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Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, E-mail: cooneycraiga{at}uams.edu
Harnwell, Box: 834, 3820 Locust Walk, Philadelphia, PA 19104
Division of Biometry, University of Arkansas for Medical Sciences, Little Rock, AR 72205
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079
Dear Editor:
Dr. Waterland makes two general points about study design in reference to our recent article (1
). First he says that it is necessary to demonstrate an effect using all offspring (carrying the gene of interest) from several litters in a study. While this could be a useful design, it is only one of several and we disagree that it is required. Both Zorilla (2
) and Holson and Pearce (3
) have discussed the design and analysis of experiments in multiparous species. Both caution strongly against measuring all the offspring in a few litters and suggest instead that choosing a limited number of offspring from each of many litters is preferable. Our method of relating DNA methylation to maternal diet via phenotype is less direct than assaying mice for DNA methylation by a sampling method that is blinded to phenotype. Dr. Waterland’s approach is widely applicable and could be used with any rodent strain, and for that matter with most experimental organisms, regardless of whether they show obvious epigenetic phenotype. It should be possible to incorporate Dr. Waterland’s design as a separate study arm or as a predesignated subset of a larger study (e.g., all offspring from the first 10 litters per group to appear after a certain predesignated date). In choosing a study design it would be useful to know a priori the variance within litters and the variance between litters. Unless these are very different, it may be desirable to use multiple nested sampling methods.
Dr. Waterland’s second general point is that accurately quantifying site- or region-specific CpG methylation is technically demanding. We certainly agree and would point out that our methods achieve decile resolution in a system with a full range of variation (from <10% to >90%). Our methods are applicable to assays of methylation at many loci provided they show a broad range of methylation levels. It should be mentioned that others, using other methods, have also shown a broad range of methylation at Avy and some other agouti alleles of mice (4
–6
).
In further discussion, Dr. Waterland offers one possible alternative explanation for the developmental establishment of epigenetic variation that we measured in adult mice. Certainly a variety of mechanisms are possible, and although we and probably others (6
) would argue for an important role of DNA methylation, few of the developmental and intermediary steps involved in establishing adult epigenetic variation have been described (however, see references 6
and 7
). Establishing developmental mechanisms is certainly technically demanding, and although we plan to investigate some relevant aspects, elucidation of these mechanisms will likely require considerable effort on the part of numerous laboratories.
We are considering Dr. Waterland’s points in our design of upcoming studies and are considering future publication detailing our methods, especially those for site-specific CpG methylation. Previously, we provided Dr. Waterland, at his request, prepublication DNA sequence (including the Avy LTR and surrounding sequences) as well as unpublished technical details for the design of methyl supplemented diets. With this information Dr. Waterland has undertaken a similar line of investigation (8
), and we look forward to his detailed findings.
Manuscript received 17 September 2002. Revision accepted 23 September 2002.
LITERATURE CITED
1. Cooney, C. A., Dave, A. A. & Wolff, G. L. (2002) Maternal methyl supplements in mice affect epigenetic variation and DNA methylation of offspring. J. Nutr. 132:2393S-2400S.
2. Zorrilla, E. P. (1997) Multiparous species present problems (and possibilities) to developmentalists. Dev. Psychobiol. 30:141-150.[Medline]
3. Holson, R. R. & Pearce, B. (1992) Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species. Neurotoxicol. Teratol. 14:221-228.[Medline]
4. Argeson, A. C., Nelson, K. K. & Siracusa, L. D. (1996) Molecular basis of the pleiotropic phenotype of mice carrying the hypervariable yellow (Ahvy) mutation at the agouti locus. Genetics 142:557-567.[Abstract]
5. Michaud, E. J., van Vugt, M. J., Bultman, S. J., Sweet, H. O., Davisson, M. T. & Woychik, R. P. (1994) Differential expression of a new dominant agouti allele (Aiapy) is correlated with methylation state and is influenced by parental lineage. Genes Dev. 8:1463-1472.
6. Morgan, H. D., Sutherland, H. G., Martin, D. I. & Whitelaw, E. (1999) Epigenetic inheritance at the agouti locus in the mouse. Nat. Genet. 23:314-318.[Medline]
7. Whitelaw, E. & Martin, D. I. (2001) Retrotransposons as epigenetic mediators of phenotypic variation in mammals. Nat. Genet. 27:361-365.[Medline]
8. Waterland, R. A. & Jirtle, R. L. (2002) Maternal dietary methyl donor supplementation affects offspring phenotype by increasing cytosine methylation at the agouti locus in Avy mice. FASEB J. 16:A228.
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