An increasing number of studies have explored the pathogenesis of GDM from the perspective of epigenetics. However, most of these were small (< 30 GDM cases), and they mainly observed the associations between GDM occurrence and the DNA methylation level of cord blood or placental tissue. 20 21 In this study, we evaluated the DNA methylation status of GDM-related CpG sites in the peripheral blood of women in early pregnancy using MethylTarget™ sequencing. In addition, we verified the associations between target CpG sites and GDM using a larger sample size (n = 80). Overall, we identified 13 CpG sites with significant differences in DNA methylation levels between the GDM and control groups based on quantitative analysis. The AUCs of the ROC curve for each methylation level of the significant CpG sites ranged from 0.593 to 0.650 predictive utility in relation to GDM. The methylation status of eight individual CpG sites were identified as differing significantly between GDM and control groups by qualitative analysis, and these were located in the promoter regions of RDH 12, HAPLN3, NFATC4, YAP1, and DNAJB6, and the intron region of C5orf34. Importantly, we found that the methylation statuses of four CpG sites were significantly associated with GDM occurrence, namely cg89438648 (HAPLN3), cg68167324 (RDH12), cg157130156 (DNAJB6), and cg24837915 (NFATC4), using conditional logistic regression analysis.
In this study, hypermethylation of the CpG site cg89438648, located in the promoter region of HAPLN3, was found to suggest a lower risk of GDM (OR = 0.206; 95%CI: 0.065 ~ 0.655). HAPLN3 codes for hyaluronan and proteoglycan link protein 3 (HAPLN3), and the connexin 3 belong to the hyaluronic acid and proteoglycan connexin (HAPLN) family, which plays roles in the aggregation of proteoglycans and hyaluronic acid, and in cell adhesion.22 HAPLN3 is involved in the organization and stability of the hyaluronic acid (HA)-dependent extracellular matrix (ECM) in many tissues. HA is one component of the ECM within the islet tissue of humans and mice.23 It can cause islet amyloid deposition, which is associated with decreased β-cell area and an increase in β cell apoptosis.24 Hull et al. suggested that islet amyloid deposition could reduce the number of β-cells.24 25 Hypermethylation of the CpG site cg89438648 located in the HAPLN3 promoter region, could reduce the level of HAPLN3, in turn reducing the stability of the HA-ECM, and consequently reducing the impact amyloid deposition on β cells.
We found that the hypermethylation status of CpG site cg68167324 located in RDH 12, can increase the risk of GDM (OR = 3.168; 95%CI: 1.038–9.666). RDH 12 encodes retinol dehydrogenase 12 (RDH12), a member of the short-chain dehydrogenases/reductases (SDRs) family,26 which participates in steroid and retinol metabolism.27 RDH12, a NADPH-dependent all-trans retinol dehydrogenase, is the key enzyme in the metabolism of retinoids.28 Two oxidation products of retinoids, 9-cis-retinoic acid and all-trans retinoic acid, function to stimulate insulin secretion.29 In adipocytes, retinoic acid induces the expression of the insulin signaling gene PDK-1 and that of the glucose transporter GLUT4. Activating retinoic acid induces the expression of genes involved in lipid and glucose metabolism, thereby improving insulin action.30 Thus, hypermethylation of the CpG cg68167386 located upstream of the promoter region of RDH12 may inhibit its transcriptional activity and reduce RDH12 levels in peripheral blood. Subsequently, the retinoic acid metabolic pathway would be inhibited, affecting insulin secretion, and reducing its effectiveness.
The DNAJB6 (DnaJ homolog, subfamily B, member 6) protein is a member of the heat shock protein 40 (HSP40) family31 and acts as a molecular chaperone for various cellular processes. While observing insulin resistant and diabetic patients, Kurucz et al. found that HSP expression was significantly changed without diabetes, and that the mRNA level of HSP72-inducible subtypes was significantly reduced in patients with type 2 diabetes.32 Additionally, the expression of HSP70 in the skeletal muscle of patients with type 2 diabetes is reduced and has been shown to correlate with the degree of insulin resistance.33 These HSP molecular chaperones are related to diabetes.34 However, the exact association between DNAJB6 and type 2 diabetes needs further study. In this study, hypermethylation of CpG sites cg157130156, located in the promoter region of DNAJB6, was observed in the GDM group. This might result in increased DNAJB6 levels via the up-regulation of DNABJ6 transcription, thereby reducing the risk of GDM (OR = 0.361; 95%CI: 0.135–0.966).
NFATC4 codes the nuclear factor of activated T cells 4 (NFATC4), which is a member of the transcription factor family under the control of calcineurin (a Ca2+-dependent phosphatase) .35 In adipose tissue, NFATC4 has been shown to promote the secretion of inflammatory factors,36 and to act as a transcriptional repressor in regulating adiponectin gene expression, suggesting that adiponectin expression is down-regulated in obesity and type 2 diabetes.37 In this study, hypermethylation of the CpG site cg24837915 located in the promoter region of NFATC4, was associated with the presence of GDM (OR = 5.232; 95% CI, 1.659–16.506).
During pregnancy, early anabolism increases and mild insulin resistance occurs.38 When insulin secretion fails to balance insulin resistance, impaired glucose tolerance develops, which might subsequently lead to GDM.39 Therefore, impaired secretion by β cells is also a key factor in GDM pathogenesis. Here, we explored the pathogenesis of GDM from an epigenetic perspective and identified 13 CpG sites that had methylation levels showing associations with GDM pathogenesis. Furthermore, conditional logistic regression analysis showed that the methylation status of four CpG sites located in the promoter regions of four genes was associated with GDM pathogenesis. These CpG sites are located in genes that could contribute to the development of GDM. Of these four CpG sites, hypermethylation of cg24837915 and cg68167324 was shown to be associated with GDM, whereas that of cg89438648 and cg157130156 could indicate reduced risk of GDM. Thus, the methylation status of these genes may function as predictors of GDM. No publications reporting on the relationship between methylation of these four CpG sites and GDM have been found, so our suggestion of such a relationship is based on the known modes of action of the genes concerned.
However, the study also had some limitations. First, our limited sample size, further verification of our results using a larger sample size is needed. Second, the selection of our target CpG sites is based on published literature, and we did not screen for differential CpG sites in the same population in this study, so there may be other CpG sites related to the pathogenesis of GDM that have not been verified.