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Aim: We evaluated five methods for detecting differentially methylated regions (DMRs): DMRcate, comb-p, seqlm, GlobalP and dmrff. Materials & methods: We used a simulation study and real data analysis to evaluate performance. Additionally, we evaluated the use of an ancestry-matched reference cohort to estimate correlations between CpG sites in cord blood. Results: Several methods had inflated Type I error, which increased at more stringent significant levels. In power simulations with 1–2 causal CpG sites with the same direction of effect, dmrff was consistently among the most powerful methods. Conclusion: This study illustrates the need for more thorough simulation studies when evaluating novel methods. More work must be done to develop methods with well-controlled Type I error that do not require individual-level data.

The genetic determinants of fasting glucose (FG) and fasting insulin (FI) have been studied mostly through genome and exome arrays, resulting in over 100 associated variants. We extended this work with a high-coverage whole genome sequencing (WGS) analysis from fifteen cohorts in the NHLBI Trans-Omics for Precision Medicine (TOPMed) program. More than 23,000 non-diabetic individuals from five self-reported race/ethnicities (African, Asian, European, Hispanic and Samoan) were included for each trait. We analyzed 60M variants in race/ethnicity-specific and pooled single variant and rare variant aggregate tests. Twenty-two variants across sixteen gene regions were found significantly associated with FG or FI, eight of which were rare (Minor Allele Frequency, MAFG6PC2locus we identified a distinct FG signal at rare variant rs2232326 (MAF=0.01) after conditioning on known common variants. Functional annotations show rs2232326 to be disruptive and likely damaging while being weakly transcribed in islets. A pair of FG-associated variants were identified near theSLC30A8locus. These variants, one of which was rare (MAF=0.001) and Asian race/ethnicity-specific, were shown to be in islet-specific active enhancer regions. Other associated regions include rare variants nearROBO1andPTPRT, and common variants nearMTNR1B, GCK, GCKR, FOXA2, APOB, TCF7L2, andADCY5. We provide a catalog of nucleotide-resolution genomic variation spanning intergenic and intronic regions down to a minor allele count of 20, creating a foundation for future sequencing-based investigation of glycemic traits.

Background DNA methylation has been shown to be associated with adiposity in adulthood. However, whether similar DNA methylation patterns are associated with childhood and adolescent body mass index (BMI) is largely unknown. More insight into this relationship at younger ages may have implications for future prevention of obesity and its related traits. Methods We examined whether DNA methylation in cord blood and whole blood in childhood and adolescence was associated with BMI in the age range from 2 to 18 years using both cross-sectional and longitudinal models. We performed meta-analyses of epigenome-wide association studies including up to 4133 children from 23 studies. We examined the overlap of findings reported in previous studies in children and adults with those in our analyses and calculated enrichment. Results DNA methylation at three CpGs (cg05937453, cg25212453, and cg10040131), each in a different age range, was associated with BMI at Bonferroni significance, P −7, with a 0.96 standard deviation score (SDS) (standard error (SE) 0.17), 0.32 SDS (SE 0.06), and 0.32 BMI SDS (SE 0.06) higher BMI per 10% increase in methylation, respectively. DNA methylation at nine additional CpGs in the cross-sectional childhood model was associated with BMI at false discovery rate significance. The strength of the associations of DNA methylation at the 187 CpGs previously identified to be associated with adult BMI, increased with advancing age across childhood and adolescence in our analyses. In addition, correlation coefficients between effect estimates for those CpGs in adults and in children and adolescents also increased. Among the top findings for each age range, we observed increasing enrichment for the CpGs that were previously identified in adults (birth Penrichment = 1; childhood Penrichment = 2.00 × 10−4; adolescence Penrichment = 2.10 × 10−7). Conclusions There were only minimal associations of DNA methylation with childhood and adolescent BMI. With the advancing age of the participants across childhood and adolescence, we observed increasing overlap with altered DNA methylation loci reported in association with adult BMI. These findings may be compatible with the hypothesis that DNA methylation differences are mostly a consequence rather than a cause of obesity.

