New targeted approaches for epigenetic age predictions

Han, Yang; Franzen, Julia; Stiehl, Thomas; Gobs, Michael; Kuo, Chao-Chung; Nikolić, Miloš; Hapala, Jan; Koop, Barbara; Strathmann, Klaus; Ritz‐Timme, Stefanie; Wagner, Wolfgang

doi:10.1186/s12915-020-00807-2

Cited by 62 publications

(98 citation statements)

References 77 publications

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“…Droplet digital PCR (ddPCR) is based on parallel PCR reactions in thousands of micro-droplets and therefore DNAm analysis with this technology may reduce PCR bias for methylated/non-methylated strands that may occur in pyrosequencing ( Figure S1a) (Han et al, 2020). Therefore, we have designed ddPCR assay for the same three amplicons for Prima1, Hsf4, and Kcns1.…”

Section: Age-prediction With Droplet Digital Pcrmentioning

confidence: 99%

“…Interestingly, the DNAm pattern of neighboring CpGs was not coherently modified on individual strands, as might be anticipated upon binding of an epigenetic writer, but rather seemed to be evoked by stochastic modifications (Han et al, 2020). Based on this, we developed an epigenetic age-predictor for BBA-seq data of human blood, which was based on the binary sequel of methylated and non-methylated sites in individual reads (Han et al, 2020). This approach might reflect heterogeneity of epigenetic aging within a sample.…”

Section: Introductionmentioning

confidence: 96%

“…Droplet Digital PCR (ddPCR) is a relatively novel targeted approach for DNAm measurement that was reported to provide precise results with less PCR bias (Han et al, 2020;Zemmour et al, 2018). Furthermore, barcoded bisulfite amplicon sequencing (BBA-seq), which is based on massive-parallel-sequencing, facilitates DNAm analysis of longer amplicons with more neighboring CpGs and provides insight into the DNAm pattern on individual DNA strands (Franzen et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, barcoded bisulfite amplicon sequencing (BBA-seq), which is based on massive-parallel-sequencing, facilitates DNAm analysis of longer amplicons with more neighboring CpGs and provides insight into the DNAm pattern on individual DNA strands (Franzen et al, 2017). We have recently demonstrated in BBA-seq data of human blood that the correlation of age with DNAm levels at neighboring CpGs follows a bell-shaped curved (Han et al, 2020). Interestingly, the DNAm pattern of neighboring CpGs was not coherently modified on individual strands, as might be anticipated upon binding of an epigenetic writer, but rather seemed to be evoked by stochastic modifications (Han et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…We have recently demonstrated in BBA-seq data of human blood that the correlation of age with DNAm levels at neighboring CpGs follows a bell-shaped curved (Han et al, 2020). Interestingly, the DNAm pattern of neighboring CpGs was not coherently modified on individual strands, as might be anticipated upon binding of an epigenetic writer, but rather seemed to be evoked by stochastic modifications (Han et al, 2020). Based on this, we developed an epigenetic age-predictor for BBA-seq data of human blood, which was based on the binary sequel of methylated and non-methylated sites in individual reads (Han et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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Epigenetic age-predictions in mice using pyrosequencing, droplet digital PCR or barcoded bisulfite amplicon sequencing

Han

Nikolić

Gobs

et al. 2020

Preprint

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Age-associated DNA methylation reflects aspects of biological aging - therefore epigenetic clocks for mice can help to elucidate the impact of treatments or genetic background on the aging process in this model organism. Initially, age-predictors for mice were trained on genome-wide DNA methylation profiles, whereas we have recently described a targeted assay based on pyrosequencing of DNA methylation at only three CG dinucleotides (CpGs). Here, we have re-evaluated pyrosequencing approaches in comparison to droplet digital PCR (ddPCR) and barcoded bisulfite amplicon sequencing (BBA-seq). At individual CpGs the correlation of DNA methylation with chronological age was slightly higher for pyrosequencing and ddPCR as compared to BBA-seq. On the other hand, BBA-seq revealed that neighboring CpGs tend to be stochastically modified in murine age-associated regions. Furthermore, the binary sequel of methylated and non-methylated CpGs in individual reads can be used for single-read predictions, which may reflect heterogeneity in epigenetic aging. In comparison to C57BL/6 mice the epigenetic age-predictions using BBA-seq were also accelerated in the shorter-lived DBA/2 mice, and in C57BL/6 mice with a lifespan quantitative trait locus of DBA/2 mice. Taken together, we describe further optimized and alternative targeted methods to determine epigenetic clocks in mice.

