Myeloperoxidase (MPO) is an enzymatic mediator of several inflammatory cascades and higher serum levels have been associated with increased risk of adverse cardiovascular events. We investigated the association of serum MPO with the ankle–brachial index (ABI) and peripheral arterial disease (PAD) in a bi-ethnic cohort of African-Americans and non-Hispanic white individuals. Participants included 1324 African-Americans (mean age 64 years, 71% women) and 1237 non-Hispanic white individuals (mean age 59 years, 57% women) belonging to hypertensive sibships. Serum levels of MPO were measured by solid phase sandwich immunoassay. ABI was measured using a standard protocol and PAD was defined as an ABI < 0.90. Multivariable regression analysis using generalized estimating equations were performed to assess whether serum MPO levels were associated with ABI and the presence of PAD. After adjustment for age and sex, higher MPO levels were significantly associated with lower ABI and the presence of PAD in African-Americans (p = 0.004 and p = 0.005, respectively) and in non-Hispanic white individuals (p = 0.001 and p = 0.016, respectively). After additional adjustment for conventional risk factors (diabetes, smoking status, total and high-density lipoprotein cholesterol, waist circumference, hypertension), prior history of myocardial infarction or stroke, and medication use (statins, aspirin, estrogen), higher MPO levels remained significantly associated with lower ABI and the presence of PAD in both African-Americans (p = 0.008 and p = 0.010, respectively) and non-Hispanic white individuals (p = 0.001 and p = 0.018, respectively). We conclude that higher MPO levels are associated with lower ABI and the presence of PAD in African-Americans and non-Hispanic white individuals.
ankle–brachial index; inflammation; myeloperoxidase; peripheral arterial disease
Pulse pressure (an indirect measure of arterial stiffness) is a robust predictor of cardiovascular events, but its pathophysiology remains poorly understood. To gain insight into the pathophysiology of arterial stiffness we conducted an exploratory investigation of the associations of 47 circulating biomarkers in etiologic pathways of arteriosclerosis with brachial artery pulse pressure.
Participants included 1,193 African-Americans and 1,145 non-Hispanic whites belonging to hypertensive sibships. Blood pressure (BP) was measured with a random-zero sphygmomanometer. Multivariable linear regression was employed to assess the associations of biomarkers with pulse pressure after adjustment for age, sex, conventional risk factors, mean arterial pressure, heart rate, and use of aspirin, statins, estrogens, and antihypertensives. Statistical significance was set at P ≤ 0.001 (Bonferroni correction for multiple testing).
Log N-terminal probrain natriuretic peptide (NT-proBNP) (African-Americans: β = 2.11 ± 0.52, non-Hispanic whites: β = 2.65 ± 0.55), log midregional proatrial natriuretic peptide (African-Americans: β = 4.83 ± 0.70, non-Hispanic whites: β = 3.70 ± 0.67), and log osteoprotegerin (African-Americans: β = 4.64 ± 1.02, non-Hispanic whites: β = 4.19 ±0.99) were independently associated with pulse pressure (P < 0.001 for all) in both ethnicities. Log C-reactive protein (CRP) (β = 1.56 ± 0.35), log midregional proadrenomedullin (MR-proADM) (β = 5.53 ± 1.19) and log matrix metalloproteinase-2 (β = 3.89 ± 1.06) were associated with greater pulse pressure in African-Americans only (P ≤ 0.001 for all), whereas higher fibrinogen was associated with pulse pressure in non-Hispanic whites only (β = 0.02 ± 0.004. P < 0.001).
Our results suggest that hemodynamic stress, vascular inflammation and calcification, and matrix remodeling may have a role in the pathogenesis and/or adverse consequences of increased pulse pressure.
adrenomedullin; arterial stiffness; atrial natriuretic peptide; biomarkers; blood pressure; brain natriuretic peptide; fibrinogen; hypertension; matrix metalloproteinase-2; osteoprotegerin; pulse pressure
To better understand the basis for previously reported ethnic differences in ankle brachial index (ABI), we investigated whether these differences were present in individuals without known peripheral arterial disease (PAD).
We used data from National Health and Nutrition Examination surveys (NHANES 1999–2004) to determine whether ethnic differences were present in respondents without PAD (1≤ABI≤1.3). We assessed whether ethnicity was an independent predictor of ABI and ankle systolic blood pressure (SBP) in linear regression models that adjusted for conventional and novel cardiovascular risk factors. To minimize effects of atherosclerosis on ABI, we studied adults aged ≤ 60 years, and also repeated our analyses in a subset aged ≤ 50 years that did not have risk factors for PAD.
3348 participants aged ≤ 60 years were included in the study. Mean ABI was 1.11 in non-Hispanic Blacks (NHB) and 1.13 in non-Hispanic Whites (NHW) (P <0.0001). In multivariable linear regression analysis that adjusted for age, gender, ethnicity, smoking, height, diabetes, brachial SBP, dyslipidemia, diabetes, renal function, concurrent cardiovascular disease, and plasma levels of homocysteine, fibrinogen and C-reactive protein, NHB had lower ABI than NHW (β= − 0.03± 0.004, P < 0.00001). Although, NHBs had higher ankle SBP than NHWs (by 5.3 mm Hg), NHBs had a lower mean ankle SBP (β= − 3.663 mm Hg ± 0.500, P <.0001) after adjusting for clinical covariates, including brachial SBP, in multivariable analysis.
Ethnic differences in ABI are present in middle-aged adults at low risk for peripheral atherosclerosis.
ankle brachial index; ethnicity
Left ventricular hypertrophy, a marker for adverse cardiovascular events, is more common in blacks than non-Hispanic whites. Mechanisms leading to left ventricular hypertrophy and mediating its clinical sequelae in blacks are not fully understood. We investigated the associations of 39 candidate biomarkers in distinct biological pathways with left ventricular mass and geometry in blacks. Participants included 1193 blacks (63 ± 9 years, 72% women, 78% hypertensive) belonging to hypertensive sibships. Left ventricular mass was measured by transthoracic echocardiography and indexed to height2.7. Left ventricular geometry was categorized as: normal, concentric remodeling, concentric hypertrophy and eccentric hypertrophy. Generalized estimating equations were employed to assess associations of the 39 biomarkers with left ventricular mass index after adjustment for age, sex, and conventional risk factors. After adjustment for potential confounders, log-transformed levels of the following biomarkers were independently associated with left ventricular mass index: N-terminal pro-brain natriuretic peptide (β±SE= 0.07±0.01 pg/mL, P< 0.0001), mid-regional pro-atrial natriuretic peptide (β±SE= 0.08±0.02 pmol/L, P< 0.0001), mid-regional pro-adrenomedullin (β±SE= 0.09±0.03 nmol/L, P= 0.0006), C-terminal pro-endothelin (β±SE= 0.05±0.02 pmol/L, P=0.0009) and osteoprotegerin (β±SE=0.07±0.02 pg/mL, P=0.0005). The associations of these biomarkers with left ventricular mass index were mainly due to their association with eccentric hypertrophy. Higher circulating levels of natriuretic peptides, adrenomedullin, endothelin and osteoprotegerin were associated with increased left ventricular mass index, providing insights into the pathophysiology of left ventricular hypertrophy in blacks.
left ventricular hypertrophy; biomarkers; brain natriuretic peptide; atrial natriuretic peptide; adrenomedullin; endothelin; osteoprotegerin
Increased red cell distribution width (RDW), a marker of anisocytosis, has been associated with adverse outcomes in multiple settings. Whether RDW is predictive of mortality in patients with peripheral artery disease (PAD) is unknown. We studied 13,039 consecutive outpatients (age 69.5 ±12.0 years, 60.9% men, 97.6% white) with PAD identified by non-invasive lower-extremity arterial testing at Mayo Clinic from 1/97 to 12/07, with follow-up through 9/09. We defined PAD as low (≤0.9) or high (≥1.4) ankle-brachial index (ABI). Cardiovascular risk factors and comorbidities were ascertained using electronic medical record (EMR)-based algorithms. RDW was obtained from the complete blood count drawn around the time of arterial evaluation. Mortality was ascertained using the Mayo EMR and Accurint® database. The association of RDW with all-cause mortality was analyzed by multivariable Cox proportional hazards regression. During a median follow-up of 5.5 years, 4039 (31.0 %) deaths occurred (28.7% in low and 38.9% in high ABI subsets). After adjustment for age, sex, cardiovascular risk factors and comorbidities, patients in the highest quartile of RDW (>14.5%) had 66% greater risk of mortality compared to the lowest quartile (<12.8%) (P<0.0001); a 1% increment in RDW was associated with 10% greater risk of all-cause mortality (hazard ratio, 1.10, 95% confidence interval [CI], 1.08 to 1.12, P<0.0001). The adjusted hazard ratio was similar in the low (1.10, 1.08-1.12) and high (1.09, 1.06-1.12) ABI subsets. In conclusion, RDW, a routinely available measure, is an independent prognostic marker in patients with PAD.
Increased arterial stiffness has been associated with greater risk of cardiovascular events. We investigated whether aortic augmentation index (AIx), a measure of arterial stiffness and wave reflection, was associated with the ankle-brachial index (ABI), a measure of peripheral arterial disease (PAD).
