BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci

Abstract

The molecular mechanism involved in the process of antigen-driven somatic hypermutation of Ig genes is unknown, but it is commonly believed that this mechanism is restricted to the Ig loci. B cell lymphomas commonly display multiple somatic mutations clustering in the 5'-regulatory region of BCL-6, a proto-oncogene encoding for a POZ/Zinc finger transcriptional repressor expressed in germinal center (GC) B cells and required for GC formation. To determine whether BCL-6 mutations represent a tumor-associated phenomenon or reflect a physiologic mechanism, we screened single human tonsillar GC B cells for mutations occurring in the BCL-6 5'-noncoding region and in the Ig variable heavy chain sequences. Thirty percent of GC B cells, but not naive B cells, displayed mutations in the 742 bp region analyzed within the first intron of BCL-6 (overall frequency: 5 x 10(-4)/bp). Accordingly, an expanded survey in lymphoid malignancies showed that BCL-6 mutations are restricted to B cell tumors displaying GC or post-GC phenotype and carrying mutated Ig variable heavy chain sequences. These results indicate that the somatic hypermutation mechanism active in GC B cells physiologically targets non-Ig sequences.

Figure 1

Distribution of mutations within the BCL-6 5′-noncoding sequences of 37 single GC B cells. (Upper) Schematic representation of the BCL-6 gene. Coding and noncoding exons are indicated by filled and empty boxes, respectively. The PCR fragment amplified for mutational analysis is approximately positioned below the map and blown up in the lower panel to show the distribution of mutations. Each line represents a 20-bp interval of the BCL-6 sequence amplified, and the first nucleotide of the BCL-6 cDNA is designated as position +1. Mutations included single base pair substitutions (closed ovals) and deletions (brackets). For each cell, identified by its code number, the type and exact position of the mutation are specified on the right column (Δ, deletion). Note that all of the 68 nucleotide exchanges, including deletions, were found in heterozygosis when both alleles were amplified (cells marked by an asterisk).

Figure 2

Comparative analysis of the mutation frequency in the BCL-6 5′-noncoding region and IgV_H segments of B cell lymphoid malignancies. Five cases representative of the major histologic subtypes and showing BCL-6 alterations upon SSCP analysis were randomly selected, along with five mantle cell lymphomas. A 742-bp region of the BCL-6 first intron and the rearranged IgV_H genes from the same patients were amplified and directly sequenced. Filled and empty circles represent the frequency of mutations in the two loci. The majority of cases in each category harbored one to two nucleotide substitutions (0.068–0.13%); particularly high values were found in one FL (n = 9, 0.6%), one BL (n = 16, 1.1%), and various DLCL cases (n = 5–24, 0.34–1.6%).