It is not known if the genes involved with endurance performance during young adulthood are also involved with changes in performance. We examined the associations of gene variants with symptom-limited exercise test duration at baseline and decrease in duration over 20 years.
Methods and Results
3,783 (1,835 Blacks 1,948 Whites) and 2,335 (1,035 Blacks 1,300 Whites) participants from CARDIA were included in the baseline and 20 year models, respectively. 217 SNPs in Blacks and 171 SNPs in Whites from 17 genes were genotyped. In Blacks, five SNPs in the ATP1A2, HIF1A, NOS3, and PPARGC1A loci tended to be associated (p<0.05) with baseline duration in a multivariate regression model. Blacks (n=99) with at least four of the most-favorable genotypes at these loci had approximately two minutes longer baseline duration than those with only two such genotypes (P<0.0001). In Whites, the HIF1A rs1957757 and PPARGC1A rs3774909 markers tended to be associated with baseline duration, but the association of a multimarker construct of the most-favorable genotypes at both SNPs with baseline duration was not statistically significant. In Whites, four SNPs in the AGT, AMPD1, ANG, and PPARGC1A loci tended to be associated with decrease in exercise duration over 20 years, and those (n=40) with all four favorable genotypes had 0.8 min less decline in duration compared to those with none or one (n=232) (P<0.0001).
In multimarker constructs, alleles at genes related to skeletal muscle Na+/K+ transport, hypoxia, and mitochondrial metabolism are associated with symptom-limited exercise test duration over time in adults.
cardiorespiratory fitness; genotype; prospective study
The cell/environment interface is composed of the proteins of plasma membrane which face the extracellular space and by the proteins secreted directly by the cell of origin or by neighboring cells. The secreted proteins can act as extracellular matrix proteins and/or autocrine/paracrine proteins. This report discusses the technical aspects involved in the identification and characterization of the secreted proteins of specific cell types that comprise the heart. These aspects include the culturing of the cells, cell co-culturing and quantitative labeling, conditioned media collection and dealing with high abundant serum proteins, post-translational modification enrichment, the use of protein separation methods and mass spectrometry, protein identification and validation and the incorporation of pathway analysis to better understand the novel discovery on the background of already known experimental biological systems. The proteomic methods have the solid emplacement in cardiovascular research and the identification of proteins secreted by cardiac cells has been used in various applications such as determination the specificity between secretomes of different cell types, e.g. cardiac stem cells and cardiac myocytes, for the global secretome screening of e.g. human arterial smooth muscle cells, for the mapping of the beneficial effect of conditioned medium of one cell type on the other cell type, e.g. conditioned medium of human mesenchymal stem cells on cardiac myocytes, and for the searching the candidate paracrine factors and potential biomarkers.
secretomes; proteomics; technology; cardiovascular diseases
Survival following myocardial infarction (MI) has improved substantially over the last 40 years; however, the incidence of subsequent congestive heart failure has dramatically increased as a consequence. Discovering plasma markers that signify adverse cardiac remodeling may allow high-risk patients to be recognized earlier and may provide an improved way to assess treatment efficacy. Alterations in extracellular matrix (ECM) regulate cardiac remodeling following MI and potentially provide a large array of candidate indicators.
The field of cardiac proteomics has progressed rapidly over the past 20 years, since publication of the first two-dimensional electrophoretic gels of left ventricle proteins. Proteomic approaches are now routinely utilized to better understand how the left ventricle responds to injury.
In this review, we will discuss how methods have developed to allow comprehensive evaluation of the ECM proteome. We will explain how ECM proteomic data can be used to predict adverse remodeling for an individual patient and highlight future directions. Although this review will focus on the use of ECM proteomics to better understand post-MI remodeling responses, these approaches have applicability to a wide-range of cardiac pathologies, including pressure overload hypertrophy, viral myocarditis, and non-ischemic heart failure.
proteomics; remodeling; myocardial infarction; biomarker; MMP
Drug-induced long QT syndrome (diLQTS) is an adverse drug effect that has an important impact on drug use, development, and regulation. Here, we tested the hypothesis that common variants in key genes controlling cardiac electrical properties modify the risk of diLQTS.