AIx and ABI were measured in a community-based sample of 475 adults without prior history of heart attack or stroke (mean age 59.3 years, 46.5% men). Radial artery pulse waveforms were obtained by applanation tonometry and an ascending aortic pressure waveform derived by a transfer function. AIx is the difference between the first and second systolic peak of the ascending aortic pressure waveform indexed to the central pulse pressure. ABI was measured using a standard protocol, and subjects with non-compressible vessels (ABI >1.5) were excluded from the analyses. Multivariable linear and logistic generalized estimating equations (GEE) analyses were used to assess whether AIx was associated with ABI and ABI <1.00 respectively, independent of conventional risk factors.
Mean (± SD) values were: AIx, 29.3±11.6 %; ABI, 1.12±0.13; 59 (12.4%) participants had an ABI <1.00. Variables associated with a lower ABI (and ABI <1.00) included older age, shorter height, female sex, higher total cholesterol, hypertension medication use, history of smoking, and higher AIx. After adjustment for mean arterial pressure and the above variables, higher AIx remained associated with a lower ABI (P=0.015) and ABI <1.00 (P=0.002). A significant interaction (P=0.007) was present between AIx and age in the prediction of ABI; the (inverse) association of AIx with ABI was stronger in older subjects (>65 years).
AIx, a measure of arterial stiffness and wave reflection, was independently associated with a lower ABI in asymptomatic subjects from the community, and this association was modified by age.
arterial stiffness; ankle-brachial index; arteries
Osteocalcin has been reported to influence insulin secretion in experimental animals. We investigated whether serum osteocalcin was associated with measures of insulin resistance, circulating adipokine levels, and the presence of metabolic syndrome (MetSyn).
Methods and Results
Serum osteocalcin was measured by solid-phase sandwich immunoassay in 1284 blacks (64±9 years; 71% women) and 1209 non-Hispanic whites (59±10 years; 57% women) belonging to hypertensive sibships. MetSyn was defined per Adult Treatment Panel III criteria. The prevalence of MetSyn was 50% in blacks and 49% in non-Hispanic whites. In each ethnic group, after adjustment for age and gender, osteocalcin levels were inversely correlated with body mass index, fasting glucose and insulin, homeostasis model assessment of insulin resistance, triglycerides, and leptin, and positively correlated with adiponectin (P<0.001 for each variable). In multivariable regression analyses that adjusted for age, gender, smoking, serum creatinine, and statin and estrogen use, osteocalcin levels in the highest quartile (compared with the lowest quartile) were associated with a lower odds ratio (OR) of having MetSyn: OR (95% CI) in blacks, 0.33 (0.23 to 0.46); OR in non-Hispanic whites, 0.43 (0.31 to 0.63).
Serum osteocalcin is associated with measures of insulin resistance, adipokine levels, and the presence of MetSyn, suggesting a novel cross-talk between bone and adipose tissue.
adiponectin; insulin resistance; leptin; metabolic syndrome; osteocalcin
To determine whether serum levels of N-terminal (NT) pro–B-type natriuretic peptide (pro-BNP) are higher in patients with poorly compressible arteries (PCA) than in patients with peripheral artery disease (PAD) and control subjects without PCA or PAD.
Methods and Results
Medial arterial calcification in the lower extremities results in PCA and may be associated with increased arterial stiffness and hemodynamic/myocardial stress. PCA was defined as having an ankle-brachial index >1.4 or an ankle blood pressure >255 mm Hg, whereas PAD was defined as having an ankle-brachial index ≤0.9. Study participants with PCA (n=100; aged 71±10 years; 70% men) and age- and sex-matched patients with PAD (n=300) were recruited from the noninvasive vascular laboratory. Age- and sex-matched controls (n=300) were identified from a community-based cohort and had no history of PAD. NT pro-BNP levels were approximately 2.5-fold higher in patients with PCA than in patients with PAD and approximately 4-fold higher than in age- and sex-matched controls. In multivariable regression analyses that adjusted for age, sex, smoking, hypertension, history of coronary heart disease/stroke, systolic blood pressure, and serum creatinine, NT pro-BNP levels remained significantly higher in patients with PCA than in patients with PAD and controls (P<0.001).
Patients with medial arterial calcification and PCA have higher serum levels of NT pro-BNP than patients with PAD and controls, which is suggestive of an adverse hemodynamic milieu and increased risk for adverse cardiovascular outcomes.
medial arterial calcification; poorly compressible arteries; peripheral artery disease; NT pro-BNP; arterial stiffness; natriuretic peptides; risk factors
African-Americans with hypertension are susceptible to left ventricular hypertrophy (LVH). Serum osteoprotegerin level has been reported to be associated with LVH. We investigated the association of osteoprotegerin with LV mass (LVM) in 898 African-Americans with hypertension (mean age 65 years, 71% women).
Osteoprotegerin levels were measured in serum by an immunoassay and log-transformed for analyses. LVM index (LVMi; LVM/height2.7) was estimated using M-mode echocardiography. Linear regression analyses using generalized estimating equations were used to assess the association of osteoprotegerin with LVMi.
