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by Christian H. Ottensmeier, and Freda K. Stevenson

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1 by Christian H. Ottensmeier, and Freda K. Stevenson
Isotype switch variants reveal clonally related subpopulations in diffuse large B-cell lymphoma by Christian H. Ottensmeier, and Freda K. Stevenson Blood Volume 96(7): October 1, 2000 ©2000 by American Society of Hematology

2 Alignment of the deduced amino acid sequences of the dominant tumor clone to the closest germline VH and JH segments for 4 cases of DLBCL.Dashes indicate homology to germline. Alignment of the deduced amino acid sequences of the dominant tumor clone to the closest germline VH and JH segments for 4 cases of DLBCL.Dashes indicate homology to germline. Capital or lower-case mismatch letters indicate replacement or silent mutations, respectively. Full JH alignment was made by taking into account the result of sequencing of the constant region transcripts. Christian H. Ottensmeier, and Freda K. Stevenson Blood 2000;96: ©2000 by American Society of Hematology

3 Intraclonal variation of the clonally related VHDJH sequences obtained from case 5334.Individual clonally-related sequences are compared to the closest germline VH gene segments V1-02 andJH5a. Intraclonal variation of the clonally related VHDJH sequences obtained from case 5334.Individual clonally-related sequences are compared to the closest germline VH gene segments V1-02 andJH5a. Sequence identity is shown by dashes, and mutations are shown by mismatched characters. Two sequences were repeated, and all others were unique. The rate of intraclonal heterogeneity was calculated at 11.8 of 1000 nucleotides. Christian H. Ottensmeier, and Freda K. Stevenson Blood 2000;96: ©2000 by American Society of Hematology

4 Analysis of VH constant region transcripts from case 5049
Analysis of VH constant region transcripts from case 5049.Alignment is made to the closest germline gene segments V4-34 and JH4a and to the LHJH sequences. Analysis of VH constant region transcripts from case 5049.Alignment is made to the closest germline gene segments V4-34 and JH4a and to the LHJH sequences. Using VH primers together with constant region primers specific for IgM, IgD, IgG, and IgA, transcripts for IgM, IgD, IgG1, IgG3, and IgA were identified. Mutational patterns indicated 2 distinct clusters of transcripts: IgM/D, IgG3, and IgA on one hand and IgG1 and IgA on the other. Only nonidentical sequences are shown; the numbers at the left of each line indicate the frequency of the repeated sequences. Constant region sequences are not shown. In total, 59 tumor-related sequences were analyzed. Christian H. Ottensmeier, and Freda K. Stevenson Blood 2000;96: ©2000 by American Society of Hematology

5 Analysis of VH constant region transcripts from case 5126
Analysis of VH constant region transcripts from case 5126.Alignment is made to the germline segments V3-21 and JH4b and shown from CDR2. Analysis of VH constant region transcripts from case 5126.Alignment is made to the germline segments V3-21 and JH4b and shown from CDR2. Transcripts for IgM, IgD, IgG3, and IgA were detected with highly similar sequences. Only nonidentical clones are shown. In total 52 tumor-related sequences were analyzed. Constant region sequences are not shown. Christian H. Ottensmeier, and Freda K. Stevenson Blood 2000;96: ©2000 by American Society of Hematology

6 Analysis of VH constant region transcripts from case 5184
Analysis of VH constant region transcripts from case 5184.LHJH sequences are shown from FWR3 for reference. Analysis of VH constant region transcripts from case 5184.LHJH sequences are shown from FWR3 for reference. Transcripts for IgM, IgD, IgG1, and IgA were detected using a CDR3-specific primer together with constant region primers. As for case 5049, IgG1 sequences diverged from the IgM/D population. Mutations in codons 105 and 106 were shared with the α-transcripts, perhaps indicating a second switch event in a cell population that had switched from IgM/D to IgG1. The figure presents the 33 sequences that were analyzed. Constant region sequences are not shown. Christian H. Ottensmeier, and Freda K. Stevenson Blood 2000;96: ©2000 by American Society of Hematology

7 Analysis of VH constant region transcripts from case 5334
Analysis of VH constant region transcripts from case 5334.LHJH sequences are shown for reference from FWR3. Analysis of VH constant region transcripts from case 5334.LHJH sequences are shown for reference from FWR3. IgM, IgD, IgG1, and IgA transcripts were detected using a CDR3-specific primer together with constant region primers. IgM and IgD sequences were virtually identical. IgG1 transcripts differed by a further mutation in codon 100c. Some IgA transcripts appeared to be related to the IgM/D group, whereas others had acquired the additional mutation in codon 100c, which was observed in the IgG1 group. All sequences are shown for the constant region transcripts. In total, 43 tumor-related sequences were analyzed. Christian H. Ottensmeier, and Freda K. Stevenson Blood 2000;96: ©2000 by American Society of Hematology


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