The Effect of rs77275268 on Gene Expression and CTCF Binding Within Locus 6q25.1

Approximately 80% of breast carcinomas present as oestrogen receptor positive (ER+), a subtype most commonly treated with oestrogen-receptor response modulators such as tamoxifen, which deprive cells of oestrogen signalling. The oestrogen receptor gene (ESR1) is located at the 6q25.1 locus, downstream of three genes CCDC170, ARMT1 and RMND1. A significant correlation has been seen between expression of these three genes and ESR1 in ER+ve breast primary tumours and further strengthened in response to aromatase inhibitor treatment. Clarifying the interplay between ESR1, ARMT1, CCDC170 and RMND1 is of great clinical significance for the development of potential breast cancer therapies. Numerous breast cancer GWAS have acknowledged the association of a number of genetic variants and breast cancer risk. Variants within 6q25.1 have displayed a highly significant association with breast cancer risk. One such SNP, rs77275268 at the 6q25.1 locus, exists in two forms, the ancestral cytosine (C) allele or as a thymine (T), with an allele frequency of 11.7%. Previous literature, has shown that this SNP disrupts a partially methylated CpG sequence that also served as a CTCF binding site. CTCF has a crucial genome-wide role in transcriptional regulation and chromatin structure, the binding of which is confirmed to be partly mediated by methylation at CpG sites. Thus, illustrating a potential mechanism by which SNPs in the 6q25.1 locus are associated with increased breast cancer risk. This project sought to investigate the mechanism that confers increased breast cancer risk in individuals who possess the thymine allele at rs77275268. To analyse the effect of this SNP in vitro, CRISPR/Cas9 genome edited MCF-7 cell lines were generated to harbour the thymine variant. The effect of altered rs77275268 SNP status on the expression of genes found within 6q25.1 (ESR1, ARMT1, CCDC170 and RMND1) was examined. Additionally, cell proliferation analysis and response to tamoxifen treatment were analysed. Chromatin Immunoprecipitation (ChIP) was also utilised to quantify variation in CTCF induced by the thymine allele at rs77275268. Finally, expression of genes in 6q25.1 were examined following treatment of a demethylating agent, decitabine. Overall, this study showed that MCF-7 cells possessing the rs77275268 thymine variant displayed decreased expression of correlated genes in locus 6q25.1; ARMT1, CCDC170 and RMND1. Phenotypically, downregulated expression did not affect cellular proliferation rates, or response to tamoxifen treatment. The, genome edited cells also exhibited a non-significant pattern of increased CTCF binding at rs77275268; seemingly caused by a reduction in methylation due to the loss of cytosine at the CpG site. Decitabine treatment, was employed as a crude mechanism of assessing methylation changes at locus 6q25.1. Interestingly demethylation following demethylation failed to restore wild-type gene expression levels at 6q25.1; thereby inferring that methylation was not the cause of reduced ARMT1, CCDC170 and RMND1 expression. In conclusion, no substantial evidence was discovered to explain the increased risk of breast cancer susceptibility previously associated with rs77275268. Further analysis is required to clarify whether reduced expression of ARMT1, CCDC170 and RMND1 is attributed to increased breast cancer risk. These findings suggest that it is possible for rs77275268 to not be the causal variant at 6q25.1, and instead be in linkage disequilibrium with another SNP, that predisposes breast cancer risk