Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Randomized Controlled Trial
. 2024 Feb 27;16(1):35.
doi: 10.1186/s13148-024-01644-8.

Improvements in lung function following vitamin C supplementation to pregnant smokers are associated with buccal DNA methylation at 5 years of age

Lyndsey E Shorey-Kendrick  1 , Cindy T McEvoy  2 , Kristin Milner  3 , Julia Harris  3 , Julie Brownsberger  3 , Robert S Tepper  4 , Byung Park  5 , Lina Gao  5 , Annette Vu  6 , Cynthia D Morris  6 , Eliot R Spindel  7
Affiliations
Randomized Controlled Trial

Improvements in lung function following vitamin C supplementation to pregnant smokers are associated with buccal DNA methylation at 5 years of age

Lyndsey E Shorey-Kendrick et al. Clin Epigenetics. .

Erratum in

Abstract

Background: We previously reported in the "Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function" randomized clinical trial (RCT) that vitamin C (500 mg/day) supplementation to pregnant smokers is associated with improved respiratory outcomes that persist through 5 years of age. The objective of this study was to assess whether buccal cell DNA methylation (DNAm), as a surrogate for airway epithelium, is associated with vitamin C supplementation, improved lung function, and decreased occurrence of wheeze.

Methods: We conducted epigenome-wide association studies (EWAS) using Infinium MethylationEPIC arrays and buccal DNAm from 158 subjects (80 placebo; 78 vitamin C) with pulmonary function testing (PFT) performed at the 5-year visit. EWAS were performed on (1) vitamin C treatment, (2) forced expiratory flow between 25 and 75% of expired volume (FEF25-75), and (3) offspring wheeze. Models were adjusted for sex, race, study site, gestational age at randomization (≤ OR > 18 weeks), proportion of epithelial cells, and latent covariates in addition to child length at PFT in EWAS for FEF25-75. We considered FDR p < 0.05 as genome-wide significant and nominal p < 0.001 as candidates for downstream analyses. Buccal DNAm measured in a subset of subjects at birth and near 1 year of age was used to determine whether DNAm signatures originated in utero, or emerged with age.

Results: Vitamin C treatment was associated with 457 FDR significant (q < 0.05) differentially methylated CpGs (DMCs; 236 hypermethylated; 221 hypomethylated) and 53 differentially methylated regions (DMRs; 26 hyper; 27 hypo) at 5 years of age. FEF25-75 was associated with one FDR significant DMC (cg05814800), 1,468 candidate DMCs (p < 0.001), and 44 DMRs. Current wheeze was associated with 0 FDR-DMCs, 782 candidate DMCs, and 19 DMRs (p < 0.001). In 365/457 vitamin C FDR significant DMCs at 5 years of age, there was no significant interaction between time and treatment.

Conclusions: Vitamin C supplementation to pregnant smokers is associated with buccal DNA methylation in offspring at 5 years of age, and most methylation signatures appear to be persistent from the prenatal period. Buccal methylation at 5 years was also associated with current lung function and occurrence of wheeze, and these functionally associated loci are enriched for vitamin C associated loci. Clinical trial registration ClinicalTrials.gov, NCT01723696 and NCT03203603.

Keywords: Airway; DNA methylation; Lung function; MSDP: maternal smoking during pregnancy; MethylationEPIC; Nicotine; RCT: randomized clinical trial; Vitamin C; Wheeze.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Summary of EWAS of vitamin C vs placebo in buccal DNAm and 5 years. The Manhattan plot A shows the –log10(P) on the y-axis and chromosome position on the x-axis; red line indicates Bonferroni adjusted p value < 0.05 and blue line indicates FDR p < 0.05; labels annotate the top 3 DMCs. The volcano plot B shows the –log10(P value) on the y-axis and effect size (logFC) on the x-axis. Hypermethylated CpGs (i.e., higher methylation level in participants with vitamin C compared to those with placebo) are shown in red; hypomethylated CpGs in blue. The red line indicates FDR adjusted p value < 0.05 and blue line indicates candidates with nominal p < 0.001. The dot plot C shows enrichment of traits previously associated with the differentially methylated CpGs identified in this study following prenatal vitamin C supplementation
Fig. 2
Fig. 2
Overlap of vitamin C DMCs with lung function DMCs and mediation analysis. The Venn diagram (top) shows the overlap of 62 CpGs associated with vitamin C supplementation (in blue) and FEF25–75 (in pink) both at p < 0.001. Mediation analysis was performed for each of the 62 CpGs associated with both exposure and outcome as indicated in the schematic
Fig. 3
Fig. 3
The majority of buccal DNAm signatures at 5 years are persistent from early life. Out of 457 vitamin C DMCs at 5 years, panel A shows the 365 CpGs with no significant interaction between time and treatment. The axes on the left panel are the vitamin C treatment effect sizes (logFC) at the indicated ages and the ellipses show the correlation (between 0 and 1) of treatment logFC between each pair of timepoints. The right panel is a heatmap of treatment effect sizes in these stable/persistent CpGs with hierarchical clustering. Panel B shows the remaining 92 CpGs with one or more significant interaction between treatment and time, repeating the correlation of logFC between ages in the left panel and heatmap clustering on the right
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Anderson TM, Lavista Ferres JM, Ren SY, Moon RY, Goldstein RD, Ramirez JM, et al. Maternal smoking before and during pregnancy and the risk of sudden unexpected infant death. Pediatrics. 2019; 143(4). PMID: 30858347. - PMC - PubMed
    1. Berlanga Mdel R, Salazar G, Garcia C, Hernandez J. Maternal smoking effects on infant growth. Food Nutr Bull. 2002;23(3 Suppl):142–145. doi: 10.1177/15648265020233S128. - DOI - PubMed
    1. Kalinka J, Hanke W. Tobacco smoking–a risk factor for intrauterine growth retardation, preterm delivery and low birth weight. Ginekol Pol. 1996;67(2):75–81. - PubMed
    1. Horta BL, Victora CG, Menezes AM, Halpern R, Barros FC. Low birthweight, preterm births and intrauterine growth retardation in relation to maternal smoking. Paediatr Perinat Epidemiol. 1997;11(2):140–151. doi: 10.1046/j.1365-3016.1997.d01-17.x. - DOI - PubMed
    1. Kalinka J, Hanke W. The role of tobacco smoking as a risk factor for intrauterine growth retardation and preterm delivery. Przegl Epidemiol. 1996;50(3):309–313. - PubMed

Publication types

Associated data