A Very Short Mutational Hot-Spot in the IGH 5′Sμ Region Suggests That the Acquisition of a Class Switch Recombination Defect Is an Important Step Toward Lymphomagenesis.

Abstract

The t(14;18), which occurs during VDJ joining, is usually seen as the primary genetic event in follicular lymphoma (FL). Pre-lymphoma cells would then migrate to the secondary lymphoid organs and acquire secondary genetic abnormalities. The mechanisms leading to these secondary lesions are unclear, but both somatic hypermutation (SHM) and class switch recombination (CSR) processes may be involved. Surprisingly, while many FL still express an IgM, aberrant switch junctions were reported on both IGH alleles, indicating dysfunctions of CSR during the onset of the neoplasm. Recently, AID mediated point mutations were shown to accumulate in the switch μ (Sμ) regions of stimulated B cells undergoing CSR. To get insights into a possible CSR deregulation in FL, we sequenced the 5′ end of the Sμ region for 73 t(14;18) positive tumours. 152 point mutations and 5 deletions were identified, for a mutation frequency of 0.31/100 bp. Notably, mutations were found for a majority of cases (49/73, 67.1%) rather than being restricted to few tumours. Significant targeting of RGYW motifs (p<10−4) and bias for transitions over transvertions (p<10−4) both suggested the involvement of AID. More importantly, our data showed a highly significant clustering effect, with 22 mutations (13,9%) found on a 4 bp cluster on the GTAA splicing donor site of the Iμ exon (p<10−6). Those 22 mutations were found in different tumors so that 30.1% of the lymphomas in our series had a mutated splicing site. Analysis of whole blood genomic DNA from 14 patients and 20 healthy donors indicated that these mutations are not polymorphisms but are instead acquired by tumor cells. Importantly, allele specific PCR experiments showed no clustering effect on the translocated alleles, while 15/79 (19%) mutations on the functional alleles were found on the Iμ splicing site (p<10−6). Such mutations were also found, but on the translocated alleles, for two additional lymphomas with complex t(3;14;18)(q27;q32;q21) and t(8;14;18)(q24;q32;q21) translocations, suggesting that they may be linked to downstream rearrangements on the IGH locus. Finally, RT-PCR experiments showed that a mutation of the Iμ splicing site leads to the expression of aberrant germline transcripts. Together, our data thus indicate that mutations leading to an incorrect splicing of the germline Sμ transcripts are probably positively selected during lymphomagenesis, and therefore that an acquired perturbation of the CSR process may have a major impact during the onset of the disease, maybe by preventing pre-lymphoma cells from switching their surface immunoglobulin and/or by favouring subsequent genetic lesions.