Strong Heart Study Research Articles

Plasma epoxyeicosatrienoic acids and dihydroxyeicosatrieonic acids, insulin, glucose and risk of diabetes: The strong heart study

BackgroundEpoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid with multiple biological functions. Rodent experiments suggest EETs play a role in insulin sensitivity and diabetes, but evidence in humans is limited. To address this knowledge gap, we conducted a case-cohort study in the Strong Heart Family Study, a prospective cohort among American Indians. MethodsWe measured 4 EET species and 4 species of corresponding downstream metabolites, dihydroxyeicosatrieonic acids (DHETs), in plasma samples from 1161 participants, including 310 with type 2 diabetes. We estimated the associations of total (esterified and free) EETs and DHETs with incident diabetes risk, adjusting for known risk factors. We also examined cross-sectional associations with plasma fasting insulin and glucose in the case-cohort and in 271 participants without diabetes from the older Strong Heart Study cohort, and meta-analyzed the results from the 2 cohorts. FindingsWe observed no significant association of total EET or DHET levels with incident diabetes. In addition, plasma EETs were not associated with plasma insulin or plasma glucose. However, higher plasma 14,15-DHET was associated with lower plasma insulin and lower plasma glucose. InterpretationIn this first prospective study of EETs and diabetes, we found no evidence for a role of total plasma EETs in diabetes. The novel associations of 14,15-DHET with insulin and glucose warrant replication and exploration of possible mechanisms. FundingUS National Institutes of Health

DNA methylation and cancer incidence: lymphatic\u2013hematopoietic versus solid cancers in the Strong Heart Study

BackgroundEpigenetic alterations may contribute to early detection of cancer. We evaluated the association of blood DNA methylation with lymphatic–hematopoietic cancers and, for comparison, with solid cancers. We also evaluated the predictive ability of DNA methylation for lymphatic–hematopoietic cancers.MethodsBlood DNA methylation was measured using the Illumina Infinium methylationEPIC array in 2324 Strong Heart Study participants (41.4% men, mean age 56 years). 788,368 CpG sites were available for differential DNA methylation analysis for lymphatic–hematopoietic, solid and overall cancers using elastic-net and Cox regression models. We conducted replication in an independent population: the Framingham Heart Study. We also analyzed differential variability and conducted bioinformatic analyses to assess for potential biological mechanisms.ResultsOver a follow-up of up to 28 years (mean 15), we identified 41 lymphatic–hematopoietic and 394 solid cancer cases. A total of 126 CpGs for lymphatic–hematopoietic cancers, 396 for solid cancers, and 414 for overall cancers were selected as predictors by the elastic-net model. For lymphatic–hematopoietic cancers, the predictive ability (C index) increased from 0.58 to 0.87 when adding these 126 CpGs to the risk factor model in the discovery set. The association was replicated with hazard ratios in the same direction in 28 CpGs in the Framingham Heart Study. When considering the association of variability, rather than mean differences, we found 432 differentially variable regions for lymphatic–hematopoietic cancers.ConclusionsThis study suggests that differential methylation and differential variability in blood DNA methylation are associated with lymphatic–hematopoietic cancer risk. DNA methylation data may contribute to early detection of lymphatic–hematopoietic cancers.

Novel DNA methylation signatures of tobacco smoking with trans-ethnic effects

BackgroundSmoking remains one of the leading preventable causes of death. Smoking leaves a strong signature on the blood methylome as shown in multiple studies using the Infinium HumanMethylation450 BeadChip. Here, we explore novel blood methylation smoking signals on the Illumina MethylationEPIC BeadChip (EPIC) array, which also targets novel CpG-sites in enhancers.MethodA smoking-methylation meta-analysis was carried out using EPIC DNA methylation profiles in 1407 blood samples from four UK population-based cohorts, including the MRC National Survey for Health and Development (NSHD) or 1946 British birth cohort, the National Child Development Study (NCDS) or 1958 birth cohort, the 1970 British Cohort Study (BCS70), and the TwinsUK cohort (TwinsUK). The overall discovery sample included 269 current, 497 former, and 643 never smokers. Replication was pursued in 3425 trans-ethnic samples, including 2325 American Indian individuals participating in the Strong Heart Study (SHS) in 1989–1991 and 1100 African-American participants in the Genetic Epidemiology Network of Arteriopathy Study (GENOA).ResultsAltogether 952 CpG-sites in 500 genes were differentially methylated between smokers and never smokers after Bonferroni correction. There were 526 novel smoking-associated CpG-sites only profiled by the EPIC array, of which 486 (92%) replicated in a meta-analysis of the American Indian and African-American samples. Novel CpG sites mapped both to genes containing previously identified smoking-methylation signals and to 80 novel genes not previously linked to smoking, with the strongest novel signal in SLAMF7. Comparison of former versus never smokers identified that 37 of these sites were persistently differentially methylated after cessation, where 16 represented novel signals only profiled by the EPIC array. We observed a depletion of smoking-associated signals in CpG islands and an enrichment in enhancer regions, consistent with previous results.ConclusionThis study identified novel smoking-associated signals as possible biomarkers of exposure to smoking and may help improve our understanding of smoking-related disease risk.

