Assessing the use of GEE methods for analyzing binary outcomes in family studies: the Strong Heart Family Study.

The generalized estimating equations method (GEE) is commonly applied to analyze data obtained from family studies. GEE is well known for its robustness on misspecification of correlation structure. However, the unbalanced distribution of family sizes and complicated genetic relatedness structure within each family may challenge GEE performance. We focused our research on binary outcomes. To evaluate the performance of GEE, we conducted a series of simulations, on data generated adopting the kinship matrix (correlation structure within each family) from the Strong Heart Family Study (SHFS). We performed a fivefold cross-validation to further evaluate the GEE predictive power on data from the SHFS. A Bayesian modeling approach, with direct integration of the kinship matrix, was also included to contrast with GEE. Our simulation studies revealed that GEE performs well on a binary outcome from families having a relatively simple kinship structure. However, data with a binary outcome generated from families with complex kinship structures, especially with a large genetic variance, can challenge the performance of GEE.

Association of Achievement of the American Heart Association's Life's Essential 8 Goals With Incident Cardiovascular Diseases in the SHFS.

BACKGROUND: Cardiovascular disease (CVD) is a leading cause of morbidity and mortality in American Indian people. In 2022, the American Heart Association developed the Life's Essential 8 goals to promote cardiovascular health (CVH) for Americans, composed of diet, physical activity, nicotine exposure, sleep, body mass index, blood lipids, blood pressure, and blood glucose. We examined whether achievement of Life's Essential 8 goals was associated with incident CVD among SHFS (Strong Heart Family Study) participants. METHODS AND RESULTS: A total of 2139 SHFS participants without CVD at baseline were included in analyses. We created a composite CVH score based on achievement of Life's Essential 8 goals, excluding sleep. Scores of 0 to 49 represented low CVH, 50 to 69 represented moderate CVH, and 70 to 100 represented high CVH. Incident CVD was defined as incident myocardial infarction, coronary heart disease, congestive heart failure, or stroke. Cox proportional hazard models were used to examine the relationship of CVH and incident CVD. The incidence rate of CVD at the 20-year follow-up was 7.43 per 1000 person-years. Compared with participants with low CVH, participants with moderate and high CVH had a lower risk of incident CVD; the hazard ratios and 95% CIs for incident CVD for moderate and high CVH were 0.52 (95% CI, 0.40-0.68) and 0.25 (95% CI, 0.14-0.44), respectively, after adjustment for age, sex, education, and study site. CONCLUSIONS: Better CVH was associated with lower CVD risk which highlights the need for comprehensive public health interventions targeting CVH promotion to reduce CVD risk in American Indian communities.

Dyslipidemia in American Indian Adolescents and Young Adults: Strong Heart Family Study.

BACKGROUND: Although many studies on the association between dyslipidemia and cardiovascular disease (CVD) exist in older adults, data on the association among adolescents and young adults living with disproportionate burden of cardiometabolic disorders are scarce. METHODS AND RESULTS: The SHFS (Strong Heart Family Study) is a multicenter, family-based, prospective cohort study of CVD in an American Indian populations, including 12 communities in central Arizona, southwestern Oklahoma, and the Dakotas. We evaluated SHFS participants, who were 15 to 39 years old at the baseline examination in 2001 to 2003 (n=1440). Lipids were measured after a 12-hour fast. We used carotid ultrasounds to detect plaque at baseline and follow-up in 2006 to 2009 (median follow-up=5.5 years). We identified incident CVD events through 2020 with a median follow-up of 18.5 years. We used shared frailty proportional hazards models to assess the association between dyslipidemia and subclinical or clinical CVD, while controlling for covariates. Baseline dyslipidemia prevalence was 55.2%, 73.6%, and 78.0% for participants 15 to 19, 20 to 29, and 30 to 39 years old, respectively. Approximately 2.8% had low-density lipoprotein cholesterol ≥160 mg/dL, which is higher than the recommended threshold for lifestyle or medical interventions in young adults of 20 to 39 years old. During follow-up, 9.9% had incident plaque (109/1104 plaque-free participants with baseline and follow-up ultrasounds), 11.0% had plaque progression (128/1165 with both baseline and follow-up ultrasounds), and 9% had incident CVD (127/1416 CVD-free participants at baseline). Plaque incidence and progression were higher in participants with total cholesterol ≥200 mg/dL, low-density lipoprotein cholesterol ≥160 mg/dL, or non-high-density lipoprotein cholesterol ≥130 mg/dL, while controlling for covariates. CVD risk was independently associated with low-density lipoprotein cholesterol ≥160 mg/dL. CONCLUSIONS: Dyslipidemia is a modifiable risk factor that is associated with both subclinical and clinical CVD, even among the younger American Indian population who have unexpectedly high rates of significant CVD events. Therefore, this population is likely to benefit from a variety of evidence-based interventions including screening, educational, lifestyle, and guideline-directed medical therapy at an early age.

