Investigation of imprinted differentially methylated regions implicates placenta- specific haplotypes as potential internal reference markers for non-invasive prenatal testing

Abstract Background: Imprinted genes play important roles in placental development and fetal growth. The human placenta exhibits a unique pattern in genomic imprinting compared to that in somatic tissues. Here, we investigated imprinted differentially methylated regions (iDMRs) in the placenta and their changes associated with preeclampsia. Methods: We analyzed 3362 CpG sites in 62 iDMRs using BisCap targeted bisulfite resequencing (BisCap-seq) assays of maternal blood, healthy and preeclamptic placenta samples. We constructed 185 imprinted methylation haplotype blocks (iMHBs) using these coupled CpG sites in the iDMRs. We accessed the methylation status of each iMHB and CpG site in the maternal blood, healthy and preeclamptic placenta samples. Results: We found that the CpG sites in the CpG islands of the iDMRs tended to show tightly coupled intermediate methylation (methylation levels: 0.35–0.65, D’ > 0.8). In addition to 60 placenta-specific iMHBs, 38 somatic iMHBs were differentially methylated (p < 2.70 ×10-4) in the placenta compared with maternal blood. We identified 27 iMHBs with differentially variable methylation patterns in the placenta, suggesting polymorphic imprinting on the sub-iDMRs scale. Moreover, iMHB methylation did not change in the preeclamptic placentas. However, we detected a significant association between preeclampsia and 27 differentially methylated cytosines (DMCs, p < 1.49 ×10-5), which were located outside the iMHB structures in the placenta. We found that placenta-specific haplotypes (PSHs) of iMHBs could quantify the placental compositions of maternal plasma circulating DNA. Conclusions: Our results demonstrated additional genomic imprinting features in the placenta and suggested that PSHs are potential internal reference markers of the placenta that may be used for non-invasive prenatal testing.