Glomerular filtration function decline, mortality, and cardiovascular events: data from the Strong Heart Study.

Rapid kidney decline is associated with mortality and cardiovascular disease, even in the absence of chronic kidney disease. American Indians (AI) have particularly high burden of kidney disease, cardiovascular disease, and stroke. This study aims to examine extreme loss in glomerular function in this population in association with clinical outcomes. The Strong Heart Study, a large longitudinal cohort of adult AI participants, collected plasma creatinine at 3 examination visits between 1989-1999. Intraindividual regressions of estimated glomerular filtration rate (eGFR) provided linear estimates of change in kidney function over this time period. Surveillance with physician adjudication identified mortality and cardiovascular events between visit 3 through 2017. Mean change in eGFR was loss 6.8 mL/min over the ten year baseline (range: -66.0 to +28.9 mL/min). The top 1 percentile lost approximately 5.7 mL/min/year. Participants with extreme eGFR loss were more likely to have diabetes (95% vs 71%), hypertension (49% vs 33%), or longer smoking history, among smokers (19 pack years vs 17 pack years). CKD (eGFR<60 mL/min) was associated only with mortality, independent of slope: HR 1.1 (95% CI 1.0-1.3). However, extreme loss in eGFR (>20 mL/min over baseline period) was associated with mortality, independent of baseline eGFR: HR 3.5 (95% CI 2.7-4.4), and also independently associated with composite CVD events and CHF: HR 1.4 and 1.7 (95% CI 1.1-1.9 and 1.2-2.6), respectively. This is the first examination of decline in eGFR in association with mortality and CVD among AIs. The implications of these findings are broad: clinical evaluation may benefit from evaluating change in eGFR over time in addition to dichotomous eGFR. Also, these findings suggest there may be aspects of renal function that are not well-marked by clinical CKD, but which may have particular relevance to long-term renal and vascular health.

Urinary Arsenic and Cadmium Associations with Findings from Cranial MRI in American Indians: Data from the Strong Heart Study.

Background:Arsenic and cadmium are known cardiovascular toxicants that pose disproportionate risk to rural communities where environmental exposures are high. American Indians have high vascular risk, which may be attributable in part to these exposures.Objective:We examined urine metal concentrations in association with magnetic resonance imaging findings of vascular brain injury or cerebral atrophy in adult American Indians.Methods:We measured arsenic and cadmium in American Indian participants from the Strong Heart Study (1989–1991) and evaluated these associations with later (2010–2013) measures of infarct, hemorrhage, white matter hyperintensity (WMH) grade, brain and hippocampal volume, and sulcal and ventricle atrophy using nested multivariate regression analyses.Results:Among participants with available data (), the median urine arsenic:creatinine ratio was [interquartile range (IQR): 4.90–11.93] and the cadmium:creatinine ratio was (IQR: 0.61–1.51). Median time between metal measurement and brain imaging was 21 y (range: 18–25 y). Statistical models detected significant associations between arsenic and higher burden of WMH [ (95% CI: 0.000, 0.028) per 10% increase in arsenic]; and between cadmium and presence of lacunar infarcts [ (95% CI: 1.004, 1.045) per 10% increase in cadmium].Discussion:This population-based cohort of American Indian elders had measured values of urine arsenic and cadmium several times higher than previous population- and clinic-based studies in the United States and Mexico, and comparable values with European industrial workers. Our findings of associations for arsenic and cadmium exposures with vascular brain injury are consistent with established literature. Environmental toxicant accumulation is modifiable; public health policy may benefit from focusing on reductions in environmental metals. https://doi.org/10.1289/EHP6930

APOE genotype, brain imaging, and neuropsychological testing markers of Alzheimer’s disease: Data from the Strong Heart Study

AbstractBackgroundCarrying an APOE ε4 allele increases risk for Alzheimer’s disease and related dementias. However, risk effects and prevalence of risk alleles are not consistent across races/ethnicities. Prevalence of cognitive impairment or dementia and attributable risk from APOE ε4 genotype are unknown in American Indians (AI). This project evaluated associations of APOE genotype with cranial imaging and cognitive testing markers of vascular and Alzheimer’s dementias in a large, prospective, population‐based cohort of AI.MethodThe Strong Heart Study recruited 4,549 AI aged 45‐75 from 13 tribal communities across 3 major geographic regions in 1989‐1991. In 2010‐2013, all eligible surviving participants, then aged 65‐95, underwent cognitive examination including 3MS, WAIS, COWA, and CVLT‐II long delay free recall and cranial magnetic resonance imaging (MRI). APOE was genotyped from blood using ABI PRISM Linkage Mapping and DNA sequencing.ResultMost participants carried either 3.3 (73%) or 4.3 genotypes (23%). Clinical risk factors for dementia were prevalent, including high body mass index (mean&gt;30), hypertension (80%), diabetes (48%), high LDL (39‐44%), chronic kidney disease (27%), and prior stroke (8‐9%). APOE ε4 carriers did not differ from non‐carriers in clinical factors, with the exception of serum LDL (103.9 mg/dL vs 96.7 mg/dL, P=0.015). Findings from MRI were also similar between genotypes, including presence of infarcts or hemorrhages, brain volume, and grade (0‐9) for white matter hyperintensities, sulcal widening, and ventricle enlargement. APOE ε4 carriers had marginally more WMH volume than non‐carriers. Cognitive examination scores were also similar, including Modified Mini Mental Status Examination (3MSE), Weschler Adult Intelligence Scale coding test (WAIS), Controlled Oral Word Association test (COWA), California Verbal Learning Test long delay free recall (CVLT).ConclusionDespite evidence for higher risk of Alzheimer’s and related dementias from presence of APOE ε4 allele among individuals from other populations, our study found little evidence of such an effect in elderly AIs. These findings raise intriguing questions about the presence of as‐yet‐unknown protective factors. Limitations of this study include cross‐sectional analyses and possibility of selective survival before the MRI examination. Future research should directly examine whether longitudinal changes in cognition and brain structure are associated with APOE genotype.

Abstract 14265: Physical Activity Lowers Cardiovascular Mortality by Mediating Systemic Inflammation: The Strong Heart Study

Background: Prior work has shown that fibrinogen, a well-known inflammatory marker, is associated with cardiovascular (CV) events. While it is known that physical activity (PA) imparts CV health benefits, there remains a knowledge gap on the interactions of PA with systemic inflammation on risk of CV death among American Indians. Hypothesis: To investigate the relationship between PA, inflammation, and CV death, we tested the hypotheses that: (i) PA is associated with lower risk of CV death, and (ii) the CV benefit of PA is mediated via an inflammatory pathway. Methods: We examined the association between PA (leisure-time plus occupational as METs[Metabolic Equivalent of Task]-hours per week via a validated questionnaire) and CV death among 3,135 adults (mean age 56 ± 8) without baseline CV disease in Strong Heart Study (1989-1991), a longitudinal study among American Indians. Preacher and Hayes bootstrap method was used for the model assessing mediation by fibrinogen. Results: During 26 years (mean 17.3 ± 8 years) of follow-up, there were 378 CV deaths. Compared to participants with minimal activity, subjects with higher levels of PA had a lower risk of CV death. Odds ratios were 0.91 (95% confidence interval [CI]: 0.65-1.21), 0.69 (95% CI: 0.51-0.92), and 0.56 (95% CI: 0.39-0.71) (p-trend=0.003), for increasing quartile of activity compared to the lowest, after adjustment for age, sex, study site, education, smoking, diabetes, hypertension, BMI, LDL cholesterol, and urine albumin-creatinine ratio. Mediation analysis showed the effect of PA on CV death was fully mediated via fibrinogen level (correlation coefficient: 0.998, 95% CI 0.995-0.999; Sobel test z= -3.11, p=0.03) (Figure). Conclusion: In a population-based study of American Indians, PA compared to inactivity, was associated with lower risk of CV death. This study highlights that PA is associated with improved CV outcomes and this effect is largely driven by inflammation.

Common DNA methylation signatures of arsenic exposure and incident cardiovascular disease in the Strong Heart Study

Background: Arsenic (As) has been related to increased cardiovascular disease (CVD) risk in the Strong Heart Study (SHS), a population-based cohort in American Indian communities. Epigenetic dysregulations have been proposed as potential mechanisms for As-related CVD. We used a method that allows simultaneously modeling large numbers of CpGs to evaluate the association of As-related differentially methylated positions (DMPs) with incident CVD. Methods: 2325 American Indian men and women 45-74 years old in 1989-1991 were followed through 2017. Blood DNA methylation measured at baseline using the EPIC array was pre-processed and corrected for batch effects and cell heterogeneity. Medical ascertainment of clinical events included all CVD, coronary heart disease (CHD), congestive heart failure (CHF), and stroke. Arsenic exposure was measured as the sum of inorganic and methylated species in urine. We used GLMnet penalized regression (elastic-net) to identify DMPs associated with CVD, CHD, CHF, stroke and inorganic As in separate models. Results: A total of 1023 participants had CVD events. The median (IQR) of urinary As was 8.5 (5.2, 14.4) µg/g creatinine. The elastic net model selected 510 CpGs for all CVD, 635 for CHD, 1099 for CHF and 319 for stroke. For urinary As, 321 CpGs were selected. One CpG annotated to TXNIP, which encodes thioredoxin interacting protein, regulates cellular redox signaling, and protects against oxidative stress, was selected for the four CVD outcomes as well as for urinary As. Two CpGs annotated to ARHGAP22 and PRR4 were selected for As and CHD. Three CpGs (one annotated to SLC16A3 and two intergenic) were selected for As and CHF. Conclusions: Common epigenetic signals were jointly associated with urinary As and all and specific CVD endpoints. As arsenic has been related to thioredoxin pathways in experimental models, the role of TXNIP on As-related CVD merits investigation.

Blood-based DNA methylation biomarkers for cumulative lead exposure: associations with cardiovascular disease incidence and mortality in the Strong Heart Study

Lead is a ubiquitous toxicant associated with increased cardiovascular disease (CVD) risk. Blood and bone (patella and tibia) lead are established biomarkers of cumulative lead exposure, but because specialized technologies are needed for analysis, they are typically not available for clinical and epidemiological applications. We recently developed and validated three polygenic biomarkers that estimate lead exposure in blood, patella, and tibia samples based only on blood DNA methylation (DNAm) data. Whether these methylation-biomarkers predict lead-related CVD risk is unknown. We thus investigated the associations of these DNAm lead biomarkers with CVD incidence and mortality in the Strong Heart Study (SHS), a population-based cohort study of CVD in American Indian adults recruited in 1989-1991. The three DNAm lead biomarkers were computed from Illumina EPIC BeadChip blood DNAm data available at baseline among 2,321 SHS participants aged 45-75 years (~41% male). During a median follow-up of ~20 years, 1,023 participants had incident CVD events and 452 died due to CVD. After adjustment for sociodemographic and cardiovascular risk factors, the three DNAm lead biomarkers were associated with CVD mortality, but not with non-fatal CVD events. The hazard ratios (95% CI) for CVD mortality were 1.15 (1.01 - 1.31), 1.12 (1.01 - 1.25), and 1.20 (1.06 - 1.37) per interquartile increase in DNAm-estimated blood, patella, and tibia lead biomarkers. All biomarkers were also associated with coronary heart disease mortality. In subgroup analyses, the associations of lead biomarkers with CVD mortality were stronger in males and ever smokers. These newly developed DNAm lead biomarkers may aid in identifying individuals at risk for CVD mortality resulted from cumulative lead exposure.

Low-level exposure to multiple metals associated with spirometry-defined lung disease in American Indians: Evidence from the Strong Heart Study

Background/Aim: American Indians have a higher burden of chronic lung disease compared to the national average. Several metals are known to induce chronic lung disease at high levels of exposure; however, less is known on the role of low- to moderate-level metal exposure in chronic lung disease. We investigated respiratory effects of environmental exposure to metals and metal-mixtures in American Indians who participated in the Strong Heart Study.Methods: We included participants with data on 6 metals measured from baseline urine samples (1989-1991) and who underwent spirometry testing at visit 2 (1993-1995). We used generalized linear regression to asses associations between metals with spirometry-defined measures of airflow limitation and restrictive ventilatory pattern, and continuous spirometry. In sensitivity analyses, we further adjusted for other metals, accounted for multiple comparisons, and stratified by smoking status. We will use Bayesian Kernel Machine Regression to investigate the joint effects of metal mixtures on lung function and disease.Results: Of the 2,077 participants included, 40% were male, median age was 55 years, 21% had spirometry-defined airflow limitation, and 14% had a restrictive ventilatory pattern. After adjustment for smoking and sociodemographic variables, arsenic, molybdenum, selenium, tungsten, and zinc were associated with increased odds of restrictive ventilatory pattern [OR (95% CI) per interquartile increase: arsenic, 1.31 (1.09, 1.58); molybdenum, 1.51 (1.24, 1.83); selenium, 1.34 (1.05, 1.71); tungsten, 1.22 (1.01, 1.47); zinc, 1.59 (1.25, 1.94)]. Arsenic, cadmium, and zinc were associated with increased odds of airflow limitation, OR (95% CI): 1.31 (1.09, 1.58); 1.19 (0.99, 1.42); 1.14 (0.98, 1.33), respectively. Results stratified by smoking status and further adjusted for other metals were consistent.Conclusions: Low-moderate level exposure to several metals is associated with higher odds of spirometry-defined lung disease in an American Indian population. Environmental exposure to metals may have an under-recognized adverse role on the respiratory system.

