Mutation study of the BCL10 gene in lymphoma with both RNA and DNA

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TO THE EDITOR

The BCL10 gene has been cloned from the chromosomal translocation t(1;14)(p22;q32) in a low-grade mucosa-associated tissue (MALT) lymphoma.1 The function of the protein is not yet clear, and it also remains to be proved whether BCL10 is an oncogene or a tumor suppressor gene.1,2 Willis et al1 suggested that BCL10 mRNA exhibited hypermutations in MALT lymphomas with t(1;14), as well as frequent mutations in 45% of B and T cell lineage lymphomas without 1q22 chromosomal rearrangements. In a recent issue of Leukemia, Luminari et al3 reported, however, that BCL10 mutations are rare, when they examined genomic DNA of various types of lymphoid malignancies. Some BCL10 gene abnormalities were proposed to be found only in RNA and not in genomic DNA,4,5 but no previous studies investigated DNA and RNA at the same time.4,5,6,7,8 To make the matter clear, we first analyzed the mutations of BCL10 RNA from 43 lymphomas, and for those lymphomas with RNA alterations we investigated the genomic DNA extracted from the same tumor and normal tissues from the same patients.

We amplified the RNA from 11 follicular lymphomas, which are reported to be more frequently involved with BCL10 gene mutations, by reverse transcription PCR (RT-PCR) with two sets of primers covering the full-length cording region of the BCL10 gene, then recovered bands after agarose gel electrophoresis, and sequenced with the direct sequencing method. In one patient a distinct band of a smaller size was detected by agarose gel electrophoresis and ethidium bromide staining of the RT-PCR product, together with a faint band of the expected size, and sequence analysis revealed an 81 base pair deletion (Figure 1). This 81 base pair deletion should cause a 27 amino acid in-frame deletion (aa 206–232) at the C-terminal portion of the BCL10 protein. In another patient we found C/T heterozygosity of the second base of codon 162 resulting in the substitution of an amino acid (threonine to methionine). We also studied RNA from these 11 follicular lymphomas and 32 diffuse lymphomas by reverse transcription PCR-SSCP (RT-PCR-SSCP) with four sets of primer pairs covering the full length of the coding region of the BCL10 gene, but detected no more abnormalities except for those mentioned above (data not shown).

Figure 1
figure1

BCL10 gene mutations in follicular lymphomas. (a) Results of RT-PCR analysis. Total cellular RNA was isolated from fresh materials by standard methods and the RNA was reverse-transcripted and amplified by the PCR method. To make the difference in size clear between deletion mutant and normal gene, we used four different primer pairs from primers for sequence analysis. The RT-PCR reactions were run on a 3.0% agarose gel. Lanes 1 and 2: follicular lymphomas without RNA alterations showed single band. Lane 3: an abnormal band of small molecular size was detected only in a follicular lymphoma, and its density was higher than the normal band. M.M indicates molecular size marker of 100 bp ladder. (b) Results of DNA analysis of tumor and normal tissue of the patient with deletion mutation. DNA from bone marrow without lymphoma and tumor were extracted and examined with PCR followed by agarose gel electrophoresis. The target band was excised, and sequenced. Lanes 1 and 2: follicular lymphomas without RNA alterations showed a single band. Lane 3 BM: Only the normal band was detected in bone marrow without lymphoma involvement from the patient with a deletion mutation. Lane 3 LN: the abnormal band of small size was observed only in the tumor sample of the patient with a deletion mutation, and it proved to be the 81 bp deletion mutant (data not shown). His abnormal band and normal bands had almost even den-sity. M.M indicates molecular size marker of 100 bp ladder. (c) Result of sequence analysis of the deletion mutation. We amplified the RNA by reverse transcription PCR (RT-PCR) with two sets of primers covering the full-length of the cording region of BCL10 gene, and eluted the products from agarose gel after electrophoresis. Then, sequencing analysis was carried out using the ABI Big Dye Terminator Cycle sequencing Ready Reaction, followed by fractionations in the ABI 310 sequencer. Eighty-one base pair deletion from nucleotide 616 to 696 was revealed. The underlined sequence was deleted. 702 indicates the end of the coding sequence.

For the two follicular lymphoma patients with sequence alterations of BCL10 RNA we investigated the mutations of the DNA samples isolated from the same lymphoma tissue and bone marrow without documented lymphoma invasion by PCR and direct sequencing. In the patient with C/T heterozygosity, identical C/T heterozygosity was detected in both DNA from the same lymphoma sample and that from the bone marrow without lymphoma involvement, thus this single nucleotide substitution may probably be a polymorphism. In the lymphoma of this patient, which is considered to be monoclonal, both C485 and T485 alleles (according to numbering described by Willis et al1), appeared to be evenly expressed. This observation may indicate that BCL10 is biallelically expressed, as most genes are. In the patient with an 81 base pair deletion in RNA, the abnormal and normal alleles were detected evenly in the genomic DNA from the same tumor sample, but the mutated allele was not detected in his bone marrow without lymphoma (Figure 1b). Therefore, this deletion mutation is tumor-specific. Furthermore, as the RNA analysis of the tumor showed that more mutated allele was expressed than normal allele (Figure 1a), this mutant allele may predominantly be expressed in the lymphoma cells.

BCL10 RNA clones with post-transcriptional sequence modification, which Willis et al1 and Takahashi et al8 showed to be frequently found in lymphoma and normal peripheral blood lymphocytes by cDNA cloning method, could not be demonstrated in this study with either RT-PCR SSCP or direct sequencing of RNA. It is suggested that the amount of each RNA clone with post-transcriptional sequence modification may be too small for detection with RT-PCR SSCP or the direct sequencing method. So it is a question whether such a small population of BCL10 RNA has a biological significance on lymphomagenesis.8

In conclusion, mutations of BCL10 gene may be infrequent in follicular lymphomas and even rarer in diffuse lymphomas. The deletion mutation we found should cause an in-frame deletion at the C-terminal portion of the BCL10 protein, which is similar to the mutations reported in previous studies,1,2 confirming that the abnormality of the C-terminal portion is important for lymphomagenesis. Our study revealed that BCL10 gene mutations detected distinctly in RNA by RT-PCR SSCP or direct sequencing were confirmed in genomic DNA, and BCL10 transcript with post-transcriptional sequence modification may be minor populations.

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Tsushita, K., Ohashi, H., Utsumi, M. et al. Mutation study of the BCL10 gene in lymphoma with both RNA and DNA. Leukemia 15, 1139–1140 (2001) doi:10.1038/sj.leu.2402154

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