Methods and Results
In a case-control setting, we included 176 patients of European descent from North America and Europe with diLQTS, defined as documented torsades de pointes during treatment with a QT prolonging drug. Control samples were obtained from 207 patients of European ancestry who displayed <50 msec QT lengthening during initiation of therapy with a QT-prolonging drug, and 837 controls from the population based KORA study. Subjects were successfully genotyped at 1,424 single nucleotide polymorphisms (SNPs) in 18 candidate genes including 1,386 SNPs tagging common haplotype blocks, and 38 non-synonymous ion channel gene SNPs. For validation we used a set of cases (n=57) and population-based controls of European descent. The SNP KCNE1 D85N (rs1805128), known to modulate an important potassium current in the heart, predicted diLQTS with an odds ratio of 9.0 (95% confidence interval: 3.5–22.9). The variant allele was present in 8.6% of cases, 2.9% of drug-exposed controls, and 1.8% of population controls. In the validation cohort the variant allele was present in 3.5% of cases, and in 1.4% of controls.
This high-density candidate SNP approach identified a key potassium channel susceptibility allele that may be associated with the rare adverse drug reaction torsades de pointes.
candidate genes; death, sudden; SNP; torsade de pointes; adverse drug events
Carotid-femoral pulse wave velocity (CFPWV) is a heritable measure of aortic stiffness that is strongly associated with increased risk for major cardiovascular disease events.
Methods and Results
We conducted a meta-analysis of genome-wide association data in 9 community-based European ancestry cohorts consisting of 20,634 participants. Results were replicated in 2 additional European ancestry cohorts involving 5,306 participants. Based on a preliminary analysis of 6 cohorts, we identified a locus on chromosome 14 in the 3′-BCL11B gene desert that is associated with CFPWV (rs7152623, minor allele frequency = 0.42, beta=−0.075±0.012 SD/allele, P = 2.8 x 10−10; replication beta=−0.086±0.020 SD/allele, P = 1.4 x 10−6). Combined results for rs7152623 from 11 cohorts gave beta=−0.076±0.010 SD/allele, P=3.1x10−15. The association persisted when adjusted for mean arterial pressure (beta=−0.060±0.009 SD/allele, P = 1.0 x 10−11). Results were consistent in younger (<55 years, 6 cohorts, N=13,914, beta=−0.081±0.014 SD/allele, P = 2.3 x 10−9) and older (9 cohorts, N=12,026, beta=−0.061±0.014 SD/allele, P=9.4x10−6) participants. In separate meta-analyses, the locus was associated with increased risk for coronary artery disease (hazard ratio [HR]=1.05, confidence interval [CI]=1.02 to 1.08, P=0.0013) and heart failure (HR=1.10, CI=1.03 to 1.16, P=0.004).
Common genetic variation in a locus in the BCL11B gene desert that is thought to harbor one or more gene enhancers is associated with higher CFPWV and increased risk for cardiovascular disease. Elucidation of the role this novel locus plays in aortic stiffness may facilitate development of therapeutic interventions that limit aortic stiffening and related cardiovascular disease events.
aorta; arterial stiffness; pulse wave velocity; genetics; cardiovascular disease
Multiple genetic loci have been associated with blood lipid levels. We tested the hypothesis that people with an unfavorable lipid gene profile would experience a greater increase in lipid levels and a higher incidence of abnormal lipid levels, relative to those with more favorable lipid gene profiles.
