Conserved patterns of somatic mutation and secondary V_H gene rearrangement create aberrant lg-encoding genes in Epstein-Barr virus-transformed and normal human B lymphocytes

Abstract

Expression of N-terminal-truncated Ig heavy chains without normal light chain expression has been shown to occur in human B cell tumor lines, and to be due to diverse types of structural alteration within the expressed Ig heavy and light chain genes. Due to the tumor cell origin of these lines, generation of aberrant Ig-encoding genes may only occur after malignant transformation, reflecting the release of the tumor B cell from the need to express functional Ig for continued clonal proliferation. The genetic basis for expression of VH-truncated mu chains without light chains in several Epstein-Barr virus (EBV)-transformed human B cell lines was investigated with the aim that this information would lead to detection of similarly aberrant Ig genes in normal human B lymphocytes. Analysis of the productive mu genes in three truncated mu-only human B cell lines showed a consistent structural change where a secondary VH-VHDJH gene rearrangement had occurred. The site of VH-VH joining was suggestive of a V(D)J recombinase-mediated event. A consistent pattern of mutation was also observed in the normal-sized, but non-functional kappa light chain transcripts, making them incapable of coding for a functional kappa chain. Using genomic DNA from peripheral blood lymphocytes of a donor whose B cells were originally used to make one of the truncated mu-only EBV B cell lines, a similarly mutated V kappa gene was detected and a similar composite VH-VH gene was cloned. The lack of such aberrant VH and V kappa genes in non-lymphoid cells of this individual showed that the structural abnormalities in the expressed Ig genes arose somatically during development of that B cell clone. The presence of these altered Ig heavy and light chain genes in the genomic DNA of untransformed lymphocytes also shows that generation of such variant B cell clones can be a pre-neoplastic event and occurs by genetic mechanisms active during normal B cell development.

Immunoglobulin Heavy Chain Variable Region Gene Replacement as a Mechanism for Receptor Revision in Rheumatoid Arthritis Synovial Tissue B Lymphocytes

“…In the course of studying the clonally amplified B lymphocytes that develop V gene point mutations in the synovial tissues of RA patients, we identified a series of clones that exhibited V H DJ H variants that were apparently generated by V H replacement. However, unlike previous studies of V H replacement in murine B cell lines 39404142 and transgenic mice 67843 and in transformed 444546 and normal human B lymphocytes 1845, these secondary V H rearrangements occurred relatively frequently and involved several heptamer-like sequences at four distinct locations within V H . Our data suggest that these rearrangements can occur in situ and could be mediated by products of the recombinase activating genes (RAGs) in an RSS-specific manner.…”

“…1). However, unlike previous examples of this phenomenon that involved rearrangements to a single site in FR3 678183940414243444546, the rearrangements detected in the synovial tissue cDNA clones occurred at three distinct sites spanning FR3 (see Fig. 2Fig.…”

Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections.

“…The fact that the two sequences harbour at least two mutations ( Figure 5) is an additional indication that these unusual sequences represent true VHDHJH rearrangements as somatic hypermutation is largely restricted to rearranged V genes (reviewed by Kocks and Rajewsky, 1989). Aberrant VH-VH genes have been described before (Deane and Norton, 1990;Brokaw et al, 1992) and, interestingly, a recent report (Chou and Morrison, 1993) indicates that two specific tetranucleotide sequences, which are also found near the recombination sites in the sequences shown here, may be important in non-homologous recombinations involving immunoglobulin sequences ( Figure 5). However, it is also possible that this unusual VH sequence did not arise somatically but represents a rearrangement of a pseudogene encoded in the germline.…”

VH replacement in rearranged immunoglobulin genes

“…1c). However, we have now shown (unpublished observation) that the three published examples 33 result from a duplication within one V H .…”