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No Association Between the MDM2 –309 T/G Promoter Polymorphism and Breast Cancer in African-Americans or Whites

Department of Epidemiology, School of Public Health and Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina

Requests for reprints: Robert Millikan, Department of Epidemiology, CB 7435 School of Public Health, University of North Carolina, Chapel Hill, NC 27599-7435. Phone: 215-440-9300; Fax: 215-440-9337. E-mail: millikan{at}email.unc.edu

	Introduction

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MDM2, a protein that binds and inactivates the tumor suppressor p53, is overexpressed in a variety of human cancers (1). Bond et al. (2) recently identified a single nucleotide polymorphism in the MDM2 gene, –309 T/G within the MDM2 promoter (database for single nucleotide polymorphism reference sequence number 2279744; http://snp500cancer.nci.nih.gov). The G allele showed increased affinity for the transcriptional activator Sp1, resulting in elevated MDM2 transcription, higher MDM2 protein levels, and enhanced p53 inhibition. Among 88 members of Li-Fraumeni syndrome families who carried germ line mutations in p53, persons with one or two copies of the MDM2 –309 G allele showed earlier onset of cancer, including breast cancer, and G/G homozygous individuals showed increased frequency of multiple primary cancers. We examined the association of MDM2 genotype and breast cancer in the Carolina Breast Cancer Study, a population-based case-control study of African-Americans and Whites in North Carolina.

	Materials and Methods

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Study Population
Details regarding the Carolina Breast Cancer Study have previously been described (3, 4), including extraction of germ line DNA from peripheral blood lymphocytes (5), analysis of p53 mutations in tumor blocks (6), p53 immunohistochemistry (7), human epidermal growth factor receptor 2 immunohistochemistry (5), and determination of estrogen receptor and progesterone receptor status (8). p53 mutational status was determined in the subset of Carolina Breast Cancer Study cases enrolled between 1993 and 1996 (6).

MDM2 Genotyping
Genotyping was conducted on germ line DNA using a Minor Groove Binding Eclipse assay developed by Nanogen, Inc. (Bothell, WA). The (G) allele probe was labeled on the 3' end with the FAM reporter dye (nucleotide sequence, 5'-CCCGCGCCGcAG-3', variant site in lower case) and the (T) allele probe was labeled on the 3' end with the TET reporter dye (5'-CCCGCGCCGaA*G-3'). The asterisk denotes a Superbase. Superbases are modified nucleotides that permit highly specific binding to GC-rich DNA templates. The forward PCR primer was 5'-ACCTGCGATCATCCGGACCT-3' and the reverse primer was 5'-TGCGG*GGCCGCT-3'. Probes and primers were designed for the complementary DNA strand. PCR amplification was done on a GenAmp 9700 thermocycler (Perkin-Elmer, Wellesley, MA) under the following conditions: 1 cycle of 50.0°C for 2 minutes, 1 cycle of 95.0°C for 2 minutes, followed by 50 cycles of 95.0°C for 5 seconds (denature), 28.0°C for 20 seconds (anneal/detection), and 76.0°C for 30 seconds (extension). Post-PCR melt curve analysis was done on the ABI Prism 7700, and data was analyzed using the Minor Groove Binding Eclipse Melt Macro for Microsoft Excel (EclipseMeltMacro_v2.332_050519.xls) provided by Nanogen. Further details are available from the authors upon request.

Genomic DNA samples obtained from the Coriell Cell Repositories (Camden, NJ) were sequenced to determine MDM2 –309 genotype status and used as positive controls. Positive controls were Coriell sample number NA12749 for G/G (FAM) and NA11587 for T/T (TET). DNA samples that did not amplify or could not be scored were repeated. Samples that did not amplify on the third PCR run were designated as "missing" (n = 8). A randomly selected 10% of samples were repeated, and all results matched the initial analysis.

Statistical Analysis
Genotype frequencies were compared using the Cochran Armitage test for trend (9), and allele frequencies were compared using ² tests. Odds ratios (OR) and 95% confidence intervals were calculated using SAS (SAS Institute, Cary, NC) and incorporated offset terms to account for sampling probabilities for cases and controls. Among cases, age at onset of breast cancer was compared across strata defined by MDM2 genotype using ANOVA for means and the Kruskal-Wallis test for medians.

	Results

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MDM2 –309 allele and genotype frequencies did not differ between cases and controls, and ORs for breast cancer were close to the null in African-Americans and Whites (Table 1). The frequency of the G allele in Whites was slightly higher than 0.33 [see Bond et al. (2)]. Genotype frequencies did not show significant departures from Hardy-Weinberg equilibrium among African-American (P = 0.22) or White (P = 0.28) cases, but there were more G/G homozygotes than expected among African-American (P = 0.001) and White (P = 0.001) controls. ORs did not differ according to menopausal status or for subgroups of cases defined by in situ versus invasive breast cancer, p53 mutational status, p53 immunohistochemistry, human epidermal growth factor receptor 2 immunochemistry, estrogen receptor, or progesterone receptor status (data not shown). ORs were unchanged when we adjusted for or stratified on family history of cancer as well as other breast cancer risk factors (data not shown). ORs were close to the null for breast cancer diagnosed before age 40, age 40 to 50, and over age 50 (Table 1).

	Discussion

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	Footnotes

 
Grant support: Specialized Program of Research Excellence in Breast Cancer (NIH/National Cancer Institute P50-CA58223), Lineberger Cancer Center Core Grant (P30-CA16086), and the Center for Environmental Health and Susceptibility (NIEHS P30-ES10126) at the University of North Carolina at Chapel Hill.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/ 8/05; revised 10/19/05; accepted 11/ 9/05.

	References

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1. Freedman D, Levine A. Regulation of the P53 protein by the MDM2 oncoprotein—38th G.H.A. Clowes Memorial Award Lecture. Cancer Res 1999;59:1–7.[Free Full Text]
2. Bond G, Hu W, Bond E, et al. A single nucleotide polymorphism in the MDM2 promoter attenuates the P53 tumor suppressor pathway and accelerates tumor formation in humans. Cell 2004;119:591–602.[CrossRef][Medline]
3. Newman B, Moorman PG, Millikan R, et al. The Carolina Breast Cancer Study: integrating population-based epidemiology and molecular biology. Breast Cancer Res Treat 1995;35:51–60.[CrossRef][Medline]
4. Hall I, Moorman P, Millikan R, Newman B. Comparative analysis of breast cancer risk factors among African-American women and white women. Am J Epidemiol 2005;161:40–51.[Abstract/Free Full Text]
5. Millikan R, Eaton A, Worley K, et al. HER2 codon 655 polymorphism and risk of breast cancer in African Americans and whites. Breast Cancer Res Treat 2003;79:355–64.[CrossRef][Medline]
6. Conway K, Edmiston S, Cui L, et al. Prevalence and spectrum of p53 mutations associated with smoking in breast cancer. Cancer Res 2002;62:1987–95.[Abstract/Free Full Text]
7. Furberg H, Millikan RC, Geradts J, et al. Environmental factors in relation to breast cancer characterized by p53 protein expression. Cancer Epidemiol Biomarkers Prev 2002;11:829–35.[Abstract/Free Full Text]
8. Huang W-Y, Newman B, Millikan R, Schell M, Hulka B, Moorman P. Hormone-related factors and risk of breast cancer by estrogen receptor and progesterone receptor status. Am J Epidemiol 2000;151:703–14.[Abstract/Free Full Text]
9. Schaid D, Jacobsen S. Biased tests of association: comparisons of allele frequencies when departing from Hardy-Weinberg equilibrium. Am J Epidemiol 1999;149:706–11.[Abstract/Free Full Text]
10. Gibson G. Population genomics: finding the variants of mass disruption. Curr Biol 2003;13:R901–3.[Medline]

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