Methods and Results
9,328 European-descent individuals in ARIC (ages 45–64 y) were followed for 9 years. Separate gene scores were created for each lipid phenotype based on 95 loci identified in a published GWAS of >100,000 European-descent individuals. Adjusted linear and survival models were used to estimate associations with lipid levels and incidence of lipid-lowering medication or abnormal lipid levels. Age and sex interactions were also explored. The cross-sectional difference (mg/dL) per one standard deviation (SD) was −1.89 for HDL-C, 9.5 for LDL-C, and 22.8 for triglycerides (p<5 × 10−34 for all). Longitudinally, overall triglyceride levels rose over time, and each SD greater triglyceride gene score was associated with an average increase in triglyceride levels of 0.3 mg/dL (p=0.003) over 3-years. The HDL-C, LDL-C and total cholesterol gene scores were not related to change. All lipid gene scores were positively related to incidence of abnormal lipid levels over follow-up (HRs per SD ranged from 1.15–1.36).
Associations of genetic variants with lipid levels over time are complex, with the triglyceride gene score positively related to change in triglycerides levels. Similar longitudinal results were not observed for LDL-C or HDL-C levels. Unfavorable gene scores were nevertheless related to higher incidence of abnormal levels.
lipids; longitudinal trends; gene score; Atherosclerosis Risk in Communities (ARIC)
Rare variant accumulation studies can implicate genes in disease susceptibility when a significant burden is observed in patients versus controls. Such analyses might be particularly useful for candidate genes that are selected based on experiments other than genome-wide association studies (GWAS). We sought to determine whether rare variants in non-GWAS candidate genes identified from mouse models and human Mendelian syndromes of hypertriglyceridemia (HTG) accumulate in patients with polygenic adult-onset HTG.
Methods and Results
We resequenced protein coding regions of 3 genes with established roles (APOC2, GPIHBP1, LMF1) and 2 genes recently implicated (CREB3L3 and ZHX3) in TG metabolism. We identified 41 distinct heterozygous rare variants, including 29 singleton variants, in the combined sample; in total, we observed 47 rare variants in 413 HTG patients versus 16 in 324 controls (OR=2.3; P=0.0050). Post hoc assessment of genetic burden in individual genes using three different tests suggested that the genetic burden was most prominent in the established genes LMF1 and APOC2, and also in the recently identified CREB3L3 gene.
These extensive resequencing studies show a significant accumulation of rare genetic variants in non-GWAS candidate genes among patients with polygenic HTG, and indicate the importance of testing specific hypotheses in large-scale resequencing studies.
hyperlipoproteinemia; genetics; apolipoproteins; lipoproteins; cardiovascular diseases
Limited data exist regarding the use of a genetic risk score for predicting risk of incident cardiovascular disease (CVD) in US based samples.
Methods and Results
Using findings from recent GWAS, we constructed genetic risk scores (GRS) comprised of 13 genetic variants associated with myocardial infarction (MI) or other manifestations of CHD and 102 genetic variants associated with CHD or its major risk factors. We also updated the 13 SNP GRS with 16 SNPs recently discovered by GWAS. We estimated the association, discrimination and risk reclassification of each GRS for incident cardiovascular events and for prevalent coronary artery calcium (CAC).
In analyses adjusted for age, sex, CVD risk factors and parental history of CVD, the 13 SNP GRS was significantly associated with incident hard CHD (HR 1.07, 95% CI 1.00-1.15, p=0.04), CVD (hazard ratio [HR] per-allele 1.05, 95% confidence interval [CI] 1.01-1.09; p=0.03), and high CAC (defined as >75th age and sex-specific percentile; odds ratio [OR] per-allele 1.18, 95% CI 1.11-1.26, p=3.4 × 10-7). The GRS did not improve discrimination for incident CHD or CVD but led to modest improvements in risk reclassification. However, significant improvements in discrimination and risk reclassification were observed for the prediction of high CAC. The addition of 16 newly discovered SNPs to the 13 SNP GRS did not significantly modify these results.
A GRS comprised of 13 SNPs associated with coronary disease is an independent predictor of cardiovascular events and of high CAC, modestly improves risk reclassification for incident CHD and significant improves discrimination for high CAC. The addition of recently discovered SNPs did not significantly improve the performance of this GRS.