The Cost-Efficacy of a Healthy Food Box for Managing Hypertension Within a Native American Population: The Chickasaw Healthy Eating Environment Research Study.

Background Dietary interventions are used for the treatment of hypertension. We evaluated the cost-efficacy of delivering boxes of healthy, culturally tailored foods and checks that can only be spent on produce in a Native American population. Methods We conducted a group randomized controlled trial from 2018-2020 with N = 2 treatment counties and N = 2 control counties and a total of N = 160 Native American adults with baseline stage 1 or stage 2 hypertension. Participants in the intervention group received monthly boxes of food that adheres to the Dietary Approaches to Stop Hypertension diet as well as checks that could only be spent on produce for 6 months. We measured blood pressure and quality of life at baseline and at a 6-month follow-up in both intervention and control groups. We used ordered logistic regression to estimate the effect of treatment on probability of blood pressure improvements. We then conducted a cost-efficacy analysis. Results We found that treatment was effective in women with stage 1 hypertension at baseline. Based on this finding, we also estimate that this intervention satisfies normative cost-effectiveness thresholds, even when lifetime treatment is needed to preserve the impact, so long as treatment is only continued in those who respond to treatment. Conclusions Direct delivery of healthy foods and checks that can only be spent on produce are a potentially cost-effective intervention for the management of hypertension among Native American women with stage 1 hypertension. Further research is needed to understand why we found an impact only for this group.

Longitudinal Lipidomic Signature of Coronary Heart Disease in American Indian People.

BACKGROUND: Dyslipidemia is an independent risk factor for coronary heart disease (CHD). Standard lipid panel cannot capture the complexity of the blood lipidome (ie, all molecular lipids in the blood). To date, very few large-scale epidemiological studies have assessed the full spectrum of the blood lipidome on risk of CHD, especially in a longitudinal setting. METHODS AND RESULTS: Using an untargeted liquid chromatography-mass spectrometry, we repeatedly measured 1542 lipid species from 1835 unique American Indian participants who attended 2 clinical visits (≈5.5 years apart) and followed up to 17.8 years in the Strong Heart Family Study (SHFS). We first identified baseline lipid species associated with risk of CHD, followed by replication in a European population. The model adjusted for age, sex, body mass index, smoking, hypertension, diabetes, low-density lipoprotein cholesterol, estimated glomerular filtration rate, education, and physical activity at baseline. We then examined the longitudinal association between changes in lipid species and changes in cardiovascular risk factors during follow-up. Multiple testing was controlled by the false discovery rate. We found that baseline levels of multiple lipid species (eg, phosphatidylcholines, phosphatidylethanolamines, and ceramides) were associated with the risk of CHD and improved the prediction accuracy over conventional risk factors in American Indian people. Some identified lipids in American Indian people were replicated in European people. Longitudinal changes in multiple lipid species (eg, acylcarnitines, phosphatidylcholines, and triacylglycerols) were associated with changes in cardiovascular risk factors. CONCLUSIONS: Baseline plasma lipids and their longitudinal changes over time are associated with risk of CHD. These findings provide novel insights into the role of dyslipidemia in CHD.

Genome-wide study investigating effector genes and polygenic prediction for kidney function in persons with ancestry from Africa and the Americas.

Chronic kidney disease is a leading cause of death and disability globally and impacts individuals of African ancestry (AFR) or with ancestry in the Americas (AMS) who are under-represented in genome-wide association studies (GWASs) of kidney function. To address this bias, we conducted a large meta-analysis of GWASs of estimated glomerular filtration rate (eGFR) in 145,732 AFR and AMS individuals. We identified 41 loci at genome-wide significance (p < 5 × 10-8), of which two have not been previously reported in any ancestry group. We integrated fine-mapped loci with epigenomic and transcriptomic resources to highlight potential effector genes relevant to kidney physiology and disease, and reveal key regulatory elements and pathways involved in renal function and development. We demonstrate the varying but increased predictive power offered by a multi-ancestry polygenic score for eGFR and highlight the importance of population diversity in GWASs and multi-omics resources to enhance opportunities for clinical translation for all.

The Contribution of Declines in Blood Lead Levels to Reductions in Blood Pressure Levels: Longitudinal Evidence in the Strong Heart Family Study.

