Genealogical trees for the VH and VL rearrangements of case 1 to analyze the clonal relationship among the individual cells of the 3 different follicles.
Genealogical trees are based on multiple sequence alignments obtained with the MacVector software 7.0 (Accelrys). “z” denotes the presumed precursers. Assumed intermediates are included in the genealogical tree and marked as “x.” When a branch in the tree is defined by more than one mutation, it may well be that they occurred in separate steps. However, this would not change the basic structure of the tree and the relationship of the distinct cells identified. Cells from the same follicle are marked with a follicle-specific color: follicle 1, yellow; follicle 2, orange; and follicle 3, blue. There were 9 cells with a VH as well as a Vκ gene amplificate (the names of these cells are given in the trees). We made some corrections of the VH tree generated by the MacVector software 7.0: at 2 positions the software regarded different point mutations habored by all cells at the same position of the sequences as a shared mutation (97 C to A or 97 C to G, 277 C to A or 277 C to G; see sequences available from the EMBL data library). As these were clearly distinct mutation events, the tree was accordingly corrected, including introduction of one backmutation into 2 VHsequences at position 97 (the most right branch with 2 f1 cells). There were 2 point mutations shared by 2 sequences that were regarded as independent mutation events; importantly, the number of mutation events remained the same with this modification. The branch with f2-9 and f2-10 could also originate from f3-22, but to aquire congruence with the Vκ tree origination from f2-48 with the same number of mutations was assumed. Additional details are available from the authors upon request. f1 indicates follicle 1; f2, follicle 2; f3, follicle 3; mut, mutation.
