Abstract
We characterized DNA methylation quantitative trait loci (mQTLs) in a large collection (n = 166) of human fetal brain samples spanning 56–166 d post-conception, identifying >16,000 fetal brain mQTLs. Fetal brain mQTLs were primarily cis-acting, enriched in regulatory chromatin domains and transcription factor binding sites, and showed substantial overlap with genetic variants that were also associated with gene expression in the brain. Using tissue from three distinct regions of the adult brain (prefrontal cortex, striatum and cerebellum), we found that most fetal brain mQTLs were developmentally stable, although a subset was characterized by fetal-specific effects. Fetal brain mQTLs were enriched amongst risk loci identified in a recent large-scale genome-wide association study (GWAS) of schizophrenia, a severe psychiatric disorder with a hypothesized neurodevelopmental component. Finally, we found that mQTLs can be used to refine GWAS loci through the identification of discrete sites of variable fetal brain methylation associated with schizophrenia risk variants.
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Acknowledgements
We thank M. Weale for providing eQTL data from the BRAINEAC database. This work was supported by grants from the UK Medical Research Council (MRC; MR/K013807/1 to J.M. and MR/L010674/1 to N.J.B.) and the US National Institutes of Health (AG036039) to J.M. R.P. and H.S. were funded by MRC PhD studentships. The human embryonic and fetal material was provided by the Joint MRC/Wellcome Trust (grant #099175/Z/12/Z) Human Developmental Biology Resource.
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J.M. and N.J.B. conceived and supervised the study and obtained funding. E.H. undertook primary data analysis and bioinformatics. L.C.S. provided analytical support. H.S., J.V., R.P., T.M.M. and J.B. performed laboratory work. C.T. and G.T. provided samples for analysis. M.C.O'D. provided support for GWAS enrichment analyses. E.H., N.J.B. and J.M. drafted the manuscript. All of the authors read and approved the final submission.
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Integrated supplementary information
Supplementary Figure 1 The distribution of effect sizes across all Bonferroni significant fetal brain mQTLs.
Shown is the DNA methylation difference (% DNA methylation) associated with each minor allele for all fetal brain mQTLs, cis-acting mQTLs, and trans-acting mQTLs. The average effect size for trans-mQTLs is significantly lower than that observed for cis-mQTLs (two-sided Wilcoxon rank sum test, P = 6.74x10−7).
Supplementary Figure 2 Frequency distribution of mQTL SNPs and associated DNA methylation sites.
A) mQTL SNPs are associated with DNA methylation at relatively few sites, but B) genetically-mediated DNA methylation sites are often associated with several mQTL SNPs.
Supplementary Figure 3 The statistical significance of association between genotype and DNA methylation is related to the distance between the Illumina 450K array probe and mQTL SNP.
Shown is the relationship between mQTL significance (–log10 P-value) and distance between the DNA methylation site and genetic variant in fetal brain samples.
Supplementary Figure 4 There is a highly-significant correlation of individual mQTL effects between fetal brain and each of the individual adult brain regions.
Shown are scatterplots showing the relationship in effect size (% DNA methylation change per allele) for all fetal brain mQTLs between fetal brain and a) PFC (r = 0.911, P < 2.2x10−16), b) STR (r = 0.899, P < 2.2x10−16) and c) CER (r = 0.835, P < 2.2x10−16).
Supplementary Figure 5 The correlation of mQTL effect sizes (% DNA methylation change per allele) between fetal brain and adult brain is stronger for replicating variants (left) than non-replicating variants (right).
Of note, fetal brain mQTLs that do not replicate in adult brain are characterized by significantly lower effect sizes across all brain regions (Supplementary Fig. 6).
Supplementary Figure 7 SNPs associated with DNA methylation are more significantly associated with gene expression than non-mQTL variants.
Density plot comparing the distribution of the minimum brain eQTL P value across all fetal brain mQTLs (red) and non-mQTLs (blue) in the mQTL dataset (Wilcoxon rank sum test P < 2.2x10−16).
Supplementary Figure 8 The overlap between independent fetal brain mQTL signals with the sentinelized subsignals from a brain eQTL dataset.
Histogram of overlap between simulated mQTL SNPs set (n = 1,000,000 simulations, matched for allele frequency) with the sentinelized eQTL set published by Ramasamy et al.. The vertical red line represents the true overlap, which is greater than any of the simulations (fold change = 4.23, P < 1.00x10−6 after 1 million simulations).
