ImprintCap, a powerful NGS-based technology to investigate the molecular background of imprinting disorders.

Introduction: Imprinting disorders (IDs) are a rare class of diseases caused by the disruption of imprinted genes, i.e., genes with a specific pattern of expression from only one allele. Currently, 48 loci are known to show parent-of-origin dependent, imprinted, expression in humans, some of which are disease-associated (da) whereas most of them are non-disease-associated (nda) loci. A subset of patients with an imprinting disorder exhibits aberrant imprinting in at least one differentially methylated region (DMR) in addition to the da loci. Correlation between multilocus imprinting disturbance (MLID), phenotype, variants in maternal effect proteins and fertility problems are currently under investigation. There is a need for a reliable, cost-effective method to detect low mosaic levels of methylation changes in all DMRs. To this end, a targeted NGS panel named ImprintCap was developed using the TWIST method for 48 DMRs. To validate the technique, 13 patients with known methylation changes were analyzed, and these were compared to 30 control samples.

Results: Methylation ranges of mean + / - 3SD were determined in 41/48 DMRs in the capture, including all da DMRs. The mean relative coverage was used to detect CNVs in each DMR. The diagnostic findings were confirmed using ImprintCap in all patients' samples, including methylation changes and deletions. From four samples with genome-wide uniparental disomy (UPD), we determined a detection level of at least 30% mosaic aberrant cells. Three patients were known to show MLID in one or more da DMRs. These changes were confirmed, and in addition, methylation changes were found in 17-32 da or nda DMRs.

Conclusion: By employing ImprintCap, methylation changes can be detected in 41 DMRs, with an overall detection level of 30% mosaic. The DMRs are located on 20 different chromosomes, enabling the detection of UPD in these regions, in addition to CNV in DMRs. The combination of these characteristics renders the methods highly suitable as a diagnostic test for all IDs, detecting UPD, methylation changes and CNVs. The panel is also a reliable tool for the detection of MLID involving both da and nda DMRs.