Application of COLD-PCR for Improved Detection of NF2 Mosaic Mutations Irene Paganini, Irene Mancini, Marta Baroncelli, Guido Arena, Francesca Gensini, Laura Papi, Roberta Sestini  The Journal of Molecular Diagnostics  Volume 16, Issue 4, Pages 393-399 (July 2014) DOI: 10.1016/j.jmoldx.2014.02.007 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Evaluation of sensitivity of COLD-PCR in detecting mosaicism. Serial dilutions of a WT DNA with genomic DNA that carries a c.169C>T mutation (arrow) in exon 2, deriving from a patient with NF2 mosaicism, were prepared. Dilutions corresponding to a 1:1, 1:4, 1:10, 1:20, and 1:100 ratio of patient/normal DNA were amplified via PCR and COLD-PCR and analyzed by Sanger sequencing. Mutations that are not visible on the sequencing chromatograms after PCR become readily visible after COLD-PCR, and the mutated allele was easily detectable until a dilution of 1:20, corresponding to 1.25% of mutated allele. The Journal of Molecular Diagnostics 2014 16, 393-399DOI: (10.1016/j.jmoldx.2014.02.007) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Comparison of conventional PCR and COLD-PCR for detection of c. 592C>T in exon 6 of the NF2 gene in a patient with NF2 mosaicism. NF2 exon 6 amplicon was evaluated via HRM after conventional PCR and COLD-PCR amplification in control (green) and patient 241 (pink) DNAs. A: Mutation-containing sample differs appreciably from WT melting curves. Sequence analyses of DNA extracted from blood (241) and two tumors (TP653, TP654) of patient with NF2 mosaicism. B: Mutation in blood DNA is reliably detected in the COLD-PCR sequence but not in the conventional PCR sequence. In tumor DNAs c.592C>T mutation (arrow) is detectable also in conventional PCR sequences; however, COLD-PCR allowed us to enrich the mutated allele percentage. The Journal of Molecular Diagnostics 2014 16, 393-399DOI: (10.1016/j.jmoldx.2014.02.007) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A: Application of COLD-PCR in patients with mosaicism. Detection of c.586C>T mutation (arrow) in exon 6 of the NF2 gene. NF2 exon 6 amplicon was evaluated via HRM after conventional PCR. Mutation-containing sample varies appreciably from WT melting curves. Sequences of DNA extracted from blood (144) and tumor (T632) are presented. Mutation in blood DNA is reliably detected in the COLD-PCR sequence but not in the conventional PCR sequence. In tumor DNA c.586C>T mutation is detectable also in conventional PCR sequences; however, COLD-PCR allowed us to enrich the mutated allele percentage. B: Detection of c.193C>T mutation (arrow) in exon 2 of the NF2 gene. Conventional PCR and HRM analyses of NF2 exon 2 amplicon shows an alteration on the melting profile of 65 samples. Any mutation is identified in blood DNA by using conventional PCR amplification. COLD-PCR sequencing allows for the detection of c.193C>T mutation in blood DNA. In tumor DNA COLD-PCR confirms the capability of mutated allele enrichment. The Journal of Molecular Diagnostics 2014 16, 393-399DOI: (10.1016/j.jmoldx.2014.02.007) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Comparison of standard PCR and COLD-PCR for detection of c.1396C>T mutation (arrow) in exon 13 of the NF2 gene in a patient with NF2 mosaicism. NF2 exon 13 amplicon was evaluated with HRM after conventional PCR. Mutation-containing sample differs appreciably from WT melting curves, but blood DNA sequence is not able to detect the mutation. COLD-PCR amplification allowed us to identify the c.1396C>T mutation in blood DNA. The Journal of Molecular Diagnostics 2014 16, 393-399DOI: (10.1016/j.jmoldx.2014.02.007) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions