Maternal smoking DNA methylation risk score associated with health outcomes in offspring of European and South Asian ancestry.

Background: Maternal smoking has been linked to adverse health outcomes in newborns but the extent to which it impacts newborn health has not been quantified through an aggregated cord blood DNA methylation (DNAm) score. Here, we examine the feasibility of using cord blood DNAm scores leveraging large external studies as discovery samples to capture the epigenetic signature of maternal smoking and its influence on newborns in White European and South Asian populations. Methods: We first examined the association between individual CpGs and cigarette smoking during pregnancy, and smoking exposure in two White European birth cohorts (n=744). Leveraging established CpGs for maternal smoking, we constructed a cord blood epigenetic score of maternal smoking that was validated in one of the European-origin cohorts (n=347). This score was then tested for association with smoking status, secondary smoking exposure during pregnancy, and health outcomes in offspring measured after birth in an independent White European (n=397) and a South Asian birth cohort (n=504). Results: Several previously reported genes for maternal smoking were supported, with the strongest and most consistent association signal from the GFI1 gene (6 CpGs with p<5 × 10-5). The epigenetic maternal smoking score was strongly associated with smoking status during pregnancy (OR = 1.09 [1.07, 1.10], p=5.5 × 10-33) and more hours of self-reported smoking exposure per week (1.93 [1.27, 2.58], p=7.8 × 10-9) in White Europeans. However, it was not associated with self-reported exposure (p>0.05) among South Asians, likely due to a lack of smoking in this group. The same score was consistently associated with a smaller birth size (-0.37±0.12 cm, p=0.0023) in the South Asian cohort and a lower birth weight (-0.043±0.013 kg, p=0.0011) in the combined cohorts. Conclusions: This cord blood epigenetic score can help identify babies exposed to maternal smoking and assess its long-term impact on growth. Notably, these results indicate a consistent association between the DNAm signature of maternal smoking and a small body size and low birth weight in newborns, in both White European mothers who exhibited some amount of smoking and in South Asian mothers who themselves were not active smokers. Funding: This study was funded by the Canadian Institutes of Health Research Metabolomics Team Grant: MWG-146332.

Consistent cord blood DNA methylation signatures of gestational age between South Asian and white European cohorts.

BACKGROUND: Epigenetic modifications, particularly DNA methylation (DNAm) in cord blood, are an important biological marker of how external exposures during gestation can influence the in-utero environment and subsequent offspring development. Despite the recognized importance of DNAm during gestation, comparative studies to determine the consistency of these epigenetic signals across different ethnic groups are largely absent. To address this gap, we first performed epigenome-wide association studies (EWAS) of gestational age (GA) using newborn cord blood DNAm comparatively in a white European (n = 342) and a South Asian (n = 490) birth cohort living in Canada. Then, we capitalized on established cord blood epigenetic GA clocks to examine the associations between maternal exposures, offspring characteristics and epigenetic GA, as well as GA acceleration, defined as the residual difference between epigenetic and chronological GA at birth. RESULTS: Individual EWASs confirmed 1,211 and 1,543 differentially methylated CpGs previously reported to be associated with GA, in white European and South Asian cohorts, respectively, with a similar distribution of effects. We confirmed that Bohlin's cord blood GA clock was robustly correlated with GA in white Europeans (r = 0.71; p = 6.0 × 10-54) and South Asians (r = 0.66; p = 6.9 × 10-64). In both cohorts, Bohlin's clock was positively associated with newborn weight and length and negatively associated with parity, newborn female sex, and gestational diabetes. Exclusive to South Asians, the GA clock was positively associated with the newborn ponderal index, while pre-pregnancy weight and gestational weight gain were strongly predictive of increased epigenetic GA in white Europeans. Important predictors of GA acceleration included gestational diabetes mellitus, newborn sex, and parity in both cohorts. CONCLUSIONS: These results demonstrate the consistent DNAm signatures of GA and the utility of Bohlin's GA clock across the two populations. Although the overall pattern of DNAm is similar, its connections with the mother's environment and the baby's anthropometrics can differ between the two groups. Further research is needed to understand these unique relationships.

A method to estimate the contribution of rare coding variants to complex trait heritability.

It has been postulated that rare coding variants (RVs; MAF < 0.01) contribute to the "missing" heritability of complex traits. We developed a framework, the Rare variant heritability (RARity) estimator, to assess RV heritability (h2RV) without assuming a particular genetic architecture. We applied RARity to 31 complex traits in the UK Biobank (n = 167,348) and showed that gene-level RV aggregation suffers from 79% (95% CI: 68-93%) loss of h2RV. Using unaggregated variants, 27 traits had h2RV > 5%, with height having the highest h2RV at 21.9% (95% CI: 19.0-24.8%). The total heritability, including common and rare variants, recovered pedigree-based estimates for 11 traits. RARity can estimate gene-level h2RV, enabling the assessment of gene-level characteristics and revealing 11, previously unreported, gene-phenotype relationships. Finally, we demonstrated that in silico pathogenicity prediction (variant-level) and gene-level annotations do not generally enrich for RVs that over-contribute to complex trait variance, and thus, innovative methods are needed to predict RV functionality.

A high-resolution PheWAS approach to alcohol-related polygenic risk scores reveals mechanistic influences of alcohol reinforcing value and drinking motives.

AIMS: This study uses a high-resolution phenome-wide approach to evaluate the motivational mechanisms of polygenic risk scores (PRSs) that have been robustly associated with coarse alcohol phenotypes in large-scale studies. METHODS: In a community-based sample of 1534 Europeans, we examined genome-wide PRSs for the Alcohol Use Disorders Identification Test (AUDIT), drinks per week, alcohol use disorder (AUD), problematic alcohol use (PAU), and general addiction, in relation to 42 curated phenotypes. The curated phenotypes were in seven categories: alcohol consumption, alcohol reinforcing value, drinking motives, other addictive behaviors, commonly comorbid psychiatric syndromes, impulsivity, and personality traits. RESULTS: The PRS for each alcohol phenotype was validated via its within-sample association with the corresponding phenotype (adjusted R2s = 0.35-1.68%, Ps = 0.012-3.6 × 10-7) with the exception of AUD. All PRSs were positively associated with alcohol reinforcing value and drinking motives, with the strongest effects from AUDIT-consumption (adjusted R2s = 0.45-1.33%, Ps = 0.006-3.6 × 10-5) and drinks per week PRSs (adjusted R2s = 0.52-2.28%, Ps = 0.004-6.6 × 10-9). Furthermore, the PAU and drinks per week PRSs were positively associated with adverse childhood experiences (adjusted R2s = 0.6-0.7%, Ps = 0.0001-4.8 × 10-4). CONCLUSIONS: These results implicate alcohol reinforcing value and drinking motives as genetically-influenced mechanisms using PRSs for the first time. The findings also highlight the value of dissecting genetic influence on alcohol involvement through diverse phenotypic risk pathways but also the need for future studies with both phenotypic richness and larger samples.

Leveraging related health phenotypes for polygenic prediction of impulsive choice, impulsive action, and impulsive personality traits in 1534 European ancestry community adults.

Impulsivity refers to a number of conceptually related phenotypes reflecting self-regulatory capacity that are considered promising endophenotypes for mental and physical health. Measures of impulsivity can be broadly grouped into three domains, namely, impulsive choice, impulsive action, and impulsive personality traits. In a community-based sample of ancestral Europeans (n = 1534), we conducted genome-wide association studies (GWASs) of impulsive choice (delay discounting), impulsive action (behavioral inhibition), and impulsive personality traits (UPPS-P), and evaluated 11 polygenic risk scores (PRSs) of phenotypes previously linked to self-regulation. Although there were no individual genome-wide significant hits, the neuroticism PRS was positively associated with negative urgency (adjusted R2 = 1.61%, p = 3.6 × 10-7 ) and the educational attainment PRS was inversely associated with delay discounting (adjusted R2 = 1.68%, p = 2.2 × 10-7 ). There was also evidence implicating PRSs of attention-deficit/hyperactivity disorder, externalizing, risk-taking, smoking cessation, smoking initiation, and body mass index with one or more impulsivity phenotypes (adjusted R2 s: 0.35%-1.07%; FDR adjusted ps = 0.05-0.0006). These significant associations between PRSs and impulsivity phenotypes are consistent with established genetic correlations. The combined PRS explained 0.91%-2.46% of the phenotypic variance for individual impulsivity measures, corresponding to 8.7%-32.5% of their reported single-nucleotide polymorphism (SNP)-based heritability, suggesting a non-negligible portion of the SNP-based heritability can be recovered by PRSs. These results support the predictive validity and utility of PRSs, even derived from related phenotypes, to inform the genetics of impulsivity phenotypes.

