Daniel A. Enquobahrie

Systematic identification of trans eQTLs as putative drivers of known disease associations

Identifying the downstream effects of disease-associated SNPs is challenging. To help overcome this problem, we performed expression quantitative trait locus (eQTL) meta-analysis in non-transformed peripheral blood samples from 5,311 individuals with replication in 2,775 individuals. We identified and replicated trans eQTLs for 233 SNPs (reflecting 103 independent loci) that were previously associated with complex traits at genome-wide significance. Some of these SNPs affect multiple genes in trans that are known to be altered in individuals with disease: rs4917014, previously associated with systemic lupus erythematosus (SLE), altered gene expression of C1QB and five type I interferon response genes, both hallmarks of SLE. DeepSAGE RNA sequencing showed that rs4917014 strongly alters the 3′ UTR levels of IKZF1 in cis, and chromatin immunoprecipitation and sequencing analysis of the trans-regulated genes implicated IKZF1 as the causal gene. Variants associated with cholesterol metabolism and type 1 diabetes showed similar phenomena, indicating that large-scale eQTL mapping provides insight into the downstream effects of many trait-associated variants.

Placental microRNA expression in pregnancies complicated by preeclampsia

OBJECTIVE The role of posttranscription regulation in preeclampsia is largely unknown. We investigated preeclampsia-related placental microRNA (miRNA) expression using microarray and confirmatory quantitative real-time polymerase chain reaction experiments. STUDY DESIGN Placental expressions of characterized and novel miRNAs (1295 probes) were measured in samples collected from 20 preeclampsia cases and 20 controls. Differential expression was evaluated using Student t test and fold change analyses. In pathway analysis, we examined functions/functional relationships of targets of differentially expressed miRNAs. RESULTS Eight miRNAs were differentially expressed (1 up-regulated and 7 down-regulated) among preeclampsia cases compared with controls. These included previously identified candidates (miR-210, miR-1, and a miRNA in the 14q32.31 cluster region) and others that are novel (miR-584 and miR-34c-5p). These miRNAs target genes that participate in organ/system development (cardiovascular and reproductive system), immunologic dysfunction, cell adhesion, cell cycle, and signaling. CONCLUSION Expression of miRNAs that target genes in diverse pathophysiological processes is altered in the setting of preeclampsia.

Glucose intolerance and gestational diabetes risk in relation to sleep duration and snoring during pregnancy: a pilot study

BackgroundInsufficient sleep and poor sleep quality, considered endemic in modern society, are associated with obesity, impaired glucose tolerance and diabetes. Little, however, is known about the consequences of insufficient sleep and poor sleep quality during pregnancy on glucose tolerance and gestational diabetes.MethodsA cohort of 1,290 women was interviewed during early pregnancy. We collected information about sleep duration and snoring during early pregnancy. Results from screening and diagnostic testing for gestational diabetes mellitus (GDM) were abstracted from medical records. Generalized linear models were fitted to derive relative risk (RR) and 95% confidence intervals (95% CIs) of GDM associated with sleep duration and snoring, respectively.ResultsAfter adjusting for maternal age and race/ethnicity, GDM risk was increased among women sleeping ≤ 4 hours compared with those sleeping 9 hours per night (RR = 5.56; 95% CI 1.31-23.69). The corresponding RR for lean women (<25 kg/m2) was 3.23 (95% CI 0.34-30.41) and 9.83 (95% CI 1.12-86.32) for overweight women (≥ 25 kg/m2). Overall, snoring was associated with a 1.86-fold increased risk of GDM (RR = 1.86; 95% CI 0.88-3.94). The risk of GDM was particularly elevated among overweight women who snored. Compared with lean women who did not snore, those who were overweight and snored had a 6.9-fold increased risk of GDM (95% CI 2.87-16.6).ConclusionsThese preliminary findings suggest associations of short sleep duration and snoring with glucose intolerance and GDM. Though consistent with studies of men and non-pregnant women, larger studies that include objective measures of sleep duration, quality and apnea are needed to obtain more precise estimates of observed associations.

Differential placental gene expression in preeclampsia.

OBJECTIVE Candidate genes that are associated with preeclampsia have not been described fully. We conducted microarray and confirmatory quantitative real time polymerase chain reaction studies to investigate global placental gene expression in preeclampsia. STUDY DESIGN RNA was extracted from placental samples that were collected from 18 preeclampsia cases and 18 normotensive control subjects. Oligonucleotide probes that represented 22,000 genes were used to measure gene expression in each sample. Differential gene expression was evaluated with the Student t test, fold change assessment, and significance analysis of microarrays. Functions and functional relationships of differentially expressed genes were evaluated. RESULTS Genes (n = 58) that participated in immune system, inflammation, oxidative stress, signaling, growth, and development pathways were expressed differentially in preeclampsia. These genes included previously described candidate genes (such as leptin), potential candidate genes with related functions (such as CYP11A) and novel genes (such as CDKN1C). CONCLUSION Expression of genes (both candidate and novel) with diverse functions is associated with preeclampsia risk, which reflects the complex pathogenesis.

Vitamin D, Parathyroid Hormone, and Sudden Cardiac Death Results From the Cardiovascular Health Study

Recent studies have demonstrated greater risks of cardiovascular events and mortality among persons who have lower 25-hydroxyvitamin D (25-OHD) and higher parathyroid hormone (PTH) levels. We sought to evaluate the association between markers of mineral metabolism and sudden cardiac death (SCD) among the 2312 participants from the Cardiovascular Health Study who were free of clinical cardiovascular disease at baseline. We estimated associations of baseline 25-OHD and PTH concentrations individually and in combination with SCD using Cox proportional hazards models after adjustment for demographics, cardiovascular risk factors, and kidney function. During a median follow-up of 14 years, there were 73 adjudicated SCD events. The annual incidence of SCD was greater among subjects who had lower 25-OHD concentrations, 2 events per 1000 for 25-OHD ≥20 ng/mL and 4 events per 1000 for 25-OHD <20 ng/mL. Similarly, SCD incidence was greater among subjects who had higher PTH concentrations, 2 events per 1000 for PTH <65 pg/mL and 4 events per 1000 for PTH ≥65 pg/mL. Multivariate adjustment attenuated associations of 25-OHD and PTH with SCD. Finally, 267 participants (11.7% of the cohort) had high PTH and low 25-OHD concentrations. This combination was associated with a >2-fold risk of SCD after adjustment (hazard ratio: 2.19 [95% CI: 1.17–4.10]; P=0.017) compared with participants with normal levels of PTH and 25-OHD. The combination of lower 25-OHD and higher PTH concentrations appears to be associated independently with SCD risk among older adults without cardiovascular disease.

New-Onset Diabetes and Risk of All-Cause and Cardiovascular Mortality The Cardiovascular Health Study

OBJECTIVE—Cardiovascular risk associated with new-onset diabetes is not well characterized. We hypothesized that risk of all-cause and cardiovascular mortality would be similar among participants with and without new-onset diabetes in the first years of follow-up and rise over time for new-onset diabetes. RESEARCH DESIGN AND METHODS—The Cardiovascular Health Study (CHS) is a longitudinal study of cardiovascular risk factors in adults aged ≥65 years. We used CHS participants to define a cohort (n = 282) with new-onset diabetes during 11 years of follow-up. New-onset diabetes was defined by initiation of antidiabetes medication or by fasting plasma glucose >125 mg/dl among CHS participants without diabetes at study entry. Three CHS participants without diabetes were matched for age, sex, and race to each participant with new-onset diabetes at the time of diabetes identification (n = 837). Survival analysis provided adjusted hazard ratios (HRs) for all-cause and cardiovascular mortality. RESULTS—During a median of 5.9 years of follow-up, there were 352 deaths, of which 41% were cardiovascular. In adjusted analyses, new-onset diabetes was associated with an HR of 1.9 (95% CI 1.4–2.5) for all-cause and 2.2 (1.4–3.4) for cardiovascular mortality compared with no diabetes. Mortality risks were elevated within 2 years of onset, especially cardiovascular risk (4.3 [95% CI 1.7–10.8]), and did not increase over time. CONCLUSIONS—Our findings indicate that there may be a mortality differential soon after diabetes onset in older adults and suggest that long-term macrovascular damage from atherosclerosis may not be primarily responsible for increased risk.

Gestational Diabetes Mellitus in Relation to Maternal Dietary Heme Iron and Nonheme Iron Intake

OBJECTIVE Higher heme iron intake is associated with increased type 2 diabetes risk. However, no previous study has evaluated gestational diabetes mellitus (GDM) risk in relation to heme iron intake during pregnancy. We investigated associations of maternal preconceptional and early pregnancy heme and nonheme iron intake with subsequent GDM risk. RESEARCH DESIGN AND METHODS We conducted a prospective cohort study of 3,158 pregnant women. A food frequency questionnaire was used to assess maternal diet. Multivariable generalized linear regression models were used to derive estimates of relative risks (RRs) and 95% CIs. RESULTS Approximately 5.0% of the cohort developed GDM (n = 158). Heme iron intake was positively and significantly associated with GDM risk (Ptrend = 0.04). After adjusting for confounders, women reporting the highest heme iron intake levels (≥1.52 vs. <0.48 mg per day) experienced a 3.31-fold–increased GDM risk (95% CI 1.02–10.72). In fully adjusted models, we noted that a 1-mg per day increase in heme iron was associated with a 51% increased GDM risk (RR 1.51 [95% CI 0.99–2.36]). Nonheme iron was inversely, though not statistically significantly, associated with GDM risk, and the corresponding RRs were 1.00, 0.83, 0.62, and 0.61 across quartiles of nonheme iron intake (Ptrend = 0.08). CONCLUSIONS High levels of dietary heme iron intake during the preconceptional and early pregnancy period may be associated with increased GDM risk. Associations of GDM risk with dietary nonheme iron intake are less clear. Confirmation of these findings by future studies is warranted.

Cholesterol Ester Transfer Protein, Interleukin-8, Peroxisome Proliferator Activator Receptor Alpha, and Toll-Like Receptor 4 Genetic Variations and Risk of Incident Nonfatal Myocardial Infarction and Ischemic Stroke

Variations in candidate genes participating in oxidative stress, inflammation, and their interactions are potentially associated with diseases of atherosclerotic origin. We investigated independent and joint associations of variations in cholesterol ester transfer protein (CETP), interleukin-8 (IL8), peroxisome proliferator activator receptor-alpha (PPARA), and Toll-like receptor 4 (TLR4) genes with incident nonfatal myocardial infarction (MI) or ischemic stroke. In a population-based case-control study, patients (848 with MI and 368 with ischemic stroke) and controls (2,682) were recruited from postmenopausal women and hypertensive men/women who were members of Group Health in western Washington State. Common tag single-nucleotide polymorphisms (SNPs; n=34) representing gene-wide variations were selected from gene sequencing data using pairwise linkage disequilibrium. Haplotypes were inferred using a modified expectation maximization algorithm. Multivariate logistic regression evaluated individual haplotype and SNP-disease associations in log-additive models. Global haplotype tests assessed overall gene-disease associations. Logic regression was used to evaluate gene-gene interactions. False discovery rates and permutation tests were used for multiple testing adjustment in evaluating independent associations and interactions, respectively. Overall, gene-wide variations in PPARA and TLR4 genes were associated with MI. The minor allele of the PPARA SNP, rs4253623, was associated with a higher risk of MI (odds ratio 1.25, 95% confidence interval 1.08 to 1.46), whereas the minor allele of the TLR4 SNP, rs1927911, was associated with a lower risk of MI (odds ratio 0.88, 95% confidence interval 0.77 to 0.99). No within-gene or gene-gene interaction was associated with MI or ischemic stroke risk. In conclusion, potential SNP-disease associations identified in the present study are novel and need further investigation.

