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1.  Variation at the NFATC2 Locus Increases the Risk of Thiazolidinedione-Induced Edema in the Diabetes REduction Assessment with ramipril and rosiglitazone Medication (DREAM) Study 
Diabetes Care  2010;33(10):2250-2253.
OBJECTIVE
Thiazolidinediones are used to treat type 2 diabetes. Their use has been associated with peripheral edema and congestive heart failure—outcomes that may have a genetic etiology.
RESEARCH DESIGN AND METHODS
We genotyped 4,197 participants of the multiethnic DREAM (Diabetes REduction Assessment with ramipril and rosiglitazone Medication) trial with a 50k single nucleotide polymorphisms (SNP) array, which captures ∼2000 cardiovascular, inflammatory, and metabolic genes. We tested 32,088 SNPs for an association with edema among Europeans who received rosiglitazone (n = 965).
RESULTS
One SNP, rs6123045, in NFATC2 was significantly associated with edema (odds ratio 1.89 [95% CI 1.47–2.42]; P = 5.32 × 10−7, corrected P = 0.017). Homozygous individuals had the highest edema rate (hazard ratio 2.89, P = 4.22 × 10−4) when compared with individuals homozygous for the protective allele, with heterozygous individuals having an intermediate risk. The interaction between the SNP and rosiglitazone for edema was significant (P = 7.68 × 10−3). Six SNPs in NFATC2 were significant in both Europeans and Latin Americans (P < 0.05).
CONCLUSIONS
Genetic variation at the NFATC2 locus contributes to edema among individuals who receive rosiglitazone.
doi:10.2337/dc10-0452
PMCID: PMC2945168  PMID: 20628086
2.  Fine mapping and association studies of a high-density lipoprotein cholesterol linkage region on chromosome 16 in French-Canadian subjects 
Low levels of high-density lipoprotein cholesterol (HDL-C) are an independent risk factor for cardiovascular disease. To identify novel genetic variants that contribute to HDL-C, we performed genome-wide scans and quantitative association studies in two study samples: a Quebec-wide study consisting of 11 multigenerational families and a study of 61 families from the Saguenay–Lac St-Jean (SLSJ) region of Quebec. The heritability of HDL-C in these study samples was 0.73 and 0.49, respectively. Variance components linkage methods identified a LOD score of 2.61 at 98 cM near the marker D16S515 in Quebec-wide families and an LOD score of 2.96 at 86 cM near the marker D16S2624 in SLSJ families. In the Quebec-wide sample, four families showed segregation over a 25.5-cM (18 Mb) region, which was further reduced to 6.6 Mb with additional markers. The coding regions of all genes within this region were sequenced. A missense variant in CHST6 segregated in four families and, with additional families, we observed a P value of 0.015 for this variant. However, an association study of this single-nucleotide polymorphism (SNP) in unrelated Quebec-wide samples was not significant. We also identified an SNP (rs11646677) in the same region, which was significantly associated with a low HDL-C (P=0.016) in the SLSJ study sample. In addition, RT-PCR results from cultured cells showed a significant difference in the expression of CHST6 and KIAA1576, another gene in the region. Our data constitute additional evidence for a locus on chromosome 16q23-24 that affects HDL-C levels in two independent French-Canadian studies.
doi:10.1038/ejhg.2009.157
PMCID: PMC2824775  PMID: 19844255
HDL-C; family study; complex traits; coronary heart disease; gene identification
3.  Fine Mapping and Association Studies of a High-Density Lipoprotein Cholesterol Linkage Region on Chromosome 16 in French-Canadian Subjects 
Low levels of high-density lipoprotein cholesterol (HDL-C) are an independent risk factor for cardiovascular disease. To identify novel genetic variants that contribute to HDL-C, we performed genome-wide scans and quantitative association studies in two study samples: a Quebec-wide study consisting of 11 multi-generational families and a study of 61 families from the Saguenay–Lac St-Jean (SLSJ) region of Quebec. The heritability of HDL-C in these study samples was 0.73 and 0.49 respectively. Variance components linkage methods identified a LOD score of 2.61, at 98 cM near the marker D16S515 in the Quebec-wide families and a LOD score of 2.96 at 86 cM, near the marker D16S2624 in the SLSJ families. In the Quebec-wide sample, four families demonstrated segregation over a 25.5 cM (18 Mb) region, which was further reduced to 6.6 Mb with additional markers. The coding regions of all genes within this region were sequenced. A missense variant in CHST6, segregated in four families and with additional families we observed a p value of 0.015 for this variant. However, an association study of this SNP in unrelated Quebec-wide samples was not significant. We also identified a SNP (rs11646677) in the same region, which was significantly associated with low HDL-C (p=0.016) in the SLSJ study sample. In addition, RT-PCR results from cultured cells demonstrated a significant difference in expression of CHST6 and KIAA1576, another gene in the region. Our data constitute additional evidence for a locus on chromosome 16q23-24 that affects HDL-C levels in two independent French Canadian studies.
doi:10.1038/ejhg.2009.157
PMCID: PMC2824775  PMID: 19844255
high-density lipoprotein cholesterol; family study; complex traits; coronary heart disease; gene identification
4.  Correction of Population Stratification in Large Multi-Ethnic Association Studies 
PLoS ONE  2008;3(1):e1382.
Background
The vast majority of genetic risk factors for complex diseases have, taken individually, a small effect on the end phenotype. Population-based association studies therefore need very large sample sizes to detect significant differences between affected and non-affected individuals. Including thousands of affected individuals in a study requires recruitment in numerous centers, possibly from different geographic regions. Unfortunately such a recruitment strategy is likely to complicate the study design and to generate concerns regarding population stratification.
Methodology/Principal Findings
We analyzed 9,751 individuals representing three main ethnic groups - Europeans, Arabs and South Asians - that had been enrolled from 154 centers involving 52 countries for a global case/control study of acute myocardial infarction. All individuals were genotyped at 103 candidate genes using 1,536 SNPs selected with a tagging strategy that captures most of the genetic diversity in different populations. We show that relying solely on self-reported ethnicity is not sufficient to exclude population stratification and we present additional methods to identify and correct for stratification.
Conclusions/Significance
Our results highlight the importance of carefully addressing population stratification and of carefully “cleaning” the sample prior to analyses to obtain stronger signals of association and to avoid spurious results.
doi:10.1371/journal.pone.0001382
PMCID: PMC2198793  PMID: 18196181
5.  Sphingomyelin phosphodiesterase-1 (SMPD1) coding variants do not contribute to low levels of high-density lipoprotein cholesterol 
BMC Medical Genetics  2007;8:79.
Background
Niemann-Pick disease type A and B is caused by a deficiency of acid sphingomyelinase due to mutations in the sphingomyelin phosphodiesterase-1 (SMPD1) gene. In Niemann-Pick patients, SMPD1 gene defects are reported to be associated with a severe reduction in plasma high-density lipoprotein (HDL) cholesterol.
Methods
Two common coding polymorphisms in the SMPD1 gene, the G1522A (G508R) and a hexanucleotide repeat sequence within the signal peptide region, were investigated in 118 unrelated subjects of French Canadian descent with low plasma levels of HDL-cholesterol (< 5th percentile for age and gender-matched subjects). Control subjects (n = 230) had an HDL-cholesterol level > the 25th percentile.
Results
For G1522A the frequency of the G and A alleles were 75.2% and 24.8% respectively in controls, compared to 78.6% and 21.4% in subjects with low HDL-cholesterol (p = 0.317). The frequency of 6 and 7 hexanucleotide repeats was 46.2% and 46.6% respectively in controls, compared to 45.6% and 49.1% in subjects with low HDL-cholesterol (p = 0.619). Ten different haplotypes were observed in cases and controls. Overall haplotype frequencies in cases and controls were not significantly different.
Conclusion
These results suggest that the two common coding variants at the SMPD1 gene locus are not associated with low HDL-cholesterol levels in the French Canadian population.
doi:10.1186/1471-2350-8-79
PMCID: PMC2241771  PMID: 18088425
6.  Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study 
Voight, Benjamin F | Peloso, Gina M | Orho-Melander, Marju | Frikke-Schmidt, Ruth | Barbalic, Maja | Jensen, Majken K | Hindy, George | Hólm, Hilma | Ding, Eric L | Johnson, Toby | Schunkert, Heribert | Samani, Nilesh J | Clarke, Robert | Hopewell, Jemma C | Thompson, John F | Li, Mingyao | Thorleifsson, Gudmar | Newton-Cheh, Christopher | Musunuru, Kiran | Pirruccello, James P | Saleheen, Danish | Chen, Li | Stewart, Alexandre FR | Schillert, Arne | Thorsteinsdottir, Unnur | Thorgeirsson, Gudmundur | Anand, Sonia | Engert, James C | Morgan, Thomas | Spertus, John | Stoll, Monika | Berger, Klaus | Martinelli, Nicola | Girelli, Domenico | McKeown, Pascal P | Patterson, Christopher C | Epstein, Stephen E | Devaney, Joseph | Burnett, Mary-Susan | Mooser, Vincent | Ripatti, Samuli | Surakka, Ida | Nieminen, Markku S | Sinisalo, Juha | Lokki, Marja-Liisa | Perola, Markus | Havulinna, Aki | de Faire, Ulf | Gigante, Bruna | Ingelsson, Erik | Zeller, Tanja | Wild, Philipp | de Bakker, Paul I W | Klungel, Olaf H | Maitland-van der Zee, Anke-Hilse | Peters, Bas J M | de Boer, Anthonius | Grobbee, Diederick E | Kamphuisen, Pieter W | Deneer, Vera H M | Elbers, Clara C | Onland-Moret, N Charlotte | Hofker, Marten H | Wijmenga, Cisca | Verschuren, WM Monique | Boer, Jolanda MA | van der Schouw, Yvonne T | Rasheed, Asif | Frossard, Philippe | Demissie, Serkalem | Willer, Cristen | Do, Ron | Ordovas, Jose M | Abecasis, Gonçalo R | Boehnke, Michael | Mohlke, Karen L | Daly, Mark J | Guiducci, Candace | Burtt, Noël P | Surti, Aarti | Gonzalez, Elena | Purcell, Shaun | Gabriel, Stacey | Marrugat, Jaume | Peden, John | Erdmann, Jeanette | Diemert, Patrick | Willenborg, Christina | König, Inke R | Fischer, Marcus | Hengstenberg, Christian | Ziegler, Andreas | Buysschaert, Ian | Lambrechts, Diether | Van de Werf, Frans | Fox, Keith A | El Mokhtari, Nour Eddine | Rubin, Diana | Schrezenmeir, Jürgen | Schreiber, Stefan | Schäfer, Arne | Danesh, John | Blankenberg, Stefan | Roberts, Robert | McPherson, Ruth | Watkins, Hugh | Hall, Alistair S | Overvad, Kim | Rimm, Eric | Boerwinkle, Eric | Tybjaerg-Hansen, Anne | Cupples, L Adrienne | Reilly, Muredach P | Melander, Olle | Mannucci, Pier M | Ardissino, Diego | Siscovick, David | Elosua, Roberto | Stefansson, Kari | O'Donnell, Christopher J | Salomaa, Veikko | Rader, Daniel J | Peltonen, Leena | Schwartz, Stephen M | Altshuler, David | Kathiresan, Sekar
Lancet  2012;380(9841):572-580.
