Christopher J. O’Donnell

Systems Genetics Analysis of Genome-Wide Association Study Reveals Novel Associations Between Key Biological Processes and Coronary Artery Disease

Objective— Genome-wide association studies have identified multiple genetic variants affecting the risk of coronary artery disease (CAD). However, individually these explain only a small fraction of the heritability of CAD and for most, the causal biological mechanisms remain unclear. We sought to obtain further insights into potential causal processes of CAD by integrating large-scale GWA data with expertly curated databases of core human pathways and functional networks. Approaches and Results— Using pathways (gene sets) from Reactome, we carried out a 2-stage gene set enrichment analysis strategy. From a meta-analyzed discovery cohort of 7 CAD genome-wide association study data sets (9889 cases/11u2009089 controls), nominally significant gene sets were tested for replication in a meta-analysis of 9 additional studies (15u2009502 cases/55u2009730 controls) from the Coronary ARtery DIsease Genome wide Replication and Meta-analysis (CARDIoGRAM) Consortium. A total of 32 of 639 Reactome pathways tested showed convincing association with CAD (replication P<0.05). These pathways resided in 9 of 21 core biological processes represented in Reactome, and included pathways relevant to extracellular matrix (ECM) integrity, innate immunity, axon guidance, and signaling by PDRF (platelet-derived growth factor), NOTCH, and the transforming growth factor-&bgr;/SMAD receptor complex. Many of these pathways had strengths of association comparable to those observed in lipid transport pathways. Network analysis of unique genes within the replicated pathways further revealed several interconnected functional and topologically interacting modules representing novel associations (eg, semaphoring-regulated axonal guidance pathway) besides confirming known processes (lipid metabolism). The connectivity in the observed networks was statistically significant compared with random networks (P<0.001). Network centrality analysis (degree and betweenness) further identified genes (eg, NCAM1, FYN, FURIN, etc) likely to play critical roles in the maintenance and functioning of several of the replicated pathways. Conclusions— These findings provide novel insights into how genetic variation, interpreted in the context of biological processes and functional interactions among genes, may help define the genetic architecture of CAD.

Induced Pluripotent Stem Cell Differentiation Enables Functional Validation of GWAS Variants in Metabolic Disease

Genome-wide association studies (GWAS) have highlighted a large number of genetic variants with potential disease association, but functional analysisxa0remains a challenge. Here we describe an approach to functionally validate identified variants through differentiation of induced pluripotent stem cells (iPSCs) to study cellular pathophysiology. We collected peripheral blood cells from Framingham Heart Study participants and reprogrammed them to iPSCs. We then differentiated 68 iPSC lines into hepatocytes and adipocytes to investigate the effect of the 1p13 rs12740374 variant on cardiometabolic disease phenotypes via transcriptomics and metabolomic signatures. We observed a clear association between rs12740374 and lipid accumulation and gene expression in differentiated hepatocytes, in particular, expression of SORT1, CELSR2, and PSRC1, consistent with previous analyses of this variant using other approaches. Initial investigation of additional SNPs also highlighted correlations with gene expression. These findings suggest that iPSC-based population studies hold promise as tools for the functional validation of GWAS variants.

On the Significance of Linkage Studies of Complex Traits

To the Editor:We read with interest the recent article by Wang et al. (2004xPremature myocardial infarction novel susceptibility locus on chromosome 1P34-36 identified by genomewide linkage analysis. Wang, Q, Rao, S, Shen, G-Q, Li, L, Moliterno, DJ, Newby, LK, Rogers, WJ, Cannata, R, Zirzow, E, Elston, RC, and Topol, EJ. Am J Hum Genet. 2004; 74: 262–271Abstract | Full Text | Full Text PDF | PubMed | Scopus (142)See all References2004) reporting linkage of premature myocardial infarction (MI) to a locus on 1p34-36. The authors have recruited a large sample of families with premature coronary artery disease (CAD), as detected by catheterization, revascularization, or MI. Such large-scale approaches will be crucial to the identification of genetic variation underlying complex traits, including atherosclerotic CAD and MI, the leading killer of men and women in the Western world. We commend the authors for undertaking such an important study.Although their article reports the nominal LOD score of 11.68 for linkage of premature MI to 1p34-36 and a corrected pointwise P value of .00011, we note that the genomewide significance of the linkage statistic is not clear. We have some methodological concerns regarding the initial emphasis on results presented in their table 2 and figure 1: 1.The finding of 11 independent −log10P values >3.13 in the multipoint linkage approach (regions identified in single-point and multipoint W2 analyses overlap significantly) raises questions regarding the assertion that such a threshold corresponds to a LOD score >2.2, as proposed by Lander and Kruglyak (1995xGenetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Lander, E and Kruglyak, L. Nat Genet. 1995; 11: 241–247Crossref | PubMed | Scopus (4037)See all References1995). A LOD score >2.2 should occur once in a maximally informative genomewide scan, under the null hypothesis that no genetic linkage is present. It seems unlikely that 11 true genetic loci influencing a complex phenotype would be detected in a single study. It is more likely that the asymptotic P value statistic generated by the authors’ modified Haseman-Elston regression model is inflated.2.The marked attenuation of the multipoint P value of <10−12 to 10−4 on pointwise permutation testing at the 1p34-36 locus suggests that the nominal asymptotic P values are inflated. It is possible that the method of linkage analysis may have inflated the P value estimates. In particular, the treatment of the dichotomous MI phenotype as a continuous variable may not be appropriate. The assumption of equal variances required for a quantitative trait may not be valid for different numbers of affected and unaffected individuals in each family.3.The authors’ attempt to correct the pointwise empirical P value estimates for the number of markers tested is quite important for establishing the significance of their findings. However, the attempt may be inadequate to account for the testing of multiple markers. The authors refer to the simulation analyses by Wiltshire et al. (2002xEvaluating the results of genomewide linkage scans of complex traits by locus counting. Wiltshire, S, Cardon, LR, and McCarthy, MI. Am J Hum Genet. 2002; 71: 1175–1182Abstract | Full Text | Full Text PDF | PubMed | Scopus (65)See all References2002), which explored the influence of experimental deviations from the Lander-Kruglyak assumptions of completely informative linkage analyses. We are uncertain whether the empirical genomewide P value estimates derived from the Wang et al. (2004xPremature myocardial infarction novel susceptibility locus on chromosome 1P34-36 identified by genomewide linkage analysis. Wang, Q, Rao, S, Shen, G-Q, Li, L, Moliterno, DJ, Newby, LK, Rogers, WJ, Cannata, R, Zirzow, E, Elston, RC, and Topol, EJ. Am J Hum Genet. 2004; 74: 262–271Abstract | Full Text | Full Text PDF | PubMed | Scopus (142)See all References2004) data correspond to the same nominal LOD-score thresholds identified in the Wiltshire et al. (2002xEvaluating the results of genomewide linkage scans of complex traits by locus counting. Wiltshire, S, Cardon, LR, and McCarthy, MI. Am J Hum Genet. 2002; 71: 1175–1182Abstract | Full Text | Full Text PDF | PubMed | Scopus (65)See all References2002) study using simulated data. Permutation testing of the Wang et al. (2004xPremature myocardial infarction novel susceptibility locus on chromosome 1P34-36 identified by genomewide linkage analysis. Wang, Q, Rao, S, Shen, G-Q, Li, L, Moliterno, DJ, Newby, LK, Rogers, WJ, Cannata, R, Zirzow, E, Elston, RC, and Topol, EJ. Am J Hum Genet. 2004; 74: 262–271Abstract | Full Text | Full Text PDF | PubMed | Scopus (142)See all References2004) data by use of the Wiltshire approach might provide greater confidence regarding the genomewide significance of the study findings, but this approach admittedly represents a significant computational burden.4.Genomewide linkage analyses at 10 cM may not extract maximally the identity-by-descent information for the sample under study. A fine-mapping study at higher density across a region of interest may show a change in the maximum-LOD-score estimate. An increase in the LOD-score estimate with better information extraction might be reassuring, but a fall in the LOD score may signal a false-positive finding. We would encourage the authors to perform and publish the results of a higher-density map.5.The study cohort was recruited on the basis of a composite definition of premature CAD. Was the broader CAD phenotype (including MI) the primary phenotype prespecified in the linkage analysis? The reported MI linkage analysis represents a subgroup of the subjects enrolled; the “less-restrictive” CAD phenotype was also tested and revealed no suggestive or significant linkage evidence. Could the authors clarify their original primary analysis and whether additional subgroups were analyzed? A true empirical P value would also account for the multiple phenotypes tested.On review of the study, the declaration of a finding of genomewide significance may not be as strongly supported as suggested by the authors. The results of this linkage analysis do not contain much overlap with those of similar analyses, and this certainly could result from differences in phenotype definition, environmental exposures, or study design (Pajukanta et al. 2000xTwo loci on chromosomes 2 and X for premature coronary heart disease identified in early- and late-settlement populations of Finland. Pajukanta, P, Cargill, M, Viitanen, L, Nuotio, I, Kareinen, A, Perola, M, Terwilliger, JD, Kempas, E, Daly, M, Lilja, H, Rioux, JD, Brettin, T, Viikari, JS, Ronnemaa, T, Laakso, M, Lander, ES, and Peltonen, L. Am J Hum Genet. 2000; 67: 1481–1493Abstract | Full Text | Full Text PDF | PubMed | Scopus (126)See all References2000; Francke et al. 2001xA genome-wide scan for coronary heart disease suggests in Indo-Mauritians a susceptibility locus on chromosome 16p13 and replicates linkage with the metabolic syndrome on 3q27. Francke, S, Manraj, M, Lacquemant, C, Lecoeur, C, Lepretre, F, Passa, P, Hebe, A, Corset, L, Yan, SL, Lahmidi, S, Jankee, S, Gunness, TK, Ramjuttun, US, Balgobin, V, Dina, C, and Froguel, P. Hum Mol Genet. 2001; 10: 2751–2765Crossref | PubMedSee all References2001; Broeckel et al. 2002xA comprehensive linkage analysis for myocardial infarction and its related risk factors. Broeckel, U, Hengstenberg, C, Mayer, B, Holmer, S, Martin, LJ, Comuzzie, AG, Blangero, J, Nurnberg, P, Reis, A, Riegger, GA, Jacob, HJ, and Schunkert, H. Nat Genet. 2002; 30: 210–214Crossref | PubMed | Scopus (252)See all References2002; Harrap et al. 2002xGenome-wide linkage analysis of the acute coronary syndrome suggests a locus on chromosome 2. Harrap, SB, Zammit, KS, Wong, ZY, Williams, FM, Bahlo, M, Tonkin, AM, and Anderson, ST. Arterioscler Thromb Vasc Biol. 2002; 22: 874–878Crossref | PubMed | Scopus (101)See all References2002; Chiodini and Lewis 2003xMeta-analysis of 4 coronary heart disease genome-wide linkage studies confirms a susceptibility locus on chromosome 3q. Chiodini, BD and Lewis, CM. Arterioscler Thromb Vasc Biol. 2003; 23: 1863–1868Crossref | PubMed | Scopus (63)See all References2003). Replication of linkage analyses for complex cardiovascular traits has often proven challenging, and the difficulty in achieving replication for MI underscores the many difficulties in the conduct and interpretation of such linkage analyses (Altmuller et al. 2001xGenomewide scans of complex human diseases: true linkage is hard to find. Altmuller, J, Palmer, LJ, Fischer, G, Scherb, H, and Wjst, M. Am J Hum Genet. 2001; 69: 936–950Abstract | Full Text | Full Text PDF | PubMed | Scopus (378)See all References2001).Identifying genetic factors underlying linkage peaks in this and related studies of MI will require considerable expenditure of resources and should proceed on the basis of the strongest possible evidence. We encourage the systematic comparison of available and accruing linkage data across studies in various CAD phenotypes, including continued assessment of the most appropriate linkage methods.

Loss of cardioprotective effects at the ADAMTS7 locus as a result of gene-smoking interactions

Background: Common diseases such as coronary heart disease (CHD) are complex in etiology. The interaction of genetic susceptibility with lifestyle factors may play a prominent role. However, gene-lifestyle interactions for CHD have been difficult to identify. Here, we investigate interaction of smoking behavior, a potent lifestyle factor, with genotypes that have been shown to associate with CHD risk. Methods: We analyzed data on 60u2009919 CHD cases and 80u2009243 controls from 29 studies for gene-smoking interactions for genetic variants at 45 loci previously reported to be associated with CHD risk. We also studied 5 loci associated with smoking behavior. Study-specific gene-smoking interaction effects were calculated and pooled using fixed-effects meta-analyses. Interaction analyses were declared to be significant at a P value of <1.0×10–3 (Bonferroni correction for 50 tests). Results: We identified novel gene-smoking interaction for a variant upstream of the ADAMTS7 gene. Every T allele of rs7178051 was associated with lower CHD risk by 12% in never-smokers (P=1.3×10–16) in comparison with 5% in ever-smokers (P=2.5×10–4), translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (interaction P value=8.7×10–5). The protective T allele at rs7178051 was also associated with reduced ADAMTS7 expression in human aortic endothelial cells and lymphoblastoid cell lines. Exposure of human coronary artery smooth muscle cells to cigarette smoke extract led to induction of ADAMTS7. Conclusions: Allelic variation at rs7178051 that associates with reduced ADAMTS7 expression confers stronger CHD protection in never-smokers than in ever-smokers. Increased vascular ADAMTS7 expression may contribute to the loss of CHD protection in smokers.

Reducing Cardiovascular Risk Using Genomic Information in the Era of Precision Medicine

With the pace of current advances in genomics technologies, we are fast approaching an era when patients will have complete genome sequence information on which clinicians will need to act when making routine clinical decisions. Precision medicine is “an approach to disease treatment and prevention that seeks to maximize effectiveness by taking into account individual variability in genes, environment and lifestyle.”1 One central aim of the recently launched US Precision Medicine Initiative is the return of genetic results for clinical utility. Atherosclerotic cardiovascular disease (CVD), the leading cause of death in men and women, is a chronic disease influenced by lifelong exposure to inherited and environmental risk factors. The major clinical and biochemical atherosclerosis risk factors for coronary heart disease (CHD) and other forms of CVD have been well defined over the past 50 years by prospective population cohorts like the Framingham Heart Study and resulting randomized, controlled treatment trials (RCTs). Genetics for CVD risk prediction provides the opportunity to more precisely identify individuals at high risk for developing disease for whom preventive therapy can be directed.nnArticle, see p 1181 nnOur initial understanding regarding genetic risk for myocardial infarction and other forms of CHD has focused on rare ( 50 common (>1:20 carrier rate) gene variants with strong evidence for modest increases in CHD risk,6 and >150 common genetic variants with strong evidence for modest alterations of levels of key lipid fractions.7nnDespite many novel discoveries, …

901-79 Borderline Isolated Systolic Hypertension and Subsequent Risk of Cardiovascular Disease

Guidelines are unclear about treatment of borderline isolated systolic hypertension, defined as both systolic BP 140-159xa0mmHg and diastolic BPxa0lxa090xa0mmHg. We investigated the association between borderline isolated systolic hypertension and subsequent risk of myocardial infarction (Ml), stroke, cardiovascular death, and the combined endpoint of important cardiovascular events (nonfatal MI, nonfatal stroke or cardiovascular death) among 22,071 men age 40-84 years followed prospectively for an average of 10.7 years in the Physicians Health Study. Baseline BP and cardiovascular risk factors were available from completed questionnaires on 16,678 men among whom 960 subsequently had an incident important cardiovascular event. In proportional hazards models adjusting for other cardiovascular risk factors, the relative risks were calculated for those with borderline isolated systolic hypertension relative to those with both systolic BPxa0lxa0140xa0mmHg and diastolic BPxa0lxa090xa0mmHg. These relative risks were 1.05 (95% confidence interval Cl 0.80 to 1.36) for Ml, 1.68 (95% Cl 1.24 to 2.26) for stroke, 1.61 (95% Cl 1.19 to 2.18) for cardiovascular death, and 1.32 (95% Cl 1.11 to 1.58) for the combined endpoint of important cardiovascular events. These associations were not altered by an analysis excluding participants who reported baseline treatment for hypertension. Conclusion Borderline isolated systolic hypertension is associated with a significantly increased risk of subsequent important cardiovascular events.