Sviluppo di un nuovo metodo di screening genetico, per la ricerca di mutazioni nei geni BRCA1 e BRCA2 associati al carcinoma della mammella.

I carcinomi della mammella e dell’ovaio rappresentano le prime cause di mortalità per tumore nel sesso femminile in tutto il mondo; in Italia ed in Europa sono diagnosticati ogni anno, rispettivamente, circa 38.000 e 430.000 nuovi casi di tumore della mammella. Il 90-95% di tutti i casi di carcinoma della mammella e dell’ovaio è da considerarsi di natura sporadica, il restante 5-10% è costituito da forme ereditarie a trasmissione autosomica dominante definite Hereditary Breast and Ovarian Cancer, HBOC. I geni che conferiscono il più elevato rischio di sviluppare questa sindrome tumorale ereditaria sono BRCA1 e BRCA2, identificati nei primi anni ’90. I soggetti che presentano la forma HBOC si distinguono da quelli con il carcinoma di tipo sporadico, soprattutto per la giovane età di insorgenza della malattia e la presenza in famiglia di numerosi casi di carcinoma, non solo della mammella, ma anche dell’ovaio e/o di altri organi. Inoltre, per quanto riguarda il carcinoma della mammella, è possibile riscontrarlo, seppure raramente, anche in soggetti di sesso maschile. Nelle famiglie con mutazioni a carico di tali geni, il rischio cumulativo di sviluppare il carcinoma mammario e/o ovarico entro i 70 anni di età, è pari rispettivamente all’80% ed al 40-60%. BRCA1 è un gene situato sul cromosoma 17, è composto da 24 esoni distribuiti su una regione genica di circa 100 kb e codifica una proteina di 1863 amminoacidi. BRCA2 si trova sul cromosoma 13, è costituito da 27 esoni che coprono una regione genica di circa 70 kb e produce una proteina di 3418 amminoacidi. Ad oggi, sono riportate più di 3000 differenti varianti di sequenza di BRCA1 e BRCA2 (patogeniche e non) distribuite uniformemente su tutta la lunghezza di entrambi i geni (BIC database). In particolare, le mutazioni più frequentemente associate ad aumentato rischio di sviluppare carcinoma della mammella sono costituite dalle mutazioni frameshift e da quelle nonsense, che causano la produzione di una proteina tronca e quindi non funzionale. In seguito alla scoperta, negli anni ’90, di BRCA1 e BRCA2, la possibilità di identificare i soggetti che sono predisposti a sviluppare carcinomi della mammella e/o dell’ovaio ha portato all’implementazione di test molecolari per la rilevazione di mutazioni deleterie in BRCA1 e BRCA2 e la successiva consulenza genetica dei soggetti portatori di mutazione. Fino ad oggi, il sequenziamento diretto costituisce il metodo d’elezione per l’analisi completa dei due geni in questione, tuttavia esso risulta alquanto dispendioso sia in termini economici che di tempo e per questo motivo nel corso degli anni sono state utilizzate delle tecniche di indagine molecolare volte ad uno screening pre-sequenziamento. Tra le varie tecniche che sono state adottate, ricordiamo la Single Strand Conformation Polymorphism (SSCP), il Protein Truncation Test (PTT), la Conformation-Sensitive Gel Electrophoresis (CSGE) e la Denaturing High Performance Liquid Chromatography (DHPLC). La DHPLC risulta senza dubbio la migliore in termini di sensibilità e costi, per cui negli ultimi anni la DHPLC si è affermata come tecnica principale ai fini di screening molecolare di BRCA1 e BRCA2. Presso i laboratori del CEINGE è stata messa a punto una metodica per l’identificazione di mutazioni in BRCA1 e BRCA2. Sono stati utilizzati, ai fini dell’ottimizzazione, 53 soggetti di controllo dell’archivio di Fibrosi Cistica del CEINGE di Napoli e 15 pazienti HBOC provenienti dall’Istituto Oncologico Giovanni Paolo II di Bari, che erano stati precedentemente sottoposti a screening per mutazioni in 6 BRCA1 e BRCA2. Dopo aver ottimizzato le condizioni per l’amplificazione di BRCA1 e BRCA2, i campioni di DNA, estratti da sangue periferico e appartenenti ai 53 soggetti di controllo di Napoli ed ai 15 soggetti HBOC di Bari, sono stati analizzati utilizzando la metodica combinata DHPLC/ nucleasi SURVEYOR®, che è basata sul metodo di sizing in condizioni non-denaturanti, piuttosto che sulle Temperature di Melting (Tm). In pratica, gli ampliconi sono analizzati in DHPLC alla temperatura di 45°C, e sono visualizzati, se wild type, con un singolo picco o, in presenza di mutazioni, con multipli picchi corrispondenti all’amplicone wild type non digerito dall’enzima e ai frammenti di amplicone con eteroduplex che derivano dal taglio enzimatico. La metodologia combinata DHPLC/SURVEYOR® è stata ottimizzata prima su singoli ampliconi, poi è stato ideato un protocollo che consentisse di analizzare più ampliconi simultaneamente, secondo un approccio “multiamplicon”. Tutti i campioni che hanno presentato cromatogrammi compatibili con la presenza di variazioni di sequenza sono stati analizzati mediante sequenziamento diretto allo scopo di caratterizzare le varianti di sequenza identificate. In seguito all’analisi dei campioni che sono stati sequenziati, possiamo affermare che la metodica combinata DHPLC/SURVEYOR® si è rivelata estremamente sensibile, poiché è stata confermata la presenza di polimorfismi e mutazioni in tutti i campioni il cui cromatogramma risultava alterato. In particolare, sono state identificate nei soggetti di controllo 42 varianti geniche, 19 su BRCA1 e 23 su BRCA2, tra cui 26 polimorfismi, 10 UnClassified Variants (UCVs) e 6 nuove varianti, 3 introniche, una esonica, una in 5’UTR ed una in 3’UTR. Nei 15 pazienti HBOC di Bari sono state rilevate tutte le 10 varianti di sequenza che erano precedentemente state identificate presso il centro di Bari. Allo stato attuale, è in corso una fase di reclutamento di pazienti della regione Campania sia con diagnosi clinica di HBOC, sia con carcinoma della mammella di tipo sporadico, da utilizzare come controlli. Fondamentali si sono rivelate, in questa fase progettuale, le collaborazioni con l’Istituto Nazionale Tumori- Fondazione G. Pascale di Napoli ed il Dipartimento Universitario di Chirurgia Generale, Geriatrica, Oncologica e Tecnologie Avanzate della Facoltà di Medicina e Chirurgia della Università degli Studi di Napoli “Federico II”. Per poter ottenere quante più informazioni possibili riguardo le pazienti reclutate, è stato creato un apposito questionario anamnestico, da sottoporre ai soggetti che daranno il consenso a partecipare a questo studio epidemiologico a scopo di ricerca. Le 10 pazienti reclutate finora sono già state sottoposte ad indagine molecolare per la ricerca di mutazioni in BRCA1 e BRCA2. In una di queste pazienti è stata identificata una mutazione deleteria, la p.K2013X, nota sia in letteratura che sul database BIC. La mutazione p.K2013X causa la creazione di un codone di stop, per cui ne risulta la produzione di una proteina tronca e non funzionale e la probabile soppressione di un sito di legame con la proteina Rad51, legame necessario per l’attività di ricombinazione omologa e riparazione dei danni al DNA. Poiché, in base a dati di letteratura, sono state riscontrate mutazioni su BRCA1 e BRCA2 soltanto nel 20% di tutti i casi di HBOC, intendiamo approfondire gli studi molecolari sul carcinoma della mammella e dell’ovaio in particolare sui soggetti clinicamente affetti da HBOC che risulteranno negativi allo screening di BRCA1 e BRCA2, prendendo in considerazione anche altri tipi di target molecolare come ad esempio i microRNA ed il trascrittoma

