CpG Methylation Patterns in Placenta and Neonatal Blood are Differentially Associated with Neonatal Inflammation
Eaves LA, Enggasser AE, Camerota M, Gogcu S, Gower WA, Hartwell H, Jackson WM, Jensen E, Joseph RM, Marsit CJ, Roell K, Santos HP Jr, Shenberger JS, Smeester L, Yanni D, Kuban KCK, O'Shea TM, Fry RC.
Pediatr Res. 2022 Jun 28. doi: 10.1038/s41390-022-02150-4. Epub ahead of print. PMID: 35764815.
Background: Infants born extremely premature are at increased risk for health complications later in life for which neonatal inflammation may be a contributing biological driver. Placental CpG methylation provides mechanistic information regarding the relationship between prenatal epigenetic programming, prematurity, neonatal inflammation, and later-in-life health.
Methods: We contrasted CpG methylation in the placenta and neonatal blood spots in relation to neonatal inflammation in the Extremely Low Gestational Age Newborn (ELGAN) cohort. Neonatal inflammation status was based on the expression of six inflammation-related proteins, assessed as (1) day-one inflammation (DOI) or (2) intermittent or sustained systemic inflammation (ISSI, inflammation on ≥2 days in the first 2 postnatal weeks). Epigenome-wide CpG methylation was assessed in 354 placental samples and 318 neonatal blood samples.
Results: Placental CpG methylation displayed the strongest association with ISSI (48 CpG sites) but was not associated with DOI. This was in contrast to CpG methylation in blood spots, which was associated with DOI (111 CpG sites) and not with ISSI (one CpG site).
Conclusions: Placental CpG methylation was strongly associated with ISSI, a measure of inflammation previously linked to later-in-life cognitive impairment, while day-one neonatal blood methylation was associated with DOI.
Impact: Neonatal inflammation increases the risk of adverse later-life outcomes, especially in infants born extremely preterm. CpG methylation in the placenta and neonatal blood spots were evaluated in relation to neonatal inflammation assessed via circulating proteins as either (i) day-one inflammation (DOI) or (ii) intermittent or sustained systemic inflammation (ISSI, inflammation on ≥2 days in the first 2 weeks). Tissue specificity was observed in epigenetic-inflammatory relationships: placental CpG methylation was associated with ISSI, neonatal blood CpG methylation was associated with DOI. Supporting the placental origins of disease framework, placental epigenetic patterns are associated with a propensity for ISSI in neonates.
A mixed-effects two-part model for twin-data and an application on identifying important factors associated with extremely preterm children’s health disorders
Zou B, Santos HP, Xenakis JG, O'Shea MM, Fry RC, Zou F.
PLoS One. 2022 Jun 13;17(6):e0269630. doi: 10.1371/journal.pone.0269630. PMID: 35696398; PMCID: PMC9191696.
Abstract
Our recent studies identifying factors significantly associated with the positive child health index (PCHI) in a mixed cohort of preterm-born singletons, twins, and triplets posed some analytic and modeling challenges. The PCHI transforms the total number of health disorders experienced (of the eleven ascertained) to a scale from 0 to 100%. While some of the children had none of the eleven health disorders (i.e., PCHI = 1), others experienced a subset or all (i.e., 0 ≤PCHI< 1). This indicates the existence of two distinct data processes-one for the healthy children, and another for those with at least one health disorder, necessitating a two-part model to accommodate both. Further, the scores for twins and triplets are potentially correlated since these children share similar genetics and early environments. The existing approach for analyzing PCHI data dichotomizes the data (i.e., number of health disorders) and uses a mixed-effects logistic or multiple logistic regression to model the binary feature of the PCHI (1 vs. < 1). To provide an alternate analytic framework, in this study we jointly model the two data processes under a mixed-effects two-part model framework that accounts for the sample correlations between and within the two data processes. The proposed method increases power to detect factors associated with disorders. Extensive numerical studies demonstrate that the proposed joint-test procedure consistently outperforms the existing method when the type I error is controlled at the same level. Our numerical studies also show that the proposed method is robust to model misspecifications and it is applicable to a set of correlated semi-continuous data.
Differential placental CpG methylation is associated with chronic lung disease of prematurity
Jackson WM, Santos HP Jr, Hartwell HJ, Gower WA, Chhabra D, Hagood JS, Laughon MM, Payton A, Smeester L, Roell K, O'Shea TM, Fry RC.
Pediatr Res. 2022 May;91(6):1428-1435. doi: 10.1038/s41390-021-01868-x. Epub 2021 Dec 2. PMID: 34857876; PMCID: PMC9160210.
Background: Chronic lung disease (CLD) is the most common pulmonary morbidity in extremely preterm infants. It is unclear to what extent prenatal exposures influence the risk of CLD. Epigenetic variation in placenta DNA methylation may be associated with differential risk of CLD, and these associations may be dependent upon sex.
Methods: Data were obtained from a multi-center cohort of infants born extremely preterm (<28 weeks’ gestation) and an epigenome-wide approach was used to identify associations between placental DNA methylation and CLD (n = 423). Associations were evaluated using robust linear regression adjusting for covariates, with a false discovery rate of 0.05. Analyses stratified by sex were used to assess differences in methylation-CLD associations.
Results: CLD was associated with differential methylation at 49 CpG sites representing 46 genes in the placenta. CLD was associated with differential methylation of probes within genes related to pathways involved in fetal lung development, such as p53 signaling and myo-inositol biosynthesis. Associations between CpG methylation and CLD differed by sex.
Conclusions: Differential placental methylation within genes with key roles in fetal lung development may reflect complex cell signaling between the placenta and fetus which mediate CLD risk. These pathways appear to be distinct based on fetal sex.
Impact: In extremely preterm infants, differential methylation of CpG sites within placental genes involved in pathways related to cell signaling, oxidative stress, and trophoblast invasion is associated with chronic lung disease of prematurity. DNA methylation patterns associated with chronic lung disease were distinctly based on fetal sex, suggesting a potential mechanism underlying dimorphic phenotypes. Mechanisms related to fetal hypoxia and placental myo-inositol signaling may play a role in fetal lung programming and the developmental origins of chronic lung disease. Continued research of the relationship between the placental epigenome and chronic lung disease could inform efforts to ameliorate or prevent this condition.
Maternal Social Risk, Gestational Age at Delivery, and Cognitive Outcomes among Adolescents Born Extremely Preterm
Robert M. Joseph, Stephen R. Hooper, Tim Heeren, Hudson P. Santos Jr, Jean A. Frazier, Lauren Venuti, Ann Foley, Caitlin K. Rollins, Karl C. K. Kuban, Rebecca C. Fry, Thomas M. O’Shea, for the ELGAN Study Investigators
Paediatr Perinat Epidemiol. 2022; 36: 654- 664. doi: 10.1111/ppe.12893
Background
Children born extremely preterm (EP) are at increased risk of cognitive deficits that persist into adulthood. Few large cohort studies have examined differential impairment of cognitive function in EP-born adolescents in relation to early life risk factors, including maternal social disadvantage, gestational age at delivery, and neonatal morbidities prevalent among EP neonates.
Objectives
To assess cognitive abilities in relation to early life risk factors in an EP-born cohort at 15 years of age.
Methods
681 of 1198 surviving participants (57%) enrolled from 2002 to 2004 in the Extremely Low Gestational Age Newborn Study returned at age 15 years for an assessment of cognitive abilities with the Wechsler Abbreviated Scale of Intelligence-II and the NIH Toolbox Cognition Battery (NTCB) verbal cognition and fluid processing composites, the latter of which measured executive functions and processing speed. Three cognitive outcomes, WASI-II IQ, NTCB verbal cognition, and NTCB fluid processing, were analysed for associations with maternal social disadvantage and gestational age. Mediation of maternal social disadvantage by gestational age and mediation of gestational age by neonatal morbidities were also examined.
Results
Test scores were lower for NTCB fluid processing relative to IQ and NTCB verbal abilities. Social disadvantage and gestational age were associated with all three cognitive outcomes. Mediation analyses indicated partial mediation of gestational age associations with all three outcomes by neonatal morbidities but did not support mediation by gestational age of social risk associations with cognitive outcomes.
Conclusions
Greater maternal social disadvantage and lower gestational age are associated with less favourable cognitive outcomes among EP-born adolescents at 15 years of age. Neonatal morbidities partially mediate associations between lower gestational age and cognitive outcomes. These findings highlight the need for improved medical and remedial interventions to mitigate the risk of poor cognitive outcomes among EP-born adolescents.
Innovative computational approaches shed light on genetic mechanisms underlying cognitive impairment among children born extremely preterm
Liu W, Sun Q, Huang L, Bhattacharya A, Wang GW, Tan X, Kuban KCK, Joseph RM, O'Shea TM, Fry RC, Li Y, Santos HP Jr.
J Neurodev Disord. 2022 Mar 3;14(1):16. doi: 10.1186/s11689-022-09429-x. PMID: 35240980; PMCID: PMC8903548.
Background: Although survival rates for infants born extremely preterm (gestation < 28 weeks) have improved significantly in recent decades, neurodevelopmental impairment remains a major concern. Children born extremely preterm remain at high risk for cognitive impairment from early childhood to adulthood. However, there is limited evidence on genetic factors associated with cognitive impairment in this population.
Methods: First, we used a latent profile analysis (LPA) approach to characterize neurocognitive function at age 10 for children born extremely preterm. Children were classified into two groups: (1) no or low cognitive impairment, and (2) moderate-to-severe cognitive impairment. Second, we performed TOPMed-based genotype imputation on samples with genotype array data (n = 528). Third, we then conducted a genome-wide association study (GWAS) for LPA-inferred cognitive impairment. Finally, computational analysis was conducted to explore potential mechanisms underlying the variant x LPA association.
Results: We identified two loci reaching genome-wide significance (p value < 5e-8): TEA domain transcription factor 4 (TEAD4 at rs11829294, p value = 2.40e-8) and syntaxin 18 (STX18 at rs79453226, p value = 1.91e-8). Integrative analysis with brain expression quantitative trait loci (eQTL), chromatin conformation, and epigenomic annotations suggests tetraspanin 9 (TSPAN9) and protein arginine methyltransferase 8 (PRMT8) as potential functional genes underlying the GWAS signal at the TEAD4 locus.
Conclusions: We conducted a novel computational analysis by utilizing an LPA-inferred phenotype with genetics data for the first time. This study suggests that rs11829294 and its LD buddies have potential regulatory roles on genes that could impact neurocognitive impairment for extreme preterm born children.
Placental genomics mediates genetic associations with complex health traits and disease
Bhattacharya A, Freedman AN, Avula V, Harris R, Liu W, Pan C, Lusis AJ, Joseph RM, Smeester L, Hartwell HJ, Kuban KCK, Marsit CJ, Li Y, O'Shea TM, Fry RC, Santos HP Jr.
Nat Commun. 2022 Feb 4;13(1):706. doi: 10.1038/s41467-022-28365-x. PMID: 35121757; PMCID: PMC8817049
As the master regulator in utero, the placenta is core to the Developmental Origins of Health and Disease (DOHaD) hypothesis but is historically understudied. To identify placental gene-trait associations (GTAs) across the life course, we perform distal mediator-enriched transcriptome-wide association studies (TWAS) for 40 traits, integrating placental multi-omics from the Extremely Low Gestational Age Newborn Study. At [Formula: see text], we detect 248 GTAs, mostly for neonatal and metabolic traits, across 176 genes, enriched for cell growth and immunological pathways. In aggregate, genetic effects mediated by placental expression significantly explain 4 early-life traits but no later-in-life traits. 89 GTAs show significant mediation through distal genetic variants, identifying hypotheses for distal regulation of GTAs. Investigation of one hypothesis in human placenta-derived choriocarcinoma cells reveal that knockdown of mediator gene EPS15 upregulates predicted targets SPATA13 and FAM214A, both associated with waist-hip ratio in TWAS, and multiple genes involved in metabolic pathways. These results suggest profound health impacts of placental genomic regulation in developmental programming across the life course.
Neonatal Cranial Ultrasound Findings among Infants Born Extremely Preterm: Associations with Neurodevelopmental Outcomes at 10 Years of Age
Heather Campbell, Jennifer Check, Karl C K Kuban, Alan Leviton, Robert M Joseph, Jean A Frazier, Laurie M Douglass, Kyle Roell, Elizabeth N Allred, Lynn Ansley Fordham, Stephen R Hooper, Hernan Jara, Nigel Paneth, Irina Mokrova, Hongyu Ru, Hudson P Santos Jr, Rebecca C Fry, T Michael O'Shea
J Pediatr. 2021 Oct;237:197-205.e4. doi: 10.1016/j.jpeds.2021.05.059. Epub 2021 Jun 4. PMID: 34090894; PMCID: PMC8478718.
Ultrasound-identified white matter damage (WMD) without intraventricular hemorrhage (IVH) was associated with an increased risk of cognitive impairment (OR 3.5, 95% CI 1.7, 7.4), cerebral palsy (OR 14.3, 95% CI 6.5, 31.5), and epilepsy (OR 6.9; 95% CI 2.9, 16.8). Similar associations were found for WMD accompanied by IVH. Isolated IVH was not significantly associated these outcomes. Among children born extremely preterm, CUS abnormalities, particularly those indicative of WMD, are predictive of neurodevelopmental impairments at 10 years of age. The strongest associations were found with cerebral palsy.
Comparing the Predictivity of Human Placental Gene, microRNA, and CpG Methylation Signatures in Relation to Perinatal Outcomes
Jeliyah Clark, Vennela Avula, Caroline Ring, Lauren A Eaves, Thomas Howard, Hudson P Santos, Lisa Smeester, Jacqueline T Bangma, Thomas Michael O'Shea, Rebecca C Fry, Julia E Rager
Toxicol Sci. 2021 Sep 28;183(2):269-284. doi: 10.1093/toxsci/kfab089. PMID: 34255065; PMCID: PMC8478332.
This study evaluated mRNA and microRNA (miRNA) expression and cytosine-guanine dinucleotide (CpG) methylation signatures in human placental tissues in relation to birth weight, placenta weight, placental damage, and placental inflammation. The following hypotheses were tested: (1) different molecular signatures will demonstrate varying levels of predictivity towards perinatal outcomes, and (2) these signatures will show disruptions from an example exposure (ie, cadmium) known to elicit perinatal toxicity. Epigenomic signatures (ie, miRNA and CpG methylation) consistently demonstrated the highest levels of predictivity; top-ranking predictors included miRNAs involved in injury and inflammation. These top-ranking miRNA predictors were analyzed in a separate pregnancy cohort and related to cadmium. Key predictive miRNAs demonstrated altered expression in association with cadmium exposure, including miR-210, known to impact placental cell growth, blood vessel development, and fetal weight.
Systemic Inflammation in the First 2 Weeks After Birth as a Determinant of Physical Growth Outcomes in Hospitalized Infants With Extremely Low Gestational Age
Mandy B Belfort, Sara E Ramel, Camilia R Martin, Raina Fichorova, Karl C K Kuban, Timothy Heeren, Rebecca C Fry, T Michael O'Shea
J Pediatr. 2021 Sep 8:S0022-3476(21)00879-9. doi: 10.1016/j.jpeds.2021.09.006. Epub ahead of print. PMID: 34508750.
The goal of this study was to examine associations of systemic inflammation with growth outcomes at neonatal intensive care unit (NICU) discharge/transfer among infants with extremely low gestational ages. Infants with CRP elevation on day 7 had lower weights at discharge/transfer (-0.17 z-scores, 95% CI -0.27, -0.06) than infants without CRP elevation; with similar results on day 14. Infants with CRP elevation on day 14 were also shorter (-0.21 length z-scores, 95% CI -0.38, -0.04), and had smaller head circumferences (-0.18 z-scores, 95% CI -0.33, -0.04) at discharge/transfer. IL-6 elevation on day 14 was associated with lower weight (-0.12, 95% CI -0.22, -0.02); IL-6 elevation on day 7 was associated with shorter length (-0.27, 95% CI -0.43, -0.12). TNF-∝ and IL-8 elevation on day 14 were associated with lower weight at discharge/transfer. Postnatal systemic inflammation may contribute to impaired nutrient accretion during a critical period in development in infants with extremely low gestational ages.
Prenatal tobacco smoke exposure and neurological impairment at 10 years of age among children born extremely preterm: a prospective cohort
K K Venkatesh, A Leviton, R N Fichorova, R M Joseph, L M Douglass, J A Frazier, Kck Kuban, H P Santos Jr, R C Fry, T M O'Shea
BJOG. 2021 Sep;128(10):1586-1597. doi: 10.1111/1471-0528.16690. Epub 2021 Apr 6. PMID: 33682301.
PubMed Link Of 1200 ELGAN study survivors, 856 were assessed at 10 years of age with neurological outcomes, of whom 14% (118/856) had active tobacco exposure during pregnancy and 24% (207/852) had passive tobacco exposure. Compared with children who were not exposed prenatally to tobacco, children exposed to active tobacco use during pregnancy had a higher risk of epilepsy (14% versus 5%; adjusted relative risk: 1.68, 95% CI 1.45-1.92). This risk remained after adjustment for gestational age at delivery and clinical subtype of preterm birth. Prenatal tobacco smoke exposure was not associated with other assessed neurological outcomes, including cerebral palsy and multiple measures of cognitive impairment.Among children born extremely preterm, prenatal active tobacco smoke exposure was associated with an increased risk of epilepsy at 10 years of life.