Genetics; single nucleotide polymorphisms; cardiovascular disease; coronary heart disease; risk prediction; reclassification
Genetic determinants of peripheral arterial disease (PAD) remain largely unknown. To identify genetic variants associated with the ankle-brachial index (ABI), a noninvasive measure of PAD, we conducted a meta-analysis of genome-wide association study data from 21 population-based cohorts.
Methods and Results
Continuous ABI and PAD (ABI≤0.9) phenotypes adjusted for age and sex were examined. Each study conducted genotyping and imputed data to the ~2.5 million SNPs in HapMap. Linear and logistic regression models were used to test each SNP for association with ABI and PAD using additive genetic models. Study-specific data were combined using fixed-effects inverse variance weighted meta-analyses. There were a total of 41,692 participants of European ancestry (~60% women, mean ABI 1.02 to 1.19), including 3,409 participants with PAD and with GWAS data available. In the discovery meta-analysis, rs10757269 on chromosome 9 near CDKN2B had the strongest association with ABI (β= −0.006, p=2.46x10−8). We sought replication of the 6 strongest SNP associations in 5 population-based studies and 3 clinical samples (n=16,717). The association for rs10757269 strengthened in the combined discovery and replication analysis (p=2.65x10−9). No other SNP associations for ABI or PAD achieved genome-wide significance. However, two previously reported candidate genes for PAD and one SNP associated with coronary artery disease (CAD) were associated with ABI : DAB21P (rs13290547, p=3.6x10−5); CYBA (rs3794624, p=6.3x10−5); and rs1122608 (LDLR, p=0.0026).
GWAS in more than 40,000 individuals identified one genome-wide significant association on chromosome 9p21 with ABI. Two candidate genes for PAD and 1 SNP for CAD are associated with ABI.
cohort study; genetic association; genome-wide association study; meta-analysis; peripheral vascular disease
Using the genome-wide association approach in individuals of European ancestry, we and others recently identified single nucleotide polymorphisms (SNPs) at 19 loci as associated with blood lipids; eight of these loci were novel. Whether these same SNPs associate with lipids in a broader range of ethnicities is unknown.
Methods and Results
We genotyped index SNPs at 19 loci in the Third United States National Health and Nutrition Examination Survey (n=7159), a population-based probability sample of the U.S. comprised primarily of non-Hispanic blacks, Mexican Americans, and non-Hispanic whites. We constructed ethnic-specific residual blood lipid levels after adjusting for age and gender. Ethnic-specific linear regression was used to test the association of genotype with blood lipids. To summarize the statistical evidence across three racial groups, we conducted a fixed-effects variance-weighted meta-analysis.
After exclusions, there were 1627 non-Hispanic blacks, 1659 Mexican Americans, and 2230 non-Hispanic whites. At five loci (1p13 near CELSR2/PSRC1/SORT1, HMGCR, CETP, LPL, and APOA5), the index SNP was associated with low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or triglycerides in all three ethnic groups. At the remaining loci, there was mixed evidence by ethnic group. In meta-analysis, we found that, at 14 of the 19 loci, SNPs exceeded a nominal P < 0.05.
At five loci including the recently-discovered region on 1p13 near CELSR2/PSRC1/SORT1, the same SNP discovered in whites associates with blood lipids in non-Hispanic blacks and Mexican Americans. For the remaining loci, fine-mapping and resequencing will be required to definitively evaluate the relevance of each locus in individuals of African and Hispanic ancestries.
lipids; genetics; epidemiology; risk factors
albuminuria genetics; genetic polymorphisms; genotype; population differentiation; single nucleotide polymorphism epidemiology
MicroRNAs (miRs) are small noncoding RNAs that recognize and bind to mRNAs and inhibit protein translation or degrade mRNA. Studies in animal models have suggested that miRs play a translational or post-translational regulatory role in myocardial growth, fibrosis, viability, and remodeling. However, whether specific temporal changes in miRs occur in patients during the LV remodeling process that follows a myocardial infarction (post-MI) remains unknown. The purpose of the current pilot study was to test the hypothesis that plasma miRs could be reliably measured in post-MI patients and that there is the relationship between temporal changes in specific miRs and post-MI LV structural remodeling.