Summary
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.
doi:10.1016/S0140-6736(12)60312-2
PMCID: PMC3419820  PMID: 22607825
7.  BRCA2 Variants and cardiovascular disease in a multi-ethnic study 
BMC Medical Genetics  2012;13:56.
Background
Germline mutations of BRCA1/2 are associated with hereditary breast and ovarian cancer. Recent data suggests excess mortality in mutation carriers beyond that conferred by neoplasia, and recent in vivo and in vitro studies suggest a modulatory role for BRCA proteins in endothelial and cardiomyocyte function. We therefore tested the association of BRCA2 variants with clinical cardiovascular disease (CVD).
Methods
Using data from 1,170 individuals included in two multi-ethnic population-based studies (SHARE and SHARE-AP), the association between BRCA2 variants and CVD was evaluated. 15 SNPs in BRCA2 with minor allele frequencies (MAF) > 0.01 had been previously genotyped using the cardiovascular gene-centric 50 k SNP array. 115 individuals (9.8%) reported a CVD event, defined as myocardial infarction (MI), angina, silent MI, stroke, and angioplasty or coronary artery bypass surgery. Analyses were adjusted for age and sex. The SNPs rs11571836 and rs1799943 were subsequently genotyped using the MassARRAY platform in 1,045 cases of incident MI and 1,135 controls from the South Asian subset of an international case-control study of acute MI (INTERHEART), and rs11571836 was imputed in 4,686 cases and 4500 controls from the Pakistan Risk of Myocardial Infarction Study (PROMIS).
Results
Two BRCA2 SNPs, rs11571836 and rs1799943, both located in untranslated regions, were associated with lower risk of CVD (OR 0.47 p = 0.01 and OR 0.56 p = 0.03 respectively) in the SHARE studies. Analysis by specific ethnicities demonstrated an association with CVD for both SNPs in Aboriginal People, and for rs11571836 only in South Asians. No association was observed in the European and Chinese subgroups. A non-significant trend towards an association between rs11571836 and lower risk of MI was observed in South Asians from INTERHEART [OR = 0.87 (95% CI: 0.75-1.01) p = 0.068], but was not evident in PROMIS [OR = 0.96 (95% CI: 0.90-1.03) p = 0.230]. Meta-analysis of both case-control studies resulted in a combined OR of 0.94 (95% CI: 0.89-1.004, p = 0.06).
Conclusions
Although there was an association between two SNPs in BRCA2 and CVD in a multi-ethnic population, these results were not replicated in two South Asian case-control studies of incident MI. Future studies exploring the association between BRCA variants and cardiovascular disorders are needed to clarify the role, if any, for BRCA variants in CVD pathogenesis.
doi:10.1186/1471-2350-13-56
PMCID: PMC3464815  PMID: 22809218
8.  A Genome Wide Association Study for Coronary Artery Disease Identifies a Novel Susceptibility Locus in the Major Histocompatibility Complex 
Background
Recent genome-wide association studies (GWAS) have identified several novel loci that reproducibly associate with CAD and/or MI risk. However, known common CAD risk variants explain only 10% of the predicted genetic heritability of the disease, suggesting that important genetic signals remain to be discovered.
