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© 2005 Oxford University Press
CORRESPONDENCE |
RESPONSE: Re: Prognostic Significance of a Short Sequence Insertion in the MCL-1 Promoter in Chronic Lymphocytic Leukemia
Affiliation of authors: Department of Pathology, Royal University Hospital and College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Correspondence to: Anurag Saxena, MD, Department of Laboratory Medicine, Royal University Hospital, Saskatoon SK, Canada (e-mail: saxena{at}sask.usask.ca).
The Correspondences by Freeman et al., Vargas et al., Iglesias-Serret et al., Nenning et al., and Dicker et al. address four key points regarding MCL-1 promoter nucleotide insertions.
Mutations or polymorphisms? Because our control group was too small (n = 18) to determine population prevalence, we simply referred to these promoter changes as nucleotide insertions. Based on an analysis of 55 additional control samples, we have now found that the allele frequencies in the Saskatchewan population are 0.98 for the wild-type allele and 0.01 each for 6- and 18-nucleotide insertion alleles (Fig. 1A and B). The presence of these nucleotide insertions in other healthy populations is also reported in four of the five letters. Vargas et al. refer to their presence in the healthy controls; the frequencies of the +18 allele were 11.8% in Freeman et al., 17.5% in Iglesias-Serret et al., and 27.5% in Dicker et al., and the frequencies of the +6 allele were 20.3%, 12.5%, and 23.7%, respectively. The frequency of these nucleotide insertions in 134 patients with acute lymphoblastic leukemia reported by Nenning et al. is similar to what we observed in patients with chronic lymphocytic leukemia (CLL). Interestingly, Dicker et al. also report a novel 12-nucleotide insertion with a frequency of 1.4% in CLL case patients. Although there can be exceptions, the commonly accepted definition of a mutation is that the least common allele has a frequency of less than 1%. The presence of these nucleotide insertions in more than 1% of healthy population suggests that the MCL-1 promoter nucleotide insertions are polymorphisms, not mutations.
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Location. The exact transcriptional start site of the MCL-1 promoter is controversial (1–3). The nucleotide insertions can be inferred to be either at position –190, as reported by us and (1), or at –180 (2). We are not entirely sure of the origin of the reference to position –188 in Freeman et al. Moreover, the references they cite for E2F1-mediated MCL-1 transcriptional regulation refer to promoter regions spanning –143/+10 and –117/+10, both of which are outside the nucleotide insertion-harboring region that we described.
Effect on gene expression. It has been shown that the MCL-1 promoter that contains the nucleotide insertion–harboring region has statistically significantly higher activity in response to phorbol 12-myristate 13-acetate (PMA), serum, and granulocyte-macrophage colony-stimulating factor than the promoter with this region deleted (2). Our original publication supports these findings because we found an association between the +6 and +18 nucleotide insertions (considered together) and higher mRNA and protein levels. By contrast, Freeman et al. report reduced PMA-stimulated transcription activity with the MCL-1 nucleotide insertions in two transfected human cell lines. The promoter region being evaluated and the length of the transfected segment are likely to influence the findings (2). Because we have not performed luciferase assays, we can neither confirm nor refute their findings. However, using a genetic algorithm for eukaryotic promoters (RegScan [http://wwwmgs.bionet.nsc.ru/mgs/]), we found that the predicted MCL-1 promoter activity score was 0.87 for the wild-type promoter, 0.99 for the +6 insertion, 0.96 for the +12 insertion, and 0.57 for the +18 nucleotide insertion (Fig. 1C). The validity of this algorithm is supported by a high concordance between the predicted value and luciferase activity for a polymorphic allele (G125A) of the BAX promoter in CLL (4) and publications on the computer program (5). Can a theoretical algorithm prediction be used to refute practical laboratory data? Not entirely, although it gives a reason to explore alternative explanations. Other factors may regulate the effect of nucleotide insertions on gene transcription, and these might also be tissue specific. Moreover, the +6 and +18 nucleotide insertions are likely to have different effects. Therefore, at present, it is not possible to draw definitive conclusions regarding the effect of nucleotide insertions on MCL-1 gene expression. Even if the nucleotide insertions negatively regulate gene expression, they may still be markers for other stimulatory influences on MCL-1 gene expression. Future studies focused on promoter activity and regulatory factors would help to resolve this issue.
Prognostic significance. Iglesias-Serret et al. assert that, because these insertions are polymorphisms, they are unlikely to predispose to CLL. This was never our conclusion. We simply reported the association of these insertions with clinical endpoints in CD38-negative CLL patients. The assertion by Freeman et al. that these nucleotide insertions are of no prognostic importance in CLL simply because these are polymorphisms is not consistent with available information. The effect of polymorphisms on prognosis in cancer is well known (6,7), as is acknowledged by Vargas et al. This is further supported by a recent paper (8) that confirms the critical importance of a BAX promoter single-nucleotide polymorphism, G125A, first reported by us (9), in influencing treatment response and overall survival in CLL.
REFERENCES
(1) Bingle CD, et al. Exon skipping in Mcl-1 results in a bcl-2 homology domain 3 only gene product that promotes cell death. J Biol Chem 2000;275:22136–46.
(2) Akgul C, et al. Functional analysis of the human MCL-1 gene. Cell Mol Life Sci 2000;57:684–91.[CrossRef][Web of Science][Medline]
(3) Bae J, et al. MCL-1S, a splicing variant of the antiapoptotic BCL-2 family member MCL-1, encodes a proapoptotic protein possessing only the BH3 domain. J Biol Chem 2000;275:25255–61.
(4) Moshynska O, Moshynskyy I, Misra V, Saxena A. G 125A single-nucleotide polymorphism in the human BAX promoter affects gene expression. Oncogene 2005;24: 2042–9.[Medline]
(5) Kolchanov NA, et al. Integrated databases and computer systems for studying eukaryotic gene expression. Bioinformatics 1999;15:669–86.
(6) Lenz HJ. The use and development of germline polymorphisms in clinical oncology. J Clin Oncol 2004;22:2519–21.
(7) Loktionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer Lett 2004;208:1–33.[CrossRef][Medline]
(8) Starczynski J, et al. Common polymorphism G (-248)A in the promoter region of the bax gene results in significantly shorter survival in patients with chronic lymphocytic Leukemia once treatment is initiated. J Clin Oncol 2005;23:1514–21.
(9) Saxena A, et al. Association of a novel single nucleotide polymorphism, G (-248)A, in the 5'-UTR of BAX gene in chronic lymphocytic leukemia with disease progression and treatment resistance. Cancer Lett 2002;187: 199–205.[CrossRef][Web of Science][Medline]
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