Arsenic methylation capacity is associated with locus-specific DNA methylation: an epigenome-wide association study among adults with low-to-moderate arsenic exposure in the United States

Background/Aims: Chronic Arsenic (As) exposure remains a global health problem. Adverse health effects persist decades after arsenic exposure has ended, suggesting a role for epigenetic dysregulation. Methylation of inorganic arsenic (iAs) to monomethylarsonate (MMA) and dimethylarsinate (DMA) by arsenic(III) methyltransferase (AS3MT) decreases toxicity and facilitates urinary excretion. Whether arsenic methylation is associated with DNA methylation is unknown. Methods: We measured DNA methylation at &gt;850,000 loci in 2,325 participants in the Strong Heart Study, a population-based study of American Indian adults exposed to low-moderate levels of arsenic in drinking water. Arsenic methylation was measured as the proportion of arsenic metabolites in urine: iAs%, MMA%, and DMA%. Blood DNA differentially methylated positions (DMPs) associated with each arsenic metabolite were tested using linear models. Gene ontology (GO) analysis was conducted using GOmeth adjusting for array coverage. Models were adjusted for estimated cell type proportions, age, sex, BMI, smoking, education, estimated glomerular filtration rate, and study site. Results: In adjusted models, we identified 84 and 13, 46 and 22, and 112 and 25 CpGs associated with iAs%, MMA%, and DMA% at PFDR &lt; 0.05 / PBonferroni &lt; 0.05, respectively. Seventeen FDR-significant CpGs were common between arsenic metabolites, including 4 CpGs annotated to AS3MT and 13 CpGs located within 650 kilobase pairs up- or downstream of the AS3MT gene and annotated to CNNM2, WBP1L, C10orf95, NT5C2, CALHM1, and TRIM8. Top GO terms overrepresented among genes containing iAs% DMPs were related to muscle hypertrophy; top GO terms associated with MMA% and %DMA% were related to response to tumor cells and regulation of protein catabolic processes. Conclusions: In an epigenome-wide association study, we identified CpGs associated with % arsenic metabolites within a gene region covering AS3MT. Further research is needed to understand the relationship between DNA methylation of AS3MT, gene expression, arsenic methylation capacity and health.

DNA methylation and adiposity phenotypes: an epigenome-wide association study among adults in the Strong Heart Study.

Elevated adiposity is often posited by medical and public health researchers to be a risk factor associated with cardiovascular disease, diabetes, and other diseases. These health challenges are now thought to be reflected in epigenetic modifications to DNA molecules, such as DNA methylation, which can alter gene expression. Here we report the results of three Epigenome Wide Association Studies (EWAS) in which we assessed the differential methylation of DNA (obtained from peripheral blood) associated with three adiposity phenotypes (BMI, waist circumference, and impedance-measured percent body fat) among American Indian adult participants in the Strong Heart Study. We found differential methylation at 8264 CpG sites associated with at least one of our three response variables. Of the three adiposity proxies we measured, waist circumference had the highest number of associated differentially methylated CpGs, while percent body fat was associated with the lowest. Because both waist circumference and percent body fat relate to physiology, we focused interpretations on these variables. We found a low degree of overlap between these two variables in our gene ontology enrichment and Differentially Methylated Region analyses, supporting that waist circumference and percent body fat measurements represent biologically distinct concepts. We interpret these general findings to indicate that highly significant regions of the genome (DMR) and synthesis pathways (GO) in waist circumference analyses are more likely to be associated with the presence of visceral/abdominal fat than more general measures of adiposity. Our findings confirmed numerous CpG sites previously found to be differentially methylated in association with adiposity phenotypes, while we also found new differentially methylated CpG sites and regions not previously identified.

Modified Pooled Cohort Atherosclerotic Cardiovascular Disease Risk Prediction Equations in American Indians

American Indians (AI) have a high prevalence of diabetes, obesity, cardiovascular disease (CVD), and chronic kidney disease. Inclusion of kidney function and other population-specific characteristics in equations used to predict atherosclerotic CVD (ASCVD) risk may help define risk more accurately in populations with these chronic diseases. We used data from the Strong Heart Study (SHS), a population-based longitudinal cohort study of AI, to modify the American College of Cardiology/American Heart Association (ACC/AHA) Pooled Cohort ASCVD risk equations and then explored the performance of the new equations in predicting ASCVD in AI. The study included baseline SHS exam data from 4213 individuals between 45 and 75 years of age, collected in 13 communities from 3 geographic areas in the United States and spanning a wide range of tribal backgrounds, with continuous follow-up data from 1989 to 2015. Using SHS data for blood pressure, diabetes, cholesterol, smoking, and renal function, Cox proportional hazard models were developed to predict ASCVD-free time for AI men and women. ASCVD risk in AI calculated using the SHS-modified equations were compared to risk calculated using the ACC/AHA pooled cohort equations for African Americans (AAs) and Whites. Goodness-of-fit measures for ASCVD risk prediction showed that the SHS-modified equations fit the data from the SHS better than the ACC/AHA equations for AAs and Whites. Adjusting risk prediction equations using population data from the SHS and including measures of renal function significantly improved ASCVD risk prediction in our AI cohort.

Cadmium, Smoking, and Human Blood DNA Methylation Profiles in Adults from the Strong Heart Study.

Background:The epigenetic effects of individual environmental toxicants in tobacco remain largely unexplored. Cadmium (Cd) has been associated with smoking-related health effects, and its concentration in tobacco smoke is higher in comparison with other metals.Objectives:We studied the association of Cd and smoking exposures with human blood DNA methylation (DNAm) profiles. We also evaluated the implication of findings to relevant methylation pathways and the potential contribution of Cd exposure from smoking to explain the association between smoking and site-specific DNAm.Methods:We conducted an epigenome-wide association study of urine Cd and self-reported smoking (current and former vs. never, and cumulative smoking dose) with blood DNAm in 790,026 CpGs (methylation sites) measured with the Illumina Infinium Human MethylationEPIC (Illumina Inc.) platform in 2,325 adults 45–74 years of age who participated in the Strong Heart Study in 1989–1991. In a mediation analysis, we estimated the amount of change in DNAm associated with smoking that can be independently attributed to increases in urine Cd concentrations from smoking. We also conducted enrichment analyses and in silico protein–protein interaction networks to explore the biological relevance of the findings.Results:At a false discovery rate (FDR)-corrected level of 0.05, we found 6 differentially methylated positions (DMPs) for Cd; 288 and 17, respectively, for current and former smoking status; and 77 for cigarette pack-years. Enrichment analyses of these DMPs displayed enrichment of 58 and 6 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes gene sets, respectively, including biological pathways for cancer and cardiovascular disease. In in silico protein-to-protein networks, we observed key proteins in DNAm pathways directly and indirectly connected to Cd- and smoking-DMPs. Among DMPs that were significant for both Cd and current smoking (annotated to PRSS23, AHRR, F2RL3, RARA, and 2q37.1), we found statistically significant contributions of Cd to smoking-related DNAm.Conclusions:Beyond replicating well-known smoking epigenetic signatures, we found novel DMPs related to smoking. Moreover, increases in smoking-related Cd exposure were associated with differential DNAm. Our integrative analysis supports a biological link for Cd and smoking-associated health effects, including the possibility that Cd is partly responsible for smoking toxicity through epigenetic changes. https://doi.org/10.1289/EHP6345

Plasma Ceramide Species Are Associated with Diabetes Risk in Participants of the Strong Heart Study

Few studies have assessed the associations of ceramides and sphingomyelins (SMs) with diabetes in humans. We assessed associations of 15 circulating ceramides and SM species with incident diabetes in 2 studies. The analysis included 435 American-Indian participants from the Strong Heart Study (nested case-control design for analyses; mean age: 57 y; 34% male; median time until diabetes 4.3 y for cases) and 1902 participants from the Strong Heart Family Study (prospective design for analyses; mean age: 37 y; 39% male; median 12.5 y of follow-up). Sphingolipid species were measured using stored plasma samples by sequential LC and MS. Using logistic regression and parametric survival models within studies, and an inverse-variance-weighted meta-analysis across studies, we examined associations of 15 ceramides and SM species with incident diabetes. There were 446 cases of incident diabetes across the studies. Higher circulating concentrations of ceramides containing stearic acid (Cer-18), arachidic acid (Cer-20), and behenic acid (Cer-22) were each associated with a higher risk of diabetes. The RRs for incident diabetes per 1 SD of each log ceramide species (μM) were 1.22 (95% CI: 1.09, 1.37) for Cer-18, 1.18 (95% CI: 1.06, 1.31) for Cer-20, and 1.20 (95% CI: 1.08, 1.32) for Cer-22. Although the magnitude of the risk estimates for the association of ceramides containing lignoceric acid (Cer-24) with diabetes was similar to those for Cer-18, Cer-20, and Cer-22 (RR=1.13; 95% CI: 1.01, 1.26), the association was not statistically significant after correction for multiple testing (P=0.007). Ceramides carrying palmitic acid (Cer-16), SMs, glucosyl-ceramides, or a lactosyl-ceramide were not associated with diabetes risk. Higher concentrations of circulating Cer-18, Cer-20, and Cer-22 were associated with a higher risk of developing diabetes in 2 studies of American-Indian adults. This trial was registered at clinicaltrials.gov as NCT00005134.

Depression Symptoms and Cognitive Test Performance in Older American Indians: The Strong Heart Study.

American Indians have excess risk of depression, which can contribute to cerebrovascular and cognitive disability, with effects on memory, processing speed, executive function, and visuospatial ability. However, studies examining depression and cognition in American Indians are limited; this study aims to report associations of depression with general cognition, verbal fluency and memory, and processing speed. Cohort study. The Cerebrovascular Disease and its Consequences in American Indians study was an ancillary examination of Strong Heart Study participants from 3 U.S. regions. All eligible were included in this analysis (N=818). Participants completed evaluations for depressive symptomology, cognition, and physical function-including Center for Epidemiologic Studies Depression (CESD), Modified Mini-Mental State Examination (3MSE), Wechsler Adult Intelligence Scale-Fourth Edition coding (WAIS), Controlled Oral Word Association (COWA), California Verbal and Learning Test, Halstead finger tapping, grip strength, and Short Physical Performance Battery (SPPB) tests. Linear mixed models were adjusted for site, age, sex, education, income, marital status, alcohol, smoking, diabetes, hypertension, obesity, cholesterol, stroke, infarct, and hemorrhage. Symptoms of depression were common, with 20% (N=138) endorsing CES-D scores of 16+. More depressive symptoms were associated with older age, female sex, lower education, lower income, non-married status, not using alcohol, not smoking, hypertension, diabetes, and stroke. In adjusted analyses, processing speed (WAIS: β -0.13, 95%CI -0.25, -0.03), general cognition (3MSE: β -0.10, 95%CI -0.17, -0.03), verbal fluency (COWA: β -0.10, 95%CI -0.19, -0.01), and motor function (SPPB: β -0.05, 95%CI -0.07, -0.03) were significantly associated with more symptoms of depression. These findings maybe informative for health disparities populations, especially those with depressive risk. Clinicians may require particular training in cultural humility. Future studies should validate use of the CES-D scale in this population; longitudinal studies may focus on causal mechanisms and potential secondary prevention, such as social support. J Am Geriatr Soc 68:1739-1747, 2020.

Abstract MP31: Blood DNA Methylation Signatures of Incident Coronary Heart Disease: An Epigenome-wide Analysis in the Strong Heart Study

Background: In the US, American Indians suffer a disproportionate burden of CHD compared to other racial/ethnic groups. Additional strategies are needed to identify individuals at risk. Objectives: Investigate the association of blood DNA methylation (DNAm) with incident CHD in the Strong Heart Study and the prediction ability of DNAm beyond traditional risk factors. We maximized prediction ability using Bayesian Hierarchical Cox (BHCox) and Survival Random Forest models, which allow large numbers of CpGs in a single model, instead of considering them individually. Methods: Among 2325 men and women 45-74 years old in 1989-1991, 557 CHD events were identified over 20 years of follow-up. DNAm was measured in 790,026 CpGs, pre-processed and corrected for batch effects. We ran adjusted BHCox models for subsets of CpGs selected from similarly adjusted Cox models for individual CpGs. Prediction ability of CpGs in BHCox was further evaluated with Survival Random Forest, which is robust to overfitting. We also conducted a targeted analysis using Cox regression. Results: 26 CpGs associated with CVD in previous studies were nominally associated with incident CHD in a targeted analysis. The cross-validated C index for the model with traditional risk factors was 0.703. In BHCox, further entering 30K CpGs in a single model, resulted in 231 CpGs being significantly associated with incident CHD, with a cross-validation C statistic of 0.855. In Survival Random Forest, further entering the 231 CpGs from the BHCox model resulted in a cross-validation C statistic of 0.771 and 182 CpGs with variable importance (VIMP) greater than zero. The top CpGs in BHCox were also VIMP&gt;0 in random forest models and were located in SLC24A1 (calcium exchanger), SHBG (sex hormone binding globulin), and LINC00346 (non-protein coding RNA 346). Conclusions: We found novel CpG sites prospectively associated with incident CHD and confirmed signals from previous studies. DNAm might help to identify individuals at high risk of developing CHD.

Abstract P156: Recruitment and Retention of an American Indian Cohort - The Strong Heart Study Reducing Disparities Through Research

Tribal communities have legitimate reasons for being wary of research. The Strong Heart Study works to coordinate multi-disciplinary efforts towards bidirectional partnerships, with an ethos of respect and capacity development. Best practices are being developed through iterative processes including co-development of survey research through translating/communicating data analysis and applying CBPR practices. Information translation and CBPR throughout the research process also includes participating in community-based research symposia, developing site specific information through informational venues (e.g. Red Talks), formal reporting to Tribal councils and health committees, informal interaction by participating in local Native American community events, engaging Community Advisory groups, and supporting local students through hands-on internships. These activities also enhance transition of traditional public health research to a support a more autonomous, individualized system for the Tribal partners toward greater self-determining their own health. This approach encourages health literacy, promotes Institutional memory and automates longitudinal surveillance of local health issues to reduce disparities in Tribal nations.