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Section: Age-prediction With Droplet Digital Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Epigenetic age-predictions in mice using pyrosequencing, droplet digital PCR or barcoded bisulfite amplicon sequencing

Han

Nikolić

Gobs

et al. 2020

Preprint

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

Schlaich,

Hubens,

Eggermann

2023

Molec Gen & Gen Med

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BackgroundBeckwith‐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.MethodsWe 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.ResultsMS‐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.ConclusionWe 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.

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Forensische DNA-Methylierungsanalyse

Naue¹,

Pfeifer²,

Augustin³

et al. 2021

Rechtsmedizin

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ZusammenfassungDie quantitative Analyse der relativen DNA-Methylierung gilt als eine der vielversprechendsten Methoden der molekularen Altersschätzung. Viele Studien der letzten Jahre identifizierten geeignete Positionen im Genom, deren DNA-Methylierung sich altersabhängig verändert. Für den Einsatz dieser Methode in der Routine- bzw. Fallarbeit ist es von großer Bedeutung, angewandte Analysetechniken zu validieren. Als ein Teilaspekt dieser Validierung sollte die Vergleichbarkeit der Analyseergebnisse zur DNA-Methylierung mithilfe der Mini- und Pyrosequenzierung zwischen verschiedenen Laboren evaluiert werden. Die Arbeitsgruppe „Molekulare Altersschätzung“ der Deutschen Gesellschaft für Rechtsmedizin (DGRM) führte hierzu den ersten, technischen Ringversuch durch, der 4 Positionen in den Genen PDE4C, EDARADD, SST und KLF14 umfasste. Diese Marker waren in vorangegangenen Studien als altersabhängige Biomarker charakterisiert worden. Am Ringversuch nahmen 12 Labore teil, wobei jedes die Wahl zwischen der Minisequenzierung und/oder der Pyrosequenzierung für die quantitative Methylierungsanalyse hatte. Jedem teilnehmenden Labor wurden Blut- und Speichelproben von 3 Personen unterschiedlichen Alters übersandt. Die Wahl der Reagenzien für die Probenbearbeitung wurde den Teilnehmern freigestellt.Die Ergebnisse der Minisequenzierung zeigten systematische Abweichungen zwischen den Laboren, die am ehesten auf die Verwendung unterschiedlicher Reagenzien und Analyseplattformen zurückzuführen sein können. Die Resultate der Pyrosequenzierung hingegen wiesen nicht auf systematische Abweichungen zwischen den Laboren hin, hier zeigte sich jedoch die Tendenz einer markerabhängigen Abweichung. Darüber hinaus konnten Unterschiede hinsichtlich technischer Probleme zwischen Laboren mit mehr Erfahrung in der jeweiligen Sequenzierungsmethode und Laboren mit weniger Erfahrung festgestellt werden. Sowohl die Beobachtung von systematischen als auch die von markerabhängigen Abweichungen lässt den Schluss zu, dass eine Übertragung von Analysemethoden zwischen Laboren grundsätzlich möglich ist, eine Anpassung des jeweiligen Modells zur Altersschätzung jedoch notwendig sein kann.

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New targeted approaches for epigenetic age predictions

Cited by 62 publications

References 77 publications

Epigenetic age-predictions in mice using pyrosequencing, droplet digital PCR or barcoded bisulfite amplicon sequencing

Epigenetic age-predictions in mice using pyrosequencing, droplet digital PCR or barcoded bisulfite amplicon sequencing

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

Forensische DNA-Methylierungsanalyse

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