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%0 Thesis %A Schlaich, Elia %T First-time application of droplet digital PCR for methylation testing of the 11p15.5 imprinting regions %I Rheinisch-Westfälische Technische Hochschule Aachen %V Dissertation %C Aachen %M RWTH-2025-08145 %P 1 Online-Ressource : Diagramme %D 2025 %Z Veröffentlicht auf dem Publikationsserver der RWTH Aachen University 2026 %Z Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2025 %X Beckwith-Wiedemann syndrome and Silver-Russell syndrome belong to the group of congenital imprinting disorders characterised by abnormal regulation of imprinted genes. These genes are expressed monoallelically, depending on whether they are inherited from the mother or father. Mutations or imprinting defects, such as epimutations (methylation disorders), cause these genes to be expressed either biallelically, with impaired function, or not at all, thereby altering gene dosage. In Beckwith-Wiedemann syndrome and Silver-Russell syndrome, opposite changes occur in so-called differentially methylated regions on chromosome 11p15.5. The most common changes in Beckwith-Wiedemann syndrome are epimutations, especially loss of methylation in maternal imprinting centre 2 (IC2) and gain of methylation in maternal imprinting centre 1 (IC1), paternal uniparental disomy of 11p15.5 and mutations in the CDKN1C gene. In Silver-Russell syndrome, there is usually a loss of methylation in paternal IC1 or a maternal uniparental disomy of chromosome 7. The molecular diagnosis of these imprinting disorders is challenging with currently available diagnostic tests due to the molecular heterogeneity and mosaic-like occurrence of the most common changes. Since the determination of the exact (epi)genotype of patients is important as a basis for personalised therapy, several approaches are needed to increase the sensitivity of diagnostic tests for imprinting disorders, besides the analysis of DNA from different tissues. We established methylation-specific digital droplet PCR (MS-ddPCR) assays for the two imprinting centres IC1 and IC2 at 11p15.5 in healthy individuals and analysed patients with paternal uniparental disomy of chromosome 11p15.5 (upd(11)pat), increased methylation in IC1, loss of methylation in IC2 and loss of methylation in IC1. We compared the results of MS-ddPCR with methylation-specific (MS) MLPA (multiplex ligation-dependent probe amplification) and MS-pyrosequencing by determining the degree of methylation for the same two CpGs in IC1 and IC2 with each method. It was found that our MS-ddPCR test identified the molecular alterations in both the healthy subjects and all the patients, and the results matched well with those of the MS-MLPA. MS-pyrosequencing results varied between runs, whereas MS-ddPCR results were reproducible. MS-MLPA is currently the most widely used test for the diagnosis of imprinting disorders and allows the study of multiple CpGs in one approach. However, its sensitivity for detecting low-grade mosaics has been questioned. MS-ddPCR analyses only one CpG, but is very flexible due to individually selectable primers and probes and reduces PCR-induced bias by absolute quantification of methylation. With this study, we show for the first time that MS-ddPCR is a reliable and easy-to-use method for identifying methylation associated changes in imprinting disorders. It thus represents an additional tool for the multimethod diagnosis of imprinting disorders, which is suitable for improving the diagnostic yield. %F PUB:(DE-HGF)11 %9 Dissertation / PhD Thesis %R 10.18154/RWTH-2025-08145 %U https://publications.rwth-aachen.de/record/1019096