Mutation screening of MIR146A/B and BRCA1/2 3′-UTRs in the GENESIS study

Abstract

Although a wide number of breast cancer susceptibility alleles associated with various levels of risk have been identified to date, about 50% of the heritability is still missing. Although the major BRCA1 and BRCA2 genes are being extensively screened for truncating and missense variants in breast and/or ovarian cancer families, potential regulatory variants affecting their expression remain largely unexplored. In an attempt to identify such variants, we focused our attention on gene regulation mediated by microRNAs (miRs). We screened two genes, MIR146A and MIR146B, producing miR-146a and miR-146b-5p, respectively, that regulate BRCA1, and the 3ʹ-untranslated regions (3ʹ-UTRs) of BRCA1 and BRCA2 in the GENESIS French national case/control study (BRCA1-and BRCA2-negative breast cancer cases with at least one sister with breast cancer and matched controls). We identified one rare variant in MIR146A, four in MIR146B, five in BRCA1 3ʹ-UTR and one in BRCA2 3ʹ-UTR in 716 index cases and 619 controls. Among these 11 rare variants, 7 were identified each in 1 index case. None of the three relevant MIR146A/MIR146B variants affected the pre-miR sequences. The potential causality of the four relevant BRCA1/BRCA2 3ʹ-UTRs variants was evaluated with luciferase reporter assays and co-segregation studies, as well as with bioinformatics analyses to predict miRs-binding sites, RNA secondary structures and RNA accessibility. This is the first study to report the screening of miR genes and of BRCA2 3ʹ-UTR in a large series of familial breast cancer cases. None of the variant identified in this study gave convincing evidence of potential pathogenicity.

Non-Coding Variants in BRCA1 and BRCA2 Genes: Potential Impact on Breast and Ovarian Cancer Predisposition

“…First, using RNAfold secondary structure prediction software, we could demonstrate that a BRCA1 5′UTR variant (c.-130del) impacts RNA conformation and it probably affects the binding of trans-acting factors and therefore mRNA translation [ 58 ]. This predicted effect was also described for some 3′UTR variants and a 5′UTR polymorphisms of BRCA1 , both with an impact in translational efficiency [ 60 , 94 ].…”

“…In addition, we reported recently the enhancer property of an intronic sequence that is located in the intron 12 of BRCA1 [ 58 ]. The BRCA1 3′ untranslated region (3′UTR) has been shown to be important for post-transcriptional regulation and this has been exemplified by a variety of variants located there that negatively regulate mRNA translation, probably by the disruption or creation of complementary MicroRNAs binding sites [ 59 , 60 , 61 , 62 ].…”

“…For now, little information about BRCA2 non-coding regions is available. A few cis-acting intronic polymorphisms that alter the binding of transcription factors at regulatory sites have been described [ 69 ], as well as one 3′UTR variant ( BRCA2 c.*172G>A), but with no clear evidence of pathogenicity [ 60 ].…”

“…One of them (c.*1340_1342del) related to the creation of a new microRNA binding site: miR-103. Another 3′UTR screening of 716 index cases that tested negative for BRCA1/2 pathogenic mutations also detected SNPs and six rare variants in these regions, three of which are novel [ 60 ].…”

Role of micro-RNAs in breast cancer surgery

“…It is thought that mir‐146 targets BRCA1 and BRCA2 such that patients with this variant allele are diagnosed at a younger age than those without any variant allele. An in vitro study confirmed that mir‐146 binds the 3′‐UTR of both genes, and showed that the binding capacity was significantly higher for the C than the G allele. This has been validated by the finding that patients with variants of the rs2190164 SNP developed breast cancer at a younger age in the setting of familial breast cancer.…”