Serum osteoprotegerin was correlated with LVMi (r = 0.21; P < 0.0001), an estimated increase in LVMi of 5.05 (95% confidence interval 2.93, 7.17) g/m2.7 in the highest compared to the lowest osteoprotegerin quartile. This association remained statistically significant after adjustment for conventional cardiovascular risk factors (age, sex, body mass index (BMI), history of smoking, diabetes, systolic blood pressure (BP), total and high-density lipoprotein cholesterol), estimated renal function, history of myocardial infarction and stroke, lifestyle factors (physical activity score, years of education, amount of alcohol consumption), medications (aspirin, antihypertensives, statins, estrogens), and C-reactive protein (CRP) (P = 0.02). Additionally, osteoprotegerin was correlated with early/atrial (E/A) ratio (r = −0.16; P < 0.0001), LV mean wall thickness (r = 0.17; P < 0.0001) and relative wall thickness (r = 0.14; P < 0.0001) but not ejection fraction (r = 0.04; P = 0.24) or internal end-diastolic dimension (r = 0.02; P = 0.60).
In African-Americans with hypertension, a higher serum osteoprotegerin level is weakly but independently associated with a higher LVM.
African-American; biomarker; blood pressure; cardiology; echocardiography; epidemiology; hypertension; hypertrophy; osteoprotegerin
Peripheral arterial disease (PAD), a relatively common manifestation of atherosclerotic vascular disease, is associated with significant morbidity and mortality. Although conventional risk factors contribute to the onset and progression of PAD, the role of ‘novel’ biomarkers in pathways of inflammation, thrombosis, lipoprotein metabolism, and oxidative stress in determining susceptibility to PAD is being increasingly recognized. Validation of novel risk factors for PAD may allow earlier detection, an improved understanding of disease etiology and progression, and the development of new therapies. In this review, we discuss available evidence for associations between novel circulating markers and several aspects of PAD including disease susceptibility, progression, functional limitation, and adverse outcomes.
inflammation; peripheral arterial disease; risk factors
Vascular reactivity may affect the stiffness characteristics of the arterial wall. We investigated the association between forearm microcirculatory and conduit artery function and measures of arterial stiffness in 527 asymptomatic non-Hispanic white adults without known cardiovascular disease. High-resolution ultrasonography of the brachial artery (ba) was performed to assess forearm microcirculatory function (ba blood flow velocity, local shear stress, and forearm vascular resistance at rest and during reactive hyperemia) and conduit artery function (ba flow-mediated dilatation baFMD and ba nitroglycerin-mediated dilatation baNMD). Arterial stiffness was assessed by cuff-derived brachial pulse pressure and aortic pulse wave velocity (aPWV) measured by applanation tonometry. In regression analyses that adjusted for heart rate, mean arterial pressure, height, cardiovascular risk factors, and hypertension medication and statin use, higher baseline ba systolic velocity and systolic shear stress were associated with greater pulse pressure (P=0.0002 and P=0.006, respectively) and higher aPWV (each P<0.0001). During hyperemia, lower ba mean velocity and lower mean shear stress were associated with higher pulse pressure (P=0.045 and P=0.036, respectively) while both systolic and mean velocity (P<0.0001 and P=0.002, respectively) and systolic and mean shear stress (P<0.0001 and P=0.003, respectively) were inversely associated with aPWV. baFMD was not associated with pulse pressure but was inversely associated with aPWV (P=0.011). baNMD was inversely associated with pulse pressure (P=0.0002) and aPWV (P=0.008). Our findings demonstrate that impaired forearm microvascular function (in the form of elevated resting blood flow velocity and impaired flow reserve) and impaired brachial artery reactivity are associated with increased arterial stiffness.
microvascular function; flow-mediated dilatation; nitroglycerin-mediated dilatation; arterial stiffness; pulse pressure; pulse wave velocity
Proprotein convertase subtilisin-like kexin type 9 (PCSK9) is a newly discovered serine protease that plays a key role in regulating plasma low-density lipoprotein (LDL) cholesterol levels. Both rare mutations and common variants in the coding regions of PCSK9 affect LDL cholesterol levels and coronary heart disease risk, as well as response to lipid-lowering therapy.
We characterized the patterns of variation at the PCSK9 locus in African-Americans and European-Americans using resequenced data from the SeattleSNPs database (pga.gs.washington.edu). We performed a test of population differentiation and the long range haplotype (LRH) test to detect signatures of recent position selection on PCSK9.
