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

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.

Figure 1

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.

Figure 2

Statistical analyses of aberrantly methylated differentially methylated region (DMRs). (a) Comparison of the number of aberrantly methylated DMRs between gametic DMRs and somatic DMRs in KvDMR1-LOM patients. There was no statistical difference between the two DMRs (P = 0.42, Fisher's exact test). (b) Comparison of the number of aberrantly methylated DMRs between matDMRs and patDMRs in KvDMR1-LOM patients. matDMRs were aberrantly methylated more frequently than patDMRs (P = 0.042, Fisher's exact test). (c) Comparison of the number of LOMs and GOMs between matDMRs and patDMRs among the aberrantly methylated DMRs in KvDMR1-LOM patients. LOM preferentially occurred on matDMRs (P = 0.050, Fisher's exact test). GOM, gain of methylation; LOM, loss of methylation; matDMR, maternally methylated DMR; patDMR, paternally methylated DMR.

Figure 3

Methylation analysis of ZDBF2-DMR and expression analysis of the ZDBF2 gene. (a) Results of matrix-assisted laser desorption/ionization mass spectrometry analysis. Averages with SD of 24 normal controls are shown in blue. Methylation indexes of the patients showing GOM are indicated in different colors. Units 1 and 2 included two and one CpG sites, respectively. (b) Results of bisulfite sequencing. Normal controls show monoallelic differential methylation, whereas four Beckwith–Wiedemann syndrome (BWS) patients (BWS-s001, BWS-s011, BWS-s023, and BWS-s060) show biallelic methylation. Two parental alleles were distinguished by a SNP (rs1861437). Mat, maternal allele; Pat, paternal allele. (c) Results of expression analysis of the ZDBF2 gene. Three BWS patients (BWS-s001, BWS-s011, and BWS-s113) heterozygous for a coding SNP (rs10932150) with GOM clearly showed biallelic expression; by contrast, two patients with normally methylated differentially methylated region (DMRs) exhibited paternal monoallelic expression (patients BWS-s004 and BWS-s060). gDNA, genomic DNA; GOM, gain of methylation; SNP, single-nucleotide polymorphism.

Figure 4

Methylation analysis of FAM50B- and GNAS1A-DMRs and expression analysis of the FAM50B and GNAS1A genes. (a,b) Results of matrix-assisted laser desorption/ionization mass spectrometry analysis. Averages with SD of 24 normal controls are shown in blue. Methylation indexes of patients showing LOM are indicated in different colors. Ten CpG units analyzed for FAM50B-DMR covered 13 CpG sites, and 13 CpG units analyzed for GNAS1A-DMR covered 18 CpG sites. (c) Results of expression analysis of the FAM50B gene. Beckwith–Wiedemann syndrome (BWS) patient BWS-s096 was heterozygous for a coding SNP (rs6597007) with LOM and showed biallelic expression with a low peak of maternal expression, whereas monoallelic expression was seen in a patient with normally methylated differentially methylated regions (DMRs) (patient BWS-s089). In patient BWS-s096, maternal expression was noted in two independent analyses despite low-grade LOM. gDNA, genomic DNA. (d) Results of expression analysis of the GNAS1A gene. Patients BWS-s062 and BWS-s113, heterozygous for a deletion/insertion variation (rs143800311) with LOM, showed biallelic expression, whereas patient BWS-s060 possessed normally methylated DMRs and exhibited monoallelic expression. Maternal expression was noted despite low-grade LOM in patient BWS-s062. Red peaks are molecular markers. GOM, gain of methylation; LOM, loss of methylation.