Revertant mosaicism for family mutations is not observed in <i>BRCA1/2</i> phenocopies

<div><p>In <i>BRCA1/2</i> families, early-onset breast cancer (BrCa) cases may be also observed among non-carrier relatives. These women are considered phenocopies and raise difficult counselling issues concerning the selection of the index case and the residual risks estimate in negative family members. Few studies investigated the presence of potential genetic susceptibility factors in phenocopies, mainly focussing on BrCa-associated single-nucleotide polymorphisms. We hypothesized that, as for other Mendelian diseases, a revertant somatic mosaicism, resulting from spontaneous correction of a pathogenic mutation, might occur also in BRCA pedigrees. A putative low-level mosaicism in phenocopies, which has never been investigated, might be the causal factor undetected by standard diagnostic testing. We selected 16 non-carriers BrCa-affected from 15 <i>BRCA1/2</i> families, and investigated the presence of mosaicism through MALDI-TOF mass spectrometry. The analyses were performed on available tumour samples (7 cases), blood leukocytes, buccal mucosa and urine samples (2 cases) or on blood only (7 cases). In one family (n.8), real-time PCR was also performed to analyse the phenocopy and her healthy parents. On the 16 phenocopies we did not detect the family mutations neither in the tumour, expected to display the highest mutation frequency, nor in the other analysed tissues. In family 8, all the genotyping assays did not detect mosaicism in the phenocopy or her healthy parents, supporting the hypothesis of a <i>de novo</i> occurrence of the <i>BRCA2</i> mutation identified in the proband. These results suggest that somatic mosaicism is not likely to be a common phenomenon in <i>BRCA1/2</i> families. As our families fulfilled high-risk selection criteria, other genetic factors might be responsible for most of these cases and have a significant impact on risk assessment in <i>BRCA1/2</i> families. Finally, we found a <i>de novo BRCA2</i> mutation, suggesting that, although rare, this event should be taken into account in the evaluation of high-risk families.</p></div

RT-PCR analyses of group B variants.

<p>For each variant, the RT-PCR products were characterized by agarose gel electrophoresis and sequencing. Gel images: lane 1, no template; lane 2, genomic DNA used as negative control of the RT-PCR reaction; lane 3, cDNA from the <i>BRCA1/BRCA2</i> wild-type LCL used as positive control; lane 4, cDNA from LCL carrying the UV. M, molecular marker (ΦX-174 HaeIII digest). The size of the full-length (FL) and aberrant transcripts are reported. Sequencing electropherogram data: (<b>B–G</b>) the RT-PCR products were directly sequenced; (<b>A, H</b>) the sequencing was performed after band excision or cloning step. (<b>H</b>) An additional band due to improper annealing of full-length and aberrant transcripts is shown by the asterisk. The Ex5del, visible in both sample and control is a naturally occurring isoform lacking exon 5. (<b>A</b>) In addition to the full-length and the Ex14del aberrant transcript, the naturally occurring isoform lacking the first 3 bp of exon 14 (Ex14_3 bp del) was observed. Ex, exon; I, intron.</p

Functional analysis of BRCA2 p.Val2985_Thr3001del.

<p>(<b>A</b>) Schematic representation of GST-BRCA2 recombinant proteins. Wild-type and mutant <i>BRCA2</i> fragments, encoding the DBD and the N-terminal region, were cloned into pGEX4T1 vector to express GST-BRCA2 fusion proteins under the control of lacUV5 promoter. BRCA2 amino acid positions, helical domain (HD) and OB fold domains 1, 2, 3 (OB1, OB2, OB3) are indicated. (<b>B</b>) Interaction of wild-type and mutated BRCA2 DBD polypeptides with DSS1<b>.</b> Equivalent amounts of GST-tagged wild-type or mutated BRCA2 fusion proteins were immobilized on GSH-Sepharose beads and challenged with MCF7 lysates as a source of GFP-DSS1. Input (top panel) and pulled down (middle panel) GFP-DSS1 protein were visualized by Western blotting with anti-GFP antibody. GSH-Sepharose beads and GST protein were used as negative controls. GST-tagged recombinant proteins were visualized by Coomassie staining of the SDS-PAGE gel used in the pull-down experiment (bottom panel)<b>.</b> (<b>C</b>) Interaction of wild-type and mutated BRCA2 polypeptides with ssDNA. The mutated and wild-type peptides, removed from glutathione-agarose beads by thrombin digestion, were chromatographed on ssDNA agarose beads. A 200 amino acids N-terminal peptide was used as negative control. The free (F) and bound (B) fractions were separated, submitted to gel electrophoresis and visualized by Coomassie staining. Immunoblots were scanned using HP Scanjet G3010 Photo Scanner (Hewlett Packard).</p