BACKGROUND: Chronic lead exposure is associated with both subclinical and clinical cardiovascular disease. We evaluated whether declines in blood lead were associated with changes in systolic and diastolic blood pressure in adult American Indian participants from the SHFS (Strong Heart Family Study). METHODS AND RESULTS: Lead in whole blood was measured in 285 SHFS participants in 1997 to 1999 and 2006 to 2009. Blood pressure and measures of cardiac geometry and function were obtained in 2001 to 2003 and 2006 to 2009. We used generalized estimating equations to evaluate the association of declines in blood lead with changes in blood pressure; cardiac function and geometry measures were considered secondary. Mean blood lead was 2.04 µg/dL at baseline. After ≈10 years, mean decline in blood lead was 0.67 µg/dL. In fully adjusted models, the mean difference in systolic blood pressure comparing the highest to lowest tertile of decline (>0.91 versus <0.27 µg/dL) in blood lead was -7.08 mm Hg (95% CI, -13.16 to -1.00). A significant nonlinear association between declines in blood lead and declines in systolic blood pressure was detected, with significant linear associations where blood lead decline was 0.1 µg/dL or higher. Declines in blood lead were nonsignificantly associated with declines in diastolic blood pressure and significantly associated with declines in interventricular septum thickness. CONCLUSIONS: Declines in blood lead levels in American Indian adults, even when small (0.1-1.0 µg/dL), were associated with reductions in systolic blood pressure. These findings suggest the need to further study the cardiovascular impacts of reducing lead exposures and the importance of lead exposure prevention.

Plasma lipidomic markers of diet quality are associated with incident coronary heart disease in American Indian adults: the Strong Heart Family Study.

BACKGROUND: Identifying lipidomic markers of diet quality is needed to inform the development of biomarkers of diet, and to understand the mechanisms driving the diet- coronary heart disease (CHD) association. OBJECTIVES: This study aimed to identify lipidomic markers of diet quality and examine whether these lipids are associated with incident CHD. METHODS: Using liquid chromatography-mass spectrometry, we measured 1542 lipid species from 1694 American Indian adults (aged 18-75 years, 62% female) in the Strong Heart Family Study. Participants were followed up for development of CHD through 2020. Information on the past year diet was collected using the Block Food Frequency Questionnaire, and diet quality was assessed using the Alternative Healthy Eating Index-2010 (AHEI). Mixed-effects linear regression was used to identify individual lipids cross-sectionally associated with AHEI. In prospective analysis, Cox frailty model was used to estimate the hazard ratio (HR) of each AHEI-related lipid for incident CHD. All models were adjusted for age, sex, center, education, body mass index, smoking, alcohol drinking, level of physical activity, energy intake, diabetes, hypertension, and use of lipid-lowering drugs. Multiple testing was controlled at a false discovery rate of <0.05. RESULTS: Among 1542 lipid species measured, 71 lipid species (23 known), including acylcarnitine, cholesterol esters, glycerophospholipids, sphingomyelins and triacylglycerols, were associated with AHEI. Most of the identified lipids were associated with consumption of ω-3 (n-3) fatty acids. In total, 147 participants developed CHD during a mean follow-up of 17.8 years. Among the diet-related lipids, 10 lipids [5 known: cholesterol ester (CE)(22:5)B, phosphatidylcholine (PC)(p-14:0/22:1)/PC(o-14:0/22:1), PC(p-38:3)/PC(o-38:4)B, phosphatidylethanolamine (PE)(p-18:0/20:4)/PE(o-18:0/20:4), and sphingomyelin (d36:2)A] were associated with incident CHD. On average, each standard deviation increase in the baseline level of these 5 lipids was associated with 17%-23% increased risk of CHD (from HR: 1.17; 95% CI: 1, 1.36; to HR: 1.23; 95% CI: 1.05, 1.43). CONCLUSIONS: In this study, lipidomic markers of diet quality in American Indian adults are found. Some diet-related lipids are associated with risk of CHD beyond established risk factors.

Arsenic Exposure and Epigenetic Aging: The Association with Cardiovascular Disease and All-Cause Mortality in the Strong Heart Study.

