF7L2 rs7903146 and PCa risk. However, among hyperglycemic men we observed that the risk allele may increase risk of PCa. The association between rs7903146 and PCa risk may also be modified by lifestyle factors.

The chromosome 9p21 variant interacts with vegetable and wine intake to influence the risk of cardiovascular disease: a population based cohort study.

BACKGROUND: Chromosome 9p21 variants are associated with cardiovascular disease (CVD) but not with any of its known risk markers. However, recent studies have suggested that the risk associated with 9p21 variation is modified by a prudent dietary pattern and smoking. We tested if the increased risk of CVD by the 9p21 single nucleotide polymorphism rs4977574 is modified by intakes of vegetables, fruits, alcohol, or wine, and if rs4977574 interacts with environmental factors on known CVD risk markers. METHODS: Multivariable Cox regression analyses were performed in 23,949 individuals from the population-based prospective Malmö Diet and Cancer Study (MDCS), of whom 3,164 developed CVD during 15 years of follow-up. The rs4977574 variant (major allele: A; minor allele: G) was genotyped using TaqMan® Assay Design probes. Dietary data were collected at baseline using a modified diet history method. Cross-sectional analyses were performed in 4,828 MDCS participants with fasting blood levels of circulating risk factors measured at baseline. RESULTS: Each rs4977574 G allele was associated with a 16% increased incidence of CVD (95% confidence interval (CI), 1.10-1.22). Higher vegetable intake (hazard ratio (HR), 0.95 [CI: 0.91-0.996]), wine intake (HR, 0.91 [CI: 0.86-0.96]), and total alcohol consumption (HR, 0.92 [CI: 0.86-0.98]) were associated with lower CVD incidence. The increased CVD incidence by the G allele was restricted to individuals with medium or high vegetable intake (Pinteraction = 0.043), and to non- and low consumers of wine (Pinteraction = 0.029). Although rs4977574 did not associate with any known risk markers, stratification by vegetable intake and smoking suggested an interaction with rs4977574 on glycated hemoglobin and high-density lipoprotein cholesterol (Pinteraction = 0.015 and 0.049, respectively). CONCLUSIONS: Our results indicate that rs4977574 interacts with vegetable and wine intake to affect the incidence of CVD, and suggest that an interaction may exist between environmental risk factors and rs4977574 on known risk markers of CVD.

Association of low-density lipoprotein cholesterol-related genetic variants with aortic valve calcium and incident aortic stenosis.

