Frequent occurrence of deletions and duplications during somatic hypermutation: Implications for oncogene translocations and heavy chain disease

Abstract

Human naive and germinal center (GC) B cells were sorted by f low cytometry and rearranged V H region genes were amplified and sequenced from single cells. Whereas no deletions or insertions were found in naive B cells, Ϸ4% of in-frame and >40% of out-of-frame rearrangements of GC B cells harbored deletions and͞or insertions of variable length. The pattern of deletions͞insertions and their restriction to mutated V genes strongly suggests that they result from somatic hypermutation. Deletions and insertions account for Ϸ6% of somatic mutations introduced into rearranged V H region genes of GC B cells. These deletions͞insertions seem to be the main cause for the generation of heavy chain disease proteins. Furthermore, it appears that several types of oncogene translocations (like c-myc translocations in Burkitt's lymphoma) occur as a byproduct of somatic hypermutation within the GC-and not during V(D)J recombination in the bone marrow as previously thought.During B cell differentiation in the bone marrow, somatic recombination of antibody gene segments is carried out by B cell progenitors to create functional heavy and light chain variable (V) region genes (1). Antibody diversity is generated by assembling one of the different V, (D), and J genes for each V region gene and by imprecise joining of these gene segments (2). Only B cells expressing functional heavy and light chain genes are allowed to enter the peripheral B cell pool as naive IgM ϩ IgD ϩ B cells (1). If peripheral B cells are activated by cognate antigen in the course of a T cell-dependent immune response, antibody V genes are further diversified by the introduction of somatic mutations in the microenvironment of the germinal center (GC) (3). The somatic mutations, which are introduced at a high rate (Ϸ10 Ϫ3 to 10 Ϫ4 ͞bp͞generation) to a region of Ϸ2 kb from the leader region to the J-C intron into rearranged heavy and light chain genes (4), mainly have been described as single nucleotide exchanges, whereas deletions͞insertions only rarely have been observed (4, 5).However, V genes harboring deletions and͞or insertions were detected repeatedly in our studies of rearranged V region genes in various human B cell lymphomas (6-12): Deletions and͞or insertions were found in 2 of 10 V H gene rearrangements and 1 of 14 V genes amplified from Burkitt's lymphomas (9), 1 of 4 V H genes from monocytoid B cell lymphomas (7), 1 of 14 V H and 1 of 11 V L genes from diffuse large cell lymphomas (8), 3 of 13 V H region genes from classical Hodgkin's disease (6, 10, 11), and 3 of 7 V H region genes from lymphocyte-predominant Hodgkin's disease (6, 12). A common feature of all of these lymphomas is that they carry somatically mutated V region genes and are thus derived from GC or post-GC B cells.The occurrence of deletions͞insertions within rearranged V genes has not been analyzed systematically in normal human B cells, so it is unclear whether their frequent occurrence in the lymphomas is a peculiar feature of malignant B cells or whether they are also present...

Expression of a V Region-Less B Cell Receptor Confers a Tolerance-Like Phenotype on Transgenic B Cells

“…One of the reason for this is that the genetic event leading to the production of a H chain disease protein probably occurs at the mature B cell stage (48), whereas in transgenic mice the ⌬V protein is expressed at an early stage, which is more likely to induce tolerance (49). In addition, in contrast to numerous in vitro findings showing that mature spleen B cells can be stimulated to proliferate after stimulation with anti-IgM, cross-linking of BCR on mature B cells in vivo induces cell death (18,43,50), and the factors that determine, in the absence of T cells, the outcome of the responses induced by BCR ligation, namely tolerance, proliferation, or differentiation to the B1 cell subset (51), remain to be elucidated.…”

B-cell development in progressively transformed germinal centers: similarities and differences compared with classical germinal centers and lymphocyte-predominant Hodgkin disease

“…The latter is apparent from the R/S value for mutations in the framework regions, 1.1 (range, 0.8 to 1.6), which is significantly lower than the typical value for GC B cells, 1.8. 31 Indeed, the R/S value of 1.1 is in a range typical for post-GC memory B and plasma cells, 13 perhaps indicating that the PTGC microenvironment supports survival of only the "very best" B cells in terms of affinity maturation. Alternatively, at the time of tissue sampling, PTGCs may often represent "old" structures that reached a degree of maturity at which members of expanded GC B-cell clones went through many rounds of selection, so that mutants with mutation patterns typical for memory B cells are enriched.…”

In vivoandin vitrostudies of immunoglobulin gene somatic hypermutation

“…Ramos constitutively diversi¢es its immunoglobulin V regions by hypermutation. This hypermutation generates immunoglobulin-negative variants through the introduction of nonsense mutations as well as deletions and duplications, as described in vivo (Goossens et al 1998;Wilson et al 1998;Wu & Kaartinen 1995). When transfected with terminal deoxynucleotidyl transferase (TdT) this repertoire of immunoglobulin-negative cells is increased by the presence of mutants that carry short non-templated insertions in V H (but not C m ).…”