Cytosine methylation dysregulation in neonates following intrauterine growth restriction

Abstract

Background: Perturbations of the intrauterine environment can affect fetal development during critical periods of plasticity, and can increase susceptibility to a number of age-related diseases (e.g., type 2 diabetes mellitus; T2DM), manifesting as late as decades later. We hypothesized that this biological memory is mediated by permanent alterations of the epigenome in stem cell populations, and focused our studies specifically on DNA methylation in CD34+ hematopoietic stem and progenitor cells from cord blood from neonates with intrauterine growth restriction (IUGR) and control subjects.

Methods and findings: Our epigenomic assays utilized a two-stage design involving genome-wide discovery followed by quantitative, single-locus validation. We found that changes in cytosine methylation occur in response to IUGR of moderate degree and involving a restricted number of loci. We also identify specific loci that are targeted for dysregulation of DNA methylation, in particular the hepatocyte nuclear factor 4alpha (HNF4A) gene, a well-known diabetes candidate gene not previously associated with growth restriction in utero, and other loci encoding HNF4A-interacting proteins.

Conclusions: Our results give insights into the potential contribution of epigenomic dysregulation in mediating the long-term consequences of IUGR, and demonstrate the value of this approach to studies of the fetal origin of adult disease.

Figure 1. Supervised group comparisons reveal significant differences in HELP data results between IUGR and controls.

Panel (A) shows a histogram distribution of p-values calculated from an unpaired T test of IUGR (n = 5) in one group and controls (n = 5) in another. The x axis represents p values, with lower values being the more significant, while the y axis shows the frequency of occurrence of different p values. The peak observed represents a subset of loci with low p values and thus significant differences between IUGR and control subjects. For comparison, panel (B) shows the results of a random distribution of subjects into two groups, mixing IUGR and controls, demonstrating the absence of a subset of loci with significant p values.

Figure 2. A second molecular interaction network suggested by Ingenuity Pathway Analysis (IPA), with HNF4A as a central node, consists of 12 genes among the top 56 differentially methylated loci.

RefSeq IDs for 33 of the top 56 sites that mapped to genes were uploaded onto the “Core Analysis” tool of IPA. The second-highest scoring molecular interaction network was constructed by 35 nodes, 12 of which were located on the input list (shaded nodes), and is associated with the cell cycle, cellular function and maintenance, and connective tissue development and function. The nodal relationships are indicated by solid lines (direct interaction) and dashed lines (indirect interactions), with or without filled arrows indicating functional interaction or merely physical association, respectively. Additionally, filled arrows that are preceded by a terminal bar indicate inhibition as well as functional interaction. The shape of each node indicates the class of molecule: horizontal ovals are transcription factors, squares are growth factors, vertical rectangles are ion channels while horizontal rectangles are nuclear receptors, inverted triangles are kinases, vertical diamonds are enzymes while horizontal diamonds are peptidases, trapezoids are transporters, and circles correspond to “other” molecules. In alphabetical order, this network consists of BUD31, CECR1, Collagen(s), CPN1, CRY1, DHX8, FAM110B, FGF2, GIN1, GRIK4, HNF4A, INO80D, KNG1, LPIN3, MAP3K3, MPRIP, MRTO4, NOC3L, PPARA, PRICKLE4, PRSS3, RSF1, RUVBL2, SLC31A1, SLC35A1, SLC35A5, SLC39A1, SMARCA5, SPAST, TGFB1, TSKU, UXT, WRNIP1, XPNPEP2, and ZNHIT6.

Figure 3. Differential methylation at the HNF4A locus proximal promoter region.

HELP data are shown in (a) as normalized, centered log₂(HpaII/MspI) ratios. A locus with a change in methylation is marked with an asterisk. A more detailed view of this region is shown in (b), showing conservation of DNA sequences at this alternative promoter of HNF4A. In (c) the degree of difference in cytosine methylation as measured by bisulphite MassArray is shown with the locus changing to a significant degree shown with its associated p value. This CG dinucleotide is within one of the HpaII sites of the informative gragment in (a) and is located immediately beside the conserved transcription factor binding sites shown in (b). These images were derived from the UCSC Genome Browser .