Discrepancies between in silico and in vitro data in the functional analysis of a breast cancer-associated polymorphism in the XRCC6/Ku70 gene

Abstract

Abstract.Previous results from our research group have shown that the c.-1310 C¨G single nucleotide polymorphism in the promoter region of the XRCC6/Ku70 gene is significantly associated with breast cancer in a sample human patient population. In an attempt to attribute a functional meaning to this polymorphism, we performed a thorough analysis using a number of established in silico tools that strongly suggested that the c.-1310C¨G transversion would activate a cryptic splicing acceptor located upstream of the canonical promoter of Ku70, but downstream of a putative alternative promoter (PAP) of the same gene. Experimental investigation of alternative transcripts, as well as of the activity of the PAP detected in silico, did not support the initial hypothesis of a functional role of the c.-1310C¨G mutation in alternative splicing. Although a functional role of the SNP has yet to be determined, some evidence points to the linkage disequilibrium of the G variant of the polymorphism, with mutations located at critical sites within the promoter region of Ku70. IntroductionBreast cancer is a major public health issue worldwide. Globally, it is by far the most frequent cancer in women, with an estimated 1.15 million new cases in 2002 (23% of all cancers), and more than half of these cases occurring in the industrialized countries of Europe and North America (1). In Europe in 2006, breast cancer accounted for 28.9% of all cancer cases in women, and was the leading cause of cancerrelated death within the European Union (16.7%) (2).In several independent studies also conducted by our group, an enhanced in vitro chromosomal radiosensitivity was observed in a significant number of breast cancer patients (3-7). Since the most detrimental form of radiation-induced DNA damage is the double-strand break (DSB), it is plausible that DSB-initiated chromosomal instability drives breast carcinogenesis. Furthermore, the key breast cancer susceptibility genes, BRCA1 and BRCA2, ATM and TP53, play important roles in DSB repair and chromosome stability (8,9). These genetic factors are present in only 6-11% of the general breast cancer patient population, indicating that other mutations in low penetrant genes or subtle defects arising from low penetrant variations in highly penetrant genes may also create a predisposition to breast cancer (9-12). Recently, several population-based case-control studies have shown a link between single nucleotide polymorphisms (SNPs) in DSB repair genes and breast cancer risk (8,9,(12)(13)(14)(15)(16)(17). Moreover, the breast is a selected microenvironment, subjected to endogenous oxidative stress through hormone exposure. Estrogen in particular has attracted considerable attention, as it may act as a complete carcinogen (8). During the oxidative metabolism of estrogen, reactive oxygen species (ROS) and DNA adducts are formed, leading to the oxidation and depurination of the DNA (18). This type of damage can further result in clustered sites of DNA damage, including DSBs. DSBs are also induced in prolife...

Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis

“…However, the variant mutation produces a site with < 0.7% of its original binding affinity, which would substantially reduce exon recognition and lead to exon skipping 116 . Also, cryptic sites created in the promoter regions of genes should not be considered to be splicing mutations 142 . Variants that are predicted to create a cryptic site upstream or overlapping a natural site of the opposite polarity (i.e.…”