Identification of differentially methylated genes in first-trimester placentas with trisomy 16

Abstract

The presence of an extra chromosome in the embryo karyotype often dramatically affects the fate of pregnancy. Trisomy 16 is the most common aneuploidy in first-trimester miscarriages. The present study identified changes in DNA methylation in chorionic villi of miscarriages with trisomy 16. Ninety-seven differentially methylated sites in 91 genes were identified (false discovery rate (FDR) < 0.05 and Δβ > 0.15) using DNA methylation arrays. Most of the differentially methylated genes encoded secreted proteins, signaling peptides, and receptors with disulfide bonds. Subsequent analysis using targeted bisulfite massive parallel sequencing showed hypermethylation of the promoters of specific genes in miscarriages with trisomy 16 but not miscarriages with other aneuploidies. Some of the genes were responsible for the development of the placenta and embryo (GATA3-AS1, TRPV6, SCL13A4, and CALCB) and the formation of the mitotic spindle (ANKRD53). Hypermethylation of GATA3-AS1 was associated with reduced expression of GATA3 protein in chorionic villi of miscarriages with trisomy 16. Aberrant hypermethylation of genes may lead to a decrease in expression, impaired trophoblast differentiation and invasion, mitotic disorders, chromosomal mosaicism and karyotype self-correction via trisomy rescue mechanisms.

Figure 1

Results of cytogenetic analysis of chorionic villous trophoblasts of miscarriages with trisomy 16. (a) Example of the aCGH profile in chorionic villous trophoblasts with trisomy 16. (b) Example of chorionic villous trophoblasts with trisomy 16 based on the results of FISH analysis with two-color subtelomeric DNA probes specific to 16p (red) and 16q (green). Interphase nuclei were stained with DAPI (blue).

Figure 2

Functional relationships between the products of genes hypermethylated in chorionic trophoblasts of embryos with trisomy 16 (highlighted in yellow) and the products of genes involved in the development of the placenta (placental development, GO: 0001890) (highlighted in blue) (STRING database, score > 0.90). The connections of the products of the hypermethylated genes with the proteins involved in the development of the placenta are highlighted in red.

Figure 3

The CpG site methylation indices in 5 DMGs in chorionic villi in the group of miscarriages with trisomy 16 compared to miscarriages with trisomy of other chromosomes, miscarriages with a normal karyotype, and induced abortions. (a) The average methylation indices for all analyzed CpG sites in the ANKRD53, GATA3-AS1, CALCB, SCL13A4, and TRPV6 genes in the groups of miscarriages with trisomy 16 (Tri (16)), miscarriages with trisomy of other chromosomes (Tri (non16)), miscarriages with a normal karyotype (NK), and induced abortions (IAs). The line in the center of the box marks the median. The boxes mark the 25th and 75th percentiles. The whiskers extend to the minimum and maximum values. The Mann–Whitney U test was used to compare groups. *—p < 0.05. (b) A detailed profile of the methylation of CpG sites in the analyzed regions of the ANKRD53, GATA3-AS1, SCL13A4, and TRPV6 genes (indicated by a blue shaded area) in the groups of miscarriages with trisomy 16 and miscarriages with trisomy of other chromosomes compared to the induced abortion group. CpG islands are indicated by green bars. The dotted lines depict the standard deviation of the methylation profile. The figure was created using UCSC Genome Browser.

Figure 4

Correlation of the methylation indices of differentially methylated gene promoters with each other and their dependence on gestational age and chromosome mosaicism. (a) Correlation of the methylation indices of the ANKRD53, TRPV6, GATA3-AS1, SCL13A4, and CALCB genes with each other in chorionic villi of spontaneous abortions with trisomy 16. The heatmap was generated using the Spearman test and the ClustVis tool. (b) Correlation of the methylation indices of the GATA3-AS1_4-5 regions with the gestational age of miscarriages with trisomy 16, miscarriages with a normal karyotype, and induced abortions. (c) Comparison of the ANKRD53 gene methylation index in chorionic villi between groups of miscarriages with mosaic and pure trisomy 16.

Figure 5

Examples of GATA3 protein staining in frozen sections of syncytiotrophoblast and cytotrophoblast cells of miscarriages with trisomy 16 (n = 4), trisomy 22 (n = 2), and a normal karyotype (n = 4).