gJLS2: an R package for generalized joint location and scale analysis in X-inclusive genome-wide association studies.

A joint analysis of location and scale can be a powerful tool in genome-wide association studies to uncover previously overlooked markers that influence a quantitative trait through both mean and variance, as well as to prioritize candidates for gene-environment interactions. This approach has recently been generalized to handle related samples, dosage data, and the analytically challenging X-chromosome. We disseminate the latest advances in methodology through a user-friendly R software package with added functionalities to support genome-wide analysis on individual-level or summary-level data. The implemented R package can be called from PLINK or directly in a scripting environment, to enable a streamlined genome-wide analysis for biobank-scale data. Application results on individual-level and summary-level data highlight the advantage of the joint test to discover more genome-wide signals as compared to a location or scale test alone. We hope the availability of gJLS2 software package will encourage more scale and/or joint analyses in large-scale datasets, and promote the standardized reporting of their P-values to be shared with the scientific community.

Author Correction: A machine-learning heuristic to improve gene score prediction of polygenic traits.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Analytical strategies to include the X-chromosome in variance heterogeneity analyses: Evidence for trait-specific polygenic variance structure.

Genotype-stratified variance of a quantitative trait could differ in the presence of gene-gene or gene-environment interactions. Genetic markers associated with phenotypic variance are thus considered promising candidates for follow-up interaction or joint location-scale analyses. However, as in studies of main effects, the X-chromosome is routinely excluded from "whole-genome" scans due to analytical challenges. Specifically, as males carry only one copy of the X-chromosome, the inherent sex-genotype dependency could bias the trait-genotype association, through sexual dimorphism in quantitative traits with sex-specific means or variances. Here we investigate phenotypic variance heterogeneity associated with X-chromosome single nucleotide polymorphisms (SNPs) and propose valid and powerful strategies. Among those, a generalized Levene's test has adequate power and remains robust to sexual dimorphism. An alternative approach is a sex-stratified analysis but at the cost of slightly reduced power and modeling flexibility. We applied both methods to an Estonian study of gene expression quantitative trait loci (eQTL; n = 841), and two complex trait studies of height, hip, and waist circumferences, and body mass index from Multi-Ethnic Study of Atherosclerosis (MESA; n = 2,073) and UK Biobank (UKB; n = 327,393). Consistent with previous eQTL findings on mean, we found some but no conclusive evidence for cis regulators being enriched for variance association. SNP rs2681646 is associated with variance of waist circumference (p = 9.5E-07) at X-chromosome-wide significance in UKB, with a suggestive female-specific effect in MESA (p = 0.048). Collectively, an enrichment analysis using permutated UKB (p < 0.1) and MESA (p < 0.01) datasets, suggests a possible polygenic structure for the variance of human height.

A robust method to estimate regional polygenic correlation under misspecified linkage disequilibrium structure.

Complex traits can share a substantial proportion of their polygenic heritability. However, genome-wide polygenic correlations between pairs of traits can mask heterogeneity in their shared polygenic effects across loci. We propose a novel method (weighted maximum likelihood-regional polygenic correlation [RPC]) to evaluate polygenic correlation between two complex traits in small genomic regions using summary association statistics. Our method tests for evidence that the polygenic effect at a given region affects two traits concurrently. We show through simulations that our method is well calibrated, powerful, and more robust to misspecification of linkage disequilibrium than other methods under a polygenic model. As small genomic regions are more likely to harbor specific genetic effects, our method is ideal to identify heterogeneity in shared polygenic correlation across regions. We illustrate the usefulness of our method by addressing two questions related to cardiometabolic traits. First, we explored how RPC can inform on the strong epidemiological association between high-density lipoprotein cholesterol and coronary artery disease (CAD), suggesting a key role for triglycerides metabolism. Second, we investigated the potential role of PPARγ activators in the prevention of CAD. Our results provide a compelling argument that shared heritability between complex traits is highly heterogeneous across loci.