Type 2 diabetes is increasing in all ancestry groups1. Part of its genetic basis may reside among the rare (minor allele frequency 2. We analyzed high-coverage (mean depth 38.2x) whole genome sequencing from 9,639 individuals with T2D and 34,994 controls in the NHLBI’s Trans-Omics for Precision Medicine (TOPMed) program2 to show that rare, non-coding variants that are poorly captured by genotyping arrays or imputation panels contribute h2=53% (P=4.2×10−5) to the genetic component of risk in the largest (European) ancestry subset. We coupled sequence variation with islet epigenomic signatures3 to annotate and group rare variants with respect to gene expression4, chromatin state5 and three-dimensional chromatin architecture6, and show that pancreatic islet regulatory elements contribute to T2D genetic risk (h2=8%, P=2.4×10−3). We used islet annotation to create a non-coding framework for rare variant aggregation testing. This approach identified five loci containing rare alleles in islet regulatory elements that suggest novel biological mechanisms readily linked to hypotheses about variant-to-function. Large scale whole genome sequence analysis reveals the substantial contribution of rare, non-coding variation to the genetic architecture of T2D and highlights the value of tissue-specific regulatory annotation for variant-to-function discovery.

Background The study of DNA methylation quantitative trait loci (meQTLs) helps dissect regulatory mechanisms underlying genetic associations of human diseases. In this study, we conducted the first genome-wide examination of genetic drivers of methylation variation in response to a triglyceride-lowering treatment with fenofibrate (response-meQTL) by using an efficient analytic approach. Methods Subjects (n = 429) from the GAW20 real data set with genotype and both pre- (visit 2) and post- (visit 4) fenofibrate treatment methylation measurements were included. Following the quality control steps of removing certain cytosine-phosphate-guanine (CpG) probes, the post−/premethylation changes (post/pre) were log transformed and the association was performed on 208,449 CpG sites. An additive linear mixed-effects model was used to test the association between each CpG probe and single nucleotide polymorphisms (SNPs) around ±1 Mb region, with age, sex, smoke, batch effect, and principal components included as covariates. Bonferroni correction was applied to define the significance threshold (p 0.8) with rs7443270, which was previously reported to be associated with fenofibrate response (p = 5.00 × 10− 6). Conclusions By using a novel analytic approach, we efficiently identified thousands of cis re-meQTLs that provide a unique resource for further characterizing functional roles and gene targets of the SNPs that are most responsive to fenofibrate treatment. Our efficient analytic approach can be extended to large response quantitative trait locus studies with large sample sizes and multiple time points data.

Type 2 diabetes (T2D) is caused by interactions between genetic predisposition and environmental exposure. DNA methylation, a key epigenetic mark, is one measure of the molecular impact of environmental factors on DNA. We tested for associations between glycemic traits and peripheral blood cell DNA methylation in 473,669 CpG sites across the genome in individuals from multiple ancestries, and tested for causal relationships at these loci. We conducted an epigenome-wide fixed effects meta-analysis for 3 glycemic traits-fasting glucose (FG), log fasting insulin (FI), and HbA1c—in 13,543 nondiabetic individuals of African (N=3,746), European (N=9,254), and Hispanic (N=543) ancestry across 16 cohorts. All analyses were adjusted for age, sex, smoking status, cell types, BMI, and technical covariates. We used Mendelian randomization (MR) analyses to determine whether glycemic traits causally influence methylation at the CpG sites identified in the meta-analysis. We performed MR analyses in Framingham Heart Study individuals (N=6,472), using separate genetic risk scores (GRS) as instruments for each of the 3 glycemic traits. In the epigenome-wide meta-analysis of all ancestries, there were 43 CpG sites associated with FG, 89 with FI, and 54 with HbA1c, using a Bonferroni threshold of P We identified many novel candidate methylation loci associated with glycemic traits and 2 CpG sites where MR analysis indicated that methylation is causally influenced by glycemic traits. These findings support the hypothesis that variation in glycemia influences peripheral blood DNA methylation. Disclosure D.A. DiCorpo: None. S. Lent: None. W. Guan: None. M. Hivert: None. J.S. Pankow: None.

