Breast cancer is one of the polygenetic disorders with complex inheritance patterns. Recently, the determination of genetic polymorphisms provided a new approach to investigate the etiology of such complex genetic diseases . Reports indicate that breast cancer susceptibility and prediction can be influenced by the polymorphism of cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene, which encodes a classic molecule offering a co-inhibitory signal to T cell activation . Additionally, some other genes encoding novel co-inhibitors, such as PD-1 and B and T lymphocyte attenuator (BTLA), are attractive in case-control studies, as candidate genes between the gene polymorphisms and the risk of diseases [20, 21]. As another co-inhibitory molecule investigated more recently, B7-H4 has shown a potential effect on tumors, by which tumors may escape from an anti-tumor immune response. To elaborate, B7-H4 protein is over expressed in several malignant tumors while is little expressed in normal tissues [6–8, 10], and its high expression in breast cancer decreased the number of TILs and prevented tumor cells from undergoing apoptosis [6, 7, 11].
In our case-control study, we chose three potentially functional polymorphisms of B7-H4, rs10754339, rs10801935 and re3738414, and firstly detected their association with the risk and prognosis of breast cancer in Chinese Han women. The results indicate that some of the alleles, genotypes and haplotypes of these three SNPs are associated with the risk and prognosis of breast cancer, which will be discussed below respectively. According to our results, in rs10754339, women with the AG genotype and G allele are likely to have an increased risk of breast cancer (OR = 1.455, 95% CI 1.119-1.892; OR = 1.325, 95%CI 1.073-1.637, respectively), indicating that the rs10754339 G allele maybe plays a risk role in breast cancer. It has been suggested that B7-H4 mRNA is widely distributed in lymphoid and nonlymphoid tissues. However, the protein is rarely expressed in normal tissues but highly expressed in many kinds of tumor tissues including breast cancer [3, 6–10]. Also, 3'-UTR can regulate gene expression at many different levels, and cis-acting elements of 3'-UTR mediate the stability, degradation, and subcellular localization of mRNA [12, 22]. Therefore, rs10754339 located on ESE may regulate the production of mRNA molecules through above mechanism, and G allele may be associated with an increased expression of B7-H4 and then influence the susceptibility of breast cancer. In addition, most tissues contain two types of B7-H4 transcriptions which are ~2.0 kb and ~800 bp. The larger one is major and their difference is that the small one has 3'-UTR spliced out partly . Rs10754339 may also be involved in the formation and expression of different types of B7-H4 transcriptions.
In rs10801935, women with the CC genotype are likely to have a decreased risk of breast cancer (OR = 0.328, 95% CI 0.145-0.739), indicating that the rs10801935 CC genotype in intron 1 may be a protective factor in breast cancer. As we know, human gene expression requires transcription controls mediated by the presence of intron sequences, especially, the first intron, which contains various regulatory elements and splicing control elements [14, 23]. Mutations occurring in introns can induce the aberrant splicing due to the disruption of the splice site, the splicing enhancers and silencers, or the alteration of the pre-mRNA secondary structure, which results in translational prevention [16, 24, 25]. Supposed to change the secondary structure of pre-mRNA, rs10801935 can possibly influence B7-H4 translational efficiency by causing deviant splicing, and CC genotype may down-regulate the B7-H4 expression and decrease the breast cancer risk.
Our research also suggests that women with the AA genotype and the A allele in rs3738414 are less likely to have breast cancer (OR = 0.412, 95% CI 0.203-0.835; OR = 0.698, 95% CI 0.564-0.864, respectively), indicating that the A allele of rs3738414 may have a potentially protective effect on breast cancer. 5'-UTR, upon which a number of DNA binding sites of regulatory factors exist, is important for post-transcriptional modification and translation efficiency . Using Pupasview software , the rs3738414 is supposed to be located on ESE, and the mutation of rs3738414 perhaps changes the protein binding pattern, and further affects the B7-H4 expression. Consequently, rs3738414 A allele may reduce the B7-H4 expression and decrease the susceptibility of breast cancer.
In addition, according to the analysis for the association between B7-H4 gene polymorphisms and clinical presentations, we found that the rs10754339 GG genotype was significantly associated with lymph node metastasis and PR status, and the G allele and the AG genotype were respectively associated with lymph node metastasis and ER status. However, the other two SNPs in our study were found to be irrelevant to clinicopathologic features. Lymph node involvement, ER and PR status are important in predicting the long-term survival of breast cancer cases. Lymph node metastasis positive cases have higher mortality , and steroid hormone receptors are valuable as predictive markers of endocrine therapy . Therefore, rs10754339 may be significant to forecast the prognosis of breast cancer.
The association between B7-H4 gene and breast cancer was further confirmed by the analysis of haplotype. Results suggested that the AAA haplotype showed a significantly decreased risk of breast cancer, whereas GAG haplotype had a contrary outcome (OR = 0.689, 95% CI 0.539-0.881; OR = 1.511, 95% CI 1.125-2.031). To elaborate, the AAA haplotype appears to be a protective factor in breast cancer, but the GAG haplotype may be a risk factor in breast cancer. Furthermore, we found that both AAA and GCG haplotypes had significantly correlations with the clinical presentations. These haplotypes may also be the valuable prognostic factors for survival of breast cancer cases.