Detection of high-frequency and novel DNMT3A mutations in acute myeloid leukemia by high-resolution melting curve analysis

Rajesh R. Singh, Ashish Bains, Keyur P. Patel, Hamed Rahimi, Bedia A. Barkoh, Abhaya Paladugu, Tigist Bisrat, Farhad Ravandi-Kashani, Jorge E. Cortes, Hagop M. Kantarjian, L. Jeffrey Medeiros, Rajyalakshmi Luthra

Research output: Contribution to journalArticle

Abstract

DNA methyltransferase 3A (DNMT3A) is mutated in a subset of de novo acute myeloid leukemia patients and is associated with poor overall and event-free survival. Because routine Sanger sequencing of the 23 DNMT3A exons is impractical in clinical laboratories, we developed a high-throughput method using high-resolution melting (HRM) analysis, which identifies sequence variants by detecting subtle changes in the melting patterns of mutant DNA in comparison with WT sequences. DNA from 104 acute myeloid leukemia patients was tested for mutations in 12 exons encoding 3 major functional domains of DNMT3A: the PWWP (proline-tryptophan-tryptophan-proline) domain (exons 8 to 10), the ADD (ATM-DNMT3-DNMT3L) zinc finger, and the methyltransferase domains encoded by exons 15 to 23. HRM analysis identified 20 of 104 patient samples as variants, which we confirmed by Sanger sequencing. Codon 882 of exon 23 was mutated at the highest frequency with an occurrence rate of 11.5%. All HRM WT calls were confirmed to be devoid of mutations by Sanger sequencing. We also identified seven novel and previously unreported DNMT3A mutations. Structural modeling showed seven of the eight missense mutations detected in our study increased the free energy, destabilized protein, and altered solvent accessibility, suggesting their loss-of-function nature. These data demonstrate HRM analysis to be a higher throughput, sensitive, and efficient alternative to Sanger sequencing for detecting DNMT3A mutations in the clinical diagnostic laboratory.

Original languageEnglish (US)
Pages (from-to)336-345
Number of pages10
JournalJournal of Molecular Diagnostics
Volume14
Issue number4
DOIs
StatePublished - Jul 1 2012

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

Cite this

Detection of high-frequency and novel DNMT3A mutations in acute myeloid leukemia by high-resolution melting curve analysis. / Singh, Rajesh R.; Bains, Ashish; Patel, Keyur P.; Rahimi, Hamed; Barkoh, Bedia A.; Paladugu, Abhaya; Bisrat, Tigist; Ravandi-Kashani, Farhad; Cortes, Jorge E.; Kantarjian, Hagop M.; Medeiros, L. Jeffrey; Luthra, Rajyalakshmi.

In: Journal of Molecular Diagnostics, Vol. 14, No. 4, 01.07.2012, p. 336-345.

Research output: Contribution to journalArticle

Singh, RR, Bains, A, Patel, KP, Rahimi, H, Barkoh, BA, Paladugu, A, Bisrat, T, Ravandi-Kashani, F, Cortes, JE, Kantarjian, HM, Medeiros, LJ & Luthra, R 2012, 'Detection of high-frequency and novel DNMT3A mutations in acute myeloid leukemia by high-resolution melting curve analysis', Journal of Molecular Diagnostics, vol. 14, no. 4, pp. 336-345. https://doi.org/10.1016/j.jmoldx.2012.02.009
Singh, Rajesh R. ; Bains, Ashish ; Patel, Keyur P. ; Rahimi, Hamed ; Barkoh, Bedia A. ; Paladugu, Abhaya ; Bisrat, Tigist ; Ravandi-Kashani, Farhad ; Cortes, Jorge E. ; Kantarjian, Hagop M. ; Medeiros, L. Jeffrey ; Luthra, Rajyalakshmi. / Detection of high-frequency and novel DNMT3A mutations in acute myeloid leukemia by high-resolution melting curve analysis. In: Journal of Molecular Diagnostics. 2012 ; Vol. 14, No. 4. pp. 336-345.
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AU - Bains, Ashish

AU - Patel, Keyur P.

AU - Rahimi, Hamed

AU - Barkoh, Bedia A.

AU - Paladugu, Abhaya

AU - Bisrat, Tigist

AU - Ravandi-Kashani, Farhad

AU - Cortes, Jorge E.

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AU - Medeiros, L. Jeffrey

AU - Luthra, Rajyalakshmi

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