BACKGROUND: Inorganic arsenic (As) may increase the risk of cardiovascular disease (CVD) and all-cause mortality through accelerated aging, which can be estimated using epigenetic-based measures. OBJECTIVES: We evaluated three DNA methylation-based aging measures (PhenoAge, GrimAge, DunedinPACE) (epigenetic aging measures) as potential mediators of the previously reported association of As exposure with CVD incidence, CVD mortality, and all-cause mortality in the Strong Heart Study (SHS), an epidemiological cohort of American Indian adults. METHODS: Blood DNA methylation and urinary As levels were measured in 2,323 SHS participants (41.5% men, mean age of 55 years old). PhenoAge and GrimAge values were calculated using a residual-based method. We tested the association of urinary As with epigenetic aging measures using linear regression, the association of epigenetic aging measures with the three health outcomes using additive hazards models, and the mediation of As-related CVD incidence, CVD mortality, and all-cause mortality by epigenetic aging measures using the product of coefficients method. RESULTS: SHS participants with higher vs. lower urinary As levels had similar PhenoAge age, older GrimAge age, and faster DunedinPACE. An interquartile range increase in urinary As was associated with higher of PhenoAge age acceleration [mean difference (95% confidence interval)=0.48 (0.17, 0.80) years], GrimAge age acceleration [0.80 (0.60, 1.00) years], and DunedinPACE [0.011 (0.005, 0.018)], after adjusting for age, sex, center location, genetic components, smoking status, and body mass index. Of the 347 incident CVD events per 100,000 person-years associated with a doubling in As exposure, 21.3% (9.1, 57.1) and 22.6% (9.5, 56.9), were attributable to differences in GrimAge and DunedinPACE, respectively. DISCUSSION: Arsenic exposure was associated with older GrimAge and faster DunedinPACE measures of biological age. Furthermore, accelerated biological aging measured from DNA methylation accounted for a relevant fraction of As-associated risk for CVD, CVD mortality, and all-cause mortality in the SHS, supporting the role of As in accelerated aging. Research of the biological underpinnings can contribute to a better understanding of the role of aging in arsenic-related disease. https://doi.org/10.1289/EHP11981.

Dietary magnesium, C-reactive protein and interleukin-6: The Strong Heart Family Study.

OBJECTIVES: To examine the associations of dietary Mg intake with inflammatory biomarkers (C-reactive protein (CRP) and interleukin 6 (IL-6)), and the interaction of dietary Mg intake with single nucleotide polymorphism (SNP) rs3740393, a SNP related to Mg metabolism and transport, on CRP and IL-6 among American Indians (AIs). METHODS: This cross-sectional study included AI participants (n = 1,924) from the Strong Heart Family Study (SHFS). Mg intake from foods and dietary supplements was ascertained using a 119-item Block food frequency questionnaire, CRP and IL-6 were measured from blood, and SNP rs3740393 was genotyped using MetaboChip. Generalized estimating equations were used to examine associations of Mg intake, and the interaction between rs3740393 and dietary Mg, with CRP and IL-6. RESULTS: Reported Mg intake was not associated with CRP or IL-6, irrespective of genotype. A significant interaction (p-interaction = 0.018) was observed between Mg intake and rs3740393 on IL-6. Among participants with the C/C genotype, for every 1 SD higher in log-Mg, log-IL-6 was 0.04 (95% CI: -0.10 to 0.17) pg/mL higher. Among participants with the C/G genotype, for every 1 SD higher in log-Mg, log-IL-6 was 0.08 (95% CI: -0.21 to 0.05) pg/mL lower, and among participants with the G/G genotype, for every 1 SD higher in log-Mg, log-IL-6 was 0.19 (95% CI: -0.38 to -0.01) pg/mL lower. CONCLUSIONS: Mg intake may be associated with lower IL-6 with increasing dosage of the G allele at rs3740393. Future research is necessary to replicate this finding and examine other Mg-related genes that influence associations of Mg intake with inflammation.

Fine particulate matter composition in American Indian vs. Non-American Indian communities.

BACKGROUND: Fine particulate matter (PM2.5) exposure is a known risk factor for numerous adverse health outcomes, with varying estimates of component-specific effects. Populations with compromised health conditions such as diabetes can be more sensitive to the health impacts of air pollution exposure. Recent trends in PM2.5 in primarily American Indian- (AI-) populated areas examined in previous work declined more gradually compared to the declines observed in the rest of the US. To further investigate components contributing to these findings, we compared trends in concentrations of six PM2.5 components in AI- vs. non-AI-populated counties over time (2000-2017) in the contiguous US. METHODS: We implemented component-specific linear mixed models to estimate differences in annual county-level concentrations of sulfate, nitrate, ammonium, organic matter, black carbon, and mineral dust from well-validated surface PM2.5 models in AI- vs. non-AI-populated counties, using a multi-criteria approach to classify counties as AI- or non-AI-populated. Models adjusted for population density and median household income. We included interaction terms with calendar year to estimate whether concentration differences in AI- vs. non-AI-populated counties varied over time. RESULTS: Our final analysis included 3108 counties, with 199 (6.4%) classified as AI-populated. On average across the study period, adjusted concentrations of all six PM2.5 components in AI-populated counties were significantly lower than in non-AI-populated counties. However, component-specific levels in AI- vs. non-AI-populated counties varied over time: sulfate and ammonium levels were significantly lower in AI- vs. non-AI-populated counties before 2011 but higher after 2011 and nitrate levels were consistently lower in AI-populated counties. CONCLUSIONS: This study indicates time trend differences of specific components by AI-populated county type. Notably, decreases in sulfate and ammonium may contribute to steeper declines in total PM2.5 in non-AI vs. AI-populated counties. These findings provide potential directives for additional monitoring and regulations of key emissions sources impacting tribal lands.

Longitudinal lipidomic signature of carotid atherosclerosis in American Indians: Findings from the Strong Heart Family Study.

BACKGROUND AND AIMS: Dyslipidemia is an independent risk factor for atherosclerosis and atherosclerotic cardiovascular disease (ASCVD). To date, a comprehensive assessment of individual lipid species associated with atherosclerosis is lacking in large-scale epidemiological studies, especially in a longitudinal setting. We investigated the association of circulating lipid species and its longitudinal changes with carotid atherosclerosis. METHODS: Using liquid chromatograph-mass spectrometry, we repeatedly measured 1542 lipid species in 3687 plasma samples from 1918 unique American Indians attending two visits (mean ∼5 years apart) in the Strong Heart Family Study. Carotid atherosclerotic plaques were assessed by ultrasonography at each visit. We identified lipids associated with prevalence or progression of carotid plaques, adjusting age, sex, BMI, smoking, hypertension, diabetes, and eGFR. Then we examined whether longitudinal changes in lipids were associated with changes in cardiovascular risk factors. Multiple testing was controlled at false discovery rate (FDR) < 0.05. RESULTS: Higher levels of sphingomyelins, ether-phosphatidylcholines, and triacylglycerols were significantly associated with prevalence or progression of carotid plaques (odds ratios ranged from 1.15 to 1.34). Longitudinal changes in multiple lipid species (e.g., acylcarnitines, phosphatidylcholines, triacylglycerols) were associated with changes in cardiometabolic traits (e.g., BMI, blood pressure, fasting glucose, eGFR). Network analysis identified differential lipid networks associated with plaque progression. CONCLUSIONS: Baseline and longitudinal changes in multiple lipid species were significantly associated with carotid atherosclerosis and its progression in American Indians. Some plaque-related lipid species were also associated with risk for CVD events.

Evaluation of plasma sphingolipids as mediators of the relationship between kidney disease and cardiovascular events.

BACKGROUND: Sphingolipids are a family of circulating lipids with regulatory and signaling roles that are strongly associated with both eGFR and cardiovascular disease. Patients with chronic kidney disease (CKD) are at high risk for cardiovascular events, and have different plasma concentrations of certain plasma sphingolipids compared to patients with normal kidney function. We hypothesize that circulating sphingolipids partially mediate the associations between eGFR and cardiovascular events. METHODS: We measured the circulating concentrations of 8 sphingolipids, including 4 ceramides and 4 sphingomyelins with the fatty acids 16:0, 20:0, 22:0, and 24:0, in plasma from 3,463 participants in a population-based cohort (Cardiovascular Health Study) without prevalent cardiovascular disease. We tested the adjusted mediation effects by these sphingolipids of the associations between eGFR and incident cardiovascular disease via quasi-Bayesian Monte Carlo method with 2,000 simulations, using a Bonferroni correction for significance. FINDINGS: The mean (±SD) eGFR was 70 (±16) mL/min/1.73 m2; 62% of participants were women. Lower eGFR was associated with higher plasma ceramide-16:0 and sphingomyelin-16:0, and lower ceramides and sphingomyelins-20:0 and -22:0. Lower eGFR was associated with risk of incident heart failure and ischemic stroke, but not myocardial infarction. Five of eight sphingolipids partially mediated the association between eGFR and heart failure. The sphingolipids associated with the greatest proportion mediated were ceramide-16:0 (proportion mediated 13%, 95% CI 8-22%) and sphingomyelin-16:0 (proportion mediated 10%, 95% CI 5-17%). No sphingolipids mediated the association between eGFR and ischemic stroke. INTERPRETATION: Plasma sphingolipids partially mediated the association between lower eGFR and incident heart failure. Altered sphingolipids metabolism may be a novel mechanism for heart failure in patients with CKD. FUNDING: This study was supported by T32 DK007467 and a KidneyCure Ben J. Lipps Research Fellowship (Dr. Lidgard). Sphingolipid measurements were supported by R01 HL128575 (Dr. Lemaitre) and R01 HL111375 (Dr. Hoofnagle) from the National Heart, Lung, and Blood Institute (NHLBI).

