Summary

Background

We tested whether genetic factors distinctly contribute to either development of coronary atherosclerosis or, specifically, to myocardial infarction in existing coronary atherosclerosis.

Methods

We did two genome-wide association studies (GWAS) with coronary angiographic phenotyping in participants of European ancestry. To identify loci that predispose to angiographic coronary artery disease (CAD), we compared individuals who had this disorder (n=12 393) with those who did not (controls, n=7383). To identify loci that predispose to myocardial infarction, we compared patients who had angiographic CAD and myocardial infarction (n=5783) with those who had angiographic CAD but no myocardial infarction (n=3644).

Findings

In the comparison of patients with angiographic CAD versus controls, we identified a novel locus, ADAMTS7 (p=4·98×10−13). In the comparison of patients with angiographic CAD who had myocardial infarction versus those with angiographic CAD but no myocardial infarction, we identified a novel association at the ABO locus (p=7·62×10−9). The ABO association was attributable to the glycotransferase-deficient enzyme that encodes the ABO blood group O phenotype previously proposed to protect against myocardial infarction.

Interpretation

Our findings indicate that specific genetic predispositions promote the development of coronary atherosclerosis whereas others lead to myocardial infarction in the presence of coronary atherosclerosis. The relation to specific CAD phenotypes might modify how novel loci are applied in personalised risk assessment and used in the development of novel therapies for CAD.

Funding

The PennCath and MedStar studies were supported by the Cardiovascular Institute of the University of Pennsylvania, by the MedStar Health Research Institute at Washington Hospital Center and by a research grant from GlaxoSmithKline. The funding and support for the other cohorts contributing to the paper are described in the webappendix.

Summary

Introduction

By guiding initial warfarin dose, pharmacogenetic (PGx) algorithms may improve the safety of warfarin initiation. However, once INR response is known, the contribution of PGx to dose refinements is uncertain. This study sought to develop and validate clinical and PGx dosing algorithms for warfarin dose refinement on days 6–11 after therapy initiation.

Materials and Methods

An international sample of 2,022 patients at 13 medical centers on 3 continents provided clinical, INR, and genetic data at treatment days 6–11 to predict therapeutic warfarin dose. Independent derivation and retrospective validation samples were composed by randomly dividing the population (80%/20%). Prior warfarin doses were weighted by their expected effect on S-warfarin concentrations using an exponential-decay pharmacokinetic model. The INR divided by that “effective” dose constituted a treatment response index.

Results

Treatment response index, age, amiodarone, body surface area, warfarin indication, and target INR were associated with dose in the derivation sample. A clinical algorithm based on these factors was remarkably accurate: in the retrospective validation cohort its R2 was 61.2% and median absolute error (MAE) was 5.0 mg/week. Accuracy and safety was confirmed in a prospective cohort (N=43). CYP2C9 variants and VKORC1-1639 G→A were significant dose predictors in both the derivation and validation samples. In the retrospective validation cohort, the PGx algorithm had: R2= 69.1% (P<0.05 vs. clinical algorithm), MAE= 4.7 mg/week.

Conclusions

A pharmacogenetic warfarin dose-refinement algorithm based on clinical, INR, and genetic factors can explain at least 69.1% of therapeutic warfarin dose variability after about one week of therapy.

Background

Total cholesterol was among the earliest identified risk factors for coronary heart disease (CHD). We sought to identify genetic variants in six genes associated with lipid metabolism and estimate their respective contribution to risk for CHD.

Methods

For 6 lipid-associated genes (LCAT, CETP, LIPC, LPL, SCARB1, and ApoF) we scanned exons, 5′ and 3′ untranslated regions, and donor and acceptor splice sites for variants using Hi-Res Melting® curve analysis (HRMCA) with confirmation by cycle sequencing. Healthy subjects were used for SNP discovery (n=64), haplotype determination/tagging SNP discovery (n=339), and lipid association testing (n=786).

Results

In 17,840 bases of interrogated sequence, 90 variant SNPs were identified; 19 (21.1%) previously unreported. Thirty-four variants (37.8%) were exonic(16 non-synonymous), 28 (31.1%) in intron-exon boundaries, and 28 (31.1%) in the 5′ and 3′ untranslated regions. Compared to cycle sequencing, HRMCA had sensitivity of 99.4% and specificity of 97.7%. Tagging SNPs (n=38) explained >90% of the variation in the 6 genes and identified linkage disequilibrium (LD) groups. Significant beneficial lipid profiles were observed for CETP LD group 2, LIPC LD groups 1 and 7, and SCARB1 LD groups 1, 3 and 4. Risk profiles worsened for CETP LD group 3, LPL LD group 4.

Conclusions

These findings demonstrate the feasibility, sensitivity, and specificity of HRMCA for SNP discovery. Variants identified in these genes may be used to predict lipid-associated risk and reclassification of clinical CHD risk.

Background

In patients with acute coronary syndrome (ACS), elevated levels of soluble CD40 ligand (sCD40L) are associated with increased risk of cardiovascular events. We evaluated sCD40L levels and future cardiovascular events in patients not experiencing ACS.

Methods

Serum sCD40L levels were measured in 909 patients undergoing angiography. A three-way matching scheme (age, gender and catheterization time period) identified 303 patients with coronary artery disease (CAD) who experienced a cardiac event within 1 year (CAD/event), 303 patients with CAD free of events (CAD/no event) and 303 patients without CAD and free of events (no CAD).

Results

Average age was 64 ± 11 years; 74% were males. Median (± SE) sCD40L levels were higher for no CAD patients (335 ± 60 pg/ml) compared to CAD (248 ± 65 pg/ml, p = 0.01) and to CAD/event (233 ± 63 pg/ml, p < 0.001). There was no significant difference in median sCD40L levels between CAD/no event and CAD/event patients. Higher sCD40L quartiles were associated with a significant decrease in the risk of CAD/event versus no CAD (quartile 4 versus quartile 1: odds ratio = 0.59, p = 0.03). There was a nonsignificant trend towards a decreased risk of CAD as compared to no CAD, and for CAD/event versus CAD.

Conclusions

In non-ACS patients, higher sCD40L levels were associated with a decreased risk of CAD. This novel interaction of sCD40L raises interesting questions for CAD pathogenesis.

Objective

To discover common variants in 6 lipid metabolic genes and construct and validate a genetic risk score (GRS) based on the joint effects of genetic variants in multiple genes from lipid and other pathobiologic pathways.

Background

Explaining the genetic basis of coronary artery disease (CAD) is incomplete. Discovery and aggregation of genetic variants from multiple pathways may advance this objective.

Methods

Premature CAD cases (N=1,918) and CAD-free controls (N=1,032) were selected from our angiographic registry. In a discovery phase, single nucleotide polymorphisms (SNPs) at 56 loci from internal discovery and external reports were tested for associations with biomarkers and CAD: 28 promising SNPs were then tested jointly for CAD associations, and a genetic risk score (GRS) consisting of SNPs contributing independently was constructed and validated in a replication set of familial cases and population-based controls (N=1,320).

Results

Five variants contributed jointly to CAD prediction in a multigenic GRS model: odds ratio (OR) =1.24 (95% CI 1.16–1.33) per risk allele, p=8.2×10−11, adjusted OR=2.03 (1.53–2.70), 4th vs. 1st quartile. GRS5 had minor impact on area under the receiver-operating characteristic curve (p>0.05) but resulted in substantial net reclassification improvement: 0.16 overall, 0.28 in intermediate risk patients (both p<0.0001). GRS5 predicted familial CAD with similar magnitude in the validation set.

Conclusions

CorGen demonstrates the ability of a multigenic, multipathway GRS to improve discrimination of angiographic CAD. Genetic risk scores promise to increase understanding of the genetic basis of CAD and improve identification of individuals at increased CAD risk.

Background

Multiple single-nucleotide polymorphisms have been associated with low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) levels. In this paper, we evaluate a weighted and an unweighted approach for estimating the combined effect of multiple markers (using genotypes and haplotypes) on lipid levels for a given individual.

