Table_1_A novel BRCA1 splicing variant detected in an early onset triple-negative breast cancer patient additionally carrying a pathogenic variant in ATM: A case report.docx

The widespread adoption of gene panel testing for cancer predisposition is leading to the identification of an increasing number of individuals with clinically relevant allelic variants in two or more genes. The potential combined effect of these variants on cancer risks is mostly unknown, posing a serious problem for genetic counseling in these individuals and their relatives, in whom the variants may segregate singly or in combination. We report a female patient who developed triple-negative high grade carcinoma in the right breast at the age of 36 years. The patient underwent bilateral mastectomy followed by combined immunotherapy and chemotherapy (IMpassion030 clinical trial). Two years later she developed a skin recurrence on the right anterior chest wall. Despite intensive treatment, the patient died at 40-year-old due to disease progression. Gene panel testing of patient’s DNA revealed the presence of a protein truncating variant in ATM [c.1672G>T; p.(Gly558Ter)] and of a not previously reported variant in the BRCA1 exon 22 donor splice site [c.5406+6T>C], whose clinical significance was unknown. The analysis of patient’s RNA revealed the up-regulation of two alternative BRCA1 mRNA isoforms derived from skipping of exon 22 and of exons 22-23. The corresponding predicted protein products, p.(Asp1778GlyfsTer27) and p.(Asp1778_His1822del) are both expected to affect the BRCA1 C Terminus (BRCT) domain. The two variants were observed to co-occur also in the proband’s brother who, in addition, was heterozygous for a common variant (c.4837A>G) mapped to BRCA1 exon 16. This allowed to ascertain, by transcript-specific amplification, the lack of functional mRNA isoforms expressed by the c.5406+6T>C allele and provided evidence to classify the BRCA1 variant as pathogenic, according to the guidelines of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. To our knowledge, excluding two cases detected following the screening of population specific recurrent variants, only one ATM/BRCA1 double heterozygote has been reported in the literature, being the case here described the one with the youngest age at cancer onset. The systematic collection of cases with pathogenic variants in more than one cancer predisposition gene is needed to verify if they deserve ad hoc counseling and clinical management.</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 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

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

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

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

Detection of BRCA1/BARD1 interaction by GFP-fragment reassembly screening.

<p>(a) Fluorescence was observed after 24 h of growth at 30°C followed by 2 days of incubation at RT. No fluorescence is observed under non-inducing condition (right column). [L-ara, L-arabinose; IPTG, Isopropyl β-D-1-tiogalattopiranoside, IPTG]. (b) SDS-PAGE of purified, reassembled complexes by IMAC methods. The expected molecular masses are indicated on the left. [<b>*</b>Non-specific band. B^N, H₆-NfrGFPBARD1; Z^N, H₆-ZNfrGFP; Z^C, ZCfrGFP-HA]. (c) Expression of NfrGFP-BARD1 and CfrGFP-BRCA1 wild-type and mutant forms.</p

Semi-quantitative fragment analysis of the Δexon5q isoform.

<p>The upper panel shows the capillary electrophoresis patterns of the cDNA fragments spanning <i>BRCA1</i> exons 5 and 6 observed in LCLs from a <i>BRCA1</i> wild type individual, and from carriers of the c.190T>C and c.212G>A, which causes the up-regulation of the Δexon5q transcript, mutations. The Δexon5q and full-length (FL) isoforms are indicated. The lower panel shows the ratio between the peak areas of the Δexon5q and full-length isoforms. The LCLs were cultured in the presence (dark grey bars) and in the absence (light grey bar) of cycloheximide. Control bars represent the average value observed in six wild-type LCLs. c.190T>C bars represent the average value observed in four mutant LCLs. The error bars represent standard deviation.</p

Number and frequencies of <i>BRCA1</i> c.190T>C positive families among those recruited at three Italian institutions and tested for BRCA mutations.

*<p>Intake criteria for BRCA testing are described in Manoukian et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086924#pone.0086924-Manoukian1" target="_blank">[38]</a>.</p