Diet Quality and Kidney Outcomes in Adolescent and Adult American Indians: the Strong Heart Family Study.

BACKGROUND: The burden of kidney disease is exceedingly high among American Indians (AIs). We sought to examine the relationship of diet quality, a modifiable risk factor, and kidney outcomes in AI adolescents and adults, hypothesizing that healthier diets are associated with lower odds of incident albuminuria and eGFR decline. METHODS: This is an analysis from the Strong Heart Family Study, a longitudinal study of cardiovascular disease and its risk factors among AIs from Arizona, North and South Dakota, and Oklahoma (n = 1720, mean age 39 + / - 16 years, 16% adolescents at baseline). Participants completed two exams (baseline: 2001-2003; follow-up: 2007-2009). The primary exposure was diet quality, expressed as the Alternative Healthy Eating Index 2010 (AHEI), on a 110-point scale (assessed using a 119-item Block food frequency questionnaire). The primary outcomes were as follows: 1) incident albuminuria (albumin to creatinine ratio 30 mg/g or greater); and 2) eGFR decline of 30% or greater. Generalized estimating equations were used to examine the association of AHEI (in quartiles) with outcomes. RESULTS: Ten percent of participants (6% of adolescents) had incident albuminuria and 2% of participants (2% of adolescents) had eGFR decline. For those with normal fasting glucose levels, the odds ratio (OR) for incident albuminuria comparing extreme quartiles of diet quality (least healthy [reference] versus healthiest quartiles) was 0.48 (95% CI 0.28, 0.81) after adjustment for demographics and comorbidities. CONCLUSIONS: For American Indians with normal fasting glucose, higher diet quality decreases the odds of developing albuminuria. These findings inform future efforts to prevent CKD in American Indian adolescents and young adults.

Smoking, blood DNA methylation sites and lung cancer risk.

Altered DNA methylation (DNAm) might be a biological intermediary in the pathway from smoking to lung cancer. In this study, we investigated the contribution of differential blood DNAm to explain the association between smoking and lung cancer incidence. Blood DNAm was measured in 2321 Strong Heart Study (SHS) participants. Incident lung cancer was assessed as time to event diagnoses. We conducted mediation analysis, including validation with DNAm and paired gene expression data from the Framingham Heart Study (FHS). In the SHS, current versus never smoking and pack-years single-mediator models showed, respectively, 29 and 21 differentially methylated positions (DMPs) for lung cancer with statistically significant mediated effects (14 of 20 available, and five of 14 available, positions, replicated, respectively, in FHS). In FHS, replicated DMPs showed gene expression downregulation largely in trans, and were related to biological pathways in cancer. The multimediator model identified that DMPs annotated to the genes AHRR and IER3 jointly explained a substantial proportion of lung cancer. Thus, the association of smoking with lung cancer was partly explained by differences in baseline blood DNAm at few relevant sites. Experimental studies are needed to confirm the biological role of identified eQTMs and to evaluate potential implications for early detection and control of lung cancer.

Deep Learning for Inference of Hepatic Proton Density Fat Fraction From T1-Weighted In-Phase and Opposed-Phase MRI: Retrospective Analysis of Population-Based Trial Data.

BACKGROUND. The confounder-corrected chemical shift-encoded MRI (CSE-MRI) sequence used to determine proton density fat fraction (PDFF) for hepatic fat quantification is not widely available. As an alternative, hepatic fat can be assessed by a two-point Dixon method to calculate signal fat fraction (FF) from conventional T1-weighted in- and opposed-phase (IOP) images, although signal FF is prone to biases, leading to inaccurate quantification. OBJECTIVE. The purpose of this study was to compare hepatic fat quantification by use of PDFF inferred from conventional T1-weighted IOP images and deep-learning convolutional neural networks (CNNs) with quantification by use of two-point Dixon signal FF with CSE-MRI PDFF as the reference standard. METHODS. This study entailed retrospective analysis of data from 292 participants (203 women, 89 men; mean age, 53.7 ± 12.0 [SD] years) enrolled at two sites from September 1, 2017, to December 18, 2019, in the Strong Heart Family Study (a prospective population-based study of American Indian communities). Participants underwent liver MRI (site A, 3 T; site B, 1.5 T) including T1-weighted IOP MRI and CSE-MRI (used to reconstruct CSE PDFF and CSE R2* maps). With CSE PDFF as reference, a CNN was trained in a random sample of 218 (75%) participants to infer voxel-by-voxel PDFF maps from T1-weighted IOP images; testing was performed in the other 74 (25%) participants. Parametric values from the entire liver were automatically extracted. Per-participant median CNN-inferred PDFF and median two-point Dixon signal FF were compared with reference median CSE-MRI PDFF by means of linear regression analysis, intraclass correlation coefficient (ICC), and Bland-Altman analysis. The code is publicly available at github.com/kang927/CNN-inference-of-PDFF-from-T1w-IOP-MR. RESULTS. In the 74 test-set participants, reference CSE PDFF ranged from 1% to 32% (mean, 11.3% ± 8.3% [SD]); reference CSE R2* ranged from 31 to 457 seconds-1 (mean, 62.4 ± 67.3 seconds-1 [SD]). Agreement metrics with reference to CSE PDFF for CNN-inferred PDFF were ICC = 0.99, bias = -0.19%, 95% limits of agreement (LoA) = (-2.80%, 2.71%) and for two-point Dixon signal FF were ICC = 0.93, bias = -1.11%, LoA = (-7.54%, 5.33%). CONCLUSION. Agreement with reference CSE PDFF was better for CNN-inferred PDFF from conventional T1-weighted IOP images than for two-point Dixon signal FF. Further investigation is needed in individuals with moderate-to-severe iron overload. CLINICAL IMPACT. Measurement of CNN-inferred PDFF from widely available T1-weighted IOP images may facilitate adoption of hepatic PDFF as a quantitative bio-marker for liver fat assessment, expanding opportunities to screen for hepatic steatosis and nonalcoholic fatty liver disease.

Longitudinal Lipidomic Profile of Hypertension in American Indians: Findings From the Strong Heart Family Study.

BACKGROUND: Dyslipidemia is an important risk factor for hypertension and cardiovascular disease. Standard lipid panel cannot reflect the complexity of blood lipidome. The associations of individual lipid species with hypertension remain to be determined in large-scale epidemiological studies, especially in a longitudinal setting. METHODS: Using liquid chromatography-mass spectrometry, we repeatedly measured 1542 lipid species in 3699 fasting plasma samples at 2 visits (1905 at baseline, 1794 at follow-up, ~5.5 years apart) from 1905 unique American Indians in the Strong Heart Family Study. We first identified baseline lipids associated with prevalent and incident hypertension, followed by replication of top hits in Europeans. We then conducted repeated measurement analysis to examine the associations of changes in lipid species with changes in systolic blood pressure, diastolic blood pressure, and mean arterial pressure. Network analysis was performed to identify lipid networks associated with the risk of hypertension. RESULTS: Baseline levels of multiple lipid species, for example, glycerophospholipids, cholesterol esters, sphingomyelins, glycerolipids, and fatty acids, were significantly associated with both prevalent and incident hypertension in American Indians. Some lipids were confirmed in Europeans. Longitudinal changes in multiple lipid species, for example, acylcarnitines, phosphatidylcholines, fatty acids, and triacylglycerols, were significantly associated with changes in blood pressure measurements. Network analysis identified distinct lipidomic patterns associated with the risk of hypertension. CONCLUSIONS: Baseline plasma lipid species and their longitudinal changes are significantly associated with hypertension development in American Indians. Our findings shed light on the role of dyslipidemia in hypertension and may offer potential opportunities for risk stratification and early prediction of hypertension.

Metal mixtures and DNA methylation measures of biological aging in American Indian populations.

INTRODUCTION: Native American communities suffer disproportionately from elevated metal exposures and increased risk for cardiovascular diseases and diabetes. DNA methylation is a sensitive biomarker of aging-related processes and novel epigenetic-based "clocks" can be used to estimate accelerated biological aging that may underlie increased risk. Metals alter DNA methylation, yet little is known about their individual and combined impact on epigenetic age acceleration. Our objective was to investigate the associations of metals on several DNA methylation-based aging measures in the Strong Heart Study (SHS) cohort. METHODS: Blood DNA methylation data from 2,301 SHS participants was used to calculate age acceleration of epigenetic clocks (PhenoAge, GrimAge, DunedinPACE, Hannum, Horvath). Urinary metals [arsenic (As), cadmium (Cd), tungsten (W), zinc (Zn), selenium (Se), molybdenum (Mo)] were creatinine-adjusted and categorized into quartiles. We examined associations of individual metals through linear regression models and used Bayesian Kernel Machine Regression (BKMR) for the impact of the total metal mixture on epigenetic age acceleration. RESULTS: The mixture of nonessential metals (W, As, Cd) was associated with greater GrimAge acceleration and DunedinPACE, while the essential metal mixture (Se, Zn, Mo) was associated with lower epigenetic age acceleration. Cd was associated with increased epigenetic age acceleration across all clocks and BKMR analysis suggested nonlinear associations between Se and DunedinPACE, GrimAge, and PhenoAge acceleration. No interactions between individual metals were observed. The associations between Cd, Zn, and epigenetic age acceleration were greater in never smokers in comparison to current/former smokers. CONCLUSION: Nonessential metals were positively associated with greater epigenetic age acceleration, with strongest associations observed between Cd and DunedinPACE and GrimAge acceleration. In contrast, essential metals were associated with lower epigenetic aging. Examining the influence of metal mixtures on epigenetic age acceleration can provide insight into metals and aging-related diseases.

