A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing

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Presentation transcript:

A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing Amir Behdad, Helmut C. Weigelin, Kojo S.J. Elenitoba-Johnson, Bryan L. Betz The Journal of Molecular Diagnostics Volume 17, Issue 1, Pages 76-84 (January 2015) DOI: 10.1016/j.jmoldx.2014.09.007 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Assay design and optimization. A: CEBPA is a single-exon gene with a 1077-bp coding region. Primers were designed such that two overlapping fragments spanning the entire coding region are amplified by PCR. B: Optimization of the PCR required the addition of 8% dimethyl sulfoxide (DMSO) and the use of GC-rich PCR buffer. Primer design and optimization of the PCR/sequencing reactions was challenging because of the high GC content of the coding region (75%), the presence of a trinucleotide repeat (GCC)7, and problems with secondary structure. C: Amplification products after optimization. D: Sensitivity of direct sequencing for CEBPA mutations. Mutation-positive genomic DNA was diluted into wild-type DNA. Mutations could be identified reliably down to a level of 10%. DBD/ZIP, DNA binding and dimerization domain; TAD1, transactivation domain 1; TAD2, transactivation domain 2; WT, wild type. The Journal of Molecular Diagnostics 2015 17, 76-84DOI: (10.1016/j.jmoldx.2014.09.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Sequence traces of N- and C-terminal CEBPA mutations, variants of unknown significance, and the c.584_589dup, p.H195_P196dup polymorphism. A variety of sequence alterations were detected, including both length-affecting mutations and nucleotide substitution mutations. The wild-type CEBPA sequence is listed above the traces. The insertion mutation in the second panel of the left column (c.245_246insGG, p.F82fs) showed more than 90% mutant allele frequency and therefore was interpreted as homozygous. Brackets in each panel indicate the deleted/inserted/duplicated nucleotide sequence; asterisks, nucleotide substitutions. The Journal of Molecular Diagnostics 2015 17, 76-84DOI: (10.1016/j.jmoldx.2014.09.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Distribution of CEBPA mutations and variants identified in this study. The locations of the mutations are shown with respect to the CEBPA protein and functional regions. Arrows depict the two translation initiation sites at amino acids (aa) 1 and 120. For interpretative purposes, the coding region of CEBPA was divided into three regions: N-terminal (aa 1 to 120), midregion (aa 121 to 277), and C-terminal (aa 278 to 358). The majority of CEBPAdm cases harbor a combination of a truncating frameshift or nonsense mutation in the N-terminal region and an inframe insertion/deletion or missense mutation in the C-terminal region. Mutations in CEBPAsm cases are distributed in the entire coding region, with a greater portion in the midregion. Missense and inframe mutations of the N-terminal and midregions were classified as variants of unknown significance. TAD1, transactivation domain 1 (aa 70 to 97); TAD2, transactivation domain 2 (aa 127 to 200); DBD/ZIP, DNA binding and dimerization domain (aa 278 to 358). The Journal of Molecular Diagnostics 2015 17, 76-84DOI: (10.1016/j.jmoldx.2014.09.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Examples of CEBPAdm cases with equal (case 41) and discrepant (case 79) mutant allele frequencies. Brackets highlight the deleted/duplicated sequence; asterisks, the mutant peak. The Journal of Molecular Diagnostics 2015 17, 76-84DOI: (10.1016/j.jmoldx.2014.09.007) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions