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Methylation QTLs in the developing brain and their enrichment in schizophrenia risk loci

Nature Neuroscience volume 19, pages 4854 (2016) | Download Citation

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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References

  1. 1.

    et al. Spatio-temporal transcriptome of the human brain. Nature 478, 483–489 (2011).

  2. 2.

    Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet. 13, 484–492 (2012).

  3. 3.

    et al. Charting a dynamic DNA methylation landscape of the human genome. Nature 500, 477–481 (2013).

  4. 4.

    et al. The relationship between DNA methylation, genetic and expression inter-individual variation in untransformed human fibroblasts. Genome Biol. 15, R37 (2014).

  5. 5.

    et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257 (2010).

  6. 6.

    et al. Methylomic trajectories across human fetal brain development. Genome Res. 25, 338–352 (2015).

  7. 7.

    et al. Abundant quantitative trait loci exist for DNA methylation and gene expression in human brain. PLoS Genet. 6, e1000952 (2010).

  8. 8.

    et al. Enrichment of cis-regulatory gene expression SNPs and methylation quantitative trait loci among bipolar disorder susceptibility variants. Mol. Psychiatry 18, 340–346 (2013).

  9. 9.

    et al. Passive and active DNA methylation and the interplay with genetic variation in gene regulation. Elife 2, e00523 (2013).

  10. 10.

    et al. Systematic localization of common disease-associated variation in regulatory DNA. Science 337, 1190–1195 (2012).

  11. 11.

    et al. Trait-associated SNPs are more likely to be eQTLs: annotation to enhance discovery from GWAS. PLoS Genet. 6, e1000888 (2010).

  12. 12.

    Schizophrenia Working Group of the Psychiatric Genomics Consortium. Biological insights from 108 schizophrenia-associated genetic loci. Nature 511, 421–427 (2014).

  13. 13.

    & The neurodevelopmental hypothesis of schizophrenia, revisited. Schizophr. Bull. 35, 528–548 (2009).

  14. 14.

    From neuropathology to neurodevelopment. Lancet 346, 552–557 (1995).

  15. 15.

    et al. The effect of genotype and in utero environment on inter-individual variation in neonate DNA methylomes. Genome Res. 24, 1064–1074 (2014).

  16. 16.

    et al. Genome-wide associations between genetic and epigenetic variation influence mRNA expression and insulin secretion in human pancreatic islets. PLoS Genet. 10, e1004735 (2014).

  17. 17.

    et al. The presence of methylation quantitative trait loci indicates a direct genetic influence on the level of DNA methylation in adipose tissue. PLoS ONE 8, e55923 (2013).

  18. 18.

    et al. Tissue-specific effects of genetic and epigenetic variation on gene regulation and splicing. PLoS Genet. 11, e1004958 (2015).

  19. 19.

    et al. Long-range epigenetic regulation is conferred by genetic variation located at thousands of independent loci. Nat. Commun. 6, 6326 (2015).

  20. 20.

    , & Exploring the three-dimensional organization of genomes: interpreting chromatin interaction data. Nat. Rev. Genet. 14, 390–403 (2013).

  21. 21.

    , , , & Interchromosomal associations between alternatively expressed loci. Nature 435, 637–645 (2005).

  22. 22.

    ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

  23. 23.

    et al. Identification and systematic annotation of tissue-specific differentially methylated regions using the Illumina 450k array. Epigenetics Chromatin 6, 26 (2013).

  24. 24.

    Roadmap Epigenomics Consortium. et al. Integrative analysis of 111 reference human epigenomes. Nature 518, 317–330 (2015).

  25. 25.

    et al. Intermediate DNA methylation is a conserved signature of genome regulation. Nat. Commun. 6, 6363 (2015).

  26. 26.

    et al. Contribution of genetic variation to transgenerational inheritance of DNA methylation. Genome Biol. 15, R73 (2014).

  27. 27.

    et al. Widespread plasticity in CTCF occupancy linked to DNA methylation. Genome Res. 22, 1680–1688 (2012).

  28. 28.

    & CTCF: an architectural protein bridging genome topology and function. Nat. Rev. Genet. 15, 234–246 (2014).

  29. 29.

    et al. Genetic variability in the regulation of gene expression in ten regions of the human brain. Nat. Neurosci. 17, 1418–1428 (2014).

  30. 30.

    et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559–575 (2007).

  31. 31.

    et al. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease. Nat. Genet. 45, 1452–1458 (2013).

  32. 32.

    et al. Genetic studies of body mass index yield new insights for obesity biology. Nature 518, 197–206 (2015).

  33. 33.

    et al. Large-scale association analysis provides insights into the genetic architecture and pathophysiology of type 2 diabetes. Nat. Genet. 44, 981–990 (2012).

  34. 34.

    et al. Bayesian test for co-localization between pairs of genetic association studies using summary statistics. PLoS Genet. 10, e1004383 (2014).

  35. 35.

    et al. Mapping DNA methylation across development, genotype, and schizophrenia in the human frontal cortex. Nat. Neurosci. advance online publication, (2015).

  36. 36.

    , & Allele-specific methylation in the human genome: implications for genetic studies of complex disease. Epigenetics 5, 578–582 (2010).

  37. 37.

    et al. Identification of schizophrenia-associated loci by combining DNA methylation and gene expression data from whole blood. Eur. J. Hum. Genet. 23, 1106–1110 (2015).

  38. 38.

    & Evidence that schizophrenia risk variation in the ZNF804A gene exerts its effects during fetal brain development. Am. J. Psychiatry 169, 1301–1308 (2012).

  39. 39.

    et al. Expression of ZNF804A in human brain and alterations in schizophrenia, bipolar disorder, and major depressive disorder: a novel transcript fetally regulated by the psychosis risk variant rs1344706. JAMA Psychiatry 71, 1112–1120 (2014).

  40. 40.

    et al. Methylomic profiling of human brain tissue supports a neurodevelopmental origin for schizophrenia. Genome Biol. 15, 483 (2014).

  41. 41.

    , , , & methylumi: Handle Illumina methylation data. R package version 2.14.0. <> (2015).

  42. 42.

    et al. Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays. Bioinformatics 30, 1363–1369 (2014).

  43. 43.

    et al. A data-driven approach to preprocessing Illumina 450K methylation array data. BMC Genomics 14, 293 (2013).

  44. 44.

    et al. Discovery of cross-reactive probes and polymorphic CpGs in the Illumina Infinium HumanMethylation450 microarray. Epigenetics 8, 203–209 (2013).

  45. 45.

    et al. Additional annotation enhances potential for biologically relevant analysis of the Illumina Infinium HumanMethylation450 BeadChip array. Epigenetics Chromatin 6, 4 (2013).

  46. 46.

    Matrix eQTL: ultra fast eQTL analysis via large matrix operations. Bioinformatics 28, 1353–1358 (2012).

  47. 47.

    & Large-scale imputation of epigenomic datasets for systematic annotation of diverse human tissues. Nat. Biotechnol. 33, 364–376 (2015).

  48. 48.

    et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience 4, 7 (2015).

  49. 49.

    et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).

  50. 50.

    , & A linear complexity phasing method for thousands of genomes. Nat. Methods 9, 179–181 (2012).

  51. 51.

    , & Improved whole-chromosome phasing for disease and population genetic studies. Nat. Methods 10, 5–6 (2013).

  52. 52.

    , , , & Fast and accurate genotype imputation in genome-wide association studies through pre-phasing. Nat. Genet. 44, 955–959 (2012).

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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.

Author information

Author notes

    • Nicholas J Bray
    •  & Jonathan Mill

    These authors contributed equally to this work.

Affiliations

  1. University of Exeter Medical School, University of Exeter, Exeter, UK.

    • Eilis Hannon
    • , Joana Viana
    • , Joe Burrage
    • , Therese M Murphy
    •  & Jonathan Mill
  2. Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK.

    • Helen Spiers
    • , Claire Troakes
    • , Nicholas J Bray
    •  & Jonathan Mill
  3. Garvan Institute of Medical Research, Sydney, NSW, Australia.

    • Ruth Pidsley
  4. Douglas Mental Health Institute, McGill University, Montreal, QC, Canada.

    • Gustavo Turecki
  5. MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University School of Medicine, Cardiff, UK.

    • Michael C O'Donovan
    •  & Nicholas J Bray
  6. School of Biological Sciences, University of Essex, Colchester, UK.

    • Leonard C Schalkwyk

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jonathan Mill.

Integrated supplementary information

Supplementary figures

  1. 1.

    The distribution of effect sizes across all Bonferroni significant fetal brain mQTLs.

  2. 2.

    Frequency distribution of mQTL SNPs and associated DNA methylation sites.

  3. 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.

  4. 4.

    There is a highly-significant correlation of individual mQTL effects between fetal brain and each of the individual adult brain regions.

  5. 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).

  6. 6.

    Fetal brain mQTLs that do not replicate in adult brain are characterized by significantly smaller effect sizes across all brain regions, including the initial fetal brain discovery sample (P = 3.18×10−141).

  7. 7.

    SNPs associated with DNA methylation are more significantly associated with gene expression than non-mQTL variants.

  8. 8.

    The overlap between independent fetal brain mQTL signals with the sentinelized subsignals from a brain eQTL dataset.

  9. 9.

    Fetal brain mQTLs ar1e significantly enriched for schizophrenia genetic risk variants.

  10. 10.

    Boxplot of mQTL effects observed for rs2535627, an index SNP from the recent schizophrenia GWAS.

  11. 11.

    Boxplot of mQTL effect observed for rs4648845, an index SNP from a recent schizophrenia GWAS.

  12. 12.

    mQTLs identified using imputed genetic data reflected the non-imputed dataset in terms of genomic distribution and observed effect sizes.

  13. 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.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–13

  2. 2.

    Supplementary Methods Checklist

Excel files

  1. 1.

    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.

  2. 2.

    Supplementary Table 2: Summary of Bonferroni-significant fetal brain mQTLs.

  3. 3.

    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.

  4. 4.

    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.

  5. 5.

    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)].

  6. 6.

    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/).

  7. 7.

    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.

  8. 8.

    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.

  9. 9.

    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.

  10. 10.

    Supplementary Table 10: Fetal brain mQTLs with significant heterogeneity between fetal and adult datasets.

    Abbreviations: PFC = prefrontal cortex, STR = striatum, CER = cerebellum.

  11. 11.

    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.

  12. 12.

    Supplementary Table 12: Summary of overlap between fetal brain mQTLs and brain expression QTLs (eQTLs).

  13. 13.

    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.

  14. 14.

    Supplementary Table 14: Enrichment of brain eQTLs in human fetal brain mQTLs.

  15. 15.

    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.

  16. 16.

    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.

  17. 17.

    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).

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  1. 1.

    Supplementary Analysis Scripts: Rscripts

    R code for analyses and figures.

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DOI

https://doi.org/10.1038/nn.4182

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