Experimentally observed effects on mRNA splicing of group B variants and predicted protein change.

a<p>Protein change was predicted using ExPASy Proteomics Server<b>.</b> (<a href="http://www.expasy.ch/" target="_blank">http://www.expasy.ch/</a>);</p>b<p>The classification as class 5 (pathogenic) or class 4 (likely pathogenic) was based on mono- or bi-allelic expression of the normal transcript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Spurdle2" target="_blank">[23]</a>, that of class 2 (likely neutral) on A-GVGD software prediction (<a href="http://agvgd.iarc.fr/" target="_blank">http://agvgd.iarc.fr/</a>). Previously characterized variants are indicated;</p>c<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Houdayer1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Zhang1" target="_blank">[50]</a>;</p>d<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Houdayer1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Friedman1" target="_blank">[46]</a>;</p>e<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Houdayer1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Ozcelik1" target="_blank">[47]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Yang1" target="_blank">[49]</a>;</p>f<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Thomassen1" target="_blank">[21]</a>;</p>g<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Gaildrat1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Colombo1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Pensabene1" target="_blank">[45]</a>;</p>h<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Thomassen1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Brandao1" target="_blank">[26]</a>;</p>i<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Menendez1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Houdayer1" target="_blank">[22]</a>;</p>j<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Bonatti1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Acedo1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Houdayer1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Peelen1" target="_blank">[48]</a>. An asterisk indicates variants for which the observed transcript pattern differed from that reported by previous studies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173.s006" target="_blank">Table S6</a>). Abbreviations: SS, splice Site (D, donor; A, acceptor); BIC, Breast Cancer Information Core (<a href="http://research.nhgri.nih.gov/bic/" target="_blank">http://research.nhgri.nih.gov/bic/</a>); HGVS, Human Genetic Variation Society (<a href="http://www.hgvs.org/mutnomen/" target="_blank">http://www.hgvs.org/mutnomen/</a>).</p

Experimentally observed effects on mRNA splicing of group A variants and predicted protein change.

a<p>Protein change was predicted using ExPASy Proteomics Server (<a href="http://www.expasy.ch/" target="_blank">http://www.expasy.ch/</a>);</p>b<p>The classification as class 5 (pathogenic) or class 4 (likely pathogenic) was based on mono- or bi-allelic expression of the normal transcript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Spurdle2" target="_blank">[23]</a>. Previously characterized variants are indicated;</p>c<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Houdayer1" target="_blank">[22]</a>;</p>d<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Machackova1" target="_blank">[43]</a>;</p>e<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Houdayer1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Colombo1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Pensabene1" target="_blank">[45]</a>;</p>f<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Machackova1" target="_blank">[43]</a>;</p>g<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173-Acedo1" target="_blank">[18]</a>. An asterisk indicates variants for which the observed transcript pattern differed from that reported by previous studies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057173#pone.0057173.s006" target="_blank">Table S6</a>). Abbreviations: SS, splice Site (D, donor; A, acceptor); BIC, Breast Cancer Information Core (<a href="http://research.nhgri.nih.gov/bic/" target="_blank">http://research.nhgri.nih.gov/bic/</a>); HGVS, Human Genetic Variation Society (<a href="http://www.hgvs.org/mutnomen" target="_blank">http://www.hgvs.org/mutnomen</a>).</p

<i>In silico</i> predicted effect of group B variants and comparison with experimental results.