Methods and Results
LV end-diastolic volume (EDV, echocardiography) and plasma miR were measured in age matched referent controls (CTL n=12) and post-MI patients (n=12) from day 2 through day 90 post-MI. Selected miRs (miR-1, -21, -29a, 133a, 208) were measured using quantitative rt-PCR and normalized for endogenous snRNA U6. Following MI, LVEDV increased progressively compared to CTL; this was accompanied by time dependent changes in specific miRs. For example, miR-21 initially fell 2 days post-MI (0.3±0.1 fold vs. CTL, p< 0.05), increased 5 days post-MI (2±1 fold vs. CTL, p< 0.05), and returned to CTL values at later post-MI time points. In contrast, miR-29a increased 5 days post-MI (4±1 fold vs. CTL, p< 0.05) and then fell to CTL at later time points. miR-208 increased 5 days post-MI (3±1 fold vs. CTL, p< 0.05) and remained elevated up to 90 days post-MI.
A time-dependent change in miRs occurred in post-MI patients that included an early and robust rise in miRs that have been shown to affect myocardial growth, fibrosis and viability. Thus, serially profiling miRs in the plasma of post-MI patients may hold both mechanistic and prognostic significance.
Myocardial Infarction; Remodeling; microRNA
Elevated von Willebrand Factor (VWF) plasma levels are associated with an increased risk of cardiovascular disease. A meta-analysis of genome wide association studies on VWF identified novel candidate genes, i.e. syntaxin-binding protein 5 (STXBP5) and syntaxin 2 (STX2), which are possibly involved in the secretion of VWF. We investigated whether VWF antigen levels (VWF:Ag), VWF collagen-binding activity (VWF:CB), and the risk of arterial thrombosis are affected by common genetic variations in these genes.
Methods and Results
In 463 young Caucasian subjects (males ≤ 45 years, females ≤ 55 years), who were included one to three months after a first event of arterial thrombosis, and 406 controls, we measured VWF:Ag and VWF:CB. Nine haplotype tagging SNPs of STXBP5 and STX2 were selected and subsequently analysed using linear regression with additive genetic models adjusted for age, sex and ABO blood group. The minor alleles of rs9399599 and rs1039084 in STXBP5 were associated with lower VWF plasma levels and activity, whereas the minor allele of rs7978987 in STX2 was associated with higher VWF plasma levels and activity. The minor alleles of the SNPs in STX2 were associated with a reduced risk of arterial thrombosis (rs1236:OR 0.73 [95%CI 0.59, 0.89], rs7978987:OR 0.81 [95%CI 0.65, 1.00], rs11061158:OR 0.69 [95%CI 0.55, 0.88]).
Genetic variability in STXBP5 and STX2 affects both VWF concentration and activity in young individuals with premature arterial thrombosis. Furthermore, in our study genetic variability in STX2 is associated with the risk of arterial thrombosis. However, at this point the underlying mechanism remains unclear.
Von Willebrand Factor; genetics; STX2; STXBP5; cardiovascular diseases
Increasing evidence points to a direct role for altered microRNA (miRNA or miR) expression levels in cardiovascular remodeling and disease progression. While alterations in miR expression levels have been directly linked to cardiac hypertrophy, fibrosis, and remodeling, their role in regulating gene expression during thoracic aortic aneurysm (TAA) development has yet to be explored.
Methods and Results
The present study examined miR expression levels in aortic tissue specimens collected from patients with ascending TAAs by quantitative real-time PCR, and observed decreased miR expression (miRs -1, -21, -29a, -133a, and -486) as compared to normal aortic specimens. A significant relationship between miR expression levels (miRs -1, -21, -29a, and -133a) and aortic diameter was identified; as aortic diameter increased, miR expression decreased. Using a bioinformatics approach, members of the matrix metalloproteinase (MMP) family, proteins involved in TAA development, were examined for putative miR binding sites. MMP-2 and MMP-9 were identified as potential targets for miR-29a and miR-133a respectively, and MMP-2 was subsequently verified as a miR-29a target in vitro. A significant inverse relationship between miR-29a and total MMP-2 was then identified in the clinical TAA specimens.
These findings demonstrate altered miR expression patterns in clinical TAA specimens, suggesting that the loss of specific miR expression may allow for the elaboration of specific MMPs capable of driving aortic remodeling during TAA development. Importantly, these data suggest that these miRs have biological and clinical relevance to the behavior of TAAs, and may provide significant targets for therapeutic and diagnostic applications.
aneurysm; thoracic aorta; microRNA; MMP; remodeling
editorial; electrocardiography; genetics; hypertrophy; conduction
Dilated cardiomyopathy (DCM) is a heritable, genetically heterogeneous disorder, typically exhibiting autosomal dominant inheritance. Genomic strategies enable discovery of novel, unsuspected molecular underpinnings of familial DCM. We performed genome-wide mapping and exome sequencing in a unique family wherein DCM segregated as an autosomal recessive (AR) trait.
Methods and Results
Echocardiography in 17 adult descendants of first cousins revealed DCM in two female siblings and idiopathic left ventricular enlargement in their brother. Genotyping and linkage analysis mapped an AR DCM locus to chromosome 7q21, which was validated and refined by high-density homozygosity mapping. Exome sequencing of the affected sisters was then employed as a complementary strategy for mutation discovery. An iterative bioinformatics process was used to filter >40,000 genetic variants, revealing a single shared homozygous missense mutation localized to the 7q21 critical region. The mutation, absent in HapMap, 1000Genomes and 474 ethnically matched controls, altered a conserved residue of GATAD1, encoding GATA zinc finger domain-containing protein 1. Thirteen relatives were heterozygous mutation-carriers with no evidence of myocardial disease, even at advanced ages. Immunohistochemistry demonstrated nuclear localization of GATAD1 in left ventricular myocytes, yet subcellular expression and nuclear morphology were aberrant in the proband.
Linkage analysis and exome sequencing were used as synergistic genomic strategies to identify GATAD1 as a gene for AR DCM. GATAD1 binds to a histone modification site that regulates gene expression. Consistent with murine DCM caused by genetic disruption of histone deacetylases, our data implicate an inherited basis for epigenetic dysregulation in human heart failure.
Cardiomyopathy; Genetics; Genomics; Epigenetics; Next generation sequencing
After age, gender is the most important risk factor for coronary artery disease (CAD). The mechanism through which women are protected from CAD is still largely unknown, but the observed gender difference suggests the involvement of the reproductive steroid hormone signaling system. Genetic association studies of the gene encoding Estrogen Receptor alpha (ESR1) have shown conflicting results, although only a limited range of variation in the gene has been investigated.
Methods and Results
We exploited information made available by advanced new methods and resources in complex disease genetics to revisit the question of ESR1's role in risk of CAD. We performed a meta-analysis of 14 genome-wide association studies (CARDIoGRAM discovery analysis, N~87,000) to search for population-wide and gender-specific associations between CAD risk and common genetic variants throughout the coding, non-coding and flanking regions of ESR1. In additional samples from the MIGen (N~6,000), WTCCC (N~7,400) and Framingham (N~3,700) studies, we extended this search to a larger number of common and uncommon variants by imputation into a panel of haplotypes constructed using data from the 1000 Genomes project. Despite the widespread expression of ER alpha in vascular tissues, we find no evidence for involvement of common or low-frequency genetic variation throughout the ESR1 gene in modifying risk of CAD, either in the general population or as a function of gender.
We suggest that future research on the genetic basis of gender-related differences in CAD risk should initially prioritize other genes in the reproductive steroid hormone biosynthesis system.
coronary artery disease; estrogen receptor alpha; menopause; polymorphism, single nucleotide; genetic association studies; meta-analysis
Circulating levels of soluble intercellular adhesion molecule-1 (sICAM-1), soluble P-selectin (sP-selectin), and soluble E-selectin (sE-selectin) have been associated with variation at the ABO locus. To evaluate these associations and the effect sizes, we performed a meta-analysis with new and previous reported data for polymorphism rs579459.