Methods and Results
We performed a discovery meta-analysis of 5 GWASs involving 13,949 subjects (7123 cases, 6826 controls) imputed at approximately 5 million SNPs using pilot 1000 Genomes based haplotypes. Promising loci were followed up in an additional 5 studies with 11,032 subjects (5211 cases, 5821 controls). A novel CAD locus on chromosome 6p21.3 in the major histocompatibility complex (MHC) between HCG27 and HLA-C was identified and achieved genome wide significance in the combined analysis (rs3869109; pdiscovery=3.3×10−7, preplication=5.3×10−4 pcombined=1.12×10−9). A sub-analysis combining discovery GWASs showed an attenuation of significance when stringent corrections for European population structure were employed (p=4.1×10-10 versus 3.2×10-7) suggesting the observed signal is partly confounded due to population stratification. This gene dense region plays an important role in inflammation, immunity and self cell recognition. To determine whether the underlying association was driven by MHC class I alleles, we statistically imputed common HLA alleles into the discovery subjects; however, no single common HLA type contributed significantly or fully explained the observed association.
Conclusions
We have identified a novel locus in the MHC associated with CAD. MHC genes regulate inflammation and T cell responses that contribute importantly to the initiation and propagation of atherosclerosis. Further laboratory studies will be required to understand the biological basis of this association and identify the causative allele(s).
doi:10.1161/CIRCGENETICS.111.961243
PMCID: PMC3335297  PMID: 22319020
coronary artery disease; myocardial infarction; meta-analysis; genetics
9.  A Genome Wide Association Study for Coronary Artery Disease Identifies a Novel Susceptibility Locus in the Major Histocompatibility Complex 
Background
Recent genome-wide association studies (GWAS) have identified several novel loci that reproducibly associate with CAD and/or MI risk. However, known common CAD risk variants explain only 10% of the predicted genetic heritability of the disease, suggesting that important genetic signals remain to be discovered.
Methods and Results
We performed a discovery meta-analysis of 5 GWASs involving 13,949 subjects (7123 cases, 6826 controls) imputed at approximately 5 million SNPs using pilot 1000 Genomes based haplotypes. Promising loci were followed up in an additional 5 studies with 11,032 subjects (5211 cases, 5821 controls). A novel CAD locus on chromosome 6p21.3 in the major histocompatibility complex (MHC) between HCG27 and HLA-C was identified and achieved genome wide significance in the combined analysis (rs3869109; pdiscovery=3.3×10−7, preplication=5.3×10−4 pcombined=1.12×10−9). A sub-analysis combining discovery GWASs showed an attenuation of significance when stringent corrections for European population structure were employed (p=4.1×10−10 versus 3.2×10−7) suggesting the observed signal is partly confounded due to population stratification. This gene dense region plays an important role in inflammation, immunity and self cell recognition. To determine whether the underlying association was driven by MHC class I alleles, we statistically imputed common HLA alleles into the discovery subjects; however, no single common HLA type contributed significantly or fully explained the observed association.
Conclusion
We have identified a novel locus in the MHC associated with CAD. MHC genes regulate inflammation and T cell responses that contribute importantly to the initiation and propagation of atherosclerosis. Further laboratory studies will be required to understand the biological basis of this association and identify the causative allele(s).
doi:10.1161/CIRCGENETICS.111.961243
PMCID: PMC3335297  PMID: 22319020
Coronary Artery Disease; Myocardial Infarction; Meta-Analysis; Genetics
10.  Homocysteine and Coronary Heart Disease: Meta-analysis of MTHFR Case-Control Studies, Avoiding Publication Bias 
PLoS Medicine  2012;9(2):e1001177.
Robert Clarke and colleagues conduct a meta-analysis of unpublished datasets to examine the causal relationship between elevation of homocysteine levels in the blood and the risk of coronary heart disease. Their data suggest that an increase in homocysteine levels is not likely to result in an increase in risk of coronary heart disease.
Background
Moderately elevated blood levels of homocysteine are weakly correlated with coronary heart disease (CHD) risk, but causality remains uncertain. When folate levels are low, the TT genotype of the common C677T polymorphism (rs1801133) of the methylene tetrahydrofolate reductase gene (MTHFR) appreciably increases homocysteine levels, so “Mendelian randomization” studies using this variant as an instrumental variable could help test causality.
Methods and Findings
Nineteen unpublished datasets were obtained (total 48,175 CHD cases and 67,961 controls) in which multiple genetic variants had been measured, including MTHFR C677T. These datasets did not include measurements of blood homocysteine, but homocysteine levels would be expected to be about 20% higher with TT than with CC genotype in the populations studied. In meta-analyses of these unpublished datasets, the case-control CHD odds ratio (OR) and 95% CI comparing TT versus CC homozygotes was 1.02 (0.98–1.07; p = 0.28) overall, and 1.01 (0.95–1.07) in unsupplemented low-folate populations. By contrast, in a slightly updated meta-analysis of the 86 published studies (28,617 CHD cases and 41,857 controls), the OR was 1.15 (1.09–1.21), significantly discrepant (p = 0.001) with the OR in the unpublished datasets. Within the meta-analysis of published studies, the OR was 1.12 (1.04–1.21) in the 14 larger studies (those with variance of log OR<0.05; total 13,119 cases) and 1.18 (1.09–1.28) in the 72 smaller ones (total 15,498 cases).