IMPORTANCE: Plasma low-density lipoprotein cholesterol (LDL-C) has been associated with aortic stenosis in observational studies; however, randomized trials with cholesterol-lowering therapies in individuals with established valve disease have failed to demonstrate reduced disease progression. OBJECTIVE: To evaluate whether genetic data are consistent with an association between LDL-C, high-density lipoprotein cholesterol (HDL-C), or triglycerides (TG) and aortic valve disease. DESIGN, SETTING, AND PARTICIPANTS: Using a Mendelian randomization study design, we evaluated whether weighted genetic risk scores (GRSs), a measure of the genetic predisposition to elevations in plasma lipids, constructed using single-nucleotide polymorphisms identified in genome-wide association studies for plasma lipids, were associated with aortic valve disease. We included community-based cohorts participating in the CHARGE consortium (n = 6942), including the Framingham Heart Study (cohort inception to last follow-up: 1971-2013; n = 1295), Multi-Ethnic Study of Atherosclerosis (2000-2012; n = 2527), Age Gene/Environment Study-Reykjavik (2000-2012; n = 3120), and the Malmö Diet and Cancer Study (MDCS, 1991-2010; n = 28,461). MAIN OUTCOMES AND MEASURES: Aortic valve calcium quantified by computed tomography in CHARGE and incident aortic stenosis in the MDCS. RESULTS: The prevalence of aortic valve calcium across the 3 CHARGE cohorts was 32% (n = 2245). In the MDCS, over a median follow-up time of 16.1 years, aortic stenosis developed in 17 per 1000 participants (n = 473) and aortic valve replacement for aortic stenosis occurred in 7 per 1000 (n = 205). Plasma LDL-C, but not HDL-C or TG, was significantly associated with incident aortic stenosis (hazard ratio [HR] per mmol/L, 1.28; 95% CI, 1.04-1.57; P = .02; aortic stenosis incidence: 1.3% and 2.4% in lowest and highest LDL-C quartiles, respectively). The LDL-C GRS, but not HDL-C or TG GRS, was significantly associated with presence of aortic valve calcium in CHARGE (odds ratio [OR] per GRS increment, 1.38; 95% CI, 1.09-1.74; P = .007) and with incident aortic stenosis in MDCS (HR per GRS increment, 2.78; 95% CI, 1.22-6.37; P = .02; aortic stenosis incidence: 1.9% and 2.6% in lowest and highest GRS quartiles, respectively). In sensitivity analyses excluding variants weakly associated with HDL-C or TG, the LDL-C GRS remained associated with aortic valve calcium (P = .03) and aortic stenosis (P = .009). In instrumental variable analysis, LDL-C was associated with an increase in the risk of incident aortic stenosis (HR per mmol/L, 1.51; 95% CI, 1.07-2.14; P = .02). CONCLUSIONS AND RELEVANCE: Genetic predisposition to elevated LDL-C was associated with presence of aortic valve calcium and incidence of aortic stenosis, providing evidence supportive of a causal association between LDL-C and aortic valve disease. Whether earlier intervention to reduce LDL-C could prevent aortic valve disease merits further investigation.

Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study.

BACKGROUND: High plasma HDL cholesterol is associated with reduced risk of myocardial infarction, but whether this association is causal is unclear. Exploiting the fact that genotypes are randomly assigned at meiosis, are independent of non-genetic confounding, and are unmodified by disease processes, mendelian randomisation can be used to test the hypothesis that the association of a plasma biomarker with disease is causal. METHODS: We performed two mendelian randomisation analyses. First, we used as an instrument a single nucleotide polymorphism (SNP) in the endothelial lipase gene (LIPG Asn396Ser) and tested this SNP in 20 studies (20,913 myocardial infarction cases, 95,407 controls). Second, we used as an instrument a genetic score consisting of 14 common SNPs that exclusively associate with HDL cholesterol and tested this score in up to 12,482 cases of myocardial infarction and 41,331 controls. As a positive control, we also tested a genetic score of 13 common SNPs exclusively associated with LDL cholesterol. FINDINGS: Carriers of the LIPG 396Ser allele (2·6% frequency) had higher HDL cholesterol (0·14 mmol/L higher, p=8×10(-13)) but similar levels of other lipid and non-lipid risk factors for myocardial infarction compared with non-carriers. This difference in HDL cholesterol is expected to decrease risk of myocardial infarction by 13% (odds ratio [OR] 0·87, 95% CI 0·84-0·91). However, we noted that the 396Ser allele was not associated with risk of myocardial infarction (OR 0·99, 95% CI 0·88-1·11, p=0·85). From observational epidemiology, an increase of 1 SD in HDL cholesterol was associated with reduced risk of myocardial infarction (OR 0·62, 95% CI 0·58-0·66). However, a 1 SD increase in HDL cholesterol due to genetic score was not associated with risk of myocardial infarction (OR 0·93, 95% CI 0·68-1·26, p=0·63). For LDL cholesterol, the estimate from observational epidemiology (a 1 SD increase in LDL cholesterol associated with OR 1·54, 95% CI 1·45-1·63) was concordant with that from genetic score (OR 2·13, 95% CI 1·69-2·69, p=2×10(-10)). INTERPRETATION: Some genetic mechanisms that raise plasma HDL cholesterol do not seem to lower risk of myocardial infarction. These data challenge the concept that raising of plasma HDL cholesterol will uniformly translate into reductions in risk of myocardial infarction. FUNDING: US National Institutes of Health, The Wellcome Trust, European Union, British Heart Foundation, and the German Federal Ministry of Education and Research.

