The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity.

Although the spliceogenic nature of the BRCA2 c.68-7T>A variant has been demonstrated, its association with cancer risk remains ontroversial. In this study, we accurately quantified by real-time PCR and digital PCR the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case-control analysis in 83,636 individuals. Co-occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5-fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68-7T>A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 x 10-115. There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86-1.24), nor for a deleterious effect of the variant when co-occurring with pathogenic variants. Our data provide for the first time robust evidence of the non-pathogenicity of the BRCA2 c.68-7T>A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants

Quality and nutritional properties of ready-to-eat blackberry fron different cultivars and pedoclimatic conditions

Minimal processing of fruit and vegetables involves different operations that increase the perishability of the final product and often cause a decrease in its nutritional properties. In this work three blackberry (Rubus fruticosus L.) cultivars (\u2018Chester\u2019, \u2018Arapaho\u2019 and \u2018Triple Crown\u2019) grown in the hill (Valle Imagna, BG, Italy) or in the plain (Arcagna, LO, Italy) were evaluated immediately after minimal processing and after 3, 6 and 8 days of shelf-life at 3\ub0C. Following parameters were evaluated: firmness, soluble solids content (SSC), acidity (AC), color (a*, b*, Hue), total anthocyanin (TA), total polyphenols (TP) and antioxidant activity (DPPH). All the cultivars, especially if grown in the plain, showed a slight decrease in firmness during shelf-life, however, they maintained adequate firmness values until the end of the storage. Blackberries grown in the plain had higher SSC and hue and lower acidity than those coming from the hill. \u2018Chester\u2019 was the richest in TP and in TA followed by \u2018Arapaho\u2019 and \u2018Triple Crown\u2019. TA content was always higher in fruits cultivated in the plain compared to those from the hill. Minimal processing did not importantly affect TA, TP content and DPPH. The nutritional content of the fruit at the end of shelf-life was not statistically different from the harvest. All the evaluated cultivars showed good physico-chemical parameters and nutritional properties throughout the storage and, then, they have an interesting potential as ready-to-eat product

Mixed Neuroendocrine/Non-neuroendocrine Neoplasm (MiNEN) of the Ovary Arising from Endometriosis: Molecular Pathology Analysis in Support of a Pathogenetic Paradigm

Primary ovarian neuroendocrine neoplasms (Ov-NENs) are infrequent and mainly represented by well-differentiated forms (neuroendocrine tumors - NETs - or carcinoids). Poorly differentiated neuroendocrine carcinomas (Ov-NECs) are exceedingly rare and only few cases have been reported in the literature. A subset of Ov-NECs are admixed with non-neuroendocrine carcinomas, as it occurs in other female genital organs, as well (mostly endometrium and uterine cervix), and may be assimilated to mixed neuroendocrine/non-neuroendocrine neoplasms (MiNENs) described in digestive and extra-digestive sites. Here, we present a case of large cell Ov-NEC admixed with an endometrioid carcinoma of the ovary, arising in the context of ovarian endometriosis, associated with a uterine endometrial atypical hyperplasia (EAH). We performed targeted next-generation sequencing analysis, along with a comprehensive immunohistochemical study and FISH analysis for TP53 locus, separately on the four morphologically distinct lesions (Ov-NEC, endometrioid carcinoma, endometriosis, and EAH). The results of our study identified molecular alterations of cancer-related genes (PIK3CA, CTNNB1, TP53, RB1, ARID1A, and p16), which were present with an increasing gradient from preneoplastic lesions to malignant proliferations, both neuroendocrine and non-neuroendocrine components. In conclusion, our findings underscored that the two neoplastic components of this Ov-MiNEN share a substantially identical molecular profile and they progress from a preexisting ovarian endometriotic lesion, in a patient with a coexisting preneoplastic proliferation of the endometrium, genotypically and phenotypically related to the ovarian neoplasm. Moreover, this study supports the inclusion of MiNEN in the spectrum ovarian and, possibly, of all gynecological NENs, among which they are currently not classified

From (cyber)space to ground: New technologies for smart farming

Increased water demand and climate change impacts have recently enhanced the need to improve water resources management, even in those areas which traditionally have an abundant supply of water, such as the Po Valley in northern Italy. The highest consumption of water is devoted to irrigation for agricultural production, and so it is in this area that efforts have to be focused to study possible interventions. Meeting and optimizing the consumption of water for irrigation also means making more resources available for drinking water and industrial use, and maintaining an optimal state of the environment. In this study we show the effectiveness of the combined use of numerical weather predictions and hydrological modelling to forecast soil moisture and crop water requirement in order to optimize irrigation scheduling. This system combines state of the art mathematical models and new technologies for environmental monitoring, merging ground observed data with Earth observations from space and unconventional information from the cyberspace through crowdsourcing

Multilevel approach to male fertility by machine learning highlights a hidden link between haematological and spermatogenetic cells

Male infertility represents a complex clinical condition requiring an accurate multilevel assessment, in which machine learning (ML) technology, combining large data series in nonlinear and highly interactive ways, could be innovatively applied

Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples

Background BRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more sequence variants of uncertain clinical significance, some of which may affect normal splicing patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation or defects in gene function. To understand which novel splicing events are associated with splicing mutations and which are part of the normal BRCA2 splicing repertoire, a study was undertaken by members of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events. Methods mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary or agarose gel electrophoresis, followed by sequencing. Results We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines. Conclusions These naturally occurring alternate-splicing events contribute to the array of cDNA fragments that may be seen in assays for mutation-associated splicing defects. Caution must be observed in assigning alternate-splicing events to potential splicing mutations

Bilateral preaxial polydactyly in a WAGR syndrome patient

We report on a 30-month-old baby girl with typical clinical features of WAGR syndrome. In addition, the patient showed bilateral preaxial polydactyly of the feet. Cytogenetic and fluorescent in situ hybridization (FISH) analyses identified a deletion, del(11)(p13p14.1), extending from 6.1 to 21.7 Mb in size. Although the simultaneous appearance of WAGR and polydactyly has been already described, to our knowledge this is the first case in which the characterization at the cytogenetic molecular level of a patient with these phenotypes is reported. These observations indicate that preaxial polydactyly may be another feature of the WAGR syndrome and suggest the existence of a related gene in the WAGR critical region or in its proximit

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