Application of a NotI subtraction and methylation‑specific genome subtractive hybridization technique in the detection of genomic DNA methylation differences between hydatidiform moles and villi.

Previous studies indicate that epigenetic modifications play an important role in transcriptional regulation and contribute to the pathogenesis of gestational trophoblastic disease, including complete hydatidiform moles (CHMs). However, the underlying mechanisms and the critical genes have not been clearly identified. In the present study, we developed a novel technique, NotI subtraction and methylation-specific genome subtractive hybridization (MS-G-SH), as a method of screening for methylation changes between hydatidiform moles and villi. Following NotI subtraction and hybridization, three different positive DNA clones were found in 110 random clones of DNA samples. Most importantly, two DNA clones having long CpG islands and high homology with exons of insulin-like growth factor 2 (IGF2) and transforming growth factor-β (TGF-β) were identified using bioinformatic tools. After bisulfite treatment and methylation-specific PCR, the specific methylation of certain exons of IGF2 and TGF-β was identified. In addition, the mRNA expression levels of these two genes were markedly different. In conclusion, this novel MS-G-SH technique is an alternative and effective approach for the detection of specific DNA methylation.