Functional Interaction Between BRCA1 and DNA Repair in Yeast May Uncover a Role of RAD50, RAD51, MRE11A, and MSH6 Somatic Variants in Cancer Development

In this study, we determined if BRCA1 partners involved in DNA double-strand break (DSB) and mismatch repair (MMR) may contribute to breast and ovarian cancer development. Taking advantage the functional conservation of DNA repair pathways between yeast and human, we expressed several BRCA1 missense variants in DNA repair yeast mutants to identify functional interaction between BRCA1 and DNA repair in BRCA1-induced genome instability. The pathogenic p.C61G, pA1708E, p.M775R, and p.I1766S, and the neutral pS1512I BRCA1 variants increased intra-chromosomal recombination in the DNA-repair proficient strain RSY6. In the mre11, rad50, rad51, and msh6 deletion strains, the BRCA1 variants p.C61G, pA1708E, p.M775R, p.I1766S, and pS1215I did not increase intra-chromosomal recombination suggesting that a functional DNA repair pathway is necessary for BRCA1 variants to determine genome instability. The pathogenic p.C61G and p.I1766S and the neutral p.N132K, p.Y179C, and p.N550H variants induced a significant increase of reversion in the msh2Δ strain; the neutral p.Y179C and the pathogenic p.I1766S variant induced gene reversion also, in the msh6Δ strain. These results imply a functional interaction between MMR and BRCA1 in modulating genome instability. We also performed a somatic mutational screening of MSH6, RAD50, MRE11A, and RAD51 genes in tumor samples from 34 patients and identified eight pathogenic or predicted pathogenic rare missense variants: four in MSH6, one in RAD50, one in MRE11A, and two in RAD51. Although we found no correlation between BRCA1 status and these somatic DNA repair variants, this study suggests that somatic missense variants in DNA repair genes may contribute to breast and ovarian tumor development

Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants

Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in Saccharomyces cerevisiae increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants

Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants

Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in Saccharomyces cerevisiae increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants

Sclerosing Paragangliomas: Correlations of Histological Features with Patients' Genotype and Vesicular Monoamine Transporter Expression

Paragangliomas and pheochromocytomas are rare neuroendocrine tumors, carrying a germ-line mutation in 40% patients. Sclerosis is a rare histological feature in these tumors. We investigated the possible correlations between histological findings, first sclerosis, immunoreactivity for vesicular catecholamine transporters (VMAT1/VMAT2) and patients' genotype in a consecutive series of 57 tumors (30 paragangliomas and 27 pheochromocytomas) from 55 patients. The M-GAPP grading system, sclerosis (0-3 scale) and VMAT1/VMAT2 (0-6 scale) immunoreactivity scores were assessed. Germ-line mutations of Succinate Dehydrogenase genes, RET proto-oncogene and Von Hippel Lindau tumor suppressor gene were searched. A germ-line mutation was found in 25/55 (45.5%) patients, mainly with paraganglioma (N = 14/30, 46,66%). Significant (score >= 2) tumor sclerosis was found in 9 (16.1%) tumors, i.e., 7 paragangliomas and 2 pheochromocytomas, most of them (8/9) from patients with a germ-line mutation. M-GAPP score was higher in the mutation status (in 76% of patients involving the SDHx genes, in 12% the RET gene and in the remaining 12% the VHL gene) and in tumors with sclerosis (p < 0.05). Spearman's rank correlation showed a strong correlation of germ-line mutations with M-GAPP (p < 0.0001) and sclerosis (p = 0.0027) scores; a significant correlation was also found between sclerosis and M-GAPP scores (p = 0.029). VMAT1 expression was higher in paragangliomas than in pheochromocytomas (p = 0.0006), the highest scores being more frequent in mutation-bearing patients' tumors (p < 0.01). VMAT2 was highly expressed in all but two negative tumors. Sclerosis and VMAT1 expression were higher in paragangliomas than in pheochromocytomas; tumor sclerosis, M-GAPP and VMAT1 scores were associated to germ-line mutations. Sclerosis might represent a histological marker of tumor susceptibility, prompting to genetic investigations in paragangliomas

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

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

Geographical distribution of <i>BRCA1</i> c.190T>C (p.Cys64Arg) mutation carriers.

<p>Symbol (“•”) indicates the birth places of index case from families segregating the mutation.</p

Haplotype branching trees in families segregating the <i>BRCA1</i> c.190T>C (p.Cys64Arg).

<p>The six short tandem repeat markers analyzed are shown together with their position in the Marshfield genetic map. Family haplotypes are indicated with the corresponding family ID codes. The most common haplotype is indicated in bold numbers.</p

Clinical and pathological features of <i>BRCA1</i> c.190T>C related breast cancer cases.

a<p>Ductal and lobular type.</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