A Pyrosequencing-Based Assay for the Rapid Detection of IDH1 Mutations in Clinical Samples Prashanth Setty, Jennifer Hammes, Thomas Rothämel, Valentina.

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Presentation transcript:

A Pyrosequencing-Based Assay for the Rapid Detection of IDH1 Mutations in Clinical Samples Prashanth Setty, Jennifer Hammes, Thomas Rothämel, Valentina Vladimirova, Christof M. Kramm, Torsten Pietsch, Andreas Waha The Journal of Molecular Diagnostics Volume 12, Issue 6, Pages 750-756 (November 2010) DOI: 10.2353/jmoldx.2010.090237 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic representation and primer localization. Map of the pyrosequencing assay for detection of codon transversion at positions 394 and 395 within exon 4, leading to the IDH1 R132 mutation. Primer-binding sites are depicted in lower case and the sequence to be analyzed in upper case. The mutation site assayed is highlighted by a box, below are the codons with the expected changes at the first and second positions, respectively. Arrows indicate the annealing positions of primer sequences from 5′ to 3′. The Journal of Molecular Diagnostics 2010 12, 750-756DOI: (10.2353/jmoldx.2010.090237) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Pyrogram and conventional sequencing traces. Detection of IDH1 R132H mutations in DNA samples isolated from HGG specimens. (A, top) Pyrogram of a sample that contained no mutation at the second position of the hotspot codon 395 (CGT) and the chromatogram of the same fragment obtained by cycle sequencing of single clone of a cloned PCR product (A, bottom), confirming the result of the pyrosequencing assay. Of 10 clones sequenced, no mutants were found. Below (B, top), pyrosequencing result of a DNA sample that exhibits a mutation at position (395), resulting in the R132H mutation and a chromatogram obtained by cycle sequencing of a single clone of a cloned PCR product (B, bottom). Three clones out of 10 were found to be mutated. Supplemental Figure S2 (available at http://jmd.amjpathol.org) shows pyrograms of the same tumor samples generated using an alternative primer set, using a reverse pyrosequencing primer and leading to virtually identical results. Codon 132 is highlighted as boxed text. Arrows indicate the wildtype (G) and the mutant (A) base. The Journal of Molecular Diagnostics 2010 12, 750-756DOI: (10.2353/jmoldx.2010.090237) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Titration of mutant and wild type IDH1 alleles. Results of the titration assay investigating the sensitivity of pyrosequencing analysis to detect IDH1 R132H mutations. Plasmids containing mutant and wild-type alleles were mixed to generate templates of 100%, 75%, 50%, 25%, and 0% mutant alleles and 0%, 25%, 50%, 75%, and 100% wild-type alleles. A–G: Pyrograms showing the percentage of adenine and guanine at the second position of codon 132 at different concentrations of mutant and wild-type alleles. H: Graphical representation of the AQ values of triplicate titration experiments. Ratio of the mutant allele to wild type is indicated on the x axis, and the percentage of adenine values is given on the y axis. Arrows indicate the wildtype (C) and the mutant (T) base. The Journal of Molecular Diagnostics 2010 12, 750-756DOI: (10.2353/jmoldx.2010.090237) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 PCR cycle kinetics of the IDH1 pyrosequencing assay. Plasmids containing mutant (CAT) and wild-type (CGT) IDH1 alleles were used in a 1:1 ratio. The R132 hotspot region was amplified by PCR with different cycle numbers (20, 30, 40, 50, and 60). Pyrosequencing of the PCR products and subsequent allele quantification showed that the relative abundance of mutant allele (A, C) and wild type (B, D) were unaffected by increasing PCR cycle numbers. E: Graphical representation of the AQ values for IDH1 mutant and wild-type alleles at different cycle numbers. Arrows indicate the wildtype (G) and the mutant (A) base. The Journal of Molecular Diagnostics 2010 12, 750-756DOI: (10.2353/jmoldx.2010.090237) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions