MDM2 SNP309 is associated with high grade node positive breast tumours and is in linkage disequilibrium with a novel MDM2 intron 1 polymorphism

BMC Cancer BioMed Central Research article Open Access MDM2 SNP309 is associated with high grade node positive breast tumours and is in linkage disequilibrium with a novel MDM2 intron 1 polymorphism Fiona EM Paulin*1,4, Mary O'Neill1,5, Gillian McGregor2,4, Andrew Cassidy2,6, Alison Ashfield1,5, Clinton W Ali3,7, Alastair J Munro1,6, Lee Baker1,5, Colin A Purdie2,7, David P Lane1,8 and Alastair M Thompson1,6 Address: 1Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK, 2Department of Molecular & Cellular Pathology, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK, 3Department of Oncology, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK, 4National Translational Cancer Research Network, UK, 5Breast Cancer Research UK, UK, 6University of Dundee, Nethergate, Dundee, DD1 4HN, Scotland, UK , 7National Health Service Tayside HQ, Kings Cross, Clepington Road, DUNDEE, DD3 8EA and 8Cancer Research UK, UK Email: Fiona EM Paulin* - ; Mary O'Neill - ; Gillian McGregor - ; Andrew Cassidy - ; Alison Ashfield - ; Clinton W Ali - ; Alastair J Munro - ; Lee Baker - ; Colin A Purdie - ; David P Lane - ; Alastair M Thompson - * Corresponding author Published: 1 October 2008 BMC Cancer 2008, 8:281 doi:10.1186/1471-2407-8-281 Received: 10 April 2008 Accepted: 1 October 2008 This article is available from: http://www.biomedcentral.com/1471-2407/8/281 © 2008 Paulin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Introduction: A functional polymorphism within MDM2, SNP309 T>G, has been linked to early onset cancer. This study examined clinical associations of breast cancer with SNP309 in a Scottish Caucasian population and investigated additional MDM2 intron 1 polymorphisms. Methods: Intron 1 of MDM2 was PCR amplified and directly sequenced from 299 breast cancer patients and 275 cancer free controls and compared with clinical and pathological parameters. Results: SNP309 was observed, for the control and breast cancer cohorts respectively, at frequencies of: T/T = 44.7% and 39.5%; G/T = 42.2% and 47.2%; G/G = 13.1% and 13.4%, indicating that SNP309 is not a predisposing factor for breast cancer. The 309G/G genotype was associated with high grade tumours (OR = 1.64, 95%CI = 1.06–2.53, p = 0.025) and greater nodal involvement (OR = 2.51, 95%CI = 1.26–4.98, p = 0.009). SNP309 was not associated with an earlier age of cancer diagnosis. No association was observed between genotype and age of breast cancer diagnosis when patients were stratified by menopausal status and estrogen receptor status. Three additional low frequency SNPs were identified: 344T>A, 285G>C and 443G>T, the latter two novel. SNP285 was in complete linkage disequilibrium with SNP309 (D' = 1.0) with the minor alleles being in phase with each other. Moreover, the 285C/C, 309G/G double homozygous genotype was only observed in the breast cancer cohort. Conclusion: SNP309G/G is associated with poor prognostic breast cancer features in the Scottish population. Additionally, a novel SNP, SNP285, that is in linkage disequilibrium with SNP309, may also have a role in breast tumorigenesis. Page 1 of 10 (page number not for citation purposes) BMC Cancer 2008, 8:281 Introduction MDM2, encoded by the human homologue of Murine Double Minute oncogene, is the principal negative regulator of p53, a transcription factor which plays key roles in cell division and response to DNA damage [1,2]. p53 is frequently mutated in cancer resulting in defective functions, including apoptotic and cell cycle arrest programs [3]. MDM2 controls p53 levels and activity by a number of different mechanisms, including direct inhibition of the transcriptional activity of p53 [4]. In addition, MDM2 acts as an E3 ubiquitin ligase targeting p53 for nuclear export and proteosomal degradation [5]. Furthermore, as MDM2 is a transcriptional target for p53, through the P2 inducible promoter located in intron 1, a finely balanced negative feedback loop mechanism exists [6]. Development of cancer is often associated with defects in this p53-MDM2 regulatory circuit, and in cells with wild type p53 other alterations in the p53 pathway are often observed [3]. MDM2 is overexpressed in a number of different cancers and in breast cancer, where only 30% of tumours have mutated p53, some 40% display overexpression of MDM2, although amplifications are rare [7-10]. A single nucleotide polymorphism (SNP) within intron 1 of MDM2, a T to G substitution (T>G) at position 309 (SNP309) (rs2279744), has been shown to lead to enhanced binding of the Sp1 transcription factor resulting in elevated levels of both MDM2 mRNA and protein, thereby attenuating the p53 response [11]. In Li-Fraumeni patients, individuals homozygous or heterozygous for SNP309 (G/G or G/T) were shown to develop cancer at an earlier age than wild type individuals; in patients with sporadic soft tissue sarcomas, the 309G/G genotype correlated with an average 12 year earlier age of diagnosis [11]. SNP309 was thus postulated as a potential modulator of cancer susceptibility [11]. Subsequent studies of the SNP309 polymorphism have demonstrated variable frequencies of 309G/G depending on race and ethnicity [12-15]. Similarly, the association between SNP309 and development of cancer has produced conflicting data (reviewed in [16]). This study therefore sought to sequence the MDM2 intron 1 region around SNP309 in detail and determine SNP frequencies from a control cohort of Scottish Caucasians (n = 275) and a cohort of geographically matched Scottish Caucasian women with breast cancer (n = 299). The MDM2 SNP genotypes were examined to determine if they could be linked to an increased cancer susceptibility, age of cancer diagnosis, pathological variables and clinical outcome. Methods Patient & control samples Venous blood samples were obtained from otherwise unselected consenting patients (299) with a diagnosis of http://www.biomedcentral.com/1471-2407/8/281 primary breast cancer attending routine breast cancer clinics at Ninewells Hospital, Dundee between 1999 – 2005. Age at first cancer, menopausal status at diagnosis, family history of breast cancer, estrogen receptor (ER), progesterone receptor (PgR), HER-2 expression, pathological nodal status, tumour grade and Nottingham Prognostic Index (NPI) were recorded. Tumour grading was carried out by a specialist pathologist (CAP) and graded as defined by the NHS Breast Screening Programme guidelines [17]. NPI was calculated as described [18] and then classified into Poor (>5.4), Moderate (3.4–5.4) and Good (<3.4) prognosis. ER and PgR were scored according to the quickscore method [19] and a score ≥ 4/18 was considered to be pos (...truncated)