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...
5241 Introduction TP53 is the most frequently mutated tumor suppressor gene in human cancers and is usually associated with an aggressive disease course. TP53 mutation has been described in a variety of hematopoietic neoplasms, and has been suggested to play a role in leukemic transformation of myeloproliferative neoplasms (MPN). However, the incidence as well as the clinical and pathogenetic implications of TP53 mutation in each sub-category of MPN, including primary myelofibrosis, have not been described. In this study, we investigated the presence and potential clinical significance of TP53 mutations in a large series of primary myelofibrosis cases. Patients and Methods We retrieved archival bone marrow DNA from 51 consecutive patients diagnosed with primary myelofibrosis at The University of Texas MD Anderson Cancer Center between the years 2005 and 2007. Diagnosis was based on morphologic, immunophenotypic, cytogenetic and molecular evaluation of bone marrow in conjunction with clinical data. Only 2 patients had blast counts >10% (11 and 14%). Twenty nine of 50 (58%) patients showed JAK2 p.V617F mutation and all patients were negative for BCR-ABL1 translocation on routine clinical testing. DNA samples were assessed for sequence variation in exons 4 through 9 of TP53 by both high resolution melting curve (HRM) analysis using LightCycler® 480 System (Roche, Indiana IN) and bidirectional Sanger sequencing using 3730XL DNA Analyzer (Life technologies, Carlsbad CA). Results The mean overall survival was 5.7 years. Five patients developed acute leukemia, all of whom died of disease. By Sanger sequencing, only one (1.9%) case showed an amino acid-altering mutation in TP53: c.707A>G (TAC to TGC) in codon 236 (p.Y236C) of exon 7. In addition, 8 cases showed silent mutations/single nucleotide polymorphisms of unknown significance - c.36G>A (CCG to CCA) in exon 4 (n=3) and c.213A>G (CGA to CGG) in exon 6 (n=6). The p.R72P polymorphism in exon 4 which has been described in other hematopoietic neoplasms was present in 1 patient. All cases with a mutant sequence by Sanger sequencing also showed a variant melting curve pattern by HRM analysis. The patient with TP53 mutation died 2 years after presentation from progressive myelofibrosis without developing acute leukemia. Conclusion TP53 mutation is very rare in primary myelofibrosis. Disclosures: No relevant conflicts of interest to declare.
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