Methods

Using data from the Framingham Heart Study SHARe genome-wide association study, we tested genome-wide genotypes and haplotypes for association with lipid levels and constructed genetic risk scores (GRS) based on multiple markers that were weighted according to their estimated effects on LDL-C, HDL-C, and TG. These scores (GRS-LDL, GRS-HDL, and GRS-TG) were then evaluated for associations with LDL-C, HDL-C, and TG, and compared with results of an unweighted method based on risk-allele counts. For comparability of metrics, GRS variables were divided into quartiles.

Results

GRS-LDL quartiles were associated with LDL-C levels (p = 2.1 × 10-24), GRS-HDL quartiles with HDL-C (p = 5.9 × 10-22), and GRS-TG quartiles with TG (p = 5.4 × 10-25). In comparison, these p-values were considerably lower than those for the associations of the unweighted GRS quartiles for LDL-C (p = 3.6 × 10-7), HDL-C (p = 6.4 × 10-16), and TG (p = 4.1 × 10-10).

Conclusion

GRS variables were highly predictive of LDL-C, HDL-C, and TG measurements, especially when weighted based on each marker's individual association with those intermediate risk phenotypes. The allele-count GRS approach that does not weight the GRS by individual marker associations was considerably less predictive of lipid and lipoprotein measures when the same genetic markers were utilized, suggesting that substantially more risk-associated genetic marker information is encapsulated by the weighted GRS variables.

Background

A chromosome 9p21 locus is associated with coronary heart disease (CHD) in at least 25 independent populations, but multiple clinically-distinct phenotypes have been evaluated. Utilizing angiographic coronary artery disease (CAD) phenotyping, this study evaluated whether 9p21 single nucleotide polymorphisms (SNPs) predict ischemic events (e.g., myocardial infarction [MI]) among CAD patients.

Methods and Results

Patients undergoing coronary angiography during 1994-2007 (population set 1A: N=1,748; set 1B: N=1,014) were evaluated for association of a 9p21 tagging SNP (rs2383206, A→G) with incident MI and death events among patients with angiographically-significant CAD. Another hypothesis evaluated rs2383206 in two additional angiographic sets of both CAD and non-CAD patients (set 2A: N=2,122; set 2B: N=1,466) for prevalent MI vs. CAD/no MI (and for MI vs. non-CAD and CAD/no MI vs. non-CAD). No association of rs2383206 was found with events in set 1A (OR=0.95 per G allele, p-trend=0.48) and set 1B (OR=0.91 per G allele, p-trend=0.28), or with MI vs. CAD/no MI in set 2A (OR=0.96 per G allele, p-trend=0.57) and set 2B (OR=0.89 per G allele, p-trend=0.21). In contrast, rs2383206 was associated with CAD/no MI compared with non-CAD (set 2A: p-trend=0.0001; set 2B: p-trend=0.0008).

Conclusions

The chromosome 9p21 locus was not associated with incident events or prevalent MI, although it did predict CAD diagnosis. This contradicts reports of a 9p21 association with MI, likely due to differences in phenotype assignment. This suggests that high-quality phenotyping for CAD and MI is required to dissect the specific contributions of genetic variation to each stage of CHD pathophysiology.

Coronary artery disease (CAD) is common and multi-factorial. Members of the Church of Jesus Christ of Latter-day Saints (LDS, or Mormons) in Utah may have lower cardiac mortality than other Utahns and the US population. While the LDS proscription of smoking likely contributes to lower cardiac risk, it is unknown whether other shared behaviors also contribute. This study evaluated potential CAD-associated effects of fasting. Patients (N1=4,629) enrolled in the Intermountain Heart Collaborative Study registry (1994-2002) were evaluated for association of religious preference with CAD diagnosis (≥70% coronary stenosis on angiography) or no CAD (normal coronaries, <10% stenosis). Consequently, another set of patients (N2=448) were surveyed (2004-2006) for association of behavioral factors with CAD, with the primary variable being routine fasting (i.e., abstinence from food and drink). Secondary survey measures included proscription of alcohol, tea, and coffee, social support, and religious worship patterns. In population 1 (initial), 61% of LDS and 66% of all others had CAD (adjusted [including for smoking]: odds ratio [OR]=0.81; p=0.009). In population 2 (survey), fasting was associated with lower risk of CAD (64% vs. 76% CAD; OR=0.55, CI=0.35, 0.87; p=0.010) and this remained after adjustment for traditional risk factors (OR=0.46, CI=0.27, 0.81; p=0.007). Fasting was also associated with lower diabetes prevalence (p=0.048). In regression models entering other secondary behavioral measures, fasting remained significant with similar effect size. In conclusion, not only proscription of tobacco, but also routine periodic fasting was associated with lower risk of CAD.