A significantly high FST (a measure of population differentiation) between African-Americans and European-Americans was noted for SNP rs505151 (FST = 0.309). The LRH test was suggestive of non-neutral evolution of two single nucleotide polymorphisms (SNPs) (rs505151 and rs562556) in PCSK9 that are associated with elevated LDL cholesterol levels (‘gain-of-function’ mutations), with differential modes of selection in African-Americans and European-Americans. We observed signals of recent positive selection on the ancestral allele of nonsynonymous SNP rs505151 (E670G, P = 0.0227 and P = 0.0001 in theoretical and empirical distribution, respectively) and the derived allele of nonsynonymous SNP rs562556 (I474V, P = 0.0227 and 0.0001) in African-Americans, whereas in European-Americans the ancestral allele of SNP rs562556 (P = 0.1320 and 0.0370) appeared to be under positive selection.
Our findings suggest that evolutionary dynamics may underlie the gain-of-function mutations in PCSK9 that influence inter-individual variation in LDL cholesterol levels.
PCSK9; low-density lipoprotein cholesterol; natural selection
Myeloperoxidase (MPO) is an enzymatic mediator of several inflammatory cascades and higher serum levels have been associated with increased risk of adverse cardiovascular events. We investigated the association of serum MPO with the ankle-brachial index (ABI) and peripheral arterial disease (PAD), in a bi-ethnic cohort of African-Americans and non-Hispanic whites. Participants included 1324 African-Americans (64 y, 71% women) and 1237 non- Hispanic whites (59 y, 57% women) belonging to hypertensive sibships. Plasma levels of MPO were measured by solid phase sandwich immunoassay. ABI was measured using a standard protocol and PAD defined as ABI <0.90. Multivariable regression analysis using generalized estimating equations (GEE) were performed to assess whether serum MPO levels were associated with ABI and the presence of PAD. After adjustment for age and sex, higher MPO levels were significantly associated with lower ABI and presence of PAD in African-Americans (P=0.004 and P=0.005, respectively) and in non-Hispanic whites (P=0.001and P=0.021, respectively). After additional adjustment for conventional risk factors (diabetes, smoking status, total and HDL cholesterol, waist circumference, hypertension), prior history of myocardial infarction or stroke, and medication use (statins, aspirin, estrogen), higher MPO levels remained significantly associated with lower ABI and presence of PAD in both African- Americans (P=0.008 and P=0.012, respectively) and non-Hispanic whites (P=0.001and P=0.029, respectively). We conclude that higher MPO levels are associated with lower ABI and the presence of PAD in African-Americans and non-Hispanic whites.
peripheral arterial disease; inflammation; ankle-brachial index; myeloperoxidase
Higher plasma concentrations of soluble adhesion molecules have been shown to be associated with increased risk of cardiovascular events. We investigated the associations of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) with the ankle-brachial index (ABI), a measure of peripheral arterial disease (PAD), in a bi-ethnic cohort of adults without known coronary heart disease or stroke.
Participants included 1102 blacks (63 y, 74% women) and 1013 non-Hispanic whites (58 y, 59% women) belonging to hypertensive sibships. Plasma concentrations of sICAM-1 and sVCAM-1 were measured using high-sensitivity immunoassays. ABI was measured using a standard protocol and PAD was defined as ABI <0.9. Generalized estimating equations (GEE) were used to assess whether sICAM-1 and sVCAM-1 were associated with ABI and with PAD, independent of conventional risk factors.
After adjustment for conventional risk factors, blacks with sICAM-1 and sVCAM-1 concentrations in the highest quartiles had lower ABI than those in the lowest quartiles (mean ABI: 1.02 vs. 0.98, P=0.007 and 1.02 vs. 0.99, P=0.003, respectively). In multivariable logistic regression analysis, sICAM-1 and sVCAM-1 concentrations in the highest quartiles were each associated with a higher odds ratio of having PAD, compared with the lowest quartiles: odds ratio (95% CI): 5.2 (1.8–15.2) and 2.2 (1.0–4.8), respectively. In contrast, in non-Hispanic whites, sICAM-1 and sVCAM-1 concentrations were not associated with ABI or with PAD.
Higher sICAM-1 and sVCAM-1 concentrations were independently associated with a lower ABI and with PAD in blacks, but not in non-Hispanic whites.
ethnicity; sICAM-1; sVCAM-1; ankle-brachial index; peripheral arterial disease; hypertension
atherosclerosis; genetics; risk factors; genome-wide association study
Hypertensive African Americans have higher rates of coronary heart disease (CHD) than their non-Hispanic white counterparts despite having higher HDL cholesterol (HDL-C) levels and lower triglyceride levels.
The goal of the present study was to assess whether low-density lipoprotein (LDL) particle size, a correlate of the above lipid traits and a risk factor for CHD, differs between hypertensive African Americans and whites.
Participants included 1177 hypertensive African Americans from Jackson MS (60±7 years, 72.4% women) and 860 hypertensive whites from Rochester MN (58±7 years, 56.7% women). LDL particle size was measured by polyacrylamide gradient gel electrophoresis. Within each sex, we assessed whether ethnicity was significantly associated with differences in LDL particle size after adjustment for CHD risk factors (age, total cholesterol, HDL-C, triglycerides, systolic BP, diabetes, history of smoking, body mass index), statin use, and estrogen use (in women), and “lifestyle” variables (physical activity and alcohol intake).
Although HDL-C levels were higher and triglyceride levels lower in African Americans, LDL particle size (adjusted for CHD risk factors) was lower (P < 0.0001) in African American men and women than in their white counterparts (mean ± SD; men, 267.6±5.2 Å vs. 270.2±4.8 Å; women 268.7±5.1 Å vs. 271.3±5.1 Å). In both sexes, African American ethnicity was associated with lower LDL particle size after adjustment for CHD risk factors, statin use and estrogen use (in women), as well as physical activity and alcohol intake.
Hypertensive African American men and women have lower LDL particle size than their white counterparts despite having higher HDL-C and lower triglycerides.
low-density lipoprotein; LDL particle size; risk factors; ethnicity; hypertension
Peripheral artery disease; genome-wide association studies; genomics
To identify novel genetic loci influencing interindividual variation in red blood cell (RBC) traits in African-Americans, we conducted a genome-wide association study (GWAS) in 2315 individuals, divided into discovery (n = 1904) and replication (n = 411) cohorts. The traits included hemoglobin concentration (HGB), hematocrit (HCT), RBC count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Patients were participants in the electronic MEdical Records and GEnomics (eMERGE) network and underwent genotyping of ~1.2 million single-nucleotide polymorphisms on the Illumina Human1M-Duo array. Association analyses were performed adjusting for age, sex, site, and population stratification. Three loci previously associated with resistance to malaria—HBB (11p15.4), HBA1/HBA2 (16p13.3), and G6PD (Xq28)—were associated (P ≤ 1 × 10−6) with RBC traits in the discovery cohort. The loci replicated in the replication cohort (P ≤ 0.02), and were significant at a genome-wide significance level (P < 5 × 10−8) in the combined cohort. The proportions of variance in RBC traits explained by significant variants at these loci were as follows: rs7120391 (near HBB) 1.3% of MCHC, rs9924561 (near HBA1/A2) 5.5% of MCV, 6.9% of MCH and 2.9% of MCHC, and rs1050828 (in G6PD) 2.4% of RBC count, 2.9% of MCV, and 1.4% of MCH, respectively. We were not able to replicate loci identified by a previous GWAS of RBC traits in a European ancestry cohort of similar sample size, suggesting that the genetic architecture of RBC traits differs by race. In conclusion, genetic variants that confer resistance to malaria are associated with RBC traits in African-Americans.
red blood cell (RBC) traits; genome-wide association study; African-Americans; natural selection; informatics; electronic medical record
Clinical data in Electronic Medical Records (EMRs) is a potential source of longitudinal clinical data for research. The Electronic Medical Records and Genomics Network or eMERGE investigates whether data captured through routine clinical care using EMRs can identify disease phenotypes with sufficient positive and negative predictive values for use in genome wide association studies (GWAS). Using data from five different sets of EMRs, we have identified five disease phenotypes with positive predictive values of 73–98% and negative predictive values of 98–100%. A majority of EMRs captured key information (diagnoses, medications, laboratory tests) used to define phenotypes in a structured format. We identified natural language processing as an important tool to improve case identification rates. Efforts and incentives to increase the implementation of interoperable EMRs will markedly improve the availability of clinical data for genomics research.
Background and method
We investigated whether chronic kidney disease detected by increased serum creatinine (SCr) or urine albumin-to-creatinine ratio (UACR) may reflect arteriosclerosis involving the kidneys. The sample consisted of 1585 members of sibships (804 non-Hispanic whites and 781 non-Hispanic blacks) in which at least two siblings had primary hypertension. We first evaluated the correlations of increased SCr and UACR with the presence of cerebral small vessel arteriosclerosis, which was determined by increased subcortical white matter hyperintensity (WMH) volume on brain magnetic resonance imaging; and with peripheral large vessel arteriosclerosis, which was determined by decreased ankle-brachial index (ABI). After age adjustment, increased SCr and UACR correlated with increased WMH volume (0.54 and 0.52, respectively) and with decreased ABI (0.50 and 0.54, respectively; all P < 0.001). We then used logistic regression to evaluate the dependency of each measure of disease on conventional risk factors for arteriosclerosis to assess whether the risk factors’ effects were proportional across different measures of disease.
