Detection of High-Frequency and Novel DNMT3A Mutations in Acute Myeloid Leukemia by High-Resolution Melting Curve Analysis

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 highresolution 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. Acute myeloid leukemia (AML) is characterized by extensive dysregulation of gene expression due to gene mutations, chromosomal translocations, and aberrant epigenetic modification. In recent years, next-generation whole-genome sequencing of AML cases has identified novel gene mutations correlating with disease progression and clinical outcome. Ley et al 1 used massive parallel sequencing of tumor DNA from an AML patient with a normal karyotype and identified somatic mutations in 10 genes of which 2 were well-known AML-associated mutations (NPM1 and FLT3), whereas the other 8 mutations were in genes such as TP53 and BRCA2 previously unreported in AML. Yamashita et al 2 identified 11 gene mutations in AML patients, which included Janus kinase 3 (JAK3) and DNA methyltransferase 3A (DNMT3A). Subsequently, Ley et al 3 sequenced 23 exons of DNMT3A in 281 AML patients and found 22% of the patients had mutations, with the highest frequency occurring in exons 15 to 23. Missense, frameshift, nonsense, and splice-site mutations as well as deletions were detected, which correlated with poor overall survival. In a recent study involving 489 AML patients, exons 15 through 23 of DNMT3A were sequenced and a total of 90 mutations were identified in 87 (17.8%) patients with the highest frequency (27.2%) observed in AML patients with a diploid karyotype. Mutations correlated inversely with favorable overall and relapsefree survival. 4 As a result of these studies, DNMT3A mutations are thought to be a predictive marker of unfav...

Phase 2 study of low‐dose clofarabine plus cytarabine for patients with higher‐risk myelodysplastic syndrome who have relapsed or are refractory to hypomethylating agents

“…[19][20][21] Briefly, genomic DNA was Original Article extracted from bone marrow aspirates or peripheral blood. [19][20][21] Briefly, genomic DNA was Original Article extracted from bone marrow aspirates or peripheral blood.…”

Next-generation sequencing-based multigene mutational screening for acute myeloid leukemia using MiSeq: applicability for diagnostics and disease monitoring

“…Eleven additional samples were sequenced on the MiSeq instrument in parallel (blind) with routinely used platforms, including Sanger sequencing, pyrosequencing and fragment analysis by capillary electrophoresis, performed as described previously. [28][29][30] To confirm mutations detected in regions covered by TSACP but not by preexisting assays in our laboratory (AmpliSeq cancer panel, Sanger or pyro sequencing), 42 new Sanger sequencing assays were also developed.…”

The de novo DNA methyltransferase DNMT3A in development and cancer

“…89 Although previous studies clearly pointed to an oncogenic role of DNMT3A in cancer, the recent discovery of genetic mutations of DNMT3A in acute myeloid leukemia (AML) suggested that its role in cancers may be more complex than was previously believed. [11][12][13][14][15][16][17][18][19][20][21][22][23] These highly recurrent DNMT3A mutations were closely associated with poor outcome in patients with AML. However, the functional implications of these mutations are still poorly understood.…”

“…10 However, several inactivating mutations of DNMT3A in myeloid malignancies [11][12][13][14][15][16][17][18][19][20][21][22][23] and loss of DNMT3A activity at advanced tumor stages 24 were recently identified. In addition, novel roles of DNMT3A in the hematopoiesis system 25 and in the age-related decline of cognition 26 have been revealed.…”