Abstract
Background
Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements.
In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology.
Methods
Forty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed.
DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR.
Results
Using ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D.
Conclusions
In our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.
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Background
DNA methylation is an epigenetic modification occurring at 5′cytosine of CpG dinucleotides; it plays a pivotal role in genome regulation in several physiological processes such as genomic imprinting, X inactivation and hematopoietic differentiation [1]. Variations of DNA methylation contribute to tumorigenesis and tumor maintenance, and aberrant DNA methylation has been also documented in hematological malignancies [2], as the regulation of CpG methylation has been established as a crucial event for stem cells and their differentiation potential. In this perspective, the analysis of DNA methylation status may be useful to identify tumor markers and therapeutic targets in cancer patients.
According to the Human Genome Assembly GRCh37, 28,299,634 CpG islands have been annotated, and up to 25% of them are located within Alu elements [42, 52]; this may be due to a small number of patients analyzed for single cytogenetic risk groups.
Since leukemias are often associated with chromosome instability and rearrangement events, and Alu methylation prevents genomic instability, evaluating global Alu methylation level by ddPCR may be interesting to inspect the correlation between the two molecular events.
Conclusions
In summary, we demonstrate that ddPCR-based assay may be useful for inspecting the global DNA methylation of Alu repeats, in hematological malignancies and investigating possible epigenetic alterations for diagnostic/prognostic purposes.
Abbreviations
- AML:
-
acute myeloid leukemia
- ASO-PCR:
-
allele-specific oligonucleotide PCR
- AZA:
-
5-azacytidine
- BM:
-
bone marrow
- CLL:
-
chronic lymphocytic leukemia
- CMML:
-
chronic myelomonocytic leukemia
- ddPCR:
-
droplet digital PCR
- DEC:
-
decitabine
- FISH:
-
Fluorescent In Situ Hybridization
- gDNA:
-
genomic DNA
- HD:
-
healthy donor
- IPSS:
-
International Prognostic Scoring System
- MDS:
-
myelodysplastic syndromes
- MDS:
-
myelodysplastic syndromes
- PB:
-
peripheral blood
- QUAlu:
-
Quantification of Unmethylated Alu
- SINEs:
-
Short Interspersed Repetitive Elements
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Acknowledgements
The authors would like to thank Ms. MVC Pragnell, B.A. for language revision of the manuscript.
Funding
This work was supported by “Associazione Italiana contro le Leucemie (AIL) - BARI”.
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Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
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Contributions
PO and LI conceived and designed the study and wrote the manuscript; PO, LI and EP performed ddPCR assays; PO performed all bioinformatics analysis; PC performed conventional cytogenetic analysis; LA, AZ, NC and GT conducted FISH experiments and interpreted data; LI, AM, EP and CB performed diagnostic molecular analysis; AR and PC provided clinical data; GS and FA supervised the manuscript preparation. All authors read and approved the final manuscript.
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The study was approved (n. 0953) by the ethics committee of the Azienda Ospedaliero-Universitaria Consorziale Policlinico di Bari (Bari, Italy). The written informed consent was obtained from the patients included in this study.
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Additional files
Additional file 1:
Table S1. Clinical characteristics of the CLL patients included in the study. (DOCX 13 kb)
Additional file 2:
Table S2. Clinical characteristics of the MDS patients retrospectively analyzed in this study. The number of hypomethylating therapy cycles at which the analysis is performed varied according to BM sample availability. (DOCX 14 kb)
Additional file 3:
Table S3. Molecular characteristics of the CMML patients retrospectively analyzed in this study. (DOCX 12 kb)
Additional file 4:
Table S4. Genomic distribution of Alu consensus sequences, considering a Transcription starting site (TSS) range of 3000 bp. (DOCX 13 kb)
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Orsini, P., Impera, L., Parciante, E. et al. Droplet digital PCR for the quantification of Alu methylation status in hematological malignancies. Diagn Pathol 13, 98 (2018). https://doi.org/10.1186/s13000-018-0777-x
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DOI: https://doi.org/10.1186/s13000-018-0777-x