In Response to Fakruddin et al. and Apostolou et al

We agree with many of the above findings, including the frequency and nature of BCL10 polymorphisms. However, it appears that some BCL10 abnormalities may be found only in RNA and not genomic DNA, suggesting that BCL10 may undergo posttranscriptional sequence modification.Wild-type BCL10 is proapoptotic and behaves as a tumor suppressor gene (1xKoseki, T., Inohara, N., Chen, S., Carrio, R., Merino, J., Hottiger, M.O., Nabel, G.J., and Nunez, G. J. Biol. Chem. 1999; 274: 9955–9961Crossref | PubMed | Scopus (122)See all References, 2xThome, M., Martinon, F., Hofmann, K., Rubio, V., Steiner, V., Schneider, P., Mattmann, C., and Tschopp, J. J. Biol. Chem. 1999; 274: 9962–9968Crossref | PubMed | Scopus (84)See all References, 3xWillis, T.G., Jadayel, D.M., Du, M-Q, Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R.A., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all References, 4xYan, M., Lee, J., Schilbach, S., Goddard, A., and Dixit, V.M. J. Biol. Chem. 1999; 274: 10287–10292Crossref | PubMed | Scopus (93)See all References, 5xZhang, Q., Siebert, R., Yan, M., Hinzmann, B., Cui, X., Xue, L., Rakestraw, K.M., Naeve, C.W., Beckmann, G., Weisenberger, D.D., Sanger, W.G., Nowotny, H., Vesely, M., Callet-Bauchu, E., Salles, G., Dixit, V.M., Rosenthal, A., Schlegelberger, B., and Morris, S.W. Nat. Genet. 1999; 22: 63–68Crossref | PubMed | Scopus (293)See all References). BCL10 sequence abnormalities were first detected in cDNA clones from a MALT lymphoma with t(1;14) and were unusually heterogeneous. An identical spectrum of cDNA abnormalities has been detected in other t(1;14) MALT lymphomas (Zhang et al. 1999xZhang, Q., Siebert, R., Yan, M., Hinzmann, B., Cui, X., Xue, L., Rakestraw, K.M., Naeve, C.W., Beckmann, G., Weisenberger, D.D., Sanger, W.G., Nowotny, H., Vesely, M., Callet-Bauchu, E., Salles, G., Dixit, V.M., Rosenthal, A., Schlegelberger, B., and Morris, S.W. Nat. Genet. 1999; 22: 63–68Crossref | PubMed | Scopus (293)See all ReferencesZhang et al. 1999), making it unlikely that all these changes represented RT, PCR, or cloning artifacts.We initially sequenced cDNA clones from 6 malignant cell lines, including Tera-1, Tera-2, and GCT-44 and three mesothelioma lines, and in each, BCL10 abnormalities were detected (Table 2 of Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M-Q, Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R.A., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999). Additional cDNA clones from Tera-2, a cell line that exhibits no genomic PCR-SSCP abnormalities, showed (Table 1Table 1): (1) all clones had different sequences; (2) multiple abnormalities in some clones; (3) no obvious clonal relationship between all clones; and (4) abnormalities within the homopolymeric runs in 4/13 clones.Table 1Summary of Tera-2 BCL10 cDNA DataClone #Point Mutations (codon)Homopolymeric RunsSplice Varia- tionsPredicted Protein (amino acids)(A) Normal Clone1nilnilnil233 wild type(B) Clones with Insertions/Deletions within the Homopolymeric Runs Alone2nil499 ins TTnil1723nil499 ins Tnil1684nil136 del A499 ins Tnil68(C) Mutated Clones +/− Other Abnormalities5N93SAAC→AGCnilnil2336N93SAAC→AGCdel A codon 149nilnil1717H40HCAT→CAC silentK45QAAA→CAAnilnil2338M153VATG→GTGL177LCTA→CTG silentA200AGCT→GCC silentnilnil2339R58QCGA→CAAS218FTCT→TTTnilnil23310R58QCGA→CAAT168TACT→ACC silentS218FTCT→TTTnilnil23311H40RCAT→CGTL41LCTA→CTT silentN217SAAC→AGC499 ins Tnil16812A20TGCC→ACCV26VGTA→GTG silentQ92stopCAG→TAGnilnil9113A20TGCC→ACCV26VGTA→GTG silentQ92stopCAG→TAGR226KAGA→AAAnil16 bp91Summary of cDNA sequence abnormalities observed in 13 clones derived from cell line Tera-2.We have now sequenced either RT-PCR products and/or multiple cDNA clones from 18 malignant cell lines, 48 primary lymphoid tumors, and peripheral blood lymphocytes from 7 normal individuals, as well as cDNAs from 5 other normal tissues, and found complex abnormalities in all instances. To summarize the three types of deviation from the wild-type sequence observed in our own and others’ studies (Zhang et al. 1999xZhang, Q., Siebert, R., Yan, M., Hinzmann, B., Cui, X., Xue, L., Rakestraw, K.M., Naeve, C.W., Beckmann, G., Weisenberger, D.D., Sanger, W.G., Nowotny, H., Vesely, M., Callet-Bauchu, E., Salles, G., Dixit, V.M., Rosenthal, A., Schlegelberger, B., and Morris, S.W. Nat. Genet. 1999; 22: 63–68Crossref | PubMed | Scopus (293)See all ReferencesZhang et al. 1999):(A) BCL10 utilized three alternative splice sites at the exon 3/4 boundary, two of which result in deletion of either 16 or 33 base pairs from wild-type sequence. These splice variants were detected as different-sized PCR products.(B) Nucleotide insertions and deletions were common, particularly within the homopolymeric runs of 8 A’s and 7 T’s. The homopolymeric runs were also sites of point mutation (Figure 1BFigure 1B). In some instances, abnormalities within these runs were seen by direct cDNA sequencing, indicating they comprised a common transcript (Figure 1DFigure 1D). These data preclude cloning artifacts. In comparison, from 500 genomic sequences obtained in our laboratories, only 4 cases exhibited deletions or insertions within the two homopolymeric runs. These data suggest that such changes are largely nontemplated and may constitute an unusual form of RNA editing.Figure 1Reverse BCL10 cDNA Sequences Showing Presence of Alterations within both Homopolymeric Runs(A) Clone from normal lymphocytes showing 8 A’s.(B) Direct sequencing of cDNA from patient with B cell precursor acute leukemia showing complex aberrations within the 8 A’s including deletion of two adenines and A→G point mutation.(C) Direct sequencing of cDNA from myeloma cell line showing normal run of 7 T’s.(D) Direct sequence of cDNA from normal human fetal brain mRNA (Clontech) showing presence of additional thymidine in about 50% of the transcripts (arrow).Note loss of readable sequence in lanes B and D subsequent to inserted or deleted nucleotides. RT and PCR reactions performed as described (Willis et al. 1999xWillis, T.G., Jadayel, D.M., Du, M-Q, Peng, H., Perry, A.R., Abdul-Rauf, M., Price, H., Karran, L., Majekodunmi, O., Wlodarska, I., Pan, L., Crook, T., Hamoudi, R.A., Isaacson, P.G., and Dyer, M.J.S. Cell. 1999; 96: 35–45Abstract | Full Text | Full Text PDF | PubMed | Scopus (470)See all ReferencesWillis et al. 1999). Poly(A)+ RNA was reverse transcribed using Superscript II (GIBCO–BRL) or rTth (Amersham) and random hexamers and the BCL10 open reading frame amplified using either Taq or Pfu polymerase with forward primer: 5′-CCTCCTCTCCTTCTTCCCCATTACC-3′ and reverse primer 5′-GCAATAAAGTGTCATTGTCTGGAAACAGT-3′. RT-PCR products were either sequenced directly or cloned into pCR2.1 (Invitrogen). Clones were sequenced in both directions using either Licor (MWG-Biotech, Germany) or ABI-377 (Applied Biosystems) automated sequencers.View Large Image | View Hi-Res Image | Download PowerPoint Slide(C) Point mutations were multiple and ongoing. Whether all point mutations are present in genomic DNA or whether some are nontemplated is not yet clear.These cDNA abnormalities appeared with comparable frequency irrespective of reverse transcriptase, DNA polymerase, or sequencing method used. A BCL10 pseudogene has not been identified by FISH, Southern, or genomic PCR experiments.In summary, at least some of the discrepancies between our initial data and those reported above can be ascribed to our use of cDNA rather than genomic DNA and to possible posttranscriptional modification of BCL10. However, our analysis of genomic DNA from primary tumors continues to demonstrate BCL10 mutations in some tumor types (H. P. et al., submitted). The reasons for this discrepancy are not clear but may include the ongoing nature of BCL10 mutations and the presence of individual BCL10 mutations within only a subset of tumor cells.All sequences will be available via

Keywords:

• genomic DNA
• Genetics
• Exon
• Base pair
• Pseudogene
• clone (Java method)
• Point mutation
• Molecular biology
• Complementary DNA
• Reverse transcriptase
• Biology

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