Identifying clinically relevant imprinted gDMRs sensitive to a transient loss of DNA methylation in human differentiated cells

Background
Imprinted genes are autosomal, but only expressed from one parental allele and are often clustered in small groups. They play an important role in the regulation of normal mammalian development. Differentially methylated regions (DMR) on each allele are important in regulating the genes, with marks being characterised as primary or secondary DMRs, depending on whether they are inherited from the germ cells or arise later, respectively. Imprinting disorders such as Prader-Willi Syndome (PWS) and Beckwith-Weidemann Syndrome (BWS) arise either from uniparental disomy or faulty DNA methylation. We wished to determine 1) which of the loci are most sensitive to loss of methylation 2) to more precisely define the sensitive regions and 3) determine what happens at primary versus secondary imprints.

Methods
Stable knockdowns of the maintenance methyltransferase DNMT1 were generated in hTERT-immortalised adult fibroblasts using shRNA. Genome wide methylation levels were assayed using the Illumina 450k BeadChip array and analysed using bioinformatic approaches.

Results
We found that 1) the imprinted loci varied extensively in their sensitivity to loss of methylation 2) the extended locus involved in PWS was particularly sensitive 3) that loss of methylation at primary DMR appears to drive gains in methylation at secondary DMR.

Conclusion
Our results point to a mechanistic link between primary and secondary DMR which may explain why imprints are difficult to reprogram in somatic tissues.