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  The Journal of Molecular Diagnostics  Volume 14, Issue 4, Pages 336-345 (July 2012) DOI: 10.1016/j.jmoldx.2012.02.009 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 HRM analysis for mutations in exon 8. A: Differential melting properties of two patient DNA samples with variant amplicons of DNMT3A exon 8 compared with WT HL-60 DNA was evident in the normalized melting curves. B: Normalized and temperature (Temp)-shifted difference plots also show the two variants. C: Differential melting curves of these variants were also evident by the derivative plots. Bidirectional Sanger sequencing confirms the presence of nonsense mutation, resulting in premature stop codons at codons 306 (TGG>TAG) and 330 (TGG>TGA) (right). D: The sensitivity of HRM analysis for exon 8 as tested by diluting the patient DNA carrying the p.W330X mutation with WT patient DNA exhibits detection sensitivity up to 1:9 (mutated:WT) dilutions, indicating high sensitivity. The Journal of Molecular Diagnostics 2012 14, 336-345DOI: (10.1016/j.jmoldx.2012.02.009) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 HRM analysis for mutations in exon 10. Detection of two variant exon 10 sequences compared with the WT HL60 sequence by HRM analysis is evident by the normalized melting curves (A), normalized and temperature (Temp)-shifted difference plots (B), and the derivative plots (C). C: Sanger sequencing confirms the presence of missense mutations in these two variants (ATT>AGT, p.I407S; ATT>ACT, p.I407T). D: The sensitivity of HRM analysis as tested by diluting the patient DNA carrying the p.I407T mutation with WT patient DNA exhibits detection sensitivity up to 1:9 (mutated:WT) dilutions, indicating high sensitivity. The Journal of Molecular Diagnostics 2012 14, 336-345DOI: (10.1016/j.jmoldx.2012.02.009) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 HRM curve analysis for mutations in exon 15. A: The differential melting properties of WT DNA (HL60) and the two variant (mutated) amplicons of DNMT3A exon 15 are shown using normalized melting curves. B: Temperature (Temp)-shifted difference plots also exhibit the presence of two variants. C: Derivative plots and the confirmation of the mutations (TGC>TAC), resulting in p.C583Y substitution in the exonic region. D: Derivative plot confirms the presence of the second variant, and Sanger sequencing shows the CGG>CAG mutation in the intronic region. E: The sensitivity of HRM analysis as tested by diluting the patient DNA carrying the p.C583Y mutation with WT patient DNA exhibits detection sensitivity up to 1:9 (mutant:WT) dilutions, indicating high sensitivity. The Journal of Molecular Diagnostics 2012 14, 336-345DOI: (10.1016/j.jmoldx.2012.02.009) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 HRM analysis for mutations in exon 18. A: The differential melting properties of WT DNA (HL60) and the two variant amplicons of DNMT3A exon 18 are shown using normalized melting curves. B: Temperature (Temp)-shifted difference plots also exhibit the presence of two variants. C: Derivative plot and the confirmation of the mutation (TGC>AGC) at codon 710, resulting in p.C710S amino acid substitution. D: Derivative plot confirms the presence of the second variant, and bidirectional Sanger sequencing shows the AAC>AAT at codon 717, resulting in p.N717N amino acid substitution mutation. E: The sensitivity of detection of the p.N717N variant is evident by clear detection of the variant at a dilution of 1:9 (mutated:WT). The Journal of Molecular Diagnostics 2012 14, 336-345DOI: (10.1016/j.jmoldx.2012.02.009) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Normalized melting curve analysis for sequence variants in exon 23. A: The difference in the melting pattern of four patient DNA samples with mutations in exon 23 in reference to the WT (HL60) DNA is evident in their normalized melting curves, indicating the possibility of mutations in them. B: The sensitivity of variant detection for exon 23 is demonstrated by the ability to detect the most frequent mutant p.R882H at a dilution of 1:9 (mutant/WT). The Journal of Molecular Diagnostics 2012 14, 336-345DOI: (10.1016/j.jmoldx.2012.02.009) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Confirmation of DNMT3A exon 23 sequence variant HRM curve analysis results by sequencing. A: HRM curve analysis shows characteristic shift in the melting pattern of amplicons by derivative plots (left) and difference plots, and Sanger sequencing confirms the mutation as being CGC>CAC (p.R882H). B: The presence of the p.R882C variant is evident by the melting pattern derivative plot (left), difference plots (middle), and sequencing (mutant versus WT), which show the sequence change to be CGC>TCG. C: The presence of the p.R882P variant is evident by the melting pattern derivative plot (left), difference plots (middle), and sequencing (mutant versus WT), which show the sequence change to be CGC>CCC. D: HRM analysis also shows the presence of another variant as evident by the melting pattern derivative plot (left), difference plots (middle), and sequencing (mutant versus WT), which show the sequence change to be TGG>AAG, resulting in p.W893R substitution. HL60 DNA was used as WT for comparison. Arrows point to the appearance of the substituted base in Sanger chromatogram in the background of the WT and indicates the site of mutations detected. The Journal of Molecular Diagnostics 2012 14, 336-345DOI: (10.1016/j.jmoldx.2012.02.009) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions