ESPE Abstracts

Background: Beckwith-Wiedemann syndrome (BWS) is a representative imprinting disorder with characteristic clinical features such as overgrowth, macroglossia, and abdominal wall defects. The BWS-responsible imprinted region is on chromosome 11p15.5. The CpGs within the H19/IGF2:IG-differentially methylated region (H19 -DMR) at the 11p15.5 imprinted region are maternally unmethylated and paternally methylated, and the H19- DMR functions as the imprinting center of this imprinting region. Mutations or deletions of OCT4/SOX2 binding sites on the maternal allele cause hypermethylation of the H19 -DMR due to inhibition of protection from genome-wide methylation after fertilization. Gain of methylation at the H19 -DMR on maternal allele leads to biallelic IGF2 expression and silencing of H19 and causes BWS phenotype. However, the precise methylation pattern in the patients with defects of OCT4/SOX2 binding sites on maternal allele remains elusive. Long read sequencing (LRS) is able to get contiguous several kb reads and allele-specific methylation levels. In this study, we conducted sequence and methylation analyses using LRS in two families with BWS which showed the H19- DMR hypermethylation. We also compared the methylation levels of each CpG between the mothers and their offspring.

Case: Family A: Both the mother and her son showed the BWS phenotype. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) revealed that they have a gain of methylation at the H19- DMR without copy number abnormalities. LRS identified a single nucleotide variant (chr11:2023018 C>T, hg19) within the OCT4/SOX2 binding site both in the mother and the offspring. Methylation analysis showed that 239 of 247 CpGs were highly methylated in both. In most of these CpGs, the aberrant methylation levels were higher in the offspring than in the mother. Family B: Both the mother and her son also exhibited the BWS phenotype. MS-MLPA　showed that they have gain of methylation at the H19 -DMR and copy number abnormalities. Approximately 2.7 kb deletion (chr11:2022825-2025479, hg19) containing the OCT4/SOX2 binding site was detected by LRS. Methylation analysis is currently in progress.

Assessment and Conclusion: LRS detected the OCT4/SOX2 binding site defects in two BWS families exhibiting the H19 -DMR hypermethylation and showed the methylation levels in all CpGs within the H19- DMR. We detected relatively higher methylation levels in the offspring than in the mother, although the number of hypermethylated CpGs was the same in both. These findings suggest that some epigenetic factors other than genetic abnormalities accelerate the abnormal methylation levels in offspring.