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Background <p>Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with characteristic clinical features such as overgrowth and macroglossia. Hypermethylation of the <i>H19/IGF2</i>:IG-differentially methylated region (<i>H19</i>-DMR) is identified in 5% of BWS patients. Although defects in the OCT4/SOX2 binding sites (OBS) on the maternal allele have been reported in patients with hypermethylated <i>H19</i>-DMR, the precise methylation patterns in patients with OBS abnormalities remain elusive. Nanopore long-read sequencing (LRS) obtains sequence reads 10–100&#xa0;kb long together with single-molecule DNA modifications, such as 5-methylcytosines. Furthermore, LRS can separate reads into haplotypes 1 and 2 based on information from single-nucleotide variants and indels on reads. Here, we conducted LRS to elucidate the abnormal hypermethylation patterns of the <i>H19</i>-DMR in BWS patients with and without OBS abnormalities and evaluate the differences between mothers and their offspring in familial cases with OBS abnormalities.</p> Results <p>We included 14 sporadic patients and four familial patients from two families with BWS showing hypermethylation of the <i>H19</i>-DMR. Amplicon sequencing for the <i>H19</i>-DMR detected OBS abnormalities in four (28.6%) sporadic cases (three point mutations, one 21-bp microdeletion overlapping OBS), an OBS point mutation in family A, and a 2.6&#xa0;kb deletion involving OBS in family B. Subsequently, we conducted targeted LRS and examined methylation indexes for 247 CpGs in the <i>H19</i>-DMR in three sporadic patients with OBS abnormalities, two sporadic patients without OBS abnormalities, and all familial patients with OBS abnormalities. In patients with point mutations and a 21-bp microdeletion affecting a single base in OBS, CpGs in the <i>H19</i>-DMR had obvious hypermethylation in the vicinity of OBS and mild-to-moderate hypermethylation with increased distance from OBS; however, in patients with deletions including OBS, CpGs had moderate hypermethylation in the entire <i>H19</i>-DMR. In families A and B, methylation levels were higher in offspring than in their mothers.</p> Conclusion <p>Because 28.6% of sporadic patients had OBS abnormalities, genetic examination for OBS should be considered in BWS patients with hypermethylated <i>H19</i>-DMR. LRS analysis for the <i>H19</i>-DMR revealed differences in methylation patterns between patients with and without OBS abnormalities and the methylation levels of each CpG between mothers and their offspring in families with OBS abnormalities.</p>

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Methylation profile characteristics in the H19/IGF2:IG-DMR revealed by long-read sequencing analysis in patients with Beckwith-Wiedemann syndrome having defects in the OCT4/SOX2 binding site

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  • 发表日期：

    2026年2月3日

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  • Hayate Masubuchi,
  • Tatsuki Urakawa,
  • Rika Kosaki,
  • Riki Nishimura,
  • Yasunori Wada,
  • Sumito Dateki,
  • Hideaki Yagasaki,
  • Reiko Kagawa,
  • Yutaka Nishimura,
  • Hidenobu Soejima,
  • Tsutomu Ogata,
  • Maki Fukami,
  • Masayo Kagami

摘要

Background

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with characteristic clinical features such as overgrowth and macroglossia. Hypermethylation of the H19/IGF2:IG-differentially methylated region (H19-DMR) is identified in 5% of BWS patients. Although defects in the OCT4/SOX2 binding sites (OBS) on the maternal allele have been reported in patients with hypermethylated H19-DMR, the precise methylation patterns in patients with OBS abnormalities remain elusive. Nanopore long-read sequencing (LRS) obtains sequence reads 10–100 kb long together with single-molecule DNA modifications, such as 5-methylcytosines. Furthermore, LRS can separate reads into haplotypes 1 and 2 based on information from single-nucleotide variants and indels on reads. Here, we conducted LRS to elucidate the abnormal hypermethylation patterns of the H19-DMR in BWS patients with and without OBS abnormalities and evaluate the differences between mothers and their offspring in familial cases with OBS abnormalities.

Results

We included 14 sporadic patients and four familial patients from two families with BWS showing hypermethylation of the H19-DMR. Amplicon sequencing for the H19-DMR detected OBS abnormalities in four (28.6%) sporadic cases (three point mutations, one 21-bp microdeletion overlapping OBS), an OBS point mutation in family A, and a 2.6 kb deletion involving OBS in family B. Subsequently, we conducted targeted LRS and examined methylation indexes for 247 CpGs in the H19-DMR in three sporadic patients with OBS abnormalities, two sporadic patients without OBS abnormalities, and all familial patients with OBS abnormalities. In patients with point mutations and a 21-bp microdeletion affecting a single base in OBS, CpGs in the H19-DMR had obvious hypermethylation in the vicinity of OBS and mild-to-moderate hypermethylation with increased distance from OBS; however, in patients with deletions including OBS, CpGs had moderate hypermethylation in the entire H19-DMR. In families A and B, methylation levels were higher in offspring than in their mothers.

Conclusion

Because 28.6% of sporadic patients had OBS abnormalities, genetic examination for OBS should be considered in BWS patients with hypermethylated H19-DMR. LRS analysis for the H19-DMR revealed differences in methylation patterns between patients with and without OBS abnormalities and the methylation levels of each CpG between mothers and their offspring in families with OBS abnormalities.