Sequencing Analysis of SLX4/FANCP Gene in Italian Familial Breast Cancer Cases

Breast cancer can be caused by germline mutations in several genes that are responsible for different hereditary cancer syndromes. Some of the genes causing the Fanconi anemia (FA) syndrome, such as BRCA2, BRIP1, PALB2, and RAD51C, are associated with high or moderate risk of developing breast cancer. Very recently, SLX4 has been established as a new FA gene raising the question of its implication in breast cancer risk. This study aimed at answering this question sequencing the entire coding region of SLX4 in 526 familial breast cancer cases from Italy. We found 81 different germline variants and none of these were clearly pathogenic. The statistical power of our sample size allows concluding that in Italy the frequency of carriers of truncating mutations of SLX4 may not exceed 0.6%. Our results indicate that testing for SLX4 germline mutations is unlikely to be relevant for the identification of individuals at risk of breast cancer, at least in the Italian population

Common germline polymorphisms associated with breast cancer-specific survival

Abstract Introduction Previous studies have identified common germline variants nominally associated with breast cancer survival. These associations have not been widely replicated in further studies. The purpose of this study was to evaluate the association of previously reported SNPs with breast cancer-specific survival using data from a pooled analysis of eight breast cancer survival genome-wide association studies (GWAS) from the Breast Cancer Association Consortium. Methods A literature review was conducted of all previously published associations between common germline variants and three survival outcomes: breast cancer-specific survival, overall survival and disease-free survival. All associations that reached the nominal significance level of P value <0.05 were included. Single nucleotide polymorphisms that had been previously reported as nominally associated with at least one survival outcome were evaluated in the pooled analysis of over 37,000 breast cancer cases for association with breast cancer-specific survival. Previous associations were evaluated using a one-sided test based on the reported direction of effect. Results Fifty-six variants from 45 previous publications were evaluated in the meta-analysis. Fifty-four of these were evaluated in the full set of 37,954 breast cancer cases with 2,900 events and the two additional variants were evaluated in a reduced sample size of 30,000 samples in order to ensure independence from the previously published studies. Five variants reached nominal significance (P <0.05) in the pooled GWAS data compared to 2.8 expected under the null hypothesis. Seven additional variants were associated (P <0.05) with ER-positive disease. Conclusions Although no variants reached genome-wide significance (P <5 x 10−8), these results suggest that there is some evidence of association between candidate common germline variants and breast cancer prognosis. Larger studies from multinational collaborations are necessary to increase the power to detect associations, between common variants and prognosis, at more stringent significance levels

Towards population-based genetic screenings for breast and ovarian cancer: A comprehensive review from economic evaluations to patient perspectives

Genetic testing for hereditary breast and ovarian cancer following genetic counseling is based on guidelines that take into account particular features of the personal and family history, and clinical criteria conferring a probability of having a BRCA mutation greater than 10% as a threshold for accessing the test. However, besides reducing mortality and social impact, the extension of screening programs also for healthy family members would allow a huge saving of the rising costs associated with these pathologies, supporting the choice of the “Test” strategy versus a “No Test” one. Analyses of different health care systems show that by applying the “Test” strategy on patients and their families, a decrease in breast and ovarian cancer cases is achieved, as well as a substantial decrease in costs of economic resources, including the costs of the clinical management of early detected tumors.In this review, we analyzed the most recent papers published on this topic and we summarized the findings on the economic evaluations related to breast and ovarian cancer population screenings. These results proved and validated that the population-wide testing approach is a more accurate screening and preventive intervention than traditional guidelines based on personal/family history and clinical criteria to reduce breast and ovarian cancer risk

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

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

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