Methods and Results
Compared with major allele homozygotes, heterozygotes and minor allele homozygotes had 4.6% (95%CI=3.4–5.8%, p=7.3×10−14) and 7.2% (95%CI=4.7–9.7%, p=1.5×10−8), respectively, lower sICAM-1 levels (n=33,671). An allele dose dependent association also was observed for sP-selectin (n=4,921), with heterozygotes and minor allele homozygotes having 11.5% (95%CI=7.2–15.8%, p=1.7×10−7) and 18.6% (95%CI=9.1–28.1%, p=1.2×10−4), respectively, lower levels than in major allele homozygotes. A larger effect size, again consistent with an additive genetic model, was seen for sE-selectin (n=2,860) whose level was 25.6% (95%CI=19.0–32.2%, p=2.1×10−14) lower in heterozygotes and 43.3% (95%CI=36.9–49.3%, p=4.3×10−42) lower in minor allele homozygotes, than in major allele homozygotes.
The data support the association of variation at the ABO locus with sICAM-1, sP-selectin and sE-selectin levels.
Cell adhesion molecules; plasma; genetics; cardiovascular disease
Genome wide association studies identified several single nucleotide polymorphisms (SNPs) associated with prevalent coronary heart disease (CHD) but less is known of associations with incident CHD. The association of thirteen published CHD SNPs was examined in five ancestry groups of four large US prospective cohorts.
Methods and Results
The analyses included incident coronary events over 9.1 to 15.7 average follow-up times in up to 26,617 white individuals (6,626 events), 8,018 African Americans (914 events), 1,903 Hispanics (113 events), 3,669 American Indians (595 events) and 885 Asian/Pacific Islanders (66 events). We used Cox proportional hazards models (with additive mode of inheritance) adjusted for age, sex and ancestry (as needed). Nine loci were statistically associated with incident CHD events in whites: 9p21 (rs10757278, p=4.7 × 10−41), 16q23.1 (rs2549513, p=0.0004), 6p24.1 (rs499818, p=0.0002), 2q36.3 (rs2943634, p=6.7 × 10−6), MTHFDIL (rs6922269, p=5.1 × 10−10), APOE (rs429358, p=2.7 × 10−18), ZNF627 (rs4804611, p=5.0 × 10−8), CXCL12 (rs501120, p=1.4 × 10−6) and LPL (rs268, p=2.7 × 10−17). The 9p21 region showed significant between-study heterogeneity, with larger effects in individuals aged 55 years or younger and in women. Inclusion of coronary revascularization procedures among the incident CHD events introduced heterogeneity. The SNPs were not associated with CHD in African Americans and associations varied in other US minorities.
Prospective analyses of white individuals replicated several reported cross-sectional CHD-SNP associations.
9p21 locus; incident coronary heart disease; genetic polymorphisms
Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes not only identification and quantification of proteins, but also the characterization of protein modifications such as post-translational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides prior to MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications mainly due to the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a “bird’s eye” view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the non-ergodic dissociation methods such as electron capture dissociation (ECD) greatly enhances the top-down capabilities. ECD is especially useful for mapping labile post-translational modifications which are well-preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full sequence coverage, discovering unexpected modifications, and identifying and quantifying positional isomers and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of top-down methodology with a focus on its application in cardiovascular research.
Cardiovascular diseases; Proteomics; Electron Capture dissociation; Post-translational modification; Top-Down Mass Spectrometry
The left and right atria have different susceptibilities towards developing arrhythmias, with left atrial arrhythmias more commonly observed. To understand the molecular basis for such differences, we catalogued miRNA and mRNA expression differences by next generation sequencing.
Methods and Results
Four human left-right atrial pairs were subjected to whole-genome expression analyses via next generation sequencing of small RNAs, including microRNAs (miRNAs), and poly-A enriched mRNAs. Using a paired sample design, significant differences in the expression of 32 miRNAs were found in between the left and right atria at a p-value of <0.01. Hsa-miR-143 was the most highly expressed miRNA in the atria, as quantified by RNA-seq. There were 746 and 2292 differentially expressed mRNAs between the left and right atria at false discovery rates of <0.001 and <0.05, respectively. Transcription factor binding elements within 2 kb of RefSeq genes were determined and specific motifs were identified that were enriched in differentially expressed genes. Similarly, specific miRNA target sequences in 3' UTRs were also enriched in differentially expressed genes. In addition eleven novel non-coding RNAs of unknown function were found to be differentially expressed between the left and right atria.
There are significant differences in miRNA and mRNA expression profiles between the left and right atria, which may yield insight into increased the arrhythmogenesis of the left atria.
arrhythmia; atrial fibrillation; atrium; gene expression
editorials; congenital; genetics; genome-wide analysis; genome-wide association study; tetralogy of Fallot
Brugada Syndrome (BrS) is associated with mutations in the cardiac sodium channel (Nav1.5). We previously reported that the function of a trafficking-deficient BrS Nav1.5 mutation, R282H, could be restored by co-expression with the sodium channel polymorphism H558R. Here, we tested the hypothesis that peptide fragments from Nav1.5, spanning the H558R-polymorphism, can be used to restore trafficking of trafficking-deficient BrS sodium channel mutations.
Methods and Results
Whole-cell patch-clamping revealed that co-transfection in HEK293 cells of the R282H channel with either the 40 or 20 amino acid cDNA fragments of Nav1.5 containing the H558R polymorphism restored trafficking of this mutant channel. Fluorescence Resonance Energy Transfer (FRET) suggested that the trafficking-deficient R282H channel was misfolded and this was corrected upon co-expression with R558-containing peptides which restored trafficking of the R282H channel. Importantly, we also expressed the peptide spanning the H558R-polymorphism with 8 additional BrS Nav1.5 mutations with reduced currents, and demonstrated that the peptide was able to restore significant sodium currents in 4 of them.
In the present study, we demonstrated that small peptides, spanning the H558R-polymorphism, are sufficient to restore trafficking defect of BrS-associated Nav1.5 mutations. Our findings suggest that it might be possible to use short-cDNA constructs as a novel strategy that is tailored to specific disease-causing mutants of BrS.
arrhythmia; electrophysiology; gene therapy; ion channels; SCN5A
Network-based approaches may leverage genome-wide association (GWA) analysis by testing for the aggregate association across several pathway members. We aimed to examine if networks of genes that represent experimentally determined protein-protein interactions are enriched in genes associated with risk of coronary heart disease (CHD).
Methods and Results
GWA analyses of ~700,000 SNPs in 899 incident CHD cases and 1,823 age- and sex-matched controls within the Nurses’ Health and the Health Professionals Follow-Up Studies were used to assign gene-wise p-values. A large database of protein-protein interactions (PPI) was used to assemble 8,300 unbiased protein complexes and corresponding gene-sets. Superimposed gene-wise p-values were used to rank gene-sets based on their enrichment in genes associated with CHD. After correcting for the number of complexes tested, one gene-set was overrepresented in CHD-associated genes (p-value=0.002). Centered on the beta-1-adrenergic receptor gene (ADRB1), this complex included 18 protein interaction partners that, so far, have not been identified as candidate loci for CHD. Five of the 19 genes in the top-complex are reported to be involved in abnormal cardiovascular system physiology based on knock-out mice (4-fold enrichment; p-value, Fisher’s exact test= 0.006). Ingenuity pathway analysis revealed that especially canonical pathways related to blood pressure regulation were significantly enriched in the genes from the top complex.
The integration of a GWA study with PPI data successfully identifies a set of candidate susceptibility genes for incident CHD that would have been missed in single-marker GWA analysis.
Genetics of cardiovascular disease; acute myocardial infarction; epidemiology