Keyur P. Patel, Bedia A. Barkoh, Zhao Chen, Deqin Ma, Neelima Reddy, L

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Keyur P. Patel, Bedia A. Barkoh, Zhao Chen, Deqin Ma, Neelima Reddy, L

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Presentation transcript:

Diagnostic Testing for IDH1 and IDH2 Variants in Acute Myeloid Leukemia Keyur P. Patel, Bedia A. Barkoh, Zhao Chen, Deqin Ma, Neelima Reddy, L. Jeffrey Medeiros, Rajyalakshmi Luthra The Journal of Molecular Diagnostics Volume 13, Issue 6, Pages 678-686 (November 2011) DOI: 10.1016/j.jmoldx.2011.06.004 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 PCR strategy to cover important exon 4 mutations in IDH1 and IDH2. Sequences (bold letters) of (A) IDH1 exon 4 (NCBI Reference Sequence: NC_000002.11) and (B) IDH2 Exon 4 (NCBI Reference Sequence: NC_000015.9) are shown with flanking intronic sequences. The alignment of forward (gray shaded, black fonts) and reverse primers (black shaded, white fonts) show that partial IDH1 exon 4 and entire IDH2 exon 4 are amplified. The boxes mark arginine residues at the active sites for IDH1R100 (CGA), IDH1R109 (AGA), IDH1R132 (CGT), IDH2R140 (CGG), IDH2R149 (CGG), and IDH2R172 (AGG). The location of a known SNP at IDH1G105 is underlined. The Journal of Molecular Diagnostics 2011 13, 678-686DOI: (10.1016/j.jmoldx.2011.06.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 High-resolution melting curve analysis for IDH1G105 in duplicate samples. The differential melting properties of representative wild-type and polymorphic IDH1G105 amplicons are shown using (A) normalized and temperature-shifted melting curves, (B) derivative plots, and (C) normalized and temperature-shifted difference plots. WT, wild type. The Journal of Molecular Diagnostics 2011 13, 678-686DOI: (10.1016/j.jmoldx.2011.06.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Detection of IDH1 mutations by HRM analysis. Difference plot analysis representing duplicate samples shows wild-type IDH1 sequence as a flat line. The samples with R132 mutation, G105 polymorphism, and coexisting R132 mutation and polymorphism in G105 show characteristic patterns as marked. The Journal of Molecular Diagnostics 2011 13, 678-686DOI: (10.1016/j.jmoldx.2011.06.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Detection of IDH2 mutations by HRM analysis. A: The derivative plot analysis of IDH2 amplicons shows bimodal peaks. B: Mutations in IDH2R140 and (C) IDH2R172 are detected on difference plot analysis of the left peak and the right peak, respectively. The Journal of Molecular Diagnostics 2011 13, 678-686DOI: (10.1016/j.jmoldx.2011.06.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Confirmation of variant HRM sequences by Sanger sequencing. Representative examples of mutations are shown in IDH1G105 (A–D), IDH2R132 (E–H), IDH2R140 (I–L), and IDH2R172 (M–P). The HRM analysis shows a characteristic shift in the melting pattern of amplicons by derivative plots (A, E, I, M, Q; y axis depicts −(d/dt) fluorescence) and difference plots (B, F, J, N, R; y axis depicts relative signal difference). The Sanger sequencing performed in parallel confirmed the presence of mutations (C, G, K, O, S). A representative wild-type sequence from a different patient sample (D, H, L, P, T) is shown for comparison. A representative patient sample showing successful Sanger sequencing confirmation using post-HRM DNA is shown in the bottom row (Q–T). The Journal of Molecular Diagnostics 2011 13, 678-686DOI: (10.1016/j.jmoldx.2011.06.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Sensitivity of detecting IDH1 and IDH2 mutations by HRM. Normalized and temperature-shifted difference plots for (A) IDH1G105 and (B) IDH2R172 were generated by diluting mutated patient sample DNA into wild-type patient sample DNA. A wild-type patient sample DNA was used as a negative control. The sensitivity adjusted for blast count is 7.9% polymorphic alleles for IDH1G105 and 7.3% mutant alleles for IDH2R172. The Journal of Molecular Diagnostics 2011 13, 678-686DOI: (10.1016/j.jmoldx.2011.06.004) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions