CORRESPONDENCE

RESPONSE: Re: HRAS1 Rare Minisatellite Alleles and Breast Cancer in Australian Women Under Age Forty Years

John L. Hopper, Frank A. Firgaira, Gillian S. Dite, Graham G. Giles, Margaret R. E. McCredie, Melissa C. Southey, Deon J. Venter, Ram Seshadri, Christopher R. E. McEvoy

Affiliations of authors: J. L. Hopper, G. S. Dite, The University of Melbourne, Centre for Genetic Epidemiology, Carlton, Victoria, Australia; F. A. Firgaira, R. Seshadri, C. R. E. McEvoy, Department of Haematology and Genetic Pathology, Flinders University, and Flinders Medical Centre, Bedford Park, South Australia; G. G. Giles, Cancer Epidemiology Centre, Anti-Cancer Council of Victoria, Carlton, Australia; M. R. E. McCredie, Cancer and Epidemiology Research Unit, New South Wales Cancer Council, Kings Cross, Australia, and Department of Preventative and Social Medicine, University of Otago, New Zealand; M. C. Southey, D. J. Venter, Department of Pathology and Research, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia, and Department of Pathology, The University of Melbourne, Parkville, Australia.

Correspondence to: John L. Hopper, Ph.D., The University of Melbourne, Centre for Genetic Epidemiology, 200 Berkeley St., Carlton, Victoria 3053, Australia (e-mail: j.hopper{at}gpph.unimelb. edu.au).

We welcome Dr. Krontiris' offer to exchange samples for repeat genotyping between our laboratories to help resolve the issues raised by our report and his letter.

Dr. Krontiris considers our study to have two flaws. On the technical side, we do not believe that our polymerase chain reaction (PCR)-based genotyping system is failing to detect large HRAS1 variable number of tandem repeats (VNTR) alleles due to "allele steal" (dropout). We were well aware of this phenomenon when we commenced this work and undertook considerable PCR optimization to reduce or eliminate it (1). We used a proofreading enzyme system. Trials with dimethyl sulfoxide and multistage cycling protocols did not provide improvements. (Betaine was not tested.) Our technique allowed us to amplify HRAS1 allele heterozygote combinations with large size differences; i.e., a1, a4 + 3, and a1, a4 + 8 (see Table 1 from our study) and a1, a4 + 15 (unpublished results). We did not experience allele dropout.

Furthermore, although our finding of a combined case and control a4 allele frequency of 0.03 (95% confidence interval [CI] = 0.02–0.04) clearly differs from the value of 0.09 (95% CI = 0.07–0.11) quoted by Dr. Krontiris based on older studies using Southern blot analysis protocols (2), it is not inconsistent with more recent work reporting values in control subjects from 0.015 to 0.045 (35) or with the estimate derived from controls by Dr. Krontiris' laboratory (6) of 0.06 (95% CI = 0.03–0.09) (P = .15; two-sided test).

An important finding of our study was that, in control subjects, the rare allele frequency using the newer method (0.17; 95% CI = 0.14–0.20) was substantially different from that found by older Southern blot analysis (0.06; 95% CI = 0.05–0.07) (P<.001; two-sided test). Even in recent work from Dr. Krontiris' laboratory (6), the allele frequency in control subjects is more than twice as large (0.13; 95% CI = 0.09–0.18) as that found in his previous work using Southern blot analysis (P<.001), yet it is no different from our estimate, once sample sizes are taken into consideration (P = .25; two-sided test).

On the conceptual side, the argument proposed by Dr. Krontiris is predicated on the assumption that there is a real association of breast cancer with the so-called "rare alleles," as measured by Southern blot analysis. We do not presume to know why there appears to be a difference between the previous apparent association with a risk factor measured with poor resolution and the lack of association in our study using a higher resolution technique. We are not at odds with Dr. Krontiris' summation that, with or without rebinning of HRAS1 alleles, our data from women under the age of 40 years reveal no difference in frequency of rare alleles between case and control subjects.

Despite the now apparent large misclassification error of the Southern blot analysis methodology for assigning rare alleles, Dr. Krontiris seems to continue to believe that the results of the meta-analysis remain valid when the newer high-resolution technologies are applied, referring to the common alleles as "low-risk" alleles. The possible explanation for the discrepancy between our null result and that of the meta-analysis—that the putative genetic effect "may be confined to or may be stronger in cancers occurring at a later age"—was only raised by us in response to a reviewer's opinion and is not one that we would favor without having studied older onset case patients using the newer methods. In this regard, we would invite Dr. Krontiris to help resolve the issue of a possible age-related difference in association by studying with us a large number of population-based case and control subjects with later onset disease, whom we have recently collected as part of the Cooperative Family Registry for Breast Cancer Studies (CFRBCS) (7), funded by the National Institutes of Health.

REFERENCES

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4 Phelan CM, Rebbeck TR, Weber BL, Devilee P, Ruttledge MH, Lynch HT, et al. Ovarian cancer risk in BRCA1 carriers is modified by the HRAS1 variable number of tandem repeat (VNTR) locus. Nat Genet 1996;12:309–11.[Medline]

5 Ryberg D, Lindstedt BA, Zienolddiny S, Haugen A. A hereditary genetic marker closely associated with microsatellite instability in lung cancer. Cancer Res 1995;55:3996–9.[Abstract]

6 Weitzel JN, Ding S, Larson GP, Nelson RA, Goodman A, Grendys EC, et al. The HRAS1 minisatellite locus and risk of ovarian cancer. Cancer Res 2000;60:259–61.[Abstract/Free Full Text]

7 Hopper JL, Chenevix-Trench G, Jolley DJ, Dite GS, Jenkins MA, Venter DJ, et al. Design and analysis issues in a population-based, case–control–family study of the genetic epidemiology of breast cancer and the Co-operative Family Registry for Breast Cancer Studies (CFRBCS). Monogr Natl Cancer Inst 1999;26:95–100.[Medline]


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