High intakes of protein and processed meat associate with increased incidence of type 2 diabetes.

Diets high in protein have shown positive effects on short-term weight reduction and glycaemic control. However, the understanding of how dietary macronutrient composition relates to long-term risk of type 2 diabetes is limited. The aim of the present study was to examine intakes of macronutrients, fibre and protein sources in relation to incident type 2 diabetes. In total, 27 140 individuals, aged 45-74 years, from the population-based Malmö Diet and Cancer cohort, were included. Dietary data were collected with a modified diet history method, including registration of cooked meals. During 12 years of follow-up, 1709 incident type 2 diabetes cases were identified. High protein intake was associated with increased risk of type 2 diabetes (hazard ratio (HR) 1.27 for highest compared with lowest quintile; 95 % CI 1.08, 1.49; P for trend = 0.01). When protein consumption increased by 5 % of energy at the expense of carbohydrates (HR 1.20; 95 % CI 1.09, 1.33) or fat (HR 1.21; 95 % CI 1.09, 1.33), increased diabetes risk was observed. Intakes in the highest quintiles of processed meat (HR 1.16; 95 % CI 1.00, 1.36; P for trend = 0.01) and eggs (HR 1.21; 95 % CI 1.04, 1.41; P for trend = 0.02) were associated with increased risk. Intake of fibre-rich bread and cereals was inversely associated with type 2 diabetes (HR 0.84; 95 % CI 0.73, 0.98; P for trend = 0.004). In conclusion, results from the present large population-based prospective study indicate that high protein intake is associated with increased risk of type 2 diabetes. Replacing protein with carbohydrates may be favourable, especially if fibre-rich breads and cereals are chosen as carbohydrate sources.

The Odds Ratio is "portable" across baseline risk but not the Relative Risk: Time to do away with the log link in binomial regression.

OBJECTIVES: In a recent paper we suggest that the relative risk (RR) be replaced with the odds ratio (OR) as the effect measure of choice in clinical epidemiology. In response, Chu, and colleagues raise several points that argue for the status quo. In this paper, we respond to their response. STUDY DESIGNS AND SETTINGS: We use the same examples given by Chu and colleagues to recompute estimates of effect and demonstrate the problem with the RR. RESULTS: We reaffirm the following findings: a) the OR and RR measure different things and their numerical difference is only important if misinterpreted b) this potential misinterpretation is a trivial issue compared to the lack of portability of the RR c) the same examples reaffirm non-portability of the RR and demonstrate how misleading the results might be in contrast to the OR, which is independent of the baseline risk d) the concept of non-collapsibility for the OR should be expected in the presence of a non-confounding risk factor, and is not a bias e) the log link in regression models that generate RRs as well as the use of RRs in meta-analysis is shown to be problematic using the same examples. CONCLUSION: The OR should replace the RR in clinical research and meta-analyses though there should be conversion of the end product into ratios or differences of risk, solely, for interpretation. To this end we provide a Stata module (logittorisk) for this purpose.

Association of Habitual Alcohol Intake With Risk of Cardiovascular Disease.

Importance: Observational studies have consistently proposed cardiovascular benefits associated with light alcohol consumption, while recent genetic analyses (ie, mendelian randomization studies) have suggested a possible causal link between alcohol intake and increased risk of cardiovascular disease. However, traditional approaches to genetic epidemiology assume a linear association and thus have not fully evaluated dose-response estimates of risk across different levels of alcohol intake. Objectives: To assess the association of habitual alcohol intake with cardiovascular disease risk and to evaluate the direction and relative magnitude of cardiovascular risk associated with different amounts of alcohol consumption. Design, Setting, and Participants: This cohort study used the UK Biobank (2006-2010, follow-up until 2016) to examine confounding in epidemiologic associations between alcohol intake and cardiovascular diseases. Using both traditional (ie, linear) and nonlinear mendelian randomization, potential associations between alcohol consumption and cardiovascular diseases (eg, hypertension and coronary artery disease) as well as corresponding association shapes were assessed. Data analysis was conducted from July 2019 to January 2022. Exposures: Genetic predisposition to alcohol intake. Main Outcomes and Measures: The association between alcohol consumption and cardiovascular diseases, including hypertension, coronary artery disease, myocardial infarction, stroke, heart failure, and atrial fibrillation. Results: This study included 371 463 participants (mean [SD] age, 57.0 [7.9] years; 172 400 [46%] men), who consumed a mean (SD) 9.2 (10.6) standard drinks per week. Overall, 121 708 participants (33%) had hypertension. Light to moderate alcohol consumption was associated with healthier lifestyle factors, adjustment for which attenuated the cardioprotective epidemiologic associations with modest intake. In linear mendelian randomization analyses, a 1-SD increase in genetically predicted alcohol consumption was associated with 1.3-fold (95% CI, 1.2-1.4) higher risk of hypertension (P < .001) and 1.4-fold (95% CI, 1.1-1.8) higher risk of coronary artery disease (P = .006). Nonlinear mendelian randomization analyses suggested nonlinear associations between alcohol consumption and both hypertension and coronary artery disease: light alcohol intake was associated with minimal increases in cardiovascular risk, whereas heavier consumption was associated with exponential increases in risk of both clinical and subclinical cardiovascular disease. Conclusions and Relevance: In this cohort study, coincident, favorable lifestyle factors attenuated the observational benefits of modest alcohol intake. Genetic epidemiology suggested that alcohol consumption of all amounts was associated with increased cardiovascular risk, but marked risk differences exist across levels of intake, including those accepted by current national guidelines.

The prevalence of adaptive immunity to COVID-19 and reinfection after recovery - a comprehensive systematic review and meta-analysis.

This study aims to estimate the prevalence and longevity of detectable SARS-CoV-2 antibodies and T and B memory cells after recovery. In addition, the prevalence of COVID-19 reinfection and the preventive efficacy of previous infection with SARS-CoV-2 were investigated. A synthesis of existing research was conducted. The Cochrane Library, the China Academic Journals Full Text Database, PubMed, and Scopus, and preprint servers were searched for studies conducted between 1 January 2020 to 1 April 2021. Included studies were assessed for methodological quality and pooled estimates of relevant outcomes were obtained in a meta-analysis using a bias adjusted synthesis method. Proportions were synthesized with the Freeman-Tukey double arcsine transformation and binary outcomes using the odds ratio (OR). Heterogeneity was assessed using the I2 and Cochran's Q statistics and publication bias was assessed using Doi plots. Fifty-four studies from 18 countries, with around 12,000,000 individuals, followed up to 8 months after recovery, were included. At 6-8 months after recovery, the prevalence of SARS-CoV-2 specific immunological memory remained high; IgG - 90.4% (95%CI 72.2-99.9, I2 = 89.0%), CD4+ - 91.7% (95%CI 78.2-97.1y), and memory B cells 80.6% (95%CI 65.0-90.2) and the pooled prevalence of reinfection was 0.2% (95%CI 0.0-0.7, I2 = 98.8). Individuals previously infected with SARS-CoV-2 had an 81% reduction in odds of a reinfection (OR 0.19, 95% CI 0.1-0.3, I2 = 90.5%). Around 90% of recovered individuals had evidence of immunological memory to SARS-CoV-2, at 6-8 months after recovery and had a low risk of reinfection.RegistrationPROSPERO: CRD42020201234.

Discovery and systematic characterization of risk variants and genes for coronary artery disease in over a million participants.

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR-Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD.