Culture-dependent decoupling of H19 allele-specific expression from H19/IGF2:IG-DMR methylation in experimental...

Genomic imprinting is an epigenetic mechanism leading to the monoallelic expression of a subset of genes, mainly regulated by DNA methylation at imprinting control regions (ICRs). Silver-Russell syndrome (SRS) is a paradigmatic imprinting disorder in which loss of methylation at the 11p15.5 H19/IGF2:IG-DMR is associated with downregulation of IGF2 and biallelic expression of H19. Currently, the allele-specific expression of imprinted genes is widely inferred from ICR methylation without direct assessment. Here, we quantified H19 allele-specific expression together with H19/IGF2:IG-DMR methylation in control (n = 32) and SRS (n = 12) samples spanning tissues and commonly used cellular models, including fibroblasts, induced pluripotent stem cells (iPSCs) and dental pulp stem cells (DPSCs). Overall, H19 allele-specific expression was concordant with H19/IGF2:IG-DMR methylation in tissues, iPSCs and fibroblasts, with clear allelic bias in controls and predominantly biallelic expression in SRS samples. Nevertheless, some SRS-derived fibroblast lines retained an allelic bias despite loss of methylation. In addition, a subset of control DPSCs with balanced methylation exhibited biallelic H19 expression. We showed that the observed decoupling between methylation and allele-specific expression was associated with culture-related procedures, including repeated passaging and freeze-thaw cycles in DPSCs. These findings show that ICR methylation does not always faithfully reflect imprinted gene expression and therefore cannot be uncritically used as a proxy for imprinting. They further indicate that culture-related procedures can promote loss of imprinting and support the systematic combined assessment of ICR DNA methylation and allele-specific expression when establishing cellular models for imprinting disorders.