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

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.