Genome‑wide DNA methylation analysis of uterosacral ligaments in women with pelvic organ prolapse

Abstract

Pelvic organ prolapse (POP) is an increasingly serious health problem that impairs quality of life and is caused by multiple additive genetic and environmental factors. As the uterosacral ligaments (ULs) provide primary support for the pelvic organs, it was hypothesized that disruption of these ligaments (as a result of aberrant methylation) may lead to a loss of support and eventually contribute to POP. In the present study, whether there are any aberrant methylations in the ULs of patients with POP compared to those of controls was investigated. Genomic DNA was isolated from the ULs of five women with POP and four women without POP, as controls, undergoing hysterectomy for benign conditions. An Illumina Infinium Methylation EPICBeadChips Infinium Human Methylation 850 K bead array was used to investigate the total methylation in the ULs. There were 3,723 differentially methylated CpG sites (Δβ<0.14; P<0.05), including 3,576 hypermethylation and 147 hypomethylation sites in the ULs of patients with POP compared with the normal controls. There were more hypermethylated CpG sites, but a high ratio of hypomethylation between CpG islands and the N‑shelf; in the gene structure, there was more hypermethylation than hypomethylation in TSS1500 and the 5' untranslated region. Gene ontology analysis demonstrated that these differentially methylated genes were associated with 'cell morphogenesis', 'extracellular matrix', 'cell junction', 'protein binding' and 'guanosine triphosphatase activity'. Several significant pathways were identified, including 'focal adhesion' and 'extracellular matrix‑receptor interaction pathway'. This study provides evidence that there are differences in genome‑wide DNA methylation between ULs in menopausal women with and without POP, and that epigenetic mechanisms may partly contribute to POP pathogenesis.

Keywords: uterosacral ligament; methylation; pelvic organ prolapse; menopause.

Figure 1.

Characteristic methylation patterns between pelvic organ prolapse uterine ligament and control subjects. (A) The ratio of equally or differentially methylated CpG sites are presented as a pie chart. (B) The ratio of hypermethylated or hypomethylated CpG sites. (C) Hypermethylated annotation of differentially methylated CpG sites. (D) Hypomethylated annotation of differentially methylated CpG sites. UTR, untranslated region.

Figure 2.

The heat map demonstrates an overview of hierarchical clustering of the differentially methylation in POPs and control subjects. Hypermethylations are presented in red, hypomethylation is labeled green. POP, pelvic organ prolapse.

Figure 3.

GO analysis of differential methylated genes (β>0.14; P<0.05). X-axis, enrichment factor; Y-axis, GO category. The red represents small P-values and the green represents the larger ones. A round node represents a biological process, the triangle represents a cellular component and a square represents a molecular function. The top 20 GO terms are presented. GO, Gene Ontology; ATPase, adenosine triphosphatase; GTPase, guanosine triphosphatase.

Figure 4.

KEGG analysis of the differentially methylations. KEGG analysis of differential methylated genes (β>0.14; P<0.05). The red represents the smaller P-values and the green means the larger ones. X-axis represents the enrichment factor; the Y-axis, represents the KEGG category. The top 20 KEGG terms are presented. KEGG, Kyoto encyclopedia of Genes and Genomes; ECM, extracellular matrix.

Figure 5.

Protein-protein interaction network of differentially expressed genes using STRING. Network nodes represent proteins; edges represent protein-protein associations. Colored lines between the proteins indicate the various types of interaction evidence in STRING. STRING, Search Tool for the Retrieval of Interacting Genes.