<p>For all computational program except ASSA, the relative percent differences of the splice site prediction scores (SSPSs) in the wild-type and the mutated sequences are reported. For ASSA, which uses the information theory-base values (Ri), the percent differences of binding affinity in the mutated compared to the wild-type sequences are reported. Empty cells indicates natural splice site not recognized by the indicated programs, <i>In silico</i> analyses predicting spliceogenic (S) or non spliceogenic (NS) variants according to the described procedure (see text) are indicated. (C) indicates <i>in silico</i> predictions concordant with <i>in vitro</i> data; (D), discordant predictions. Abbreviations: HGVS, Human Genetic Variation Society (<a href="http://www.hgvs.org/mutnomen/" target="_blank">http://www.hgvs.org/mutnomen/</a>).</p

Comparative <em>In Vitro</em> and <em>In Silico</em> Analyses of Variants in Splicing Regions of <em>BRCA1</em> and <em>BRCA2</em> Genes and Characterization of Novel Pathogenic Mutations

<div><p>Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at <i>BRCA1</i> and <i>BRCA2</i> splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in <i>BRCA1</i> and 8 in <i>BRCA2</i>, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that <i>in silico</i> tools can be used for selecting splice site variants for <i>in vitro</i> analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts.</p> </div

Expression of miR-342 and ID4 in patients.

<p>Negative correlation of ID4 expression measured by qRT-PCR and miR-342 levels detected by miRNA arrays. Samples are represented according to their positive/negative estrogen receptor status. The dashed line was obtained using a linear regression model.</p

miR-342 Regulates BRCA1 Expression through Modulation of ID4 in Breast Cancer

<div><p>A miRNAs profiling on a group of familial and sporadic breast cancers showed that miRNA-342 was significantly associated with estrogen receptor (ER) levels. To investigate at functional level the role of miR-342 in the pathogenesis of breast cancer, we focused our attention on its “<i>in silico</i>” predicted putative target gene ID4, a transcription factor of the helix-loop-helix protein family whose expression is inversely correlated with that of ER. ID4 is expressed in breast cancer and can negatively regulate BRCA1 expression. Our results showed an inverse correlation between ID4 and miR-342 as well as between ID4 and BRCA1 expression. We functionally validated the interaction between ID4 and miR-342 in a reporter Luciferase system. Based on these findings, we hypothesized that regulation of ID4 mediated by miR-342 could be involved in the pathogenesis of breast cancer by downregulating BRCA1 expression. We functionally demonstrated the interactions between miR-342, ID4 and BRCA1 in a model provided by ER-negative MDA-MB-231 breast cancer cell line that presented high levels of ID4. Overexpression of miR-342 in these cells reduced ID4 and increased BRCA1 expression, supporting a possible role of this mechanism in breast cancer. In the ER-positive MCF7 and in the BRCA1-mutant HCC1937 cell lines miR-342 over-expression only reduced ID4. In the cohort of patients we studied, a correlation between miR-342 and BRCA1 expression was found in the ER-negative cases. As ER-negative cases were mainly BRCA1-mutant, we speculate that the mechanism we demonstrated could be involved in the decreased expression of BRCA1 frequently observed in non BRCA1-mutant breast cancers and could be implicated as a causal factor in part of the familial cases grouped in the heterogeneous class of non BRCA1 or BRCA2-mutant cases (BRCAx). To validate this hypothesis, the study should be extended to a larger cohort of ER-negative cases, including those belonging to the BRCAx class.</p></div

Down-regulation of ID4 protein through miR-342 increases BRCA1 expression in MDA-MB-231 cells.

<p>(A) Expression of ID4 (left) and miR-342 (right) measured by qRT-PCR in five breast cancer cell lines. (B) qRT-PCR evaluation of miR-342 expression in MDA-MB-231, HCC1937 and MCF7 cells after transfection of pre-miR-342 (grey) or of a scramble oligonucleotide (black). (C) Western blotting analysis of ID4 and BRCA1 proteins on the same cells. qRT-PCR data are expressed as two elevated to –ΔCt (delta cycle threshold) value which is directly related to the expression levels. β-Actin and Vinculin were used as loading control.</p