First‐time application of droplet digital PCR for methylation testing of the 11p15.5 imprinting regions

Background Beckwith‐Wiedemann syndrome and Silver‐Russel syndrome are two imprinting disorders caused by opposite molecular alterations in 11p15.5. With the current diagnostic tests, their molecular diagnosis is challenging due to molecular heterogeneity and mosaic occurrence of the most frequent al...

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Bibliographic Details
Published in:Molecular genetics & genomic medicine 2023-12, Vol.11 (12), p.e2264-n/a
Main Authors: Schlaich, Elia, Hubens, Wouter H. G., Eggermann, Thomas
Format: Article
Language:English
Subjects:
Beckwith-Wiedemann syndrome
Chromosome 11
Diagnostic tests
Disorders
DNA methylation
Droplets
Genotypes
Heterogeneity
imprinting disorders
Laboratories
Methylation
mosaicism
MS‐ddPCR
MS‐MLPA
MS‐pyrosequencing
Original
Patients
Sensitivity analysis
Tumors
Underpotential deposition
Uniparental disomy
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Summary:Background Beckwith‐Wiedemann syndrome and Silver‐Russel syndrome are two imprinting disorders caused by opposite molecular alterations in 11p15.5. With the current diagnostic tests, their molecular diagnosis is challenging due to molecular heterogeneity and mosaic occurrence of the most frequent alterations. As the determination of precise (epi)genotype of patients is relevant as the basis for a personalized treatment, different approaches are needed to increase the sensitivity of diagnostic testing of imprinting disorders. Methods We established methylation‐specific droplet digital PCR approaches (MS‐ddPCR) for the two imprinting centers in 11p15.5, and analyzed patients with paternal uniparental disomy of chromosome 11p15.5 (upd(11)pat) and other imprinting defects in the region. The results were compared to those from MS‐MLPA (multiplex ligation‐dependent probe amplification) and MS‐pyrosequencing. Results MS‐ddPCR confirmed the molecular alterations in all patients and the results matched well with MS‐MLPA. The results of MS‐pyrosequencing varied between different runs, whereas MS‐ddPCR results were reproducible. Conclusion We show for the first time that MS‐ddPCR is a reliable and easy applicable method for determination of MS‐associated changes in imprinting disorders. It is therefore an additional tool for multimethod diagnostics of imprinting disorders suitable to improve the diagnostic yield. We describe for the first time the application of droplet digital PCR to identify aberrant methylation in imprinting disorders. As a proof of principle of the diagnostic suitability of the method to detect aberrant epigenetic patterns, we have chosen DNA samples with specific aberrant imprinting patterns. We could show the reliability of the technique by comparing it with two other well established methods (MS‐MLPA, MS‐pyrosequencing).
ISSN:2324-9269
2324-9269
DOI:10.1002/mgg3.2264