Comparison of the sensitivity of restriction analysis and PCR with high-resolution melting curve analysis for the detection of the R882H mutation in the DNMT3A gene
- Authors: Kulaeva E.D.1, Muzlaeva E.S.1, Lipilkin P.V.2,3, Mashkina E.V.1
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Affiliations:
- Southern Federal University, Academy of Biology and Biotechnology
- Rostov State Medical University, Department of Hematology and Transfusiology (with courses in clinical laboratory diagnostics, genetics and laboratory genetics)
- Don State Technical University, Department of Biology and General Pathology
- Issue: Vol 21, No 6 (2023)
- Pages: 27-32
- Section: Reviews
- URL: https://journals.eco-vector.com/1728-2918/article/view/624951
- DOI: https://doi.org/10.29296/24999490-2023-06-04
- ID: 624951
Cite item
Abstract
Introduction. DNMT3A is one of the most frequently mutated genes in acute myeloid leukemia (AML), and the R882H (G>A) substitution is one of the most common mutations in this gene. Restriction analysis and high resolution melting (HRM) analysis are the most common methods used to detect this mutation, but the sensitivity of these approaches varies greatly from study to study.
Purpose of the study. Determine the sensitivity of commonly used methods for assessing the R882H mutation content of a sample under controlled conditions.
Methods. We compared the sensitivity of restriction analysis and HRM on presynthesized DNA samples with varying levels of mutant template in the sample and provided detailed protocols for reproducing our results by other researchers.
Results. We found that the detection limit of the R882H mutation in the DNMT3A gene was 20% for restriction analysis and 40% for HRM.
Conclusion. The results obtained in this work are important for identifying restriction analysis and HRM as suitable methods for use in laboratory diagnosis of the R882H mutation for patients with AML with a high mutational load.
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About the authors
Elizaveta D. Kulaeva
Southern Federal University, Academy of Biology and Biotechnology
Email: ekulaeva@sfedu.ru
ORCID iD: 0000-0001-5886-7975
Research Assistant, Research Laboratory «Developmental Biology and Genome Organization» of Genetics Department
Russian Federation, Stachki Ave. 194/1, Rostov-on-Don, 344090Elizaveta S. Muzlaeva
Southern Federal University, Academy of Biology and Biotechnology
Author for correspondence.
Email: ekulaeva@sfedu.ru
ORCID iD: 0000-0002-4344-4221
Bachelor student, Human and Animal Genetics Research Laboratory
Russian Federation, Stachki Ave. 194/1, Rostov-on-Don, 344090Pavel V. Lipilkin
Rostov State Medical University, Department of Hematology and Transfusiology (with courses in clinical laboratory diagnostics, genetics and laboratory genetics); Don State Technical University, Department of Biology and General Pathology
Email: plipilkin@donstu.ru
ORCID iD: 0000-0002-3220-2753
Graduate student, Department of Hematology and Transfusiology (with courses of clinical laboratory diagnostics, genetics and laboratory genetics), Senior Lecturer, Department of Biology and General Pathology
Russian Federation, per. Nakhichevansky, 29, Rostov-on-Don, 344022; pl. Gagarina, 1, Rostov-on-Don, 344000Elena V. Mashkina
Southern Federal University, Academy of Biology and Biotechnology
Email: lenmash@sfedu.ru
ORCID iD: 0000-0002-4424-9508
Head of Research Laboratory «Human and Animal Genetics», Doctor of Biological Sciences, Professor
Russian Federation, Stachki Ave. 194/1, Rostov-on-Don, 344090References
- De Kouchkovsky I., Abdul-Hay M. Acute myeloid leukemia: a comprehensive review and 2016 update. Blood Cancer J. 2016; 6 (7): e441. https://doi.org/10.1038/bcj.2016.50.
- Hou H.A., Tien H.F. Genomic landscape in acute myeloid leukemia and its implications in risk classification and targeted therapies. J Biomed Sci. 2020; 27 (1): 81. https://doi.org/10.1186/s12929-020-00674-7.
- Que Y., Li H., Lin L., Zhu X., Xiao M., Wang Y., Zhu L., Li D. Study on the Immune Escape Mechanism of Acute Myeloid Leukemia With DNMT3A Mutation. Front Immunol. 2021; 12: 653030. https://doi.org/10.3389/fimmu.2021.653030.
- Venugopal K., Feng Y., Shabashvili D., Guryanova O.A. Alterations to DNMT3A in Hematologic Malignancies. Cancer Res. 2021; 81 (2): 254–63. https://doi.org/10.1158/0008-5472.CAN-20-3033.
- Rajavelu A., Jurkowska R.Z., Fritz J., Jeltsch A. Function and disruption of DNA Methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation. Nucleic Acids Res. 2012; 40 (2): 569–80. https://doi.org/10.1093/nar/gkr753.
- Anteneh H., Fang J., Song J. Structural basis for impairment of DNA methylation by the DNMT3A R882H mutation. Nat. Commun. 2020; 11 (1): 1–12. https://doi.org/10.1038/s41467-020-16213-9.
- Guryanova O.A., Shank K., Spitzer B., Luciani L., Koche R.P., Garrett-Bakelman F.E., Ganzel C., Durham B.H., Mohanty A., Hoermann G., Rivera S.A., Chramiec A.G., Pronier E., Bastian L., Keller M.D., Tovbin D., Loizou E., Weinstein A.R., Gonzalez A.R,. Lieu Y.K., Rowe J.M., Pastore F., McKenney A.S., Krivtsov A.V., Sperr W.R., Cross J.R., Mason C.E., Tallman M.S., Arcila M.E., Abdel-Wahab O., Armstrong S.A., Kubicek S., Staber P.B., Gönen M., Paietta E.M., Melnick A.M., Nimer S.D., Mukherjee S., Levine R.L. DNMT3A mutations promote anthracycline resistance in acute myeloid leukemia via impaired nucleosome remodeling. Nat. Med. 2016; 22 (12): 1488–95. https://doi.org/10.1038/nm.4210.
- Hou H.A., Kuo Y.Y., Liu C.Y., Chou W.C., Lee M.C., Chen C.Y., Lin LI, Tseng M.H., Huang C.F., Chiang Y.C., Lee F.Y., Liu M.C., Liu C.W., Tang J.L., Yao M., Huang S.Y., Ko B.S., Hsu S.C., Wu S.J., Tsay W., Chen Y.C., Tien H.F. DNMT3A mutations in acute myeloid leukemia: stability during disease evolution and clinical implications. Blood. 2012; 119 (2): 559–68. https://doi.org/10.1182/blood-2011-07-369934.
- Berenstein R., Blau I.W., Suckert N., Baldus C., Pezzutto A., Dörken B., Blau O. Quantitative detection of DNMT3A R882H mutation in acute myeloid leukemia. J. Exp. Clin. Cancer Res. 2015; 34 (1): 55. https://doi.org/10.1186/s13046-015-0173-2.
- Singh R.R., Bains A., Patel K.P., Rahimi H., Barkoh B.A., Paladugu A., Bisrat T., Ravandi-Kashani F., Cortes J.E., Kantarjian H.M., Medeiros L.J., Luthra R. Detection of High-Frequency and Novel DNMT3A Mutations in Acute Myeloid Leukemia by High-Resolution Melting Curve Analysis. J. Mol. Diagn. 2012; 14 (4): 336–45. https://doi.org/10.1016/j.jmoldx.2012.02.009.
- Berenstein R., Blau I.W., Kar A., Cay R., Sindram A., Seide C., Blau O. Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia. J. Exp. Clin. Cancer Res. 2014; 33 (1): 44. https://doi.org/10.1186/1756-9966-33-44.
- Gonzalez-Bosquet J., Calcei J., Wei J.S., Garcia-Closas M., Sherman M.E., Hewitt S., Vockley J., Lissowska J., Yang H.P., Khan J., Chanock S. Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers. PLoS One. 2011; 6 (1): 14522. https://doi.org/10.1371/journal.pone.0014522.
- Jeziskova I., Musilova M., Culen M., Foltankova V., Dvorakova D., Mayer J., Racil Z. Distribution of mutations in DNMT3A gene and the suitability of mutations in R882 codon for MRD monitoring in patients with AML. Int. J. Hematol. 2015; 102 (5): 553–7. https://doi.org/10.1007/s12185-015-1856-3.
- Do H., Dobrovic A. Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies. Mol Cancer. 2009; 8: 82. https://doi.org/10.1186/1476-4598-8-82.
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