Alignment of clonally related platelet autoantibody heavy-chain amino acid sequences and their putative ontogenic trees.

The H and L nomenclature is the same as in Figure 1. (A) Groups of related sequences comprising expanded heavy-chain clones in each patient library (clone A and clone B) are enclosed in boxes. The coalignment with the rest of the 16 unique heavy chains is available on the Blood website; see the Supplemental Data Set link at the top of the online article. For clone A, the putative intermediate heavy-chain sequences are also shown (1, 2, and 3 asterisks). The number of nucleotide differences from germline V_H is tabulated to the right of each sequence. Because D segments showed poor homology with known D genes, mutations were not scored in these regions. Replacement mutations are indicated by letters, identities as “.”, and insertions as −, and + to maintain spacing due to variability in CDR3 length. Sequences derived from the 5′ V-region primers used for library construction22 are marked as >. CDR-region designations are according to the system of Kabat et al29; numbering and hypervariable loop designations are according to the system of Chothia et al.30 (B) Analysis of nucleotide data in each patient revealed a distinct set of somatically mutated heavy chains sharing common V_HDJ_H rearrangements of V_H3-30, D1-26, and J_H4b gene segments. Circles represent isolated and sequenced clones (Figures 1 and 2A); diamonds (for ITP patient A only) represent putative intermediates. For each member of a patient's clone, the number of nucleotide mutations from its germline V_H gene is shown in parentheses, and the resulting number of replacement (R) or silent mutations (S) is shown in brackets. For each patient clone ontogenic tree, the distance in the horizontal direction represents the extent of mutation from the proposed germline origin within the constraints of the diagram.