Conclusions
The CI for the overall result from large unpublished datasets shows lifelong moderate homocysteine elevation has little or no effect on CHD. The discrepant overall result from previously published studies reflects publication bias or methodological problems.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Coronary heart disease (CHD) is the leading cause of death among adults in developed countries. With age, fatty deposits (atherosclerotic plaques) coat the walls of the coronary arteries, the blood vessels that supply the heart with oxygen and nutrients. The resultant restriction of the heart's blood supply causes shortness of breath, angina (chest pains that are usually relieved by rest), and sometimes fatal heart attacks. Many established risk factors for CHD, including smoking, physical inactivity, being overweight, and eating a fat-rich diet, can be modified by lifestyle changes. Another possible modifiable risk factor for CHD is a high blood level of the amino acid homocysteine. Methylene tetrahydofolate reductase, which is encoded by the MTHFR gene, uses folate to break down and remove homocysteine so fortification of cereals with folate can reduce population homocysteine blood levels. Pooled results from prospective observational studies that have looked for an association between homocysteine levels and later development of CHD suggest that the reduction in homocysteine levels that can be achieved by folate supplementation is associated with an 11% lower CHD risk.
Why Was This Study Done?
Prospective observational studies cannot prove that high homocysteine levels cause CHD because of confounding, the potential presence of other unknown shared characteristics that really cause CHD. However, an approach called “Mendelian randomization” can test whether high blood homocysteine causes CHD. A common genetic variant of the MTHFR gene—the C677T polymorphism—reduces MTHFR efficiency so TT homozygotes (individuals in whom both copies of the MTHFR gene have the nucleotide thymine at position 677; the human genome contains two copies of most genes) have 25% higher blood homocysteine levels than CC homozygotes. In meta-analyses (statistical pooling of the results of several studies) of published Mendelian randomized studies, TT homozygotes have a higher CHD risk than CC homozygotes. Because gene variants are inherited randomly, they are not subject to confounding, so this result suggests that high blood homocysteine causes CHD. But what if only Mendelian randomization studies that found an association have been published? Such publication bias would affect this aggregate result. Here, the researchers investigate the association of the MTHFR C677T polymorphism with CHD in unpublished datasets that have analyzed this polymorphism incidentally during other genetic studies.
What Did the Researchers Do and Find?
The researchers obtained 19 unpublished datasets that contained data on the MTHFR C677T polymorphism in thousands of people with and without CHD. Meta-analysis of these datasets indicates that the excess CHD risk in TT homozygotes compared to CC homozygotes was 2% (much lower than predicted from the prospective observational studies), a nonsignificant difference (that is, it could have occurred by chance). When the probable folate status of the study populations (based on when national folic acid fortification legislation came into effect) was taken into account, there was still no evidence that TT homozygotes had an excess CHD risk. By contrast, in an updated meta-analysis of 86 published studies of the association of the polymorphism with CHD, the excess CHD risk in TT homozygotes compared to CC homozygotes was 15%. Finally, in a meta-analysis of randomized trials on the use of vitamin B supplements for homocysteine reduction, folate supplementation had no significant effect on the 5-year incidence of CHD.
What Do These Findings Mean?
These analyses of unpublished datasets are consistent with lifelong moderate elevation of homocysteine levels having no significant effect on CHD risk. In other words, these findings indicate that circulating homocysteine levels within the normal range are not causally related to CHD risk. The meta-analysis of the randomized trials of folate supplementation also supports this conclusion. So why is there a discrepancy between these findings and those of meta-analyses of published Mendelian randomization studies? The discrepancy is too large to be dismissed as a chance finding, suggest the researchers, but could be the result of publication bias—some studies might have been prioritized for publication because of the positive nature of their results whereas the unpublished datasets used in this study would not have been affected by any failure to publish null results. Overall, these findings reveal a serious example of publication bias and argue against the use of folate supplements as a means of reducing CHD risk.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001177.
The American Heart Association provides information about CHD and tips on keeping the heart healthy; it also provides information on homocysteine, folic acid, and CHD, general information on supplements and heart health, and personal stories about CHD
The UK National Health Service Choices website provides information about CHD, including personal stories about CHD
Information is available from the British Heart Foundation on heart disease and keeping the heart healthy
The US National Heart Lung and Blood Institute also provides information on CHD (in English and Spanish)
MedlinePlus provides links to many other sources of information on CHD (in English and Spanish)
Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1001177
PMCID: PMC3283559  PMID: 22363213
11.  The Effect of Chromosome 9p21 Variants on Cardiovascular Disease May Be Modified by Dietary Intake: Evidence from a Case/Control and a Prospective Study 
PLoS Medicine  2011;8(10):e1001106.
Ron Do and colleagues find that a prudent diet high in raw vegetables may modify the increased genetic risk of cardiovascular disease conferred by the chromosome 9p21 SNP.
Background
One of the most robust genetic associations for cardiovascular disease (CVD) is the Chromosome 9p21 region. However, the interaction of this locus with environmental factors has not been extensively explored. We investigated the association of 9p21 with myocardial infarction (MI) in individuals of different ethnicities, and tested for an interaction with environmental factors.
Methods and Findings
We genotyped four 9p21 SNPs in 8,114 individuals from the global INTERHEART study. All four variants were associated with MI, with odds ratios (ORs) of 1.18 to 1.20 (1.85×10−8≤p≤5.21×10−7). A significant interaction (p = 4.0×10−4) was observed between rs2383206 and a factor-analysis-derived “prudent” diet pattern score, for which a major component was raw vegetables. An effect of 9p21 on MI was observed in the group with a low prudent diet score (OR = 1.32, p = 6.82×10−7), but the effect was diminished in a step-wise fashion in the medium (OR = 1.17, p = 4.9×10−3) and high prudent diet scoring groups (OR = 1.02, p = 0.68) (p = 0.014 for difference). We also analyzed data from 19,129 individuals (including 1,014 incident cases of CVD) from the prospective FINRISK study, which used a closely related dietary variable. In this analysis, the 9p21 risk allele demonstrated a larger effect on CVD risk in the groups with diets low or average for fresh vegetables, fruits, and berries (hazard ratio [HR] = 1.22, p = 3.0×10−4, and HR = 1.35, p = 4.1×10−3, respectively) compared to the group with high consumption of these foods (HR = 0.96, p = 0.73) (p = 0.0011 for difference). The combination of the least prudent diet and two copies of the risk allele was associated with a 2-fold increase in risk for MI (OR = 1.98, p = 2.11×10−9) in the INTERHEART study and a 1.66-fold increase in risk for CVD in the FINRISK study (HR = 1.66, p = 0.0026).
Conclusions
The risk of MI and CVD conferred by Chromosome 9p21 SNPs appears to be modified by a prudent diet high in raw vegetables and fruits.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Cardiovascular diseases (CVDs)—diseases that affect the heart and/or the blood vessels—are a leading cause of illness and death worldwide. In the United States, for example, the leading cause of death is coronary heart disease, a CVD in which narrowing of the heart's blood vessels by fatty deposits slows the blood supply to the heart and may eventually cause a heart attack (myocardial infarction, or MI); the third leading cause of death in the US is stroke, a CVD in which the brain's blood supply is interrupted. Environmental factors such as diet, physical activity, and smoking alter a person's risk of developing CVD. In addition, certain genetic variants (alterations in the DNA that forms the body's blueprint; DNA is packed into structures called chromosomes) alter the risk of developing CVD and are passed from parent to child. Thus, in CVD, as in most common diseases, both genetics and the environment play a role.
Why Was This Study Done?
Recent studies have identified several genetic variants that are associated with an increased risk of developing CVD. One of the most robust of these genetic associations is a cluster of single nucleotide polymorphisms (SNPs, differences in a single DNA building block) in a chromosomal region (locus) called 9p21. So far, this association has been mainly studied in European populations. Moreover, the interaction of this locus with environmental factors has not been extensively studied. A better understanding of how 9p21 variants affect CVD risk in people of different ethnicities and of the interaction between this locus and environmental factors could allow the development of targeted strategies for the prevention of CVD. In this study, the researchers investigate the association of 9p21 risk variants with CVD in people of different ethnicities and test for an interaction between this locus and environmental factors.
What Did the Researchers Do and Find?
The researchers assessed four 9p21 SNPs in people enrolled in the INTERHEART study, a global retrospective case-control study that investigated potential MI risk factors by comparing people who had had an acute non-fatal MI with similar people without heart disease. All four SNP risk variants increased the risk of MI by about a fifth. However, the effect of the SNPs on MI was influenced by the “prudent” diet pattern score of the INTERHEART participants, a score that includes fresh fruit and vegetable intake as recorded in food frequency questionnaires. That is, the risk of MI in people carrying SNP risk variants was influenced by their diet. The strongest interaction was seen with an SNP called rs2383206, but although rs2383206 carriers who ate a diet poor in fruits and vegetables had a higher risk of MI than people with a similar diet who did not carry this SNP, rs2383206 carriers and non-carriers who ate a fruit- and vegetable-rich diet had a comparable MI risk. Overall, the combination of the least “prudent” diet and two copies of the risk variant (human cells contain two complete sets of chromosomes) was associated with a two-fold increase in risk for MI in the INTERHEART study. Additionally, data collected in the FINRISK study, which characterized healthy individuals living in Finland at baseline and then followed them to see whether they developed CVD, revealed a similar interaction between diet and 9p21 SNPs.
What Do These Findings Mean?
These findings suggest that the risk of CVD conferred by chromosome 9p21 SNPs may be influenced by diet in multiple ethnic groups. Importantly, they suggest that the deleterious effect of 9p21 SNPs on CVD might be mitigated by consuming a diet rich in fresh fruits and vegetables. The accuracy of these findings may be affected by recall bias in the INTERHEART study (that is, some people may not have remembered their diet accurately) and by the small number of CVD cases in the FINRISK study. Nevertheless, these findings suggest that gene–environment interactions are important drivers of CVD, and they raise the possibility that a sound diet can mediate the effects of 9p21 SNPs.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001106.
The American Heart Association provides information about many types of cardiovascular disease for patients, caregivers, and professionals and tips on keeping the heart healthy
The UK National Health Service Choices website provides information about cardiovascular disease and stroke
Information is available from the British Heart Foundation on heart disease and keeping the heart healthy
The US National Heart Lung and Blood Institute provides information on a wide range of cardiovascular diseases
MedlinePlus provides links to many other sources of information on heart diseases, vascular diseases, and stroke (in English and Spanish)
The US Centers for Disease Control and Prevention has a simple fact sheet on gene-environment interactions; the US National Institute of Environmental Health Sciences provides links to other information on gene-environment interactions
More information is available on the INTERHEART study and on the FINRISK study
doi:10.1371/journal.pmed.1001106
PMCID: PMC3191151  PMID: 22022235
12.  Exome Sequencing, ANGPTL3 Mutations, and Familial Combined Hypolipidemia 
The New England journal of medicine  2010;363(23):2220-2227.
SUMMARY
We sequenced all protein-coding regions of the genome (the “exome”) in two family members with combined hypolipidemia, marked by extremely low plasma levels of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides. These two participants were compound heterozygotes for two distinct nonsense mutations in ANGPTL3 (encoding the angiopoietin-like 3 protein). ANGPTL3 has been reported to inhibit lipoprotein lipase and endothelial lipase, thereby increasing plasma triglyceride and HDL cholesterol levels in rodents. Our finding of ANGPTL3 mutations highlights a role for the gene in LDL cholesterol metabolism in humans and shows the usefulness of exome sequencing for identification of novel genetic causes of inherited disorders. (Funded by the National Human Genome Research Institute and others.)
doi:10.1056/NEJMoa1002926
PMCID: PMC3008575  PMID: 20942659
13.  Genetic Variation at the Proprotein Convertase Subtilisin/Kexin Type 5 Gene Modulates HDL Cholesterol Levels 
Background
A low level of plasma high-density lipoprotein cholesterol (HDL-C) is a risk factor for cardiovascular disease. HDL particles are modulated by a variety of lipases, including endothelial lipase (EL), a phospholipase present on vascular endothelial cells. The proprotein convertase subtilisin/kexin type 5 (PCSK5) gene product is known to directly inactivate EL, and, indirectly, cleave and activate angiopoetin-like protein 3, a natural inhibitor of EL. We therefore investigated the effect of human PCSK5 genetic variants on plasma HDL-C levels.
Methods and Results
Haplotypes at the PCSK5 locus were examined in nine multi-generational families that included 60 individuals with HDL-C<10th percentile. Segregation with low HDL-C in one family was found. Sequencing of the PCSK5 gene in 12 probands with HDL-C<5th percentile identified seven novel variants. Using a two-stage design, we first genotyped these single-nucleotide polymorphisms (SNPs) along with 163 tagSNPs and 12 additional SNPs (n=182 total) in 457 individuals with documented coronary artery disease. We identified nine SNPs associated with HDL-C (P<0.05), with the strongest results for rs11144782 and rs11144766 (P=0.002 and P=0.005 respectively). The SNP rs11144782 was also associated with very low-density lipoprotein (P=0.039), triglycerides (P=0.049) and total apolipoprotein B levels (P=0.022). In stage 2, we replicated the association of rs11144766 with HDL-C (P=0.014) in an independent sample of Finnish low HDL-C families. In a combined analysis of both stages (n=883), region-wide significance of rs11144766 and low HDL-C was observed (unadjusted P=1.86×10−4 and Bonferroni adjusted P=0.031).
Conclusions
We conclude that variability at the PCSK5 locus influences HDL-C levels, possibly through the inactivation of EL activity and consequently, atherosclerotic cardiovascular disease risk.
doi:10.1161/CIRCGENETICS.109.877811
PMCID: PMC2901678  PMID: 20031622
cholesterol; coronary disease; genetics; lipids; lipoproteins
14.  Genetic Loci Influencing C-reactive Protein Levels and Risk of Coronary Heart Disease 
Context:
Plasma levels of C-reactive protein (CRP) are independently associated with risk of coronary heart disease, but whether CRP is causally associated with coronary heart disease or merely a marker of underlying atherosclerosis is uncertain.
Objective:
To investigate association of genetic loci with CRP levels and risk of coronary heart disease.
Design, setting and participants:
We first carried out a genome-wide association (n=17,967) and replication study (n=14,747) to identify genetic loci associated with plasma CRP concentrations. Data collection took place between 1989 and 2008 and genotyping between 2003 and 2008. We carried out a Mendelian randomisation study of the most closely associated SNP in the CRP locus and published data on other CRP variants involving a total of 28,112 cases and 100,823 controls, to investigate the association of CRP variants with coronary heart disease. We compared our finding with that predicted from meta-analysis of observational studies of CRP levels and risk of coronary heart disease. For the other loci associated with CRP levels, we selected the most closely associated SNP for testing against coronary heart disease among 14,365 cases and 32,069 controls.
