Epigenomic profiling of newborns with isolated orofacial clefts reveals widespread DNA methylation changes and implicates metastable epiallele regions in disease risk

Abstract

Cleft lip with or without cleft palate (CL/P) is a common human birth defect whose etiologies remain largely unknown. Several studies have demonstrated that periconceptional supplementation of folic acid can reduce risk of CL/P in offspring. In this study, we tested the hypothesis that the preventive effect of folic acid is manifested through epigenetic modifications by determining whether DNA methylation changes are associated with CL/P. To more readily observe the potential effects of maternal folate on the offspring epigenome, we focused on births prior to mandatory dietary folate fortification in the United States (i.e. birth year 1997 or earlier). Genomic DNA methylation levels were assessed from archived newborn bloodspots in a 182-member case-control study using the Illumina® Human Beadchip 450K array. CL/P cases displayed striking epigenome-wide hypomethylation relative to controls: 63% of CpGs interrogated had lower methylation levels in case newborns, a trend which held up in racially stratified sub-groups. 28 CpG sites reached epigenome-wide significance and all were case-hypomethylated. The most significant CL/P-associated differentially methylated region encompassed the VTRNA2-1 gene, which was also hypomethylated in cases (FWER p = 0.014). This region has been previously characterized as a nutritionally-responsive, metastable epiallele and CL/P-associated methylation changes, in general, were greater at or near putative metastable epiallelic regions. Gene Set Enrichment Analysis of CL/P-associated DMRs showed an over-representation of genes involved in palate development such as WNT9B, MIR140 and LHX8. CL/P-associated DNA methylation changes may partly explain the mechanism by which orofacial clefts are responsive to maternal folate levels.

Keywords: DNA methylation; Orofacial cleft; birth defect; epigenetics; folic acid; metastable epiallele.

Figure 1.

Regression coefficients for 319,253 CpGs in the final, filtered analytical dataset are plotted versus p value (expressed as – log₁₀ p). Left panel: All samples (94 cases, 88 controls). Middle panel: Hispanic-only samples (74 cases, 69 controls). Right panel: non-Hispanic white-only samples (20 cases, 19 controls).

Figure 2.

Directional concordance of regression coefficients between Hispanic and non-Hispanic white sample sets for subsets of CpG sites. The numbers of CpG sites in each quadrant are indicated. Left panel: All 319,253 CpG in the analytical dataset. Middle panel: 2,694 CpG sites with p value < 0.05 in both sample sets. Right panel: 262 CpG sites with p value < 0.01 in both sample sets.

Figure 3.

Distributions of DNA methylation β-values for each of the 12 CpG sites (identified by chromosomal coordinate) within the ~1 kb region surrounding the VTRNA2-1 gene. Data is from all cases (N = 94, gray-filled boxes) and all controls (N = 88, white-filled boxes). VTRNA2-1 transcript coordinates are chr5:135416187–135416286.

Figure 4.

All CpGs were binned based on proximity (kb) to putative metastable epiallele regions as defined and localized in Table S2 from ref. 20. For each distance bin shown in the figure, the average extent of case-associated methylation changes were calculated by averaging the absolute value of the regression coefficients (to equally account for hypo- and hypermethylation events). # CpGs averaged per bin is indicated. A distance of ‘0’ indicates CpGs that fall within the borders of a metastable epiallele region. Significance of pair-wise distribution comparisons was determined by the Mann Whitney U test. * p < .05; ** p < 0.005; *** p < 0.0005.

Figure 5.

The difference in mean β-values between CL/P cases relative to controls for the individual CpGs (delineated as chromosomal coordinates) that comprise DMRs described in Table 3. Plots depict the DMR CpGs plus 6 adjacent CpGs that flank the DMR on either end. Top panel: MIR140; transcription start site at coordinate 69966984. Middle panel: WNT9B promoter; transcription start site at coordinate 44928968. Bottom panel: CpG island (Chr1:75590818–75591354) upstream of LHX8 transcription start site (coordinate 75594119).