1. Ramasamy, A. et al. Genetic variability in the regulation of gene expression in ten regions of the human brain. Nat Neurosci 17, 1418-28 (2014).
Supplementary Figure 9 Fetal brain mQTLs ar1e significantly enriched for schizophrenia genetic risk variants.
Different significance thresholds (represented by different colored bars) were used to classify SNP associations from a recent large GWAS analysis of schizophrenia1. There is an enrichment of schizophrenia-associated variants amongst fetal brain mQTLs at all tested GWAS significance thresholds. Shown is the enrichment fold-change with 95% confidence intervals based on 1 million simulated SNP-sets. See also Table 1.
1. Schizophrenia Working Group of the Psychiatric Genomics Consortium,. Biological insights from 108 schizophrenia-associated genetic loci. Nature 511, 421-427 (2014).
Supplementary Figure 10 Boxplot of mQTL effects observed for rs2535627, an index SNP from the recent schizophrenia GWAS.
Shown are boxplots demonstrating the relationship between genotype and DNA methylation for rs2535627 and cg11645453. There is a significant association in the fetal brain (P = 3.05x10−13) but none of the adult brain regions tested.
Supplementary Figure 11 Boxplot of mQTL effect observed for rs4648845, an index SNP from a recent schizophrenia GWAS.
This boxplot demonstrates the relationship between genotype and DNA methylation for rs4648845 and cg02275930 in the fetal brain (P = 4.54x10−15). This mQTL was not tested in the adult brain regions because of the low minor allele frequency of rs4648845.
Supplementary Figure 12 mQTLs identified using imputed genetic data reflected the non-imputed dataset in terms of genomic distribution and observed effect sizes.
A) The genomic distribution of all imputed mQTLs in fetal brain, where the position on the x-axis indicates the location of Illumina 450K HumanMethylation array probes and the position on the y-axis indicates the location of all imputed SNPs. The color of the point corresponds to the difference in DNA methylation per allele, with the largest effects plotted in dark red. A clear positive diagonal can be observed demonstrating that the majority of mQTLs in fetal brain are associated with genotype in cis, reflecting the distribution shown in Fig. 1a. B) The distribution of effect sizes across all imputed fetal brain mQTLs. The distribution is similar to that for the non-imputed dataset (Supplementary Fig. 1).
Supplementary Figure 13 Effect sizes at fetal brain mQTLs identified in this study are highly correlated with those identified in an independent study of cortical mQTLs across development.
Effect sizes at mQTLs identified in this study (Hannon et al., X-axis) are highly correlated with those reported in a companion study (Jaffe et al., Y-axis). Each point represents a cis-mQTL identified in the current fetal brain dataset using the thresholds used by Jaffe et al [Nature Neuroscience, in press] (<20kb distance between SNP and DNA methylation site, FDR < 0.1).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–13 (PDF 2544 kb)
Supplementary Table 1: Summary of demographic data for each of the four brain mQTL datasets generated in this study.
Abbreviations: PFC = prefrontal cortex, STR = striatum, CER = cerebellum. (XLSX 98 kb)
Supplementary Table 3: Annotated list of all Bonferroni significant fetal brain mQTLs.
For each significant (P < 3.69×10-13) mQTL, associated DNA methylation sites are annotated with genomic location, Illumina gene annotation, and ENCODE transcription factor binding site or DNaseI hypersensitivity site (DHS) overlap. mQTL SNPs are annotated with genomic location. Regression coefficients and P-values are provided for each mQTL across for fetal brain, and three adult brain region datasets. A list of all significant fetal brain mQTLs generated using imputed genotypes is available for download from: http://epigenetics.essex.ac.uk/mQTL/. Abbreviations: PFC = prefrontal cortex, STR = striatum, CER = cerebellum, nt = not tested in that dataset, ns = not significant (P > 0.0001) in that dataset. (XLSX 3569 kb)
Supplementary Table 4: Many fetal brain trans-mQTLs are observed in non-neural tissues.
For each DNA methylation probe identified as being influenced by a trans-mQTL in fetal brain, the strongest corresponding trans-mQTL effect in two published genome-wide mQTL analyses in pancreatic islets [Olsson, A.H. et al. PLoS Genet 10, e1004735 (2014)] and lymphocytes [Lemire, M. et al. Nat Commun 6, 6326 (2015)] are presented. (XLSX 53 kb)
Supplementary Table 5: Overlap of fetal brain mQTLs in non-neural tissues.