Main outcome measure:
Risk of coronary heart disease.
Results:
Polymorphisms in five genetic loci were strongly associated with CRP levels (% difference per minor allele): SNP rs6700896 in LEPR (−14.7% [95% Confidence Interval {CI}], −17.5 – −11.9, P=1.6×10−21), rs4537545 in IL6R (−10.8% [95% CI, −13.8 – −7.7], P=5.1×10−11), rs7553007 in CRP locus (−20.7% [95% CI, −23.5 – −17.9], P=3.3×10−38), rs1183910 in HNF1A (−13.6% [95% CI, −16.4 – −10.6], P=1.2×10−17) and rs4420638 in APOE-CI-CII (−21.8% [95% CI, −25.4 – −18.1], P=2.1×10−25). Association of SNP rs7553007 in the CRP locus with coronary heart disease gave odds ratio (OR) 0.98 (95% CI, 0.94 – 1.01) per 20% lower CRP. Our Mendelian randomisation study of variants in the CRP locus showed no association with coronary heart disease: OR 1.00 (95% CI, 0.97 – 1.02) per 20% lower CRP, compared with OR 0.94 (95% CI, 0.94 – 0.95) predicted from meta-analysis of the observational studies of CRP levels and coronary heart disease (Z-score −3.45, P<.001). SNPs rs6700896 in LEPR (OR 1.06 [95% CI, 1.02 – 1.09] per minor allele), rs4537545 in IL6R (OR 0.94 [95% CI, 0.91 – 0.97]) and rs4420638 in the APOE-CI-CII cluster (OR 1.16 [95% CI, 1.12 – 1.21]) were all associated with risk of coronary heart disease.
Conclusions:
The lack of concordance between the effect on coronary heart disease risk of CRP genotypes and CRP levels argues against a causal association of CRP with coronary heart disease.
doi:10.1001/jama.2009.954
PMCID: PMC2803020  PMID: 19567438
15.  Meta-Analysis of the INSIG2 Association with Obesity Including 74,345 Individuals: Does Heterogeneity of Estimates Relate to Study Design? 
PLoS Genetics  2009;5(10):e1000694.
The INSIG2 rs7566605 polymorphism was identified for obesity (BMI≥30 kg/m2) in one of the first genome-wide association studies, but replications were inconsistent. We collected statistics from 34 studies (n = 74,345), including general population (GP) studies, population-based studies with subjects selected for conditions related to a better health status (‘healthy population’, HP), and obesity studies (OB). We tested five hypotheses to explore potential sources of heterogeneity. The meta-analysis of 27 studies on Caucasian adults (n = 66,213) combining the different study designs did not support overall association of the CC-genotype with obesity, yielding an odds ratio (OR) of 1.05 (p-value = 0.27). The I2 measure of 41% (p-value = 0.015) indicated between-study heterogeneity. Restricting to GP studies resulted in a declined I2 measure of 11% (p-value = 0.33) and an OR of 1.10 (p-value = 0.015). Regarding the five hypotheses, our data showed (a) some difference between GP and HP studies (p-value = 0.012) and (b) an association in extreme comparisons (BMI≥32.5, 35.0, 37.5, 40.0 kg/m2 versus BMI<25 kg/m2) yielding ORs of 1.16, 1.18, 1.22, or 1.27 (p-values 0.001 to 0.003), which was also underscored by significantly increased CC-genotype frequencies across BMI categories (10.4% to 12.5%, p-value for trend = 0.0002). We did not find evidence for differential ORs (c) among studies with higher than average obesity prevalence compared to lower, (d) among studies with BMI assessment after the year 2000 compared to those before, or (e) among studies from older populations compared to younger. Analysis of non-Caucasian adults (n = 4889) or children (n = 3243) yielded ORs of 1.01 (p-value = 0.94) or 1.15 (p-value = 0.22), respectively. There was no evidence for overall association of the rs7566605 polymorphism with obesity. Our data suggested an association with extreme degrees of obesity, and consequently heterogeneous effects from different study designs may mask an underlying association when unaccounted for. The importance of study design might be under-recognized in gene discovery and association replication so far.
Author Summary
A polymorphism of the INSIG2 gene was identified as being associated with obesity in one of the first genome-wide association studies. However, this association has since then been highly debated upon inconsistent subsequent reports. We collected association information from 34 studies including a total of 74,000 participants. In a meta-analysis of the 27 studies including 66,000 Caucasian adults, we found no overall association of this polymorphism rs7566605 with obesity, comparing subjects with a body-mass-index (BMI)≥30 kg/m2 with normal BMI subjects (BMI<30 kg/m2). Our data suggested an association of this polymorphism with extreme obesity (e.g., BMI≥37.5 kg/m2) compared to normal controls. Such an association with extreme obesity might induce heterogeneous effects from different study designs depending on the proportion of extreme obesity included by the design. However, further studies would be required to substantiate this finding. The importance of study design might be under-recognized in gene discovery and association replication so far.
doi:10.1371/journal.pgen.1000694
PMCID: PMC2757909  PMID: 19851442

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