title: Exploration of hydroxymethylation in Kagami-Ogata syndrome caused by hypermethylation of imprinting control regions.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  oxBS 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.
  
  
  CONCLUSIONS
  5hmC 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.
authors: Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535 Japan.
authors: Keiko
authors: K
authors: Matsubara
authors: Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535 Japan.
authors: Masayo
authors: M
authors: Kagami
authors: Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535 Japan.
authors: Kazuhiko
authors: K
authors: Nakabayashi
authors: Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535 Japan.
authors: Kenichiro
authors: K
authors: Hata
authors: Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535 Japan.
authors: Maki
authors: M
authors: Fukami
authors: Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535 Japan ; Department of Pediatrics, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi, Hamamatsu, Shizuoka 431-3192 Japan.
authors: Tsutomu
authors: T
authors: Ogata
authors: Department of Molecular Endocrinology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535 Japan ; Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582 Japan ; Clinical Genetics Center, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro, Tokyo 152-8902 Japan.
authors: Kazuki
authors: K
authors: Yamazawa
keywords: Hydroxymethylation; Imprinting; Kagami-Ogata syndrome; Methylation; Methylation array; Oxidative bisulfite