Maternal DNA methylation signatures of arsenic exposure is associated with adult offspring insulin resistance in the Strong Heart Study.

Exposure to low to moderate arsenic (As) levels has been associated with type 2 diabetes (T2D) and other chronic diseases in American Indian communities. Prenatal exposure to As may also increase the risk for T2D in adulthood, and maternal As has been associated with adult offspring metabolic health measurements. We hypothesized that T2D-related outcomes in adult offspring born to women exposed to low to moderate As can be evaluated utilizing a maternally-derived molecular biosignature of As exposure. Herein, we evaluated the association of maternal DNA methylation with incident T2D and insulin resistance (Homeostatic model assessment of insulin resistance [HOMA2-IR]) in adult offspring. For DNA methylation, we used 20 differentially methylated cytosine-guanine dinucleotides (CpG) previously associated with the sum of inorganic and methylated As species (ΣAs) in urine in the Strong Heart Study (SHS). Of these 20 CpGs, we found six CpGs nominally associated (p < 0.05) with HOMA2-IR in a fully adjusted model that included clinically relevant covariates and offspring adiposity measurements; a similar model that adjusted instead for maternal adiposity measurements found three CpGs nominally associated with HOMA2-IR, two of which overlapped the offspring adiposity model. After adjusting for multiple comparisons, cg03036214 remained associated with HOMA2-IR (q < 0.10) in the offspring adiposity model. The odds ratio of incident T2D increased with an increase in maternal DNA methylation at one HOMA2-IR associated CpG in the model adjusting for offspring adiposity, cg12116137, whereas adjusting for maternal adiposity had a minimal effect on the association. Our data suggests offspring adiposity, rather than maternal adiposity, potentially influences the effects of maternal DNAm signatures on offspring metabolic health parameters. Here, we have presented evidence supporting a role for epigenetic biosignatures of maternal As exposure as a potential biomarker for evaluating risk of T2D-related outcomes in offspring later in life.

Sequencing-based fine-mapping and in silico functional characterization of the 10q24.32 arsenic metabolism efficiency locus across multiple arsenic-exposed populations.

Inorganic arsenic is highly toxic and carcinogenic to humans. Exposed individuals vary in their ability to metabolize arsenic, and variability in arsenic metabolism efficiency (AME) is associated with risks of arsenic-related toxicities. Inherited genetic variation in the 10q24.32 region, near the arsenic methyltransferase (AS3MT) gene, is associated with urine-based measures of AME in multiple arsenic-exposed populations. To identify potential causal variants in this region, we applied fine mapping approaches to targeted sequencing data generated for exposed individuals from Bangladeshi, American Indian, and European American populations (n = 2,357, 557, and 648 respectively). We identified three independent association signals for Bangladeshis, two for American Indians, and one for European Americans. The size of the confidence sets for each signal varied from 4 to 85 variants. There was one signal shared across all three populations, represented by the same SNP in American Indians and European Americans (rs191177668) and in strong linkage disequilibrium (LD) with a lead SNP in Bangladesh (rs145537350). Beyond this shared signal, differences in LD patterns, minor allele frequency (MAF) (e.g., rs12573221 ~13% in Bangladesh ~0.2% among American Indians), and/or heterogeneity in effect sizes across populations likely contributed to the apparent population specificity of the additional identified signals. One of our potential causal variants influences AS3MT expression and nearby DNA methylation in numerous GTEx tissue types (with rs4919690 as a likely causal variant). Several SNPs in our confidence sets overlap transcription factor binding sites and cis-regulatory elements (from ENCODE). Taken together, our analyses reveal multiple potential causal variants in the 10q24.32 region influencing AME, including a variant shared across populations, and elucidate potential biological mechanisms underlying the impact of genetic variation on AME.