Droplet digital PCR for the quantification of Alu methylation status in hematological malignancies

Orsini, Paola; Impera, Luciana; Parciante, Elisa; Cumbo, Cosimo; Minervini, Crescenzio Francesco; Minervini, Angela; Zagaria, Antonella; Anelli, Luisa; Coccaro, Nicoletta; Casieri, Paola; Tota, Giuseppina; Brunetti, Claudia; Ricco, Alessandra; Carluccio, Paola; Specchia, Giorgina; Albano, Francesco

doi:10.1186/s13000-018-0777-x

Cited by 7 publications

(3 citation statements)

References 47 publications

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“…The global DNA methylation profile was evaluated using a droplet digital PCR (ddPCR)-based strategy, quantifying the Alu methylation level to investigate the possible effect of DNMT3A loss of function ( Additional file 1 ) ( 16 ). Although unexpected, DNMT3A’s severe loss of function at diagnosis and DR, results in a global genomic hypermethylation, as compared to CR and a statistically significant difference was observed between CR and DR (81.6% vs 86.7%, respectively, p=0.04) ( Figure 2 ).…”

Section: Case Presentationmentioning

confidence: 99%

Case report: biallelic DNMT3A mutations in acute myeloid leukemia

et al. 2023

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DNMT3A gene mutations, detected in 20-25% of de novo acute myeloid leukemia (AML) patients, are typically heterozygous. Biallelic variants are uncommon, affecting ~3% of cases and identifying a worse prognosis. Indeed, two concomitant DNMT3A mutations were recently associated with shorter event-free survival and overall survival in AML. We present an AML case bearing an unusual DNMT3A molecular status, strongly affecting its function and strangely impacting the global genomic methylation profile. A 56-year-old Caucasian male with a diagnosis of AML not otherwise specified (NOS) presented a complex DNMT3A molecular profile consisting of four different somatic variants mapping on different alleles (in trans). 3D modelling analysis predicted the effect of the DNMT3A mutational status, showing that all the investigated mutations decreased or abolished DNMT3A activity. Although unexpected, DNMT3A’s severe loss of function resulted in a global genomic hypermethylation in genes generally involved in cell differentiation. The mechanisms through which DNMT3A contributes to AML remain elusive. We present a unique AML case bearing multiple biallelic DNMT3A variants abolishing its activity and resulting in an unexpected global hypermethylation. The unusual DNMT3A behavior described requires a reflection on its role in AML development and persistence, highlighting the heterogeneity of its deregulation.

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Section: Case Presentationmentioning

confidence: 99%

Case report: biallelic DNMT3A mutations in acute myeloid leukemia

et al. 2023

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“…A further field of ddPCR application in myeloid malignancies includes the possibility of measuring methylated DNA [ 56 ], also analyzing the Alu repeats whose methylation levels are useful for evaluating the global DNA methylation. In the work performed by the Dr. Albano’s group, bone marrow samples from patients receiving azacytidine for intermediate-2/high-risk myelodysplasias were tested by ddPCR before and during treatment: as expected, a significant decrease of Alu sequences methylation after therapy compared to diagnosis was observed, so making ddPCR as an appealing instrument for DNA methylation assessment [ 57 ].…”

Section: Ddpcr Applications In Hematologymentioning

confidence: 99%

Digital Droplet PCR in Hematologic Malignancies: A New Useful Molecular Tool

et al. 2022

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Digital droplet PCR (ddPCR) is a recent version of quantitative PCR (QT-PCR), useful for measuring gene expression, doing clonality assays and detecting hot spot mutations. In respect of QT-PCR, ddPCR is more sensitive, does not need any reference curve and can quantify one quarter of samples already defined as “positive but not quantifiable”. In the IgH and TCR clonality assessment, ddPCR recapitulates the allele-specific oligonucleotide PCR (ASO-PCR), being not adapt for detecting clonal evolution, that, on the contrary, does not represent a pitfall for the next generation sequencing (NGS) technique. Differently from NGS, ddPCR is not able to sequence the whole gene, but it is useful, cheaper, and less time-consuming when hot spot mutations are the targets, such as occurs with IDH1, IDH2, NPM1 in acute leukemias or T315I mutation in Philadelphia-positive leukemias or JAK2 in chronic myeloproliferative neoplasms. Further versions of ddPCR, that combine different primers/probes fluorescences and concentrations, allow measuring up to four targets in the same PCR reaction, sparing material, time, and money. ddPCR is also useful for quantitating BCR-ABL1 fusion gene, WT1 expression, donor chimerism, and minimal residual disease, so helping physicians to realize that “patient-tailored therapy” that is the aim of the modern hematology.

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“…Another potential application of dPCR is DNA methylation analysis, which has been explored in several studies of malignancies [155]. In the hematologic field, Orsini et al recently proposed a ddPCR method to investigate Alu differential methylation for the use in profiling patients affected by hematologic malignancies for diagnostic/prognostic purposes [174].…”

Section: Other Dpcr Applications and Evolutionmentioning

confidence: 99%

Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies

Coccaro

Tota

Anelli

et al. 2020

IJMS

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The digital polymerase chain reaction (dPCR) is considered to be the third-generation polymerase chain reaction (PCR), as it yields direct, absolute and precise measures of target sequences. dPCR has proven particularly useful for the accurate detection and quantification of low-abundance nucleic acids, highlighting its advantages in cancer diagnosis and in predicting recurrence and monitoring minimal residual disease, mostly coupled with next generation sequencing. In the last few years, a series of studies have employed dPCR for the analysis of hematologic malignancies. In this review, we will summarize these findings, attempting to focus on the potential future perspectives of the application of this promising technology.

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Droplet digital PCR for the quantification of Alu methylation status in hematological malignancies

Cited by 7 publications

References 47 publications

Case report: biallelic DNMT3A mutations in acute myeloid leukemia

Case report: biallelic DNMT3A mutations in acute myeloid leukemia

Digital Droplet PCR in Hematologic Malignancies: A New Useful Molecular Tool

Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies

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