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

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 3'- untranslated regions (3'-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 3'-UTR and one in BRCA2 3'-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 3'-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 3'-UTR in a large series of familial breast cancer cases. None of the variant identified in this study gave convincing evidence of potential pathogenicity.

Occurrence of a non deleterious gene conversion event in the BRCA1 gene.

The duplication in the primate lineage of a portion of the breast and ovarian cancer susceptibility gene BRCA1 has created a BRCA1 pseudogene 45 kb away. Non-allelic homologous recombination (NAHR) between BRCA1 and BRCA1P1 has generated recurrent deleterious germ-line 37-kb deletions encompassing the first two exons of BRCA1, accounting for several breast and ovarian cancer families in various populations. In principle, NAHR intermediates resolution could also lead through a non-crossover configuration to interlocus gene conversion (IGC), but none had been described as yet. Here, we report for the first time an IGC event identified in a breast and ovarian cancer family involving exactly the same segment as that involved in the 37-kb deletions. Close examination of the consequences of this IGC event showed that it does not impact BRCA1 expression. Detailed analysis of the regions of homology between BRCA1 and its pseudogene revealed the specificity of the segment where recombination systematically occurs.

Estimation of the RNU2 macrosatellite mutation rate by BRCA1 mutation tracing.

Large tandem repeat sequences have been poorly investigated as severe technical limitations and their frequent absence from the genome reference hinder their analysis. Extensive allelotyping of this class of variation has not been possible until now and their mutational dynamics are still poorly known. In order to estimate the mutation rate of a macrosatellite, we analysed in detail the RNU2 locus, which displays at least 50 different alleles containing 5-82 copies of a 6.1 kb repeat unit. Mining data from the 1000 Genomes Project allowed us to precisely estimate copy numbers of the RNU2 repeat unit using read depth of coverage. This further revealed significantly different mean values in various recent modern human populations, favoring a scenario of fast evolution of this locus. Its proximity to a disease gene with numerous founder mutations, BRCA1, within the same linkage disequilibrium block, offered the unique opportunity to trace RNU2 arrays over a large timescale. Analysis of the transmission of RNU2 arrays associated with one 'private' mutation in an extended kindred and four founder mutations in multiple kindreds gave an estimation by maximum likelihood of 5 × 10(-3) mutations per generation, which is close to that of microsatellites.

Direct visualization of the highly polymorphic RNU2 locus in proximity to the BRCA1 gene.

Although the breast cancer susceptibility gene BRCA1 is one of the most extensively characterized genetic loci, much less is known about its upstream variable number tandem repeat element, the RNU2 locus. RNU2 encodes the U2 small nuclear RNA, an essential splicing element, but this locus is missing from the human genome assembly due to the inherent difficulty in the assembly of repetitive sequences. To fill the gap between RNU2 and BRCA1, we have reconstructed the physical map of this region by re-examining genomic clone sequences of public databases, which allowed us to precisely localize the RNU2 array 124 kb telomeric to BRCA1. We measured by performing FISH analyses on combed DNA for the first time the exact number of repeats carried by each of the two alleles in 41 individuals and found a range of 6-82 copies and a level of heterozygosity of 98%. The precise localisation of the RNU2 locus in the genome reference assembly and the implementation of a new technical tool to study it will make the detailed exploration of this locus possible. This recently neglected macrosatellite could be valuable for evaluating the potential role of structural variations in disease due to its location next to a major cancer susceptibility gene.

BRCA2 deep intronic mutation causing activation of a cryptic exon: opening toward a new preventive therapeutic strategy.

PURPOSE: Diagnostic screening of the BRCA1/2 genes in breast cancer families is mostly done on genomic DNA. For families with a very strong family history and no mutation identified in the coding sequences or the exon-intron boundaries, BRCA1/2 transcripts' analysis is an efficient approach to uncover gene inversion and pre-mRNA splicing defaults missed by conventional DNA-based protocols. EXPERIMENTAL DESIGN: We analyzed RNA from patients of negative BRCA families by reverse transcriptase PCR and identified an insertion in one family that we characterized by sequencing and by using a minigene splicing assay. More than 2,000 additional BRCA1/2 negative families were subsequently screened for this mutation using a dedicated PCR approach. RESULTS: Nine families were found to harbor a BRCA2 mutant transcript containing a 95-nucleotide cryptic exon between exons 12 and 13. This cryptic exon results from a new mutation located deep into intron 12, c.6937+594T > G, which reinforces the strength of a preexisting 5' splice site, turning it into a perfect consensus sequence. It is systematically included in transcripts produced by the mutant allele in cells from mutation carriers or produced by a mutant splicing reporter minigene. The inclusion of the cryptic exon was prevented when we cotransfected the minigene with antisense oligonucleotides complementary to the 3' or mutated 5' splice sites. CONCLUSION: This first deep intronic BRCA mutation emphasizes the importance of analyzing RNA to provide comprehensive BRCA1/2 diagnostic tests and opens the possibility of using antisense therapy in the future as an alternative strategy for cancer prevention.

Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants.

Assessing the impact of variants of unknown significance (VUS) on splicing is a key issue in molecular diagnosis. This impact can be predicted by in silico tools, but proper evaluation and user guidelines are lacking. To fill this gap, we embarked upon the largest BRCA1 and BRCA2 splice study to date by testing 272 VUSs (327 analyses) within the BRCA splice network of Unicancer. All these VUSs were analyzed by using six tools (splice site prediction by neural network, splice site finder (SSF), MaxEntScan (MES), ESE finder, relative enhancer and silencer classification by unanimous enrichment, and human splicing finder) and the predictions obtained were compared with transcript analysis results. Combining MES and SSF gave 96% sensitivity and 83% specificity for VUSs occurring in the vicinity of consensus splice sites, that is, the surrounding 11 and 14 bases for the 5' and 3' sites, respectively. This study was also an opportunity to define guidelines for transcript analysis along with a tentative classification of splice variants. The guidelines drawn from this large series should be useful for the whole community, particularly in the context of growing sequencing capacities that require robust pipelines for variant interpretation.

Down-regulation of BRCA1 expression by miR-146a and miR-146b-5p in triple negative sporadic breast cancers.

Germ-line mutations in the BRCA1 gene strongly predispose women to breast cancer (lifetime risk up to 80%). Furthermore, the BRCA1 protein is absent or present at very low levels in about one third of sporadic breast cancers. However, the mechanisms underlying BRCA1 somatic inactivation appear multiple and are still not fully understood. We report here the involvement of miR-146a and miR-146b-5p that bind to the same site in the 3'UTR of BRCA1 and down-regulate its expression as demonstrated using reporter assays. This was further confirmed with the endogenous BRCA1 gene by transfecting microRNA (miRNA) precursors or inhibitors in mammary cell lines. This down-regulation was accompanied by an increased proliferation and a reduced homologous recombination rate, two processes controlled by BRCA1. Furthermore, we showed that the highest levels of miR-146a and/or miR-146b-5p are found in basal-like mammary tumour epithelial cell lines and in triple negative breast tumours, which are the closest to tumours arising in carriers of BRCA1 mutations. This work provides further evidence for the involvement of miRNAs in sporadic breast cancer through down-regulation of BRCA1.

Comparison of nonsense-mediated mRNA decay efficiency in various murine tissues.

BACKGROUND: The Nonsense-Mediated mRNA Decay (NMD) pathway detects and degrades mRNAs containing premature termination codons, thereby preventing the accumulation of potentially detrimental truncated proteins. Intertissue variation in the efficiency of this mechanism has been suggested, which could have important implications for the understanding of genotype-phenotype correlations in various genetic disorders. However, compelling evidence in favour of this hypothesis is lacking. Here, we have explored this question by measuring the ratio of mutant versus wild-type Men1 transcripts in thirteen tissues from mice carrying a heterozygous truncating mutation in the ubiquitously expressed Men1 gene. RESULTS: Significant differences were found between two groups of tissues. The first group, which includes testis, ovary, brain and heart, displays a strong decrease of the nonsense transcript (average ratio of 18% of mutant versus wild-type Men1 transcripts, identical to the value measured in murine embryonic fibroblasts). The second group, comprising lung, intestine and thymus, shows much less pronounced NMD (average ratio of 35%). Importantly, the extent of degradation by NMD does not correlate with the expression level of eleven genes encoding proteins involved in NMD or with the expression level of the Men1 gene. CONCLUSION: Mouse models are an attractive option to evaluate the efficiency of NMD in multiple mammalian tissues and organs, given that it is much easier to obtain these from a mouse than from a single individual carrying a germline truncating mutation. In this study, we have uncovered in the thirteen different murine tissues that we examined up to a two-fold difference in NMD efficiency.

Unclassified variants identified in BRCA1 exon 11: Consequences on splicing.

Numerous mutations identified in breast/ovarian cancer families occur in splice sites of the BRCA1 gene. Splicing can also be disrupted by mutations occurring in exonic splicing enhancer (ESE) sequences. It is important to identify those mutations among the large number of nontruncating sequence variants that are identified during molecular diagnosis, as this could help to classify some of them as cancer predisposing. Several software programs have been designed to identify ESEs and can therefore be used to predict the outcome of genetic variation. However, it is not known whether these predictions are relevant in the case of BRCA1 exon 11 (3.4 kb). In this study, we assessed the consequences on splicing of 108 exon 11 variants identified in French breast/ovarian cancer families, most of them predicted to alter putative ESEs, and of nine variants located in the exon 11 alternative donor splice site. We employed a BRCA1 minigene consisting of exon 10 to 12, into which we introduced separately each of the variants to be tested. RNA was analyzed by RT-PCR after transient transfection of the resulting minigenes. None of the tested variants was found to dramatically alter splicing through disruption of an ESE. However, we identified several variants in the alternative donor splice site that are likely to be of biological significance as they appear to favor the expression of BRCA1-Delta11b over that of the full-length transcript. The results of this study will be of value to classify BRCA1 exon 11 variants of unknown significance. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Does the nonsense-mediated mRNA decay mechanism prevent the synthesis of truncated BRCA1, CHK2, and p53 proteins?

The nonsense-mediated mRNA decay (NMD) mechanism is an evolutionarily conserved process ensuring the degradation of transcripts carrying premature termination codon(s). NMD is believed to prevent the synthesis of truncated proteins that could be detrimental to the cell. However, although numerous studies have assessed the efficiency of this mechanism at the mRNA level, data are lacking in regard to whether NMD fulfills its expected goal at the protein level. In this study, we have investigated whether endogenous alleles of breast cancer predisposing genes carrying nonsense codons were able to produce detectable amounts of truncated proteins in lymphoblastoid cell lines. A total of 20 truncating BRCA1 mutations were analyzed, along with the 1100delC CHEK2 and the 770delT TP53 mutations. All the studied alleles triggered NMD, the amount of mutant transcript ranging from 16 to 63% of that of the wild-type species. We found that BRCA1 and CHK2 truncated proteins could not be detected, even when NMD was inhibited. This suggests that BRCA1 and CHK2 truncated proteins are highly unstable. Conversely, the p53 protein encoded by the 770delT allele is as abundant as the wild-type protein, as removal of the C-terminal p53 domain leads to a stabilized mutant protein, whose abundance is markedly increased when NMD is inhibited. Therefore, our results show that it is not possible to infer the presence of truncated proteins in cells from carriers of a truncated mutation without experimental verification, as each case is expected to be different.

The 185delAG mutation (c.68_69delAG) in the BRCA1 gene triggers translation reinitiation at a downstream AUG codon.

The 185delAG mutation (c.68_69delAG; ter39) in the BRCA1 gene is a founder Jewish Ashkenazi mutation that is carried by 1% of this population and has been identified in thousands of breast or ovarian cancer patients. We have previously described that transcripts bearing this mutation, as well as transcripts bearing the 188del11 mutation (c.71_81del; ter36), are not degraded by nonsense-mediated mRNA decay (NMD), contrary to our observations of other truncating mutations that introduce premature termination codons (PTCs) farther downstream in the coding sequence [Perrin-Vidoz et al., 2002]. To test the hypothesis that these two mutations fail to trigger NMD because of translation reinitiation, we have constructed BRCA1 minigenes and studied their protein expression after transient expression in HeLa cells. We show here that in the presence of a (PTC) at position 36 or 39, translation reinitiation occurs in the BRCA1 minigenes at position 128.

Interaction of the Epstein-Barr virus mRNA export factor EB2 with human Spen proteins SHARP, OTT1, and a novel member of the family, OTT3, links Spen proteins with splicing regulation and mRNA export.

The Epstein-Barr virus early protein EB2 (also called BMLF1, Mta, or SM), a protein absolutely required for the production of infectious virions, shares properties with mRNA export factors. By using a yeast two-hybrid screen, we have identified the human protein OTT3 as an EB2-interacting factor. OTT3 is a new member of the Spen (split end) family of proteins (huSHARP, huOTT1, DmSpen, and muMINT), which are characterized by several N-terminal RNA recognition motifs and a highly conserved C-terminal SPOC (Spen Paralog and Ortholog C-terminal) domain that, in the case of SHARP, has been shown to interact with SMRT/NCoR corepressors. OTT3 is ubiquitously expressed as a 120-kDa protein. Transfected OTT3 is a nonshuttling nuclear protein that co-localizes with co-transfected EB2. We also showed that EB2 interacts with the SPOC domains of both OTT1 and SHARP proteins. Although the OTT3 interaction domain maps within the 40 N-terminal amino acids of EB2, OTT1 and SHARP interact within the C-terminal half of the protein. Furthermore, we demonstrated that the capacity of the OTT3 and OTT1 SPOC domains to interact with SMRT and repress transcription is far weaker than that of SHARP. Thus there is no evidence for a role of OTT3 in transcriptional regulation. Most interestingly, however, we have found that OTT3 has a role in splicing regulation; OTT3 represses accumulation of the alternatively spliced beta-thalassemia mRNAs, but it has no effect on the beta-globin constitutively spliced mRNA. Thus our results suggested a new function for Spen proteins related to mRNA export and splicing.