A Breast Cancer-Associated SNP Adjacent to ESR1 Correlates with Oestrogen Receptor-α (ERα) Level in Invasive Breast Tumours.
Authors: Drury S. , Drury S. , Johnson N. , Hills M. , Salter J. , Salter J. , Afentakis M. , Dunbier A. , Dunbier A. , Folkerd E. , Peto J. , Peto J. , Fletcher O. , Dowsett M. , Dowsett M. , FACE Trialists . 
Published: December 2010

Abstract
Aim: To identify whether breast cancer-associated SNP rs2046210 is associated with ERα level in invasive breast tumours.BackgroundA recent genome-wide association study identified SNP rs2046210 at 6q25.1 as having a strong association with breast cancer risk1. The SNP is located 29kb upstream of the first untranslated exon of ESR1 and 180kb upstream of the transcription start site2. Rs2046210 is not in linkage disequilibrium (LD) with two of the most widely studied polymorphisms in ESR1. We test here the hypothesis that rs2046210 may be associated with altered ESR1 expression.MethodsPatients with both leukocyte DNA and invasive breast tumour paraffin blocks available were identified from two of our ongoing tissue collections: Femara Anastrazole Clinical Evaluation (FACE) and the British Breast Cancer (BBC) study. Germline DNA was extracted from bloods using the QIAamp DNA Blood Mini Kit. PCR was performed with primers spanning the SNP site, with a single basepair mismatch in the reverse sequence to generate an HhaI restriction site in the presence of the C allele. PCR product was run on a 3% agarose gel to confirm presence of a single 120bp band. Restriction digest with HhaI was then performed and products run on a 4% Metaphor agarose gel. Genotype was assigned as follows: 97bp band only = wild-type (C/C); 97bp and 120bp band = heterozygote (C/T); 120bp band only = variant (T/T). From corresponding invasive breast tumour, ERα was assessed on 4μm whole sections using clone 6F11 (Vector Labs) and quantified by H-score. Mean H-scores (left and right invasive breast tumours) were used for the BBC group. ERα- samples (H-score≤1.0) were excluded. Analysis was by ANOVA using non-parametric bias-corrected and accelerated 95% bootstrap confidence intervals (2000 replications), with genotype fitted as a score and study as a stratifying co-variate.ResultsH-score (mean) by genotypeStudyWild-typeHeterozygoteVariantFACE (n=280)171.8180.9184.9BBC (n=46)180.0178.9194.1Fitted mean172.6179.7186.7(% samples)(45)(44)(11) Minor allele frequency was 33%, which is similar to the 38% previously reported in patients of European ancestry1. Within FACE, increased ERα was seen with presence of variant SNP. In the BBC group, variant SNP patients had higher ERα than both wild-type and heterozygote. Overall, there was a significant difference in ERα score per genotype group of 7.05, (95% CI 0.7-13.5, p=0.035). This was circa 4% difference in H-score per variant allele.ConclusionThe variant genotype of SNP rs2046210 is associated with increased ERα expression. While the increase contributed by the variant allele is relatively modest, this may partly explain why the SNP is associated with increased breast cancer risk. Similar studies should be conducted in normal breast tissue.1Zheng et al. (2009) Nat. Gen. 41(3): 324-3282Kos et al. (2001) Mol. Endocrinol. 15: 2057-2063
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4138.