Background

Single nucleotide polymorphisms (SNPs) in matrix metalloproteinase (MMP) genes may be associated with myocardial infarction (MI) and coronary artery disease (CAD), but studies of multiple MMP genes and their tissue inhibitors (TIMPs) are scarce. Further, differentiation of predictive ability by endpoint (MI vs. CAD) has not been addressed. This study evaluated the association with MI of SNPs in genes encoding MMPs 1, 2, 3, and 9 and TIMPs 1, 2, and 3.

Methods

Genotypes of patients (N=5,148) with MI (n=1,693) and angiographically-defined CAD (=1 lesion of =70% stenosis, n=1,967) were compared to MI-free (n=3,455) and non-CAD patients (n=1,122), respectively. Due to linkage disequilibrium, MMP-1 and MMP-3 SNPs (chromosome 11) were combined, as were the two MMP-9 SNPs.

Results

For MI, only MMP-9 group CT/RQ (OR=1.25, p=0.007 vs. wild-type CC/RR) had greater MI risk, with TT/QQ having a weak trend (OR=1.43, p=0.10). These findings remained (CT/RQ) or were strengthened (TT/QQ) after full adjustment. For CAD, association was found for MMP-1/-3 groups 2G1G/6A6A (OR=1.45, p=0.022), 2G1G/6A5A (OR=1.49, p=0.001), 2G1G/5A5A (OR=1.64, p=0.003), and 1G1G/5A5A (OR=1.35, p=0.035) compared to wild-type.

Conclusions

Composite MMP-9 genotypes but not other SNPs were associated with MI, while MMP-1/-3 genotypes were CAD-associated. The largest MMP/TIMP gene study to date, this study suggests care in selection and definition of clinical phenotypes. Further, this suggests that the evaluated SNPs only approximately account for intra-genic variation in these genes and that comprehensive evaluation of all variation in these genes should better elucidate associations with MI and CAD phenotypes.

Background

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). More than 30% of the LQT2 mutations result in premature termination codons (PTCs). Degradation of PTC-containing mRNA transcripts by nonsense-mediated mRNA decay (NMD) is increasingly recognized as a mechanism for reducing mRNA levels in a variety of human diseases. However, the role of NMD in LQT2 mutations has not been explored.

Methods and Results

We examined the expression of hERG mRNA in lymphocytes from patients carrying the R1014X mutation using a technique of allele-specific transcript quantification. The R1014X mutation led to a reduced level of mutant mRNA compared to that of the wild-type allele. The decrease in mutant mRNA was also observed in LQT2 nonsense mutations W1001X and R1014X using hERG minigenes expressed in HEK293 cells or neonatal rat ventricular myocytes. Treatment with the protein synthesis inhibitor cycloheximide or RNAi-mediated knockdown of the Upf1 protein resulted in the restoration of mutant mRNA to levels comparable to that of the wild-type minigene, suggesting that hERG nonsense mutations are subject to NMD.

Conclusions

These results indicate that LQT2 nonsense mutations cause a decrease in mutant mRNA levels by NMD rather than production of truncated proteins. Our findings suggest that the degradation of hERG mutant mRNA by NMD is an important mechanism in LQT2 patients with nonsense or frameshift mutations.

We performed a multipoint linkage analysis for rheumatoid arthritis (RA) using high-density single-nucleotide polymorphism (SNP) data for chromosome 6 and chromosome 21 using Genetic Analysis Workshop 15 (GAW15) data. These regions were previously shown to have high LOD scores, not accounting for linkage disequilibrium (LD). We propose three novel methods to control for LD in a linkage analysis: allow for LD between markers using graphical modeling, eliminate high-LD markers by principal-component analysis (PCA) using haplotype data, and eliminate high-LD markers by PCA using genotype data. All three novel methods were compared to the previously published SNPLINK high-LD elimination method. Although all four methods verified the previous results, differences in linkage peak height and position were observed across methods. Additional work is required to further understand the effects of LD on linkage results and explore LD control methodology.

Background

High triglycerides (TG) and low high-density lipoprotein cholesterol (HDL-C) jointly increase coronary disease risk. We performed linkage analysis for TG/HDL-C ratio in the Framingham Heart Study data as a quantitative trait, using methods implemented in LINKAGE, GENEHUNTER (GH), MCLINK, and SOLAR. Results were compared to each other and to those from a previous evaluation using SOLAR for TG/HDL-C ratio on this sample. We also investigated linked pedigrees in each region using by-pedigree analysis.

Results

Fourteen regions with at least suggestive linkage evidence were identified, including some that may increase and some that may decrease coronary risk. Ten of the 14 regions were identified by more than one analysis, and several of these regions were not previously detected. The best regions identified for each method were on chromosomes 2 (LOD = 2.29, MCLINK), 5 (LOD = 2.65, GH), 7 (LOD = 2.67, SOLAR), and 22 (LOD = 3.37, LINKAGE). By-pedigree multi-point LOD values in MCLINK showed linked pedigrees for all five regions, ranging from 3 linked pedigrees (chromosome 5) to 14 linked pedigrees (chromosome 7), and suggested localizations of between 9 cM and 27 cM in size.

Conclusion

Reasonable concordance was found across analysis methods. No single method identified all regions, either by full sample LOD or with by-pedigree analysis. Concordance across methods appeared better at the pedigree level, with many regions showing by-pedigree support in MCLINK when no evidence was observed in the full sample. Thus, investigating by-pedigree linkage evidence may provide a useful tool for evaluating linkage regions.

Content

Clopidogrel, one of the most commonly prescribed medications, is a pro-drug requiring CYP450 biotransformation. Data suggest its pharmacologic effect varies based on CYP2C19 genotype, but there is uncertainty regarding the clinical risk imparted by specific genotypes.

Objective

In patients treated with clopidogrel, to define the risk of major adverse cardiovascular outcomes among carriers of one (∼26% prevalence in whites) and carriers of two (∼2% prevalence in whites) reduced-function CYP2C19 variants.

Data Sources and Study Selection

A literature search was conducted (January 2000-August 2010) of the MEDLINE, Cochrane, and EMBASE databases. Genetic studies were included where clopidogrel was initiated in predominantly invasively managed patients in a manner consistent with the current guideline recommendations and where clinical outcomes were ascertained.

Data Extraction

Investigators from nine studies evaluating CYP2C19 genotype and clinical outcomes in patients treated with clopidogrel contributed the relevant hazard ratios (HRs) and their 95% confidence intervals (CI) for specific cardiovascular outcomes by genotype.

Results

Among 9685 patients [91.3% of whom underwent percutaneous coronary intervention (PCI) and 54.5% of whom had an acute coronary syndrome (ACS)], 863 experienced the composite endpoint of cardiovascular death, myocardial infarction, or stroke; 84 patients had stent thrombosis among the 5894 evaluated for such. Overall, 71.5% were non-carriers, 26.3% had one, and 2.2% had two CYP2C19 reduced-function alleles. A significantly increased risk of the composite endpoint was evident in both carriers of one (HR 1.55, 95% CI 1.11-2.27, P=0.01) and two (HR 1.76, 95% CI 1.24-2.50, P=0.002) CYP2C19 reduced-function alleles. Similarly, there was a significantly increased risk of stent thrombosis in both carriers of one (HR 2.67, 95% CI 1.69-4.22, P<0.0001) and two (HR 3.97, 95% CI 1.75-9.02, P=0.001) CYP2C19 reduced-function alleles.

Conclusion

Among patients treated with clopidogrel for PCI, carriage of even one reduced-function CYP2C19 allele appears to be associated with a significantly increased risk of major adverse cardiovascular events, particularly stent thrombosis.