Exploration of hydroxymethylation in Kagami-Ogata syndrome caused by hypermethylation of imprinting control regions.

Keiko Matsubara,Kazuki Yamazawa,Kazuhiko Nakabayashi,Tsutomu Ogata,Maki Fukami,Kenichiro Hata,Masayo Kagami

doi:10.1186/s13148-015-0124-y

Abstract

Background5-Hydroxymethylcytosine (5hmC), converted from 5-methylcytosine (5mC) by ten-eleven translocation (Tet) enzymes, has recently drawn attention as the “sixth base” of DNA since it is considered an intermediate of the demethylation pathway. Nonetheless, it remains to be addressed how 5hmC is linked to the development of human imprinting disorders. In this regard, conventional bisulfite (BS) treatment is unable to differentiate 5hmC from 5mC. It is thus hypothesized that BS conversion-derived “hypermethylation” at imprinting control regions (ICRs), which may cause imprinting disorders, would in fact be attributable to excessively increased levels of 5hmC as well as 5mC. To test this hypothesis, we applied the newly developed oxidative BS (oxBS) treatment to detect 5hmC in blood samples from Kagami-Ogata syndrome (KOS14) patients caused by an epimutation (hypermethylation) of two differentially methylated regions (DMRs) functioning as ICRs, namely, IG-DMR and MEG3-DMR.FindingsoxBS with pyrosequencing revealed that there were few amounts of 5hmC at the hypermethylated IG-DMR and MEG3-DMR in blood samples from KOS14 patients. oxBS with genome-wide methylation array demonstrated that global levels of 5hmC were very low with similar distribution patterns in blood samples from KOS14 patients and normal controls. We also confirmed the presence of large amounts of 5hmC in the brain sample from a normal control.Conclusions5hmC is not a major component in abnormally hypermethylated ICRs or at a global level, at least in blood from KOS14 patients. As the brain sample contained large amounts of 5hmC, the neural tissues of KOS14 patients are promising candidates for analysis in elucidating the role of 5hmC in the neurodevelopmental context.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0124-y) contains supplementary material, which is available to authorized users.

Highlights

5-Hydroxymethylcytosine (5hmC), converted from 5-methylcytosine (5mC) by ten-eleven translocation (Tet) enzymes, has recently drawn attention as the “sixth base” of DNA since it is considered an intermediate of the demethylation pathway
Methylation/hydroxymethylation at the IG-differentially methylated region (DMR) and MEG3DMR The pyrosequencing results revealed that, as expected, the “methylation” values derived by BS reaction at the IG-DMR and MEG3-DMR in Kagami-Ogata syndrome (KOS14) patients were considerably high compared to those in the blood/brain samples of the normal control (Fig. 1a and Additional file 2: Figure S1)
The values derived by oxidative BS (oxBS) reaction at the IG-DMR and MEG3-DMR were increased in KOS14 patients, whereas those in the control blood/ brain were normal

Summary

Introduction

5-Hydroxymethylcytosine (5hmC), converted from 5-methylcytosine (5mC) by ten-eleven translocation (Tet) enzymes, has recently drawn attention as the “sixth base” of DNA since it is considered an intermediate of the demethylation pathway It remains to be addressed how 5hmC is linked to the development of human imprinting disorders. It is hypothesized that BS conversion-derived “hypermethylation” at imprinting control regions (ICRs), which may cause imprinting disorders, would be attributable to excessively increased levels of 5hmC as well as 5mC To test this hypothesis, we applied the newly developed oxidative BS (oxBS) treatment to detect 5hmC in blood samples from Kagami-Ogata syndrome (KOS14) patients caused by an epimutation (hypermethylation) of two differentially methylated regions (DMRs) functioning as ICRs, namely, IG-DMR and MEG3-DMR

Methods

Results

Conclusion