1. Analysis of clonotypic switch junctions reveals multiple myeloma originates from a single class switch event with ongoing mutation in the isotype-switched progeny
by Brian J. Taylor, Jitra Kriangkum, Julie A. Pittman, Michael J. Mant, Tony Reiman, Andrew R. Belch, and Linda M. Pilarski
Blood
Volume 112(5):
September 1, 2008
©2008 by American Society of Hematology

2. Clonotypic switch (CS-PCR) amplification in MM patients.
Clonotypic switch (CS-PCR) amplification in MM patients. (A) A diagram of the rearranged immunoglobulin heavy chain locus: V indicates variable; D, diversity; JH, junction segments; Eμ, intronic enhancer; Iμ, I exon; Sμ, switch region before class switching; Sμ/Sx, hybrid switch region generated after class switching; C, downstream constant region; and x, α (IgA) or γ (IgG) isotope regions. (B) CS-PCR-A and CS-PCR-B, with patient-specific CDR2 and CSR-specific Sx1 or CxB primers to enable selective amplification of clonotypic switch products. For some cases, CS-PCR-A products were used in a nested CS-PCR-B reaction, called CS-PCR-AB, to increase sensitivity. (C) Sensitivity assay for the CS-PCR-A reaction using LP1 cells diluted into normal blood. Cell concentration is shown above the panel; neg indicates no LP1 cells added.
Brian J. Taylor et al. Blood 2008;112:
©2008 by American Society of Hematology

3. CS-PCR in MM patient time point samples.
CS-PCR in MM patient time point samples. A representative CS-PCR-A in patients MM1 to MM4 is shown above the panel. BM indicates bone marrow; BL, blood. Time point number is given for each sample, and the time between samples is indicated below the panel. Molecular weight markers are shown on the right. Faint secondary bands below the strong CS-PCR band arise from nonspecific priming by the downstream Sγ1 primer.
Brian J. Taylor et al. Blood 2008;112:
©2008 by American Society of Hematology

4. Switch junction analysis in 6 MM patients.
Switch junction analysis in 6 MM patients. Homologies between switch regions and the switch junction sequence were determined by BLAST and ClustalW analysis. Switch junctions are indicated with open boxes. Upper sequence indicates Sμ switch region; center, switch junction sequence from this study; and lower, Sγ switch region. Switch base positions are shown in bold. Vertical lines indicate sequence identity; dashes, spaces introduced for optimal alignment. A switch junction microhomology of 4 to 5 bp is evident in patient MM1.
Brian J. Taylor et al. Blood 2008;112:
©2008 by American Society of Hematology

5. Detection of new mutations in the second time point sample of MM patients.
Detection of new mutations in the second time point sample of MM patients. (A) The positions of the germ-line elements in the V/D/J-S region are indicated at the top, with CS-PCR sequence from the diagnosis time point represented for patients MM1-5 below. Dashed lines represent regions that were not sequenced. New mutations in the second time point sample are shown as follows: closed circles are point mutations with base changes between first and second samples presented directly underneath; open circles, deletions, with the number of bases deleted shown above. A deletion followed by tandem duplication of downstream DNA is shown for MM3. (B) The frequency of each point mutation is summarized in the box.
Brian J. Taylor et al. Blood 2008;112:
©2008 by American Society of Hematology