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Comprehensive and quantitative multilocus methylation analysis reveals the susceptibility of specific imprinted differentially methylated regions to aberrant methylation in Beckwith-Wiedemann syndrome with epimutations.

Maeda T, Higashimoto K, Jozaki K, Yatsuki H, Nakabayashi K, Makita Y, Tonoki H, Okamoto N, Takada F, Ohashi H, Migita M, Kosaki R, Matsubara K, Ogata T, Matsuo M, Hamasaki Y, Ohtsuka Y, Nishioka K, Joh K, Mukai T, Hata K, Soejima H - Genet. Med. (2014)

Bottom Line: Biallelic expression of the genes was associated with aberrant methylation.Cis-acting pathological variations were not found in any aberrantly methylated DMR.Cis-acting variations of the DMRs are not involved in the multiple methylation defects.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Molecular Genetics and Epigenetics, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, Japan [2] Department of Pediatrics, Faculty of Medicine, Saga University, Saga, Japan.

ABSTRACT

Purpose: Expression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith-Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith-Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined.

Methods: Comprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith-Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced.

Results: Thirty-four percent of KvDMR1-loss of methylation patients and 30% of H19DMR-gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1-loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR.

Conclusion: Maternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.

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Results of methylation analyses of 29 imprinted differentially methylated regions (DMRs) in Beckwith–Wiedemann syndrome patients with epimutations. (a) Results of patients with KvDMR1-LOM. Only the results of multiple methylation defects are shown. Aberrant methylation was confirmed by two quantitative methods: matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing. The definition of aberrant methylation used here is described in the Materials and Methods section. Shaded rectangle: aberrant hypomethylation; dark gray rectangle: aberrant hypermethylation. (b) Results of all patients with H19DMR-GOM. GOM, gain of methylation; LOM, loss of methylation.
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fig1: Results of methylation analyses of 29 imprinted differentially methylated regions (DMRs) in Beckwith–Wiedemann syndrome patients with epimutations. (a) Results of patients with KvDMR1-LOM. Only the results of multiple methylation defects are shown. Aberrant methylation was confirmed by two quantitative methods: matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing. The definition of aberrant methylation used here is described in the Materials and Methods section. Shaded rectangle: aberrant hypomethylation; dark gray rectangle: aberrant hypermethylation. (b) Results of all patients with H19DMR-GOM. GOM, gain of methylation; LOM, loss of methylation.

Mentions: Among the 44 KvDMR1-LOM patients, 15 (34.1%) showed aberrantly methylated DMRs outside of KvDMR1: six showed aberrant methylation at only one DMR, and the other nine showed two or more methylated DMRs (Figure 1a and Supplementary Figure S3 online). The greatest number of aberrantly methylated DMRs was found in patient BWS-s113, who exhibited 12 DMRs. Most of the aberrantly methylated DMRs demonstrated LOM, which was seen at ARHI-CG1, ARHI-CG2, ARHI-CG3, FAM50B, ZAC, IGF2R-DMR2, MEST, NNAT, L3MBTL1, NESPAS, GNASXL, and GNAS1A. Among them, the most frequently hypomethylated DMRs were ARHI-CG1 and ARHI-CG3, found in nine (20.5%) and eight (18.2%) patients, respectively. By contrast, three DMRs, located at ZDBF2, NESP, and MCTS2, showed GOM, which was found in six (13.6%), two (4.5%), and one (2.3%) patients, respectively. GNASXL-DMR showed GOM in one patient (2.3%), whereas four patients (9.1%) showed LOM. The other 13 DMRs were not aberrantly methylated in any KvDMR1-LOM patient.


Comprehensive and quantitative multilocus methylation analysis reveals the susceptibility of specific imprinted differentially methylated regions to aberrant methylation in Beckwith-Wiedemann syndrome with epimutations.

Maeda T, Higashimoto K, Jozaki K, Yatsuki H, Nakabayashi K, Makita Y, Tonoki H, Okamoto N, Takada F, Ohashi H, Migita M, Kosaki R, Matsubara K, Ogata T, Matsuo M, Hamasaki Y, Ohtsuka Y, Nishioka K, Joh K, Mukai T, Hata K, Soejima H - Genet. Med. (2014)

Results of methylation analyses of 29 imprinted differentially methylated regions (DMRs) in Beckwith–Wiedemann syndrome patients with epimutations. (a) Results of patients with KvDMR1-LOM. Only the results of multiple methylation defects are shown. Aberrant methylation was confirmed by two quantitative methods: matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing. The definition of aberrant methylation used here is described in the Materials and Methods section. Shaded rectangle: aberrant hypomethylation; dark gray rectangle: aberrant hypermethylation. (b) Results of all patients with H19DMR-GOM. GOM, gain of methylation; LOM, loss of methylation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262761&req=5

fig1: Results of methylation analyses of 29 imprinted differentially methylated regions (DMRs) in Beckwith–Wiedemann syndrome patients with epimutations. (a) Results of patients with KvDMR1-LOM. Only the results of multiple methylation defects are shown. Aberrant methylation was confirmed by two quantitative methods: matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing. The definition of aberrant methylation used here is described in the Materials and Methods section. Shaded rectangle: aberrant hypomethylation; dark gray rectangle: aberrant hypermethylation. (b) Results of all patients with H19DMR-GOM. GOM, gain of methylation; LOM, loss of methylation.
Mentions: Among the 44 KvDMR1-LOM patients, 15 (34.1%) showed aberrantly methylated DMRs outside of KvDMR1: six showed aberrant methylation at only one DMR, and the other nine showed two or more methylated DMRs (Figure 1a and Supplementary Figure S3 online). The greatest number of aberrantly methylated DMRs was found in patient BWS-s113, who exhibited 12 DMRs. Most of the aberrantly methylated DMRs demonstrated LOM, which was seen at ARHI-CG1, ARHI-CG2, ARHI-CG3, FAM50B, ZAC, IGF2R-DMR2, MEST, NNAT, L3MBTL1, NESPAS, GNASXL, and GNAS1A. Among them, the most frequently hypomethylated DMRs were ARHI-CG1 and ARHI-CG3, found in nine (20.5%) and eight (18.2%) patients, respectively. By contrast, three DMRs, located at ZDBF2, NESP, and MCTS2, showed GOM, which was found in six (13.6%), two (4.5%), and one (2.3%) patients, respectively. GNASXL-DMR showed GOM in one patient (2.3%), whereas four patients (9.1%) showed LOM. The other 13 DMRs were not aberrantly methylated in any KvDMR1-LOM patient.

Bottom Line: Biallelic expression of the genes was associated with aberrant methylation.Cis-acting pathological variations were not found in any aberrantly methylated DMR.Cis-acting variations of the DMRs are not involved in the multiple methylation defects.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Molecular Genetics and Epigenetics, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, Japan [2] Department of Pediatrics, Faculty of Medicine, Saga University, Saga, Japan.

ABSTRACT

Purpose: Expression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith-Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith-Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined.

Methods: Comprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith-Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced.

Results: Thirty-four percent of KvDMR1-loss of methylation patients and 30% of H19DMR-gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1-loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR.

Conclusion: Maternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.

Show MeSH
Related in: MedlinePlus