Abstract P146: Telomere Length, DNA Methylation, and Risk of Cardiovascular Diseases: Meta-EWAS of Four Multi-ethnic Prospective Cohorts

Background: Biological aging assessed by both leukocyte telomere length (LTL) and DNA methylation (DNAm) has been associated with CVD and its risk factors. Moreover, LTL is epigenetically regulated. We hypothesized that LTL-associated epigenetic changes are associated with risk of CVD in the community. Objective: To test whether LTL-associated loci are associated with incident CVD, independent of standard risk factors in multi-ethnic cohorts. Method: We evaluated 3,628 participants with complete LTL and DNAm data in four prospective cohorts, including 1,531 American Indians from the Strong Heart Study (SHS, mean age 56, 60% women), 821 non-Hispanic Whites (NHW) from the Framingham Heart Study (FHS, mean age 60, 51% women), 471 NHW, 150 Hispanics, and 162 African Americans from the Multi-ethnic Study of Atherosclerosis (MESA, mean age 70 , 55% women), and 471 NHW and 342 African Americans from the Women’s Health Initiative (WHI, mean age 65 , all women). LTL was quantified by qPCR (SHS, MESA) or Southern blot (FHS, WHI). DNAm was assayed by Illumina EPIC (SHS) or 450K (FHS, MESA and WHI) arrays. We imputed 450K to 850K using random forest algorithms. CVD events included fatal and nonfatal MI, CHD, heart failure, stroke, peripheral artery diseases, and cardiovascular deaths. Cohort-specific EWAS was conducted to identify CpGs associated with LTL, adjusting for age, sex, race/ethnicity, smoking, alcohol, BMI, site, cell proportion, and batch. Multiple testing was Bonferroni-corrected (genome-wide P &lt; 2.4 x10 -7 ). Results across studies were combined by random-effects meta-analysis. To examine whether LTL-associated epigenetic loci are associated with CVD risk, we used a weighted methylation score to predict incident CVD by Cox regression, adjusting for age, sex, site, smoking, alcohol, BMI, glucose, SBP, LDL-C, and total cholesterol. Results: We ascertained 2,001 CVD events, including 986 in the SHS (average follow-up 15.2 years), 208 in FHS (average follow-up 7.7 years), 74 in MESA (average follow-up 4.9 years), and 733 in WHI (average follow-up 12.2 years). Meta-EWAS identified 22 CpGs (mapped to 17 unique genes) associated with LTL. Of these, 19 loci (15 negatively and 4 positively associated with LTL) had consistent directionality of association across four cohorts. The most significant genes harboring altered CpG sites included TLL2 (cg10549018, P= 2.42 x 10 -12 ) and TPST1 ( cg10691866, P =8.6 x 10 -10 ). A higher composite methylation score, which reflects longer LTL ( P &lt;0.0001), was significantly associated with a reduced risk of CVD in the SHS (HR=0.16, 95% CI: 0.07–0.37), FHS (HR=0.08, 95% CI: 0.01–0.40), and WHI (HR=0.32, 95%CI: 0.13–0.82), but not MESA (HR=0.47, 95% CI: 0.04–5.09). Conclusion: Altered DNA methylation at 19 CpG loci was significantly associated with LTL. Their combined effects may predict a reduced risk of CVD. The observed associations warrant further investigation.

Associations of maternal arsenic exposure with adult fasting glucose and insulin resistance in the Strong Heart Study and Strong Heart Family Study

Experimental and prospective epidemiologic evidence suggest that arsenic exposure has diabetogenic effects. However, little is known about how family exposure to arsenic may affect risk for type 2 diabetes (T2D)-related outcomes in adulthood. We evaluated the association of both maternal and offspring arsenic exposure with fasting glucose and incident T2D in 466 participants of the Strong Heart Family Study. Total arsenic (ΣAs) exposure was calculated as the sum of inorganic arsenic (iAs) and methylated (MMA, DMA) arsenic species in maternal and offspring baseline urine. Median maternal ΣAs at baseline (1989–91) was 7.6 µg/g creatinine, while median offspring ΣAs at baseline (2001–03) was 4.5 µg/g creatinine. Median offspring glucose in 2006–2009 was 94 mg/dL, and 79 participants developed T2D. The fully adjusted mean difference (95% CI) for offspring glucose was 4.40 (−3.46, 12.26) mg/dL per IQR increase in maternal ΣAs vs. 2.72 (−4.91 to 10.34) mg/dL per IQR increase in offspring ΣAs. The fully adjusted odds ratio (95%CI) of incident T2D was 1.35 (1.07, 1.69) for an IQR increase in maternal ΣAs and 1.15 (0.92, 1.43) for offspring ΣAs. The association of maternal ΣAs with T2D outcomes were attenuated with adjustment for offspring adiposity markers. Familial exposure to arsenic, as measured in mothers 15–20 years before offspring follow-up, is associated with increased odds of offspring T2D. More research is needed to confirm findings and better understand the importance of family exposure to arsenic in adult-onset diabetes.