Early pregnancy vitamin D status and risk of preeclampsia

BACKGROUND Low vitamin D status in pregnancy was proposed as a risk factor of preeclampsia. METHODS We assessed the effect of vitamin D supplementation (4,400 vs. 400 IU/day), initiated early in pregnancy (10-18 weeks), on the development of preeclampsia. The effects of serum vitamin D (25-hydroxyvitamin D [25OHD]) levels on preeclampsia incidence at trial entry and in the third trimester (32-38 weeks) were studied. We also conducted a nested case-control study of 157 women to investigate peripheral blood vitamin D-associated gene expression profiles at 10 to 18 weeks in 47 participants who developed preeclampsia. RESULTS Of 881 women randomized, outcome data were available for 816, with 67 (8.2%) developing preeclampsia. There was no significant difference between treatment (N = 408) or control (N = 408) groups in the incidence of preeclampsia (8.08% vs. 8.33%, respectively; relative risk: 0.97; 95% CI, 0.61-1.53). However, in a cohort analysis and after adjustment for confounders, a significant effect of sufficient vitamin D status (25OHD ≥30 ng/ml) was observed in both early and late pregnancy compared with insufficient levels (25OHD <30 ng/ml) (adjusted odds ratio, 0.28; 95% CI, 0.10-0.96). Differential expression of 348 vitamin D-associated genes (158 upregulated) was found in peripheral blood of women who developed preeclampsia (FDR <0.05 in the Vitamin D Antenatal Asthma Reduction Trial [VDAART]; P < 0.05 in a replication cohort). Functional enrichment and network analyses of this vitamin D-associated gene set suggests several highly functional modules related to systematic inflammatory and immune responses, including some nodes with a high degree of connectivity. CONCLUSIONS Vitamin D supplementation initiated in weeks 10-18 of pregnancy did not reduce preeclampsia incidence in the intention-to-treat paradigm. However, vitamin D levels of 30 ng/ml or higher at trial entry and in late pregnancy were associated with a lower risk of preeclampsia. Differentially expressed vitamin D-associated transcriptomes implicated the emergence of an early pregnancy, distinctive immune response in women who went on to develop preeclampsia. TRIAL REGISTRATION ClinicalTrials.gov NCT00920621. FUNDING Quebec Breast Cancer Foundation and Genome Canada Innovation Network. This trial was funded by the National Heart, Lung, and Blood Institute. For details see Acknowledgments.

Risk of Gestational Diabetes Mellitus in Relation to Maternal Egg and Cholesterol Intake

Higher egg and cholesterol intakes are associated with increased risk of type 2 diabetes mellitus. However, their association with gestational diabetes mellitus (GDM) has not been evaluated. The authors assessed such associations in both a prospective cohort study (1996-2008; 3,158 participants) and a case-control study (1998-2002; 185 cases, 411 controls). A food frequency questionnaire was used to assess maternal diet. Multivariable models were used to derive relative risks and 95% confidence intervals. Compared with no egg consumption, adjusted relative risks for GDM were 0.94, 1.01, 1.12, 1.54, and 2.52 for consumption of ≤1, 2-3, 4-6, 7-9, and ≥10 eggs/week, respectively (P for trend=0.008). Women with high egg consumption (≥7/week) had a 1.77-fold increased risk compared with women with lower consumption (95% confidence interval (CI): 1.19, 2.63). The relative risk for the highest quartile of cholesterol intake (≥294 mg/day) versus the lowest (<151 mg/day) was 2.35 (95% CI: 1.35, 4.09). In the case-control study, the adjusted odds ratio for consuming ≥7 eggs/week versus <7 eggs/week was 2.65 (95% CI: 1.48, 4.72), and the odds of GDM increased with increasing cholesterol intake (P for trend=0.021). In conclusion, high egg and cholesterol intakes before and during pregnancy are associated with increased risk of GDM.