A machine-learning heuristic to improve gene score prediction of polygenic traits.

Machine-learning techniques have helped solve a broad range of prediction problems, yet are not widely used to build polygenic risk scores for the prediction of complex traits. We propose a novel heuristic based on machine-learning techniques (GraBLD) to boost the predictive performance of polygenic risk scores. Gradient boosted regression trees were first used to optimize the weights of SNPs included in the score, followed by a novel regional adjustment for linkage disequilibrium. A calibration set with sample size of ~200 individuals was sufficient for optimal performance. GraBLD yielded prediction R 2 of 0.239 and 0.082 using GIANT summary association statistics for height and BMI in the UK Biobank study (N = 130 K; 1.98 M SNPs), explaining 46.9% and 32.7% of the overall polygenic variance, respectively. For diabetes status, the area under the receiver operating characteristic curve was 0.602 in the UK Biobank study using summary-level association statistics from the DIAGRAM consortium. GraBLD outperformed other polygenic score heuristics for the prediction of height (p < 2.2 × 10-16) and BMI (p < 1.57 × 10-4), and was equivalent to LDpred for diabetes. Results were independently validated in the Health and Retirement Study (N = 8,292; 688,398 SNPs). Our report demonstrates the use of machine-learning techniques, coupled with summary-level data from large genome-wide meta-analyses to improve the prediction of polygenic traits.

Ranking and characterization of established BMI and lipid associated loci as candidates for gene-environment interactions.

Phenotypic variance heterogeneity across genotypes at a single nucleotide polymorphism (SNP) may reflect underlying gene-environment (G×E) or gene-gene interactions. We modeled variance heterogeneity for blood lipids and BMI in up to 44,211 participants and investigated relationships between variance effects (Pv), G×E interaction effects (with smoking and physical activity), and marginal genetic effects (Pm). Correlations between Pv and Pm were stronger for SNPs with established marginal effects (Spearman's ρ = 0.401 for triglycerides, and ρ = 0.236 for BMI) compared to all SNPs. When Pv and Pm were compared for all pruned SNPs, only BMI was statistically significant (Spearman's ρ = 0.010). Overall, SNPs with established marginal effects were overrepresented in the nominally significant part of the Pv distribution (Pbinomial <0.05). SNPs from the top 1% of the Pm distribution for BMI had more significant Pv values (PMann-Whitney = 1.46×10-5), and the odds ratio of SNPs with nominally significant (<0.05) Pm and Pv was 1.33 (95% CI: 1.12, 1.57) for BMI. Moreover, BMI SNPs with nominally significant G×E interaction P-values (Pint<0.05) were enriched with nominally significant Pv values (Pbinomial = 8.63×10-9 and 8.52×10-7 for SNP × smoking and SNP × physical activity, respectively). We conclude that some loci with strong marginal effects may be good candidates for G×E, and variance-based prioritization can be used to identify them.

A method to estimate the contribution of regional genetic associations to complex traits from summary association statistics.

Despite considerable efforts, known genetic associations only explain a small fraction of predicted heritability. Regional associations combine information from multiple contiguous genetic variants and can improve variance explained at established association loci. However, regional associations are not easily amenable to estimation using summary association statistics because of sensitivity to linkage disequilibrium (LD). We now propose a novel method, LD Adjusted Regional Genetic Variance (LARGV), to estimate phenotypic variance explained by regional associations using summary statistics while accounting for LD. Our method is asymptotically equivalent to a multiple linear regression model when no interaction or haplotype effects are present. It has several applications, such as ranking of genetic regions according to variance explained or comparison of variance explained by two or more regions. Using height and BMI data from the Health Retirement Study (N = 7,776), we show that most genetic variance lies in a small proportion of the genome and that previously identified linkage peaks have higher than expected regional variance.

Contribution of large region joint associations to complex traits genetics.

A polygenic model of inheritance, whereby hundreds or thousands of weakly associated variants contribute to a trait's heritability, has been proposed to underlie the genetic architecture of complex traits. However, relatively few genetic variants have been positively identified so far and they collectively explain only a small fraction of the predicted heritability. We hypothesized that joint association of multiple weakly associated variants over large chromosomal regions contributes to complex traits variance. Confirmation of such regional associations can help identify new loci and lead to a better understanding of known ones. To test this hypothesis, we first characterized the ability of commonly used genetic association models to identify large region joint associations. Through theoretical derivation and simulation, we showed that multivariate linear models where multiple SNPs are included as independent predictors have the most favorable association profile. Based on these results, we tested for large region association with height in 3,740 European participants from the Health and Retirement Study (HRS) study. Adjusting for SNPs with known association with height, we demonstrated clustering of weak associations (p = 2x10-4) in regions extending up to 433.0 Kb from known height loci. The contribution of regional associations to phenotypic variance was estimated at 0.172 (95% CI 0.063-0.279; p < 0.001), which compared favorably to 0.129 explained by known height variants. Conversely, we showed that suggestively associated regions are enriched for known height loci. To extend our findings to other traits, we also tested BMI, HDLc and CRP for large region associations, with consistent results for CRP. Our results demonstrate the presence of large region joint associations and suggest these can be used to pinpoint weakly associated SNPs.

Does genetic heterogeneity account for the divergent risk of type 2 diabetes in South Asian and white European populations?

AIMS/HYPOTHESIS: South Asians are up to four times more likely to develop type 2 diabetes than white Europeans. It is postulated that the higher prevalence results from greater genetic risk. To evaluate this hypothesis, we: (1) systematically reviewed the literature for single nucleotide polymorphisms (SNPs) predisposing to type 2 diabetes in South Asians; (2) compared risk estimates, risk alleles and risk allele frequencies of predisposing SNPs between South Asians and white Europeans; and (3) tested the association of novel SNPs discovered from South Asians in white Europeans. METHODS: MEDLINE, Embase, the Cumulative Index to Nursing and Allied Health Literature (CINAHL) and the Cochrane registry were searched for studies of genetic variants associated with type 2 diabetes in South Asians. Meta-analysis estimates for common and novel bi-allelic SNPs in South Asians were compared with white Europeans from the DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) consortium. The population burden from predisposing SNPs was assessed using a genotype score. RESULTS: Twenty-four SNPs from 21 loci were associated with type 2 diabetes in South Asians after meta-analysis. The majority of SNPs increase odds of the disorder by 15-35% per risk allele. No substantial differences appear to exist in risk estimates between South Asians and white Europeans from SNPs common to both groups, and the population burden also does not differ. Eight of the 24 are novel SNPs discovered from South Asian genome-wide association studies, some of which show nominal associations with type 2 diabetes in white Europeans. CONCLUSIONS/INTERPRETATION: Based on current literature there is no strong evidence to indicate that South Asians possess a greater genetic risk of type 2 diabetes than white Europeans.

Temporal relationship between subclinical atrial fibrillation and embolic events.

BACKGROUND: Among patients with implantable pacemakers and defibrillators, subclinical atrial fibrillation (SCAF) is associated with an increased risk of stroke; however, there is limited understanding of their temporal relationship. METHODS AND RESULTS: The Asymptomatic Atrial Fibrillation and Stroke Evaluation in Pacemaker Patients and the Atrial Fibrillation Reduction Atrial Pacing Trial (ASSERT) enrolled 2580 pacemaker and defibrillator patients aged ≥65 years with a history of hypertension but without a history of atrial fibrillation. Pacemakers and implantable cardioverter-defibrillators precisely logged the time and duration of all episodes of SCAF and recorded electrograms that were adjudicated by experts. We examined the temporal relationship between SCAF >6 minutes in duration and stroke or systemic embolism. Of 51 patients who experienced stroke or systemic embolism during follow-up, 26 (51%) had SCAF. In 18 patients (35%), SCAF was detected before stroke or systemic embolism. However, only 4 patients (8%) had SCAF detected within 30 days before stroke or systemic embolism, and only 1 of these 4 patients was experiencing SCAF at the time of the stroke. In the 14 patients with SCAF detected >30 days before stroke or systemic embolism, the most recent episode occurred at a median interval of 339 days (25th to 75th percentile, 211-619) earlier. Eight patients (16%) had SCAF detected only after their stroke, despite continuous monitoring for a median duration of 228 days (25th to 75th percentile, 202-719) before their event. CONCLUSIONS: Although SCAF is associated with an increased risk of stroke and embolism, very few patients had SCAF in the month before their event. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT00256152.

Meta-analysis of SNPs involved in variance heterogeneity using Levene's test for equal variances.

Meta-analysis is a commonly used approach to increase the sample size for genome-wide association searches when individual studies are otherwise underpowered. Here, we present a meta-analysis procedure to estimate the heterogeneity of the quantitative trait variance attributable to genetic variants using Levene's test without needing to exchange individual-level data. The meta-analysis of Levene's test offers the opportunity to combine the considerable sample size of a genome-wide meta-analysis to identify the genetic basis of phenotypic variability and to prioritize single-nucleotide polymorphisms (SNPs) for gene-gene and gene-environment interactions. The use of Levene's test has several advantages, including robustness to departure from the normality assumption, freedom from the influence of the main effects of SNPs, and no assumption of an additive genetic model. We conducted a meta-analysis of the log-transformed body mass index of 5892 individuals and identified a variant with a highly suggestive Levene's test P-value of 4.28E-06 near the NEGR1 locus known to be associated with extreme obesity.

Associations of the FTO rs9939609 variant with discrete body fat depots and dietary intake in a multi-ethnic cohort.

The fat mass and obesity associated (FTO) gene has been implicated with obesity and dietary intake predominantly in European populations. We assessed the association between the FTO rs9939609 variant with body fat distribution and dietary intake in a multi-ethnic population. Aboriginal, Chinese, European and South Asian participants living in Canada (n = 706) were assessed for body fat and inner-abdominal fat using imaging techniques, dietary intake and genotyped for the FTO rs9939609 variant. Linear regression was used to study the associations between the minor allele of the variant and measures of adiposity and dietary intake. Minor allele frequencies were: Aboriginals (17%), Chinese (17%), Europeans (39%) and South Asians (31%). The rs9939609 variant was associated with intake of dietary macronutrients in Aboriginals and Europeans only. In the total population, there were positive associations between the rs9939609 minor allele and greater fat mass (0.94 ± 0.56 kg, P = 0.045), per cent body fat (0.7 ± 0.4%, P = 0.031), relative greater subcutaneous abdominal adipose tissue (4.9 ± 2.8%, P = 0.039) and percent daily calories from fat (0.4 ± 0.2%, P = 0.064). Our findings suggest that the FTO rs9939609 minor allele may be associated with dietary intake in adults and is positively associated with regional fat deposition.

A fast algorithm to optimize SNP prioritization for gene-gene and gene-environment interactions.

Detection of gene-environment interactions using an exhaustive search necessarily raises the multiple hypothesis problem. While frequently used to control for experiment-wise type I error, Bonferroni correction is overly conservative and results in reduced statistical power. We have previously shown that prioritizing SNPs on the basis of heterogeneity in quantitative trait variance per genotype leads to increased power to detect genetic interactions. Our proposed method, variance prioritization (VP), selects SNPs having significant heterogeneity in variance per genotype using a pre-determined P-value threshold. We now suggest prioritizing SNPs individually such that the optimal heterogeneity of variance P-value is determined for each SNP. The large number of SNPs in genome-wide studies calls for a fast algorithm to output the optimal prioritization threshold for each SNP. In this report, we present such an algorithm, the Gene Environment Wide Interaction Search Threshold (GEWIST), and show that the use of GEWIST will increase power under a variety of interaction scenarios. Furthermore, by integrating over possible interaction effect sizes, we provide a framework to optimize prioritization in situations where interactions are a priori unknown.