The set of DNA methylation probes associated with mQTL SNPs in fetal brain were compared to the set of DNA methylation probes associated with mQTL SNPs in two published genome-wide mQTL analyses undertaken in pancreatic islets [Olsson, A.H. et al. PLoS Genet 10, e1004735 (2014)] and lymphocytes [Lemire, M. et al. Nat Commun 6, 6326 (2015)]. (XLSX 29 kb)
Supplementary Table 6: Enrichment of human fetal brain mQTLs in ChIP-seq peaks for regulatory histone modifications in fetal brain.
We tested for enrichment of genetically-mediated DNA methylation sites in fetal brain histone modification ChIP-seq peaks identified by the Roadmap Epigenomics Project (http://www.roadmapepigenomics.org/). (XLSX 30 kb)
Supplementary Table 7: Enrichment of human fetal brain mQTLs in DNase hypersensitivity sites (DHSs).
We tested for enrichment of genetically-mediated DNA methylation sites in DHSs identified by the ENCODE project. (XLSX 31 kb)
Supplementary Table 8: Enrichment of human fetal brain mQTLs in transcription factor binding sites (TFBSs).
We tested for enrichment of genetically-mediated DNA methylation sites in TFBSs identified by the ENCODE project. (XLSX 41 kb)
Supplementary Table 9: Comparison of mQTLs across fetal and adult brain regions.
Summary statistics for Bonferroni significant fetal brain mQTLs are presented at a range of P-value replication thresholds. Abbreviations: PFC = prefrontal cortex, STR = striatum, CER = cerebellum. (XLSX 35 kb)
Supplementary Table 10: Fetal brain mQTLs with significant heterogeneity between fetal and adult datasets.
Abbreviations: PFC = prefrontal cortex, STR = striatum, CER = cerebellum. (XLSX 2357 kb)
Supplementary Table 11: Fetal brain mQTLs characterized by opposite direction of effects in at least one adult brain region.
Abbreviations: PFC = prefrontal cortex, STR = striatum, CER = cerebellum. (XLSX 51 kb)
Supplementary Table 13: Genetic variants associated with DNA methylation and gene expression in the human brain.
All pairs of mQTLs and eQTLs for the intersecting set of genetic variants where the same SNP was found to influence DNA methylation and gene expression in cis. (XLSX 280 kb)
Supplementary Table 15: GWAS variants identified as human fetal brain mQTLs.
All genome-wide significant variants (P < 5 ×10−8) identified for Alzheimer's disease and BMI prior to LD clumping that may mediate DNA methylation are included in this table along with their Bonferroni significant mQTLs. There were no genome-wide significant type 2 diabetes variants that were Bonferroni significant mQTLs. A comparable table identifying the overlap with mQTLs generated using imputed genotypes is available for download from: http://epigenetics.essex.ac.uk/mQTL/. Abbreviations: PFC = prefrontal cortex, STR = striatum, CER = cerebellum. (XLSX 45 kb)
Supplementary Table 16: Robustly-associated schizophrenia GWAS variants characterized by human fetal brain mQTLs.
The set of 'likely causal variants' for schizophrenia was defined as all index variants representing the 125 independently associated autosomal loci in the latest PGC schizophrenia GWAS3. Given that the causal variant at these loci is yet to be established, this list was extended to include additional variants in strong LD (r2 < 0.8 based on 1000 Genomes European populations) with these 125 autosomal index variants. For this list of 'likely causal variants', all Bonferroni significant fetal brain mQTLs were identified. A comparable table identifying the overlap with mQTLs generated using imputed genotypes is available for download from: http://epigenetics.iop.kcl.ac.uk/mQTL/. Abbreviations: PFC = prefrontal cortex, STR = striatum, CER = cerebellum. (XLSX 50 kb)
Supplementary Table 17
Genomic loci with evidence for association with both schizophrenia and DNA methylation. Co-localization analysis was performed using the publically available summary statistics from the PGC schizophrenia GWAS and imputed mQTLs generated in this study. This table contains the 306 pairs where the association for schizophrenia and a DNA methylation site overlap the same genomic region. This includes instances where either the same causal variant is associated with both phenotypes or there are separate signals for each disorder. The posterior probabilities for each hypothesis are reported (see Online Methods for details). (XLSX 98 kb)
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R code for analyses and figures. (ZIP 27 kb)
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Hannon, E., Spiers, H., Viana, J. et al. Methylation QTLs in the developing brain and their enrichment in schizophrenia risk loci. Nat Neurosci 19, 48–54 (2016). https://doi.org/10.1038/nn.4182
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DOI: https://doi.org/10.1038/nn.4182
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