Nucleotide insertions and deletions within the homopolymeric runs of adenines and thymidines of BCL10 cDNAs in normal peripheral blood leukocytes

The BCL10 gene was isolated from the breakpoint region of the t(1;14) in mucosa-associated lymphoid tissue (MALT) lymphomas,1,2 and BCL10 was found to be a cellular homologue of the equine herpesvirus-2 E10 gene because both contain an amino-terminal caspase recruitment domain, which is homologous to that seen in several apoptotic molecules.1,2 In functional assays, wild-type BCL10 was found to induce apoptosis. It is noteworthy that BCL10 in t(1;14)-bearing MALT lymphomas exhibits a variety of truncating mutations,1,2 and that truncated BCL10 fails to induce apoptosis and exhibits a transforming activity. Similar mutations were also identified in a subset of follicular lymphoma (FCL) and other tumor cell lines with a loss of heterozygosity in chromosome 1p22.1 However, several recent studies, including ours, have reported that BCL10 mutations are rare in various human tumors, thus raising questions regarding the pathological role of BCL10 as a tumor suppressor gene in these tumors.3-9 

Recently, Dyer et al suggested that at least some of the discrepancies between their data and those reported by others can be ascribed to their use of cDNA rather than genomic DNA.10 They found that nucleotide insertions and deletions within homopolymeric runs of 8 adenines and 7 thymidines are common in BCL10 cDNAs. In order to investigate whether BCL10 abnormalities occur at the RNA level, we searched for somatic mutations in BCL10 cDNAs by means of reverse transcription polymerase chain reaction (RT-PCR) and sequencing analyses in 3 normal peripheral blood leukocytes (PBLs) and 3 FCL samples. The pathological diagnosis was established in accordance with the revised European-American classification of lymphoid neoplasms (REAL). The coding region of BCL10 cDNAs (702 bp) was amplified by using Taq DNA polymerase with high fidelity and subcloned into the plasmid vectors. The inserts of 5 independent BCL10 cDNA clones from each of the normal PBLs or lymphoma samples were sequenced in both directions using an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Foster City, CA) on an ABI 373 DNA automated sequencer (Applied Biosystems, Foster City, CA). The spectrum of BCL10 cDNA abnormalities is summarized in the Table. These include multiple point mutations and nucleotide insertions or deletions within homopolymeric runs of adenines and thymidines. The spectrum of multipleBCL10 cDNA abnormalities is similar to those reported by others in certain tumors.1,2,10 It is possible that some of these multiple point mutations may be due to PCR or cloning artifacts. In our study, however, nucleotide insertions or deletions within homopolymeric runs, resulting in truncation of BCL10, were identified in the same 8 out of 15 clones both from normal PBLs and from FCL samples (Table). The possibility that these truncation-type abnormalities are due to PCR or cloning artifacts is minute, because only 4 out of 500 genomic DNA sequences were found to exhibit such insertions or deletions10 and our FCL samples exhibited an apparently normal germ-line DNA.9 Finally, a novel 118 bp deletion in exon 3 of BCL10 was identified in one FCL sample. It is not clear if this alteration contributes to the pathogenesis of FCL, but in the light of the fact that only one clone carried such a deletion, it is doubtful whether this deletion has any functional significance.

In summary, our results strongly suggest that BCL10 may undergo a novel posttranscriptional RNA modification even in normal PBL cells and that BCL10 cDNA abnormalities are not necessarily associated with the molecular pathogenesis of a wide range of human tumors.