Identification of autosomal cis expression quantitative trait methylation (cis eQTMs) in children's blood.

ABSTRACT

Background: The identification of expression quantitative trait methylation (eQTMs), defined as associations between DNA methylation levels and gene expression, might help the biological interpretation of epigenome-wide association studies (EWAS). We aimed to identify autosomal cis eQTMs in children's blood, using data from 832 children of the Human Early Life Exposome (HELIX) project. Methods: Blood DNA methylation and gene expression were measured with the Illumina 450K and the Affymetrix HTA v2 arrays, respectively. The relationship between methylation levels and expression of nearby genes (1 Mb window centered at the transcription start site, TSS) was assessed by fitting 13.6 M linear regressions adjusting for sex, age, cohort, and blood cell composition. Results: We identified 39,749 blood autosomal cis eQTMs, representing 21,966 unique CpGs (eCpGs, 5.7% of total CpGs) and 8,886 unique transcript clusters (eGenes, 15.3% of total transcript clusters, equivalent to genes). In 87.9% of these cis eQTMs, the eCpG was located at <250 kb from eGene's TSS; and 58.8% of all eQTMs showed an inverse relationship between the methylation and expression levels. Only around half of the autosomal cis-eQTMs eGenes could be captured through annotation of the eCpG to the closest gene. eCpGs had less measurement error and were enriched for active blood regulatory regions and for CpGs reported to be associated with environmental exposures or phenotypic traits. In 40.4% of the eQTMs, the CpG and the eGene were both associated with at least one genetic variant. The overlap of autosomal cis eQTMs in children's blood with those described in adults was small (13.8%), and age-shared cis eQTMs tended to be proximal to the TSS and enriched for genetic variants. Conclusions: This catalogue of autosomal cis eQTMs in children's blood can help the biological interpretation of EWAS findings and is publicly available at https//helixomics.isglobal.org/ and at Dryad (doi10.5061/dryad.fxpnvx0t0). Funding: The study has received funding from the European Community's Seventh Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project); the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project); from the European Union's Horizon 2020 research and innovation programme under grant agreement no 733206 (LIFECYCLE project), and from the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL and Instituto de Salud Carlos III) under the grant agreement no AC18/00006 (NutriPROGRAM project). The genotyping was supported by the projects PI17/01225 and PI17/01935, funded by the Instituto de Salud Carlos III and co-funded by European Union (ERDF, "A way to make Europe") and the Centro Nacional de Genotipado-CEGEN (PRB2-ISCIII). BiB received core infrastructure funding from the Wellcome Trust (WT101597MA) and a joint grant from the UK Medical Research Council (MRC) and Economic and Social Science Research Council (ESRC) (MR/N024397/1). INMA data collections were supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research. The Rhea project was financially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV-1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009- single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EU- FP7- HEALTH-2012 Proposal No 308333 HELIX), and the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in preschool children, in Heraklion district, Crete, Greece 2011-2014; "Rhea Plus" Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health 2012-15). We acknowledge support from the Spanish Ministry of Science and Innovation through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. MV-U and CR-A were supported by a FI fellowship from the Catalan Government (FI-DGR 2015 and #016FI_B 00272). MC received funding from Instituto Carlos III (Ministry of Economy and Competitiveness) (CD12/00563 and MS16/00128).

Cells can fine-tune which genes they activate, when and at which levels using a range of chemical marks on the DNA and certain proteins that help to organise the genome. One well-known example of such 'epigenetic tags' is DNA methylation, whereby a methyl group is added onto particular positions in the genome. Many factors ­ including environmental effects such as diet ­ control DNA methylation, allowing an organism to adapt to ever-changing conditions. An expression quantitative trait methylation (eQTM) is a specific position of the genome whose DNA methylation status regulates the activity of a given gene. A catalogue of eQTMs would be useful in helping to reveal how the environment and disease impacts the way cells work. Yet, currently, the relationships between most epigenetic tags and gene activity remains unclear, especially in children. To fill this gap, Ruiz-Arenas et al. studied DNA methylation in blood samples from over 800 healthy children across Europe. Amongst all tested DNA methylation sites, 22,000 (5.7% of total) were associated with the expression of a gene ­ and therefore were eQTMs; reciprocally, 9,000 genes (15.3% of all tested genes) were linked to at least one methylation site, leading to a total of 40,000 pairs of DNA methylation sites and genes. Most often, eQTMs regulated the expression of nearby genes ­ but only half controlled the gene that was the closest to them. Age and the genetic background of the individuals influenced the nature of eQTMs. This catalogue is a useful resource for the scientific community to start understanding the relationship between epigenetics and gene activity. Similar studies are now needed for other tissues and age ranges. Overall, extending our knowledge of eQTMs may help reveal how life events lead to illness, and could inform prevention efforts.