Profiling placental and fetal DNA methylation in human neural tube defects

Abstract

Background: The incidence of neural tube defects (NTDs) declined by about 40 % in Canada with the introduction of a national folic acid (FA) fortification program. Despite the fact that few Canadians currently exhibit folate deficiency, NTDs are still the second most common congenital abnormality. FA fortification may have aided in reducing the incidence of NTDs by overcoming abnormal one carbon metabolism cycling, the process which provides one carbon units for methylation of DNA. We considered that NTDs persisting in a folate-replete population may also occur in the context of FA-independent compromised one carbon metabolism, and that this might manifest as abnormal DNA methylation (DNAm). Second trimester human placental chorionic villi, kidney, spinal cord, brain, and muscle were collected from 19 control, 22 spina bifida, and 15 anencephalic fetuses in British Columbia, Canada. DNA was extracted, assessed for methylenetetrahydrofolate reductase (MTHFR) genotype and for genome-wide DNAm using repetitive elements, in addition to the Illumina Infinium HumanMethylation450 (450k) array.

Results: No difference in repetitive element DNAm was noted between NTD status groups. Using a false discovery rate <0.05 and average group difference in DNAm ≥0.05, differentially methylated array sites were identified only in (1) the comparison of anencephaly to controls in chorionic villi (n = 4 sites) and (2) the comparison of spina bifida to controls in kidney (n = 3342 sites).

Conclusions: We suggest that the distinctive DNAm of spina bifida kidneys may be consequent to the neural tube defect or reflective of a common etiology for abnormal neural tube and renal development. Though there were some small shifts in DNAm in the other tested tissues, our data do not support the long-standing hypothesis of generalized altered genome-wide DNAm in NTDs. This finding may be related to the fact that most Canadians are not folate deficient, but it importantly opens the field to the investigation of other epigenetic and non-epigenetic mechanisms in the etiology of NTDs.

Keywords: 450k array; Anencephaly; DNA methylation (DNAm); Epigenome-wide association study (EWAS); Illumina HumanMethylation450 BeadChip; Neural tube defects (NTDs); Spina bifida.

Fig. 1

Sample clustering based on array-wide DNA methylation. Unsupervised hierarchical clustering of 442,091 CpG sites clustered samples primarily by tissue type. Within a tissue, samples did not cluster by NTD status. We did note however, a cluster of eight spina bifida and one anencephaly in kidney and a cluster of 12 spina bifida and two controls in spinal cord

Fig. 2

Tissue distribution of unadjusted p values from linear modeling of differential methylation in NTDs. Distribution of p values from the comparison of DNA methylation for spina bifida to control samples at each of 442,091 CpG sites (left) and for anencephaly to controls samples at each of 442,091 CpG sites (right), including fetal gestational age and sex as additive covariates. Flat distributions indicate equal likelihood of significant and non-significant tests, while left-peaking distributions indicate greater likelihood of significant tests

Fig. 3

Spina bifida array-wide volcano plots. Volcano plots comparing the magnitude of difference in DNAm (adjusted delta beta) to statistical significance (−log10(adjusted P.Value)) for each CpG site (n = 442,091) in spina bifida vs. control samples

Fig. 4

Differentially methylated CpG sites in the chorionic villi comparison of anencephaly cases to controls. Four CpG sites were identified as significantly differentially methylated at an FDR <0.05 and deltaβ ≥0.05. Each plot is labeled with the 450k CpG site identifier, the gene which it is closest to and average difference in DNAm between anencephaly and controls (deltaβ). Box edges are plotted at the 25th and 75th percentiles (the inter-quartile range (IQR)) and whiskers are plotted to the last sample within ± 1.5*IQR

Fig. 5

Identification and investigation of persistent hits in spina bifida kidneys. a By comparing our spina bifida (SB) and control (CON) samples to an independent control group (n = 5, GEO CON), 748 of the differentially methylated kidney spina bifida CpG sites were identified as persistent hits. Persistent hits were enriched for CpG sites located in enhancers and outside of CpG islands. b Hierarchical clustering of our spina bifida and control samples based solely on persistent hits almost completely separated the spina bifida from control cases, and two groups of SB cases emerged