SOMATIC HYPERMUTATION INTRODUCES INSERTIONS AND DELETIONS INTO IMMUNOGLOBULIN GENES

During a germinal center reaction, random mutations are introduced int o immunoglobulin V genes to increase the affinity of antibody molecule s and to further diversify the B cell repertoire. Antigen-directed sel ection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertion s and deletions have been reported to occur at a low frequency. In thi s study, we describe five insertion and four deletion events in otherw ise somatically mutated V-H gene cDNA molecules. Two of these insertio ns and all four deletions were obtained through the sequencing of 395 cDNA clones (similar to 110,000 nucleotides) from CD38(+)IgD(-) germin al center, and CD3S(-)IgD(-) memory B cell populations from a single h uman tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic V (H)4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occ urred as triplets or multiples thereof, leaving the transcripts in fra me. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD(+)CD38(-) follicular mantle cDN A clones statistically associates these events to the somatic hypermut ation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions col lected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most freque ntly involve sequence motifs resembling known intrinsic hotspots of so matic hypermutation. These insertion/deletion events are consistent wi th models of somatic hypermutation involving an unstable polymerase en zyme complex lacking proofreading capabilities, and suggest a downregu lation or alteration of DNA repair at the V locus during the hypermuta tion process.