Correspondence to: Theodore G. Krontiris, M.D., Ph.D., City of Hope National Medical Center, Beckman Research Center, Division of Molecular Medicine, 1500 East Duarte Rd., Duarte, CA 91010 (e-mail: tkrontir{at}coh.org).
The report by Firgaira et al. (1) makes the potentially important observation that breast cancer in women under 40 years of age may not demonstrate association with rare alleles of the HRAS1 minisatellite. Breast cancer in younger women may differ genetically and biologically from late-onset disease and, therefore, the lack of association with genetic modifiers of modest effect would be an interesting, but not unexpected, outcome.
Although the authors mention potential biologic differences between cancers in patients of different age groups as one possible basis for the observed difference in rare allele association, they clearly favor a more prosaic explanation: that "new methods" with greater resolving power challenge the original observations and meta-analysis. Under this construction, the previously observed association is merely an artifact of the inability of previous methods to resolve more rare alleles among larger sized, common (low-risk) alleles.
I believe this study has two flaws, one technical and one conceptual, that undermine its conclusions. On the technical side, the newer methods using fluorescence-based genotyping on automated sequencing platforms do resolve alleles better. But a critical issue is the reliability of polymerase chain reaction (PCR) amplification in detecting the larger alleles that sequencers are capable of resolving. The PCR-based method described in the report by Firgaira et al. is not satisfactory in this regard. We know from our own experience that PCR strategies with straightforward cycling protocols can preferentially amplify lower molecular weight alleles at the expense of larger ones ("allele steal") or they can create artifactual rare alleles from larger rare and common alleles. Our own protocol combines additives (dimethyl sulfoxide and betaine [N,N,N-trimethylglycine]), proofreading enzymes, and a multistage cycling protocol to minimize artifactual loss of larger alleles (2,3).
It seems likely that genotyping artifacts introduced by a technique that is not robust may have affected the outcome of their study in a major way. For example, the frequency of their largest common allele, a4, is only slightly more than half of that reported in other studies. This loss is not compensated by the appearance of newly resolved alleles a4 - 3, 4 - 2, 4 - 1, 4 + 1, 4 + 2, and 4 + 3. In contrast to the statements in the "Discussion" section that provide a larger number, I compute a total allele frequency in this group from Table 1 (1) of 0.05, compared with the meta-analysis value of 0.09. Therefore, nearly half the high-molecular-weight, low-risk alleles are unaccounted for in their cohort. Since the authors maintain that the population they are studying is ethnically comparable to that of other studies in the literature (an assertion with which I concur), then there must be a technical flaw in the genotyping. Of potential relevance is the fact that only 63% of available blood samples are reported for case patients and 80% of available samples are reported for control subjects. It would be important to know if technical failures account for any of this discrepancy. Although the differences discussed above in low-risk allele frequencies are small, these differences can have a very large effect on the casecontrol distribution of rare alleles.
The authors state that their analysis reveals no deviation from HardyWeinberg equilibrium, which, if true, would argue against substantial technical artifact. However, I strongly question whether, given the sample size, such analysis is sensitive enough to detect critical differences among so many sparsely populated allele classes. Therefore, I believe that any conclusions drawn from these data must be set aside until the technical question is resolved. A blind exchange of samples for repeat genotyping between my laboratory and that of the Australian group would settle this issue.
On the conceptual side, one would predict that, if lack of resolution of more alleles in larger, low-risk allele classes were the basis for the previously noted association, then simply regrouping newly resolved alleles should reproduce the old association. In other words, pretending that a2 - 2, a2 - 1, a2, a2 + 1, and a2 + 2 are all a2 (and likewise for a3 and a4) should recreate the casecontrol differences. No rebinning of a1-related alleles is required, since they were resolved by earlier methods. Ignoring the issue of genotyping accuracy for the moment, rebinning the alleles in this report still reveals no difference in the frequency of rare alleles between case patients and control subjects. Therefore, the conclusion concerning the effect of technical resolution of alleles on cancer association is questionable.
Until the issues raised herein are addressed, the possibility of age-related differences in the association of rare HRAS1 alleles with breast cancer remains an interesting, but unproved, outcome.
REFERENCES
1
Firgaira FA, Seshadri R, McEvoy CR, Dite GS, Giles GG, McCredie MR, et al. HRAS1 rare minisatellite alleles and breast cancer in Australian women under age forty years. J Natl Cancer Inst 1999;91:210711.
2 Larson GP, Zhang G, Ding S, Foldenauer K, Udar N, Gatte RA, et al. An allelic variant at the ATM locus is implicated in breast cancer susceptibility. Genet Test 19971998;1:16570.[Medline]
3
Ding S, Larson GP, Foldenauer K, Zhang G, Krontiris TG. Distinct mutation patterns of breast cancer-associated alleles of the HRAS1 minisatellite locus. Hum Mol Genet 1999;8:51521.
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |