Abstract Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We adapted amplicon bisulfite-sequencing protocols to design IMPLICON for ICRs in adult tissues of inbred mice, validating it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.

The human chr15q11-q13 imprinted cluster is linked to several disorders, including Prader–Willi (PWS) and Angelman (AS) syndromes. Recently, disease modeling approaches based on induced pluripotent stem cells (iPSCs) have been used to study these syndromes. A concern regarding the use of these cells for imprinted disease modeling is the numerous imprinting defects found in many iPSCs. Here, by reprogramming skin fibroblasts from a control and AS individuals, we generated several iPSC lines and addressed the stability of imprinting status across the PWS/AS domain. We focused on three important regulatory DNA elements which are all differentially methylated regions (DMRs), methylated on the maternal allele: the PWS imprinting center (PWS-IC), which is a germline DMR and the somatic NDN and MKRN3 DMRs, hierarchically controlled by PWS-IC. Normal PWS-IC methylation pattern was maintained in most iPSC lines; however, loss of maternal methylation in one out of five control iPSC lines resulted in a monoallelic to biallelic switch for many imprinted genes in this domain. Surprisingly, MKRN3 DMR was found aberrantly hypermethylated in all control and AS iPSCs, regardless of the methylation status of the PWS-IC master regulator. This suggests a loss of hierarchical control of imprinting at PWS/AS region. We confirmed these results in established iPSC lines derived using different reprogramming procedures. Overall, we show that hierarchy of imprinting control in donor cells might not apply to iPSCs, accounting for their spectrum of imprinting alterations. Such differences in imprinting regulation should be taken into consideration for the use of iPSCs in disease modeling.

Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a new high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We initially designed IMPLICON to look at ICRs in adult tissues of inbred mice. Then, we validated it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles.Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions.In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.(OMIM#105830) syndromes caused by defects in the paternal or maternal chr15q11-q13 region, respectively (13,14).Most imprinted genes are located in clusters throughout the genome, containing a cisacting CpG-rich DNA element referred to as imprinting control region (ICR) (reviewed in (1)). The ICR is epigenetically marked by DNA methylation in a parent-of-origin fashion, which correlates with expression and/or silencing of the surrounding imprinted genes.Deletions of ICRs result in the loss of parental allele-specific expression within an imprinted cluster (15,16). ICRs acquire parental-specific DNA methylation in the germline, which are maintained throughout development and adulthood, resisting the global wave of demethylation and de novo methylation steps during early embryonic development (reviewed in (1,17,18)). The preservation of parental allele-specific methylation at ICRs, also known as germline differentially methylated regions (gDMRs), is fundamental for the correct maintenance of imprinted expression throughout life.Despite their importance, there is currently no robust, cost-effective and highthroughput method to assess the methylation status of ICRs across multiple imprinted regions (Suppl . Table 1; reviewed in (19)). The traditional way of measuring DNA methylation is bisulfite sequencing (20). Bisulfite treatment of DNA results in deamination of unmodified cytosines to uracils, whereas methylated cytosines remain unchanged. This is followed by PCR amplification of a region of interest followed by subcloning and Sanger sequencing. This method is laborious and not cost-effective for multiple samples or viewpoints. Alternatively, bisulfite ...