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Distal Mediator-Enriched, Placental Transcriptome-Wide Analyses Illustrate The medRxiv preprint doi: https://doi.org/10.1101/2021.04.12.21255170; this version posted April 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 1 Distal mediator-enriched, placental transcriptome-wide analyses illustrate the 2 Developmental Origins of Health and Disease 3 4 Arjun Bhattacharya, PhD1*, Anastasia N. Freedman, BA2, Vennela Avula2, Rebeca Harris, BS3, Weifang 5 Liu, BA4, Robert M. Joseph, PhD5, Lisa Smeester, PhD2,6,7, Hadley J. Hartwell, MS2, Karl C.K. Kuban, 6 MD8, Carmen J. Marsit, PhD9, Yun Li, PhD4,10,11, T. Michael O’Shea, MD, MPH12, Rebecca C. Fry, 7 PhD2,6,7*†, Hudson P. Santos Jr., PhD, RN3,6*† 8 9 1. Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of 10 California, Los Angeles, CA, USA 11 2. Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, 12 University of North Carolina, Chapel Hill, NC, USA 13 3. Biobehavioral Laboratory, School of Nursing, University of North Carolina, Chapel Hill, NC, USA 14 4. Department of Biostatistics, Gillings School of Global Public Health, University of North Carolina, 15 Chapel Hill, NC, USA 16 5. Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, MA, USA 17 6. Institute for Environmental Health Solutions, Gillings School of Global Public Health, University of 18 North Carolina, Chapel Hill, NC, USA 19 7. Curriculum in Toxicology and Environmental Medicine, University of North Carolina, Chapel Hill, NC, 20 USA 21 8. Department of Pediatrics, Division of Pediatric Neurology, Boston University Medical Center, Boston, 22 MA, USA 23 9. Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, 24 Atlanta, GA, USA 25 10. Department of Genetics, University of North Carolina, Chapel Hill, NC, USA 26 11. Department of Computer Science, University of North Carolina, Chapel Hill, NC, USA 27 12. Department of Pediatrics, School of Medicine, University of North Carolina, Chapel Hill, NC, USA 28 1 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2021.04.12.21255170; this version posted April 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 1 †Equal contribution 2 *Please direct correspondence to AB ([email protected]), RCF ([email protected]), HPS 3 ([email protected]). 4 5 ABSTRACT 6 As the master regulator of the intrauterine environment, the placenta is core to the Developmental Origins 7 of Health and Disease (DOHaD) but is understudied in relation to tissue-specific gene and trait regulation. 8 We performed distal mediator-enriched transcriptome-wide association studies (TWAS) for 40 health 9 traits across 5 physiological categories, using gene expression models trained with multi-omic data from 10 the Extremely Low Gestational Age Newborn Study (푁 = 272). At 푃 < 2.5 × 10−6, we detected 248 11 gene-trait associations (GTAs) across 176 genes, mostly for metabolic and neonatal traits and enriched 12 for cell growth and immunological pathways. Of these GTAs, 89 showed significant mediation through 13 genetic variants distal to the gene, identifying potential targets for functional validation. Functional 14 validation of a mediator gene (EPS15) in human placenta-derived JEG-3 trophoblasts resulted in 15 increased expression of its predicted targets, SPATA13 and FAM214A, both associated with the trait of 16 waist-hip ratio in TWAS. These results illustrate the profound health impacts of placental genetic and 17 genomic regulation in developmental programming across the life course. 18 19 KEYWORDS 20 transcriptome-wide association study, placental biology, in utero development, developmental origins of 21 health and disease, developmental programming, multi-omics integration 2 medRxiv preprint doi: https://doi.org/10.1101/2021.04.12.21255170; this version posted April 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 1 MAIN 2 The placenta serves as the master regulator of the intrauterine environment via nutrient transfer, 3 metabolism, gas exchange, neuroendocrine signaling, growth hormone production, and immunologic 4 control1–5. Due to strong influences on postnatal health, the placenta is central to the Developmental 5 Origins of Health and Disease (DOHaD) hypothesis, which purports that the in utero experience has 6 lifelong impacts on child health by altering developmental programming and influencing risk of common, 7 noncommunicable health conditions6. For example, placental biology has been linked to neuropsychiatric, 8 developmental, and metabolic diseases or health traits (collectively referred to as traits) that manifest 9 throughout the life course, either early- or later-in-life (Figure 1)7–10. Despite its long-lasting influences on 10 health, the placenta has not been well-studied in large consortia studies of multi-tissue gene 11 regulation11,12. Studying regulatory mechanisms in the placenta underlying biological processes in 12 developmental programming will provide novel insight into health and disease etiology. 13 14 The complex interplay between genetics and placental transcriptomics and epigenomics has strong 15 effects on gene expression that may explain variation in gene-trait associations (GTAs). Quantitative trait 16 loci (QTL) analyses have identified a strong influence of cis-genetic variants on both placental gene 17 expression and DNA methylation13,14. Furthermore, there is growing evidence that the placental 18 epigenome influences gene regulation, often distally (more than 1-3 Megabases away in the genome)15, 19 and that placental DNA methylation and microRNA (miRNA) expression are associated with health traits 20 in children16–18. Dysfunction of transcription factor regulation in the placenta has also shown profound 21 effects on childhood traits19–22. Although combining genetics, transcriptomics, and epigenomics lends 22 insight into the influence of placental genomics on complex traits23, genome-wide screens for GTAs that 23 integrate different molecular profiles and generate functional hypotheses require more sophisticated 24 computational methods. 25 26 To this end, advances in transcriptome-wide association studies (TWAS) have allowed for integration of 27 genome-wide association studies (GWAS) and eQTL datasets to boost power in identifying GTAs, 28 specific to a relevant tissue24–26. However, traditional methods for TWAS largely overlook genetic variants 3 medRxiv preprint doi: https://doi.org/10.1101/2021.04.12.21255170; this version posted April 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 1 distal to genes of interest, ostensibly mediated through regulatory biomarkers (e.g., transcription factors, 2 miRNAs, and DNA methylation sites)27,28. Not only may these distal biomarkers explain a significant 3 portion of both gene expression heritability and trait heritability on the tissue-specific expression level29–32, 4 they may also influence tissue-specific trait associations for individual genes. Due to the strong interplay 5 of regulatory elements in placental gene regulation, we sought to systematically characterize portions of 6 gene expression that are influenced by these distal regulatory elements. 7 8 Here, we investigate three broad questions: (1) which genes show associations between their placental 9 genetically-regulated expression (GReX) and various traits across the life course, (2) which traits along 10 the life course can be explained by placental GReX, in aggregate, and (3) which transcription factors, 11 miRNAs, or CpG sites potentially regulate trait-associated genes in the placenta (Figure 1). We 12 leveraged gene expression, CpG methylation, and miRNA expression data from fetal-side placenta tissue 13 from the Extremely Low Gestational Age Newborn (ELGAN) Cohort Study33. We trained predictive models 14 of gene expression, enriched for distal SNPs using MOSTWAS, a recent TWAS extension that integrates 15 multi-omic data34. Re-analyzing 40 GWAS of European-ancestry subjects from large consortia35–39, we 16 performed a series of TWAS for non-communicable health traits and disorders that may be influenced by 17 the placenta to identify GTAs and functional hypotheses for regulation (Figure 2). To our knowledge, this 18 is the first distal mediator-enriched TWAS of health traits that integrates placental multi-omics. Results 19 from our analysis can be explored at our Shiny R app, the ELGAN DOHaD Atlas: https://elgan- 20 twas.shinyapps.io/dohad/. 21 22 RESULTS 23 Genetic heritability and correlations across traits 24 From large consortia35–39, we curated GWAS summary statistics from subjects of European ancestry for 25 40 complex, non-communicable traits and
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