Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis Philippe Szankasi, David.

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Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis Philippe Szankasi, David W. Bahler The Journal of Molecular Diagnostics Volume 12, Issue 2, Pages 244-249 (March 2010) DOI: 10.2353/jmoldx.2010.090091 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Gel electrophoresis of PCR products. IgVH fragments from eight CLL samples (each positive for a different VH gene family and the VH3-21 segment) were amplified. PCR reactions with the consensus leader primers for the VH1, VH3, or VH4 gene families are labeled 1, 3, and 4, respectively. The multiplexed PCR reaction combining the VH2, VH5, and VH6 consensus leader primers and the VH3-21 leader primer is labeled C. Fragment sizes are indicated in bp. The Journal of Molecular Diagnostics 2010 12, 244-249DOI: (10.2353/jmoldx.2010.090091) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 PCR amplification of the VH3-21 variable region. Four CLL cases expressing the VH3-21 segment and 9 cases expressing other VH3 variable regions were amplified with the VH3 consensus leader primer (top) or the VH3-21 specific primer (bottom). Fragment sizes are indicated in bp. The Journal of Molecular Diagnostics 2010 12, 244-249DOI: (10.2353/jmoldx.2010.090091) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Distribution of VH segment usage. The number of times each VH segment was observed is indicated. The numbers are separated into mutated (black bars) and unmutated (gray bars) cases. The Journal of Molecular Diagnostics 2010 12, 244-249DOI: (10.2353/jmoldx.2010.090091) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Distribution of the observed percentage of germline sequence identity. The calculated percentages of sequence identity to the closest matching germline sequence from 99 CLL samples were combined into bins of 2% ranges. The number of cases falling into each bin is indicated. The Journal of Molecular Diagnostics 2010 12, 244-249DOI: (10.2353/jmoldx.2010.090091) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Detection limit by direct sequencing. Gel electrophoresis of PCR products from serial twofold dilutions of a CLL sample into a normal sample (see text for more details) is shown. The fold dilution is indicated on the left. The calculated theoretical concentration of CLL cells in percent among WBC and total B cells (B) is shown on the right. The ability to identify the expected VH3-48 variable region with 99.7% identity to the germline sequence is indicated in the far right column (Y or N). The gel lanes are labeled according to the leader primer used in the PCR reaction: 1, VH1; 3, VH3; 4, VH4; C, VH2, VH3-21, VH5, and VH6 multiplex. DNA fragment length is indicated in bp. The Journal of Molecular Diagnostics 2010 12, 244-249DOI: (10.2353/jmoldx.2010.090091) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions