CORRESPONDENCE

Re: Genetic Analysis of the {beta}-Tubulin Gene, TUBB, in Non-Small-Cell Lung Cancer

Sanja Sale, Peter J. Oefner, Branimir I. Sikic

Affiliations of authors: S. Sale, B. I. Sikic, Oncology Division, Department of Medicine, Stanford University School of Medicine, Stanford, CA; P. J. Oefner, Stanford Genome Technology Center, Palo Alto, CA.

Correspondence to: Branimir I. Sikic, M.D., Oncology Division, CCSR 1105, Stanford Medical Center, Stanford, CA 94305–5151 (e-mail: brandy{at}stanford.edu).

Recently, Kelley et al. (1) reported that there were no amino-acid-altering mutations in the class I {beta}-tubulin (also known as M40 or TUBB) among 25 lung cancer cell lines and 20 primary non-small-cell lung cancers (NSCLCs). This result contrasts with a report by Monzo et al. (2) that 33% of 49 NSCLC samples harbored mutations in the {beta}-tubulin gene and were "a strong predictor of the response to the antitubulin drug paclitaxel."

We have recently reported strong conservation of the class I {beta}-tubulin gene among human populations and an absence of mutations of this gene in ovarian and lung cancers (3). DNA was isolated from 93 control individuals representing a wide variety of ethnicities and from 79 cancer specimens or human tumor cell lines. There were 62 specimens derived from ovarian carcinomas, including 32 without prior exposure to paclitaxel, and 30 known paclitaxel-resistant specimens. We also included 17 nontreated NSCLC specimens. We screened for sequence variations in exons 1, 2, 3, the first half of exon 4, and flanking noncoding regions of human class I {beta}-tubulin by using denaturing high pressure liquid chromatography (DHPLC). The second half of exon 4 was analyzed by direct sequence analysis because the 3`-UTR region contains a run of G's that cause slippage and, consequently, formation of heteroduplexes that may affect the ability of DHPLC to detect mutations.

None of the 172 tumor samples, cell lines, xenografts, or controls had nonsynonymous (i.e., amino acid altering) mutations or polymorphisms in the coding region. We detected two silent polymorphisms in exon 4—Leu217Leu (CTG/ CTA) and Gly400Gly (GGC/GGT)— with minor allele frequencies of 17% and 0.5%, respectively. The amino acid sequence of class I {beta}-tubulin was completely conserved among chimpanzee, orangutan, gorilla, and humans. Nucleotide diversity analysis revealed that human class I {beta}-tubulin is one of the most conserved genes studied so far, with 4.4-fold less sequence diversity than that found in 106 other human genes (4).

What are the reasons for the discrepancies between the study by Monzo et al. (2) and the reports by Kelley et al. (1) and our group (3)? Specificity of the primers is one of the crucial issues in the analysis of single nucleotide polymorphisms and mutations in any gene, particularly those with more than one isotype or with pseudogenes. Sequence variations in {beta}-tubulin are especially difficult to study because there are six {beta}-tubulin isotypes and more than 20 {beta}-tubulin pseudogenes, all of which are highly homologous.

In analyzing class I {beta}-tubulin, Monzo et al. (2) designed primers TP4 and STP4, which have 100% identity not only for class I {beta}-tubulin but also for four {beta}-tubulin pseudogenes (GenBank accession numbers V00598, K00841, J00317, and M24191). Moreover, another primer for exon 4, STB4, does not have a 100% match with any gene, including class I {beta}-tubulin. The closest match is located on chromosome 16 and is not specific for the whole primer sequence. Lack of specificity for the class I {beta}-tubulin isotype could have resulted in a mixture of polymerase chain reaction (PCR) products and a false-positive detection of mutations. Kelley et al. (1) provide experimental evidence supporting this hypothesis. We used the class I {beta}-tubulin sequence retrievable from GenBank (accession number J00314) to design five pairs of primers encompassing all exons, adjacent intronic sequences, and 5`- and 3`-UTRs and to test amplicon specificity (Fig. 1Go).



View larger version (7K):
[in this window]
[in a new window]
 
Fig. 1. Schematic presentation of the class I {beta}-tubulin gene and the location of the five pairs of primers specific for this isotype (3). EnF, forward primer; EnR, reverse primer (n = 1–4). Two pairs of primers were used to amplify exon 4. (Fig. reproduced from (3) with permission from American Association for Cancer Research.)

 
The paper of Monzo et al. (2) is also problematic in its discussion of the functional domains of the {beta}-tubulin protein designating the domain encoded by exon 1 as the paclitaxel binding region. Electron crystallographic analysis has shown that the paclitaxel binding site is located in the intermediate region of the {beta}-tubulin protein encoded by exon 4 (5).

Although Kelley et al. (1) and our group used different primers and sequencing methods, we obtained similar results, thus providing convincing evidence that sequence variations in class I {beta}-tubulin in NSCLC and ovarian cancer are uncommon. The extremely high conservation of amino acid sequence in control individuals and the absence of mutations in NSCLC and ovarian carcinomas make it unlikely that variations in class I {beta}-tubulin isotype are a clinically relevant cause of resistance to taxanes in these diseases. We agree with Kelley et al. (1) that it may be wise to postpone any ongoing clinical trials in which the treatment for NSCLC is based on the {beta}-tubulin "mutation" status of the patients. The example of class I {beta}-tubulin highlights the need to verify and confirm reports of mutations in genes that are found to be associated with various clinical parameters, such as drug response or survival.

REFERENCES

1 Kelley MJ, Li S, Harpole DH. Genetic analysis of the beta-tubulin gene, TUBB, in non-small-cell lung cancer. J Natl Cancer Inst 2001;93:1886–8.[Free Full Text]

2 Monzo M, Rosell R, Sanchez JJ, Lee JS, O'Brate A, Gonzalez-Larriba JL, et al. Paclitaxel resistance in non-small-cell lung cancer associated with beta-tubulin gene mutations. J Clin Oncol 1999;17:1786–93.[Abstract/Free Full Text]

3 Sale S, Sung R, Shen P, Yu K, Wang Y, Duran GE, et al. Conservation of the class I beta-tubulin gene in human populations and lack of mutations in lung cancers and paclitaxel-resistant ovarian cancers. Mol Cancer Ther 2002;1:215–25.[Abstract/Free Full Text]

4 Cargill M, Altshuler D, Ireland J, Sklar P, Ardlie K, Patil N, et al. Characterization of single-nucleotide polymorphisms in coding regions of human genes. Nat Genet 1999;22:231–8.[Medline]

5 Nogales E, Wolf SG, Downing KH. Structure of the beta-tubulin dimer by electron crystallography. Nature 1998;391:199–203.[Medline]


This article has been cited by other articles in HighWire Press-hosted journals:


             
Copyright © 2002 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement