RNA assay identifies a previous misclassification of BARD1 c.1977A>G variant.

Case-control studies have shown an association of BARD1 with hereditary breast and/or ovarian cancer (HBOC) predisposition. BARD1 alternatively spliced isoforms are abundant and some are highly expressed in different cancer types. In addition, a number of BARD1 germline pathogenic variants have been reported among HBOC patients. In previous reports, BARD1 c.1977A>G variant has been classified as pathogenic since it produces a frameshift transcript lacking exons 2 to 9. In the present study, we sought to validate the mRNA splicing results previously published and to contribute with new evidence to refine the classification of this substitution according to ACMG/AMP guidelines. The presence of the variant was screened in patients and controls. RT-PCR was performed in order to compare the transcriptional profiles of two variant carriers and ten non-carrier controls. In addition, allele-specific expression was assessed. No differences in variant frequency were detected between patients and controls. The RNA assay confirmed the presence of the shorter transcript lacking exons 2-9, but it was detected both in carriers and non-carriers. Furthermore, allelic imbalance was discarded and no significant differences in the proportion of full-length and shorter transcript were detected between carriers and controls. The shorter transcript detected corresponds to BARD1 isoform η, constituted by exons 1, 10 and 11. Our results support that this transcript is a constitutive splicing product rather than an aberrant transcript caused by BARD1 c.1977A>G variant, and for this reason this variant should be considered as likely benign following ACMG/AMP guidelines.

Improving Genetic Testing in Hereditary Cancer by RNA Analysis: Tools to Prioritize Splicing Studies and Challenges in Applying American College of Medical Genetics and Genomics Guidelines.

RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.

Transposon activation is a major driver in the genome evolution of cultivated olive trees (Olea europaea L.).

The primary domestication of olive (Olea europaea L.) in the Levant dates back to the Neolithic period, around 6,000-5,500 BC, as some archeological remains attest. Cultivated olive trees are reproduced clonally, with sexual crosses being the sporadic events that drive the development of new varieties. In order to determine the genomic changes which have occurred in a modern olive cultivar, the genome of the Picual cultivar, one of the most popular olive varieties, was sequenced. Additional 40 cultivated and 10 wild accessions were re-sequenced to elucidate the evolution of the olive genome during the domestication process. It was found that the genome of the 'Picual' cultivar contains 79,667 gene models, of which 78,079 were protein-coding genes and 1,588 were tRNA. Population analyses support two independent events in olive domestication, including an early possible genetic bottleneck. Despite genetic bottlenecks, cultivated accessions showed a high genetic diversity driven by the activation of transposable elements (TE). A high TE gene expression was observed in presently cultivated olives, which suggests a current activity of TEs in domesticated olives. Several TEs families were expanded in the last 5,000 or 6,000 years and produced insertions near genes that may have been involved in selected traits during domestication as reproduction, photosynthesis, seed development, and oil production. Therefore, a great genetic variability has been found in cultivated olive as a result of a significant activation of TEs during the domestication process.

ERCC3, a new ovarian cancer susceptibility gene?

BACKGROUND: Hereditary breast and ovarian cancer syndrome (HBOC) is an inherited disorder with an increased risk of breast cancer (BC) and ovarian cancers (OC). Mutations in BRCA1-BRCA2 explains less than a half of cases. In the last decade several genes with different penetrance have been associated with an increased risk of BC or OC. A recurrent heterozygous ERCC3 truncating mutation increases the risk for breast cancer in patients with Ashkenazi Jewish ancestry. Our study aimed to investigate the role of ERCC3 truncating variants in a cohort of patients with suspicion of HBOC. PATIENTS AND METHODS: ERCC3 screening by multigene-panel analysis in 1311 unrelated patients after our regional consensus for genetic testing in hereditary cancer was done. In addition, 453 Spanish cancer-free individuals and 51,343 GnomAD non-Finnish, non-cancer European individuals were used as control populations. RESULTS: We identified 13 patients with heterozygous ERCC3 truncating variants (0.99%). Five of them also carried a mutation in a high- /moderate-penetrance HBOC gene (BRCA1, BRCA2, CHEK2, and TP53) being Multilocus Inherited Neoplasia Alleles syndrome (MINAS) patients. The frequency in 453 Spanish controls was of 0.22%; similar to that observed in 51,343 non-Finnish European GnomAD population (0.24%). We found an almost statistically significant association of truncating ERCC3 variants with BC (odds ratio [OR] = 2.25, confidence interval [CI] = 0.6-5.93, P = 0.11), and we observed for the first time a significant association with OC (OR = 4.74, CI = 1-14.34, P = 0.028), that holds even after removing MINAS cases. CONCLUSIONS: To our knowledge, this is the largest HBOC series comprehensively analysed for ERCC3 mutations, and the first study identifying ERCC3 as a cancer risk for OC.

ICO amplicon NGS data analysis: a Web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS Junior next-generation sequencing.

Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.

Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes.

Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.

The intracellular genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone mediates G2-M cell cycle arrest in cancer cells via modulation of the p38 signaling pathway.

The cellular actions of genistein are believed to mediate the decreased risk of breast cancer associated with high soy consumption. We have investigated the intracellular metabolism of genistein in T47D tumorigenic and MCF-10A nontumorigenic cells and assessed the cellular actions of resultant metabolites. Genistein selectively induced growth arrest and G2-M phase cell cycle block in T47D but not MCF10A breast epithelial cells. These antiproliferative effects were paralleled by significant differences in the association of genistein to cells and in particular its intracellular metabolism. Genistein was selectively taken up into T47D cells and was subject to metabolism by CYP450 enzymes leading to the formation of both 5,7,3',4'-tetrahydroxyisoflavone (THIF) and two glutathionyl conjugates of THIF. THIF inhibited cdc2 activation via the phosphorylation of p38 MAP kinase, suggesting that this species may mediate genistein's cellular actions. THIF exposure activated p38 and caused subsequent inhibition of cyclin B1 (Ser 147) and cdc2 (Thr 161) phosphorylation, two events critical for the correct functioning of the cdc2-cyclin B1 complex. We suggest that the formation of THIF may mediate the cellular actions of genistein in tumorigenic breast epithelial cells via the activation of signaling through p38.

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury.

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis.