As a third and last step, for all 43 patients with evidence of monoclonality on RT-PCR assay, the monoclonal rearrangement was directly sequenced and the IGHV-subgroup status and mutational status were assessed. We were able to identify and analyze the IGHV genes in 35 of 45 patients (78%), probably because of the limited number of clonal B cells (or possibly because of the presence of more than one clone with overlapping sequences) in these prediagnostic samples.33 Among the 35 sequenced IGHV gene rearrangements, 16 samples (46%) had IGHV3-subgroup genes and 9 (26%) had IGHV4-subgroup genes, frequencies that are similar to the expected frequency in the normal B-cell repertoire. In particular, the most prevalent IGHV genes were IGHV3–23, which were identified in six patients (17%), and IGHV4–34, which were identified in four (11%). The vast majority of the IGHV sequences (27 of 35, or 77%) were mutated (range of germ-line identity, 87.9 to 97.9%), whereas only 8 of the sequences (23%) were unmutated, including those in 6 samples with a 100% identity with the closest germ-line IGHV gene.