Age, race, sex, hypertension, diabetes, total cholesterol, and smoking made similar overall contributions to the prediction of each measure of disease, as judged by the model C-statistics, which varied in a narrow range from 0.84 to 0.85 (all P < 0.001). However, the relative contributions that the modifiable risk factors, including hypertension, diabetes, total cholesterol, and smoking made to prediction of increased SCr and UACR were disproportionate to their relative contributions to prediction of decreased ABI (P < 0.0001).
The findings support the view that chronic kidney disease detected by increased SCr or UACR primarily reflects small vessel arteriosclerosis involving the kidneys.
albuminuria; ankle-brachial blood pressure index; arteriosclerosis; blood pressure; glomerular filtration rate; hypertension; subcortical white matter hyperintensity
Genome-wide association studies (GWAS) are a useful approach in the study of the genetic components of complex phenotypes. Aside from large cohorts, GWAS have generally been limited to the study of one or a few diseases or traits. The emergence of biobanks linked to electronic medical records (EMRs) allows the efficient re-use of genetic data to yield meaningful genotype-phenotype associations for multiple phenotypes or traits. Phase I of the electronic MEdical Records and GEnomics (eMERGE-I) Network is a National Human Genome Research Institute (NHGRI)-supported consortium composed of five sites to perform various genetic association studies using DNA repositories and EMR systems. Each eMERGE site has developed EMR-based algorithms to comprise a core set of fourteen phenotypes for extraction of study samples from each site’s DNA repository. Each eMERGE site selected samples for a specific phenotype, and these samples were genotyped at either the Broad Institute or at the Center for Inherited Disease Research (CIDR) using the Illumina Infinium BeadChip technology. In all, approximately 17,000 samples from across the five sites were genotyped. A unified quality control (QC) pipeline was developed by the eMERGE Genomics Working Group and used to ensure thorough cleaning of the data. This process includes examination of sample quality, marker quality, and various batch effects. Upon completion of the genotyping and QC analyses for each site’s primary study, the eMERGE Coordinating Center merged the datasets from all five sites. This larger merged dataset re-entered the established eMERGE QC pipeline. Based on lessons learned during the process, additional analyses and QC checkpoints were added to the pipeline to ensure proper merging. Here we explore the challenges associated with combining datasets from different genotyping centers and describe the expansion to the eMERGE QC pipeline for merged datasets. These additional steps will be useful as the eMERGE project expands to include additional sites in eMERGE-II and also serve as a starting point for investigators merging multiple genotype data sets accessible through the National Center for Biotechnology Information (NCBI) in the database of Genotypes and Phenotypes (dbGaP). Our experience demonstrates that merging multiple datasets after additional QC can be an efficient use of genotype data despite new challenges that appear in the process.
quality control; genome-wide association (GWAS); eMERGE; dbGaP; merging datasets
Compare survival of patients with poorly compressible arteries (PCA) to those with a normal ankle-brachial index (ABI) and those with peripheral arterial disease (PAD).
Limited data are available regarding survival in patients with PCA identified in the clinical setting by non-invasive lower extremity arterial evaluation.
We conducted a historical cohort study of consecutive patients who underwent outpatient, non-invasive lower extremity arterial evaluation at Mayo Clinic, Rochester, Minnesota, from January 1998 through December 2007, and were followed for a mean duration of 5.8±3.1 years. An ABI 1.00-1.30 was considered normal, PAD was defined as a resting or post-exercise ABI ≤0.90, and PCA defined as an ABI ≥1.4 and/or an ankle systolic blood pressure >255 mm Hg. Patients were followed for all-cause mortality through 09/30/2009.
Of 16,493 individuals (mean age ± standard deviation = 67.8±13.0 years, 59% male); 29% had normal ABI, 54% had PAD, and 17% had PCA. During follow-up (mean duration = 5.8±3.1 years), 4365 patients (26%) died. The percent alive at the end of the study period was 88%, 70%, and 60% for normal ABI, PAD, and PCA respectively. After adjustment for age, sex, cardiovascular risk factors, comorbid conditions, and medication use, the hazard ratios (confidence interval) of death associated with PCA were 2.0 (1.8-2.2) and 1.3 (1.2-1.4) compared to normal ABI and PAD groups, respectively.
Patients identified by non-invasive vascular testing to have poorly compressible leg arteries have poor survival, worse than those with a normal ABI or those with PAD.
peripheral arterial disease; arterial calcification; mortality; ankle-brachial index
To identify common genetic variants influencing red blood cell (RBC) traits.
Patients and Methods
We performed a genomewide association study from June 2008 through July 2011 of hemoglobin, hematocrit, RBC count, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration in 12,486 patients of European ancestry from the electronic MEdical Records and Genomics (eMERGE) network. We developed an electronic medical record–based algorithm that included individuals who had RBC measurements obtained for clinical care and excluded values measured in the setting of hematopoietic disorders, comorbid conditions, or medications known to affect RBC production or a recent history of blood loss.
We identified 4 new genetic loci and replicated 11 loci previously reported to be associated with one or more RBC traits in individuals of European ancestry. Notably, genes present in 3 of the 4 newly identified loci (THRB, PTPLAD1, CDT1) and in 6 of the 11 replicated loci (KLF1, ALDH8A1, CCND3, SPTA1, FBXO7, TFR2/EPO) are implicated in erythroid differentiation and regulation of cell cycle in hematopoietic stem cells.
Genes in the erythroid differentiation and cell cycle regulation pathways influence interindividual variation in RBC indices. Our results provide insights into the molecular basis underlying variation in RBC traits.
eMERGE, electronic MEdical Records and GEnomics; EMMAX, mixed-model association-expedited; EMR, electronic medical record; eQTL, expression quantitative trait locus; GHC, Group Health Cooperative--University of Washington; GWAS, genomewide association study; HCT, hematocrit; HGB, hemoglobin; IBS, identity-by-state; LD, linkage disequilibrium; MC, Marshfield Clinic; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MIM, Mendelian Inheritance of Man; NU, Northwestern University; RBC, red blood cell; SNP, single-nucleotide polymorphism; VUMC, Vanderbilt University Medical Center
The metabolic syndrome (MetS) is defined as concomitant disorders of lipid and glucose metabolism, central obesity, and high blood pressure, with an increased risk of type 2 diabetes and cardiovascular disease. This study tests whether common genetic variants with pleiotropic effects account for some of the correlated architecture among five metabolic phenotypes that define MetS.
RESEARCH DESIGN AND METHODS
Seven studies of the STAMPEED consortium, comprising 22,161 participants of European ancestry, underwent genome-wide association analyses of metabolic traits using a panel of ∼2.5 million imputed single nucleotide polymorphisms (SNPs). Phenotypes were defined by the National Cholesterol Education Program (NCEP) criteria for MetS in pairwise combinations. Individuals exceeding the NCEP thresholds for both traits of a pair were considered affected.
Twenty-nine common variants were associated with MetS or a pair of traits. Variants in the genes LPL, CETP, APOA5 (and its cluster), GCKR (and its cluster), LIPC, TRIB1, LOC100128354/MTNR1B, ABCB11, and LOC100129150 were further tested for their association with individual qualitative and quantitative traits. None of the 16 top SNPs (one per gene) associated simultaneously with more than two individual traits. Of them 11 variants showed nominal associations with MetS per se. The effects of 16 top SNPs on the quantitative traits were relatively small, together explaining from ∼9% of the variance in triglycerides, 5.8% of high-density lipoprotein cholesterol, 3.6% of fasting glucose, and 1.4% of systolic blood pressure.
Qualitative and quantitative pleiotropic tests on pairs of traits indicate that a small portion of the covariation in these traits can be explained by the reported common genetic variants.
Peripheral arterial disease (PAD) is a relatively common manifestation of systemic atherosclerosis that leads to progressive narrowing of the lumen of leg arteries. Circulating monocytes are in contact with the arterial wall and can serve as reporters of vascular pathology in the setting of PAD. We performed gene expression analysis of peripheral blood mononuclear cells (PBMC) in patients with PAD and controls without PAD to identify differentially regulated genes.
PAD was defined as an ankle brachial index (ABI) ≤0.9 (n = 19) while age and gender matched controls had an ABI > 1.0 (n = 18). Microarray analysis was performed using Affymetrix HG-U133 plus 2.0 gene chips and analyzed using GeneSpring GX 11.0. Gene expression data was normalized using Robust Multichip Analysis (RMA) normalization method, differential expression was defined as a fold change ≥1.5, followed by unpaired Mann-Whitney test (P < 0.05) and correction for multiple testing by Benjamini and Hochberg False Discovery Rate. Meta-analysis of differentially expressed genes was performed using an integrated bioinformatics pipeline with tools for enrichment analysis using Gene Ontology (GO) terms, pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG), molecular event enrichment using Reactome annotations and network analysis using Ingenuity Pathway Analysis suite. Extensive biocuration was also performed to understand the functional context of genes.
We identified 87 genes differentially expressed in the setting of PAD; 40 genes were upregulated and 47 genes were downregulated. We employed an integrated bioinformatics pipeline coupled with literature curation to characterize the functional coherence of differentially regulated genes.
Notably, upregulated genes mediate immune response, inflammation, apoptosis, stress response, phosphorylation, hemostasis, platelet activation and platelet aggregation. Downregulated genes included several genes from the zinc finger family that are involved in transcriptional regulation. These results provide insights into molecular mechanisms relevant to the pathophysiology of PAD.
Peripheral arterial disease; Gene expression; Microarray analysis; Vascular disease; Biomarkers