Abstract
IntroductionThe absence of mutation or promoter hypermethylation in the BRCA2 gene in the majority of breast cancer cases has indicated alternative ways of its involvement, deregulated expression being one possibility. We show how a polymorphism in the 5' untranslated region (UTR) of BRCA2 can serve as one such factor. Based on the hypothesis that variants of genes involved in the same pathway can influence the risk provided for breast cancer, the status of p53 codon 72 polymorphism was also investigated and a possible interaction between the polymorphisms was examined.MethodsThe luciferase reporter assay followed by RNA secondary structure analysis was used for the functional characterization of -26 5' UTR G>A polymorphism in BRCA2. The genotype and the allele frequency for the polymorphisms were determined and relative risk adjusted for age was calculated in a case-control study of 576 individuals (243 patients and 333 controls) from north India.Results-26 G>A polymorphism in the 5' UTR of BRCA2 was found to be functional whereby the A allele increased the reporter gene expression by twice that of the G allele in MCF-7 (P = 0.003) and HeLa (P = 0.013) cells. RNA secondary structure analysis by two different programs predicted the A allele to alter the stability of a loop in the vicinity of the translation start site. Its direct implication in breast cancer became evident by a case-control study in which the heterozygous genotype was found to be protective in nature (Pheterozygote advantage model = 0.0005, odds ratio [OR] = 0.5, 95% confidence interval [CI] = 0.4 to 0.8), which was further supported by trends observed in a genomic instability study. The p53 codon 72 Arg homozygous genotype was found to be over-represented in patients (P = 0.0005, OR = 2.3, 95% CI = 1.4 to 3.6). The interaction study indicated an increased protection under simultaneous presence of protector genotypes of both the polymorphic loci (P = 0.0001, OR = 0.2, 95% CI = 0.1 to 0.4).ConclusionOur study shows that -26 5' UTR polymorphism in BRCA2 can modulate the fine-tuned regulation of the multifunctional gene BRCA2 and renders risk or protection according to the genotype status in the sporadic form of breast cancer, which is further influenced by the germline genetic backgrounds of codon 72 polymorphism of p53.
Highlights
The absence of mutation or promoter hypermethylation in the BRCA2 gene in the majority of breast cancer cases has indicated alternative ways of its involvement, deregulated expression being one possibility
Our study shows that -26 5' untranslated region (UTR) polymorphism in BRCA2 can modulate the fine-tuned regulation of the multifunctional gene BRCA2 and renders risk or protection according to the genotype status in the sporadic form of breast cancer, which is further influenced by the germline genetic backgrounds of codon 72 polymorphism of p53
Effect of 5' UTR -26 G>A polymorphism on reporter gene expression To study the effect on expression of BRCA2 5' UTR -26 G>A polymorphism, HeLa and MCF-7 cells were transiently transfected with reporter gene construct with A or G at the polymorphic site
Summary
The absence of mutation or promoter hypermethylation in the BRCA2 gene in the majority of breast cancer cases has indicated alternative ways of its involvement, deregulated expression being one possibility. Based on the hypothesis that variants of genes involved in the same pathway can influence the risk provided for breast cancer, the status of p53 codon 72 polymorphism was investigated and a possible interaction between the polymorphisms was examined. Loss of heterozygosity of the BRCA2 locus has been detected in more than 50% of sporadic breast tumors [12], somatic mutations [13,14,15] or inactivation by methylation has been either absent or rare [16]. The involvement of altered expression of BRCA2 in the development of sporadic breast cancer is a possibility
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