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

<h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusion</h4>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.