Over 100 associated genetic loci have been robustly associated with schizophrenia. Gene prioritization and pathway analysis have focused on a priori hypotheses and thus may have been unduly influenced by prior assumptions and missed important causal genes and pathways. Using a data-driven approach, we show that genes in associated loci: (1) are highly expressed in cortical brain areas; (2) are enriched for ion channel pathways (false discovery rates

Background Recent focus on studying rare variants makes imputation accuracy of rare variants an important issue. Many approaches have been proposed to increase imputation accuracy among rare variants, from reference panel selection to combinations of existing methods to multistage analyses. We aimed to bring the strengths of these new approaches together with our proposed two-stage imputation for family data. Methods Our imputation methods were tested on the region from 46.75Mb to 49.25Mb on chromosome 3. We did quality control based on the proportion of missing genotypes per variant and individual, leaving 495 individuals with 761 genome-wide association studies (GWAS) variants only, 45 with 14,077 sequence variants only, and 419 with both GWAS and sequencing data. All data were prephased using SHAPEIT2 with a duo hidden Markov model algorithm prior to performing imputation. Imputations were performed 100 times, each time masking the sequence data for 1 individual and imputing it from the GWAS data. We used well-imputed genotypes, defined as a probability of greater than 0.9, above 2 different minor allele frequency cutoffs—0.01 and 0.05—from Impute2 as input for Merlin, and compared these results to Impute2 and Merlin separately. The imputed results were evaluated using correlation measurement and the imputation quality score. Results Our method improved imputation accuracy, measured by imputation quality score, for variants with minor allele frequency between 0.01 and 0.40, but failed to improve accuracy for variants with minor allele frequency less than 0.01 when we used a minor allele frequency cutoff of 0.01 for the Impute2 results. In contrast, our 2-stage approach with a minor allele frequency cutoff of 0.05 performed the worst of all methods for variants with minor allele frequency between 0.01 and 0.40. Conclusions This method gave promising results, but may be further improved by changing the inclusion criteria of Impute2 variants. More analyses are needed on a larger region with different inclusion thresholds to assess the accuracy of this approach.

Despite existing reports on differential DNA methylation in type 2 diabetes (T2D) and obesity, our understanding of the functional relevance of the phenomenon remains limited. Because obesity is the main risk factor for T2D and a driver of methylation from previous study, we aimed to explore the effect of DNA methylation in the early phases of T2D pathology while accounting for body mass index (BMI). We performed a blood-based epigenome-wide association study (EWAS) of fasting glucose and insulin among 4,808 non-diabetic European individuals and replicated the findings in an independent sample consisting of 11,750 non-diabetic subjects. We integrated blood-based in silico cross-omics databases comprising genomics, epigenomics and transcriptomics collected by BIOS project of the Biobanking and BioMolecular resources Research Infrastructure of the Netherlands (BBMRI-NL), the Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC), the DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) consortium, and the tissue-specific Genotype-Tissue Expression (GTEx) project. We identified and replicated nine novel differentially methylated sites in whole blood (P-value < 1.27 × 10-7): sites in LETM1, RBM20, IRS2, MAN2A2 genes and 1q25.3 region were associated with fasting insulin; sites in FCRL6, SLAMF1, APOBEC3H genes and 15q26.1 region were associated with fasting glucose. The association between SLAMF1, APOBEC3H and 15q26.1 methylation sites and glucose emerged only when accounted for BMI. Follow-up in silico cross-omics analyses indicate that the cis-acting meQTLs near SLAMF1 and SLAMF1 expression are involved in glucose level regulation. Moreover, our data suggest that differential methylation in FCRL6 may affect glucose level and the risk of T2D by regulating FCLR6 expression in the liver. In conclusion, the present study provided nine new DNA methylation sites associated with glycemia homeostasis and also provided new insights of glycemia related loci into the genetics, epigenetics and transcriptomics pathways based on the integration of cross-omics data in silico.