The pharmacological perturbation of brain zinc impairs BDNF-related signaling and the cognitive performances of young mice.

Zinc (Zn2+) is a pleiotropic modulator of the neuronal and brain activity. The disruption of intraneuronal Zn2+ levels triggers neurotoxic processes and affects neuronal functioning. In this study, we investigated how the pharmacological modulation of brain Zn2+ affects synaptic plasticity and cognition in wild-type mice. To manipulate brain Zn2+ levels, we employed the Zn2+ (and copper) chelator 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol, CQ). CQ was administered for two weeks to 2.5-month-old (m.o.) mice, and effects studied on BDNF-related signaling, metalloproteinase activity as well as learning and memory performances. CQ treatment was found to negatively affect short- and long-term memory performances. The CQ-driven perturbation of brain Zn2+ was found to reduce levels of BDNF, synaptic plasticity-related proteins and dendritic spine density in vivo. Our study highlights the importance of choosing "when", "where", and "how much" in the modulation of brain Zn2+ levels. Our findings confirm the importance of targeting Zn2+ as a therapeutic approach against neurodegenerative conditions but, at the same time, underscore the potential drawbacks of reducing brain Zn2+ availability upon the early stages of development

The molecular analysis of BRCA1 and BRCA2: Next-generation sequencing supersedes conventional approaches

Abstract Background Accurate and sensitive detection of BRCA 1/2 germ-line mutations is crucial for the clinical management of women affected by breast cancer, for prevention and, notably, also for the identification of at-risk healthy relatives. The most widely used methods for BRCA1 / 2 molecular analysis are Sanger sequencing, and denaturing high performance liquid chromatography (dHPLC) followed by the Sanger method. However, recent findings suggest that next-generation sequencing (NGS)-based approaches may be an efficient tool for diagnostic purposes. In this context, we evaluated the effectiveness of NGS for BRCA gene analysis compared with dHPLC/Sanger sequencing. Methods Seventy women were screened for BRCA1/2 mutations by both dHPLC/Sanger sequencing and NGS, and the data were analyzed using a bioinformatic pipeline. Results Sequence data analysis showed that NGS is more sensitive in detecting BRCA 1/2 variants than the conventional procedure, namely, dHPLC/Sanger. Conclusion Next-generation sequencing is more sensitive, faster, easier to use and less expensive than the conventional Sanger method. Consequently, it is a reliable procedure for the routine molecular screening of the BRCA 1/2 genes

Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-\u394Np63\u3b1-RFP and R279H-\u394Np63\u3b1-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome

Crossotomy <i>vs</i> crossectomy for saphenous vein sparing surgery in patients with varicose veins due to ostial incontinence: protocol for double blind, multicenter, randomized trial

Double‑blind/multicenter/randomized trial protocol. Eligibility criteria: age 18-70 yrs; C2-C5 leg varices secondary to the Great Saphenous Vein (GSV) incontinence; GSV size 6-10mm, at 10cm from the Saphenous-Femoral Junction (SFJ); ostial reflux lasting >0.5 sec at duplex ultrasound; negative reflux elimination test; acceptance of the GSV sparing treatment plus partial/total varicose veins removal. Exclusion criteria: non-isolated GSV reflux; district already treated; pregnancy/lactation; impaired walking ability; deep vein thrombosis/insufficiency; severe comorbidities. Participants recruited from 7 Italian tertiary referral centres. Interventions: crossotomy (no SFJ’s tributaries ligation) vs crossectomy. The study aimed to verify if GSV drainage through the SFJ’s tributaries reduces groin/peripheral recurrences. Primary endpoint: 1-year GSV reflux recurrence, positive to the Valsalva maneuver, originating from the SF. Participants equally randomized. Participants, care givers, and those assessing the outcomes blinded to group assignment

Reply to: New Meta- and Mega-analyses of Magnetic Resonance Imaging Findings in Schizophrenia: Do They Really Increase Our Knowledge About the Nature of the Disease Process?

This work was supported by National Institute of Biomedical Imaging and Bioengineering Grant No. U54EB020403 (to the ENIGMA consortium)

A machine-learning based bio-psycho-social model for the prediction of non-obstructive and obstructive coronary artery disease

Background: Mechanisms of myocardial ischemia in obstructive and non-obstructive coronary artery disease (CAD), and the interplay between clinical, functional, biological and psycho-social features, are still far to be fully elucidated. Objectives: To develop a machine-learning (ML) model for the supervised prediction of obstructive versus non-obstructive CAD. Methods: From the EVA study, we analysed adults hospitalized for IHD undergoing conventional coronary angiography (CCA). Non-obstructive CAD was defined by a stenosis &lt; 50% in one or more vessels. Baseline clinical and psycho-socio-cultural characteristics were used for computing a Rockwood and Mitnitski frailty index, and a gender score according to GENESIS-PRAXY methodology. Serum concentration of inflammatory cytokines was measured with a multiplex flow cytometry assay. Through an XGBoost classifier combined with an explainable artificial intelligence tool (SHAP), we identified the most influential features in discriminating obstructive versus non-obstructive CAD. Results: Among the overall EVA cohort (n = 509), 311 individuals (mean age 67 ± 11&nbsp;years, 38% females; 67% obstructive CAD) with complete data were analysed. The ML-based model (83% accuracy and 87% precision) showed that while obstructive CAD was associated with higher frailty index, older age and a cytokine signature characterized by IL-1β, IL-12p70 and IL-33, non-obstructive CAD was associated with a higher gender score (i.e., social characteristics traditionally ascribed to women) and with a cytokine signature characterized by IL-18, IL-8, IL-23. Conclusions: Integrating clinical, biological, and psycho-social features, we have optimized a sex- and gender-unbiased model that discriminates obstructive and non-obstructive CAD. Further mechanistic studies will shed light on the biological plausibility of these associations. Clinical trial registration: NCT02737982

Cortical brain abnormalities in 4474 individuals with schizophrenia and 5098 control subjects via the enhancing neuro Imaging genetics through meta analysis (ENIGMA) Consortium

BACKGROUND: The profile of cortical neuroanatomical abnormalities in schizophrenia is not fully understood, despite hundreds of published structural brain imaging studies. This study presents the first meta-analysis of cortical thickness and surface area abnormalities in schizophrenia conducted by the ENIGMA (Enhancing Neuro Imaging Genetics through Meta Analysis) Schizophrenia Working Group. METHODS: The study included data from 4474 individuals with schizophrenia (mean age, 32.3 years; range, 11-78 years; 66% male) and 5098 healthy volunteers (mean age, 32.8 years; range, 10-87 years; 53% male) assessed with standardized methods at 39 centers worldwide. RESULTS: Compared with healthy volunteers, individuals with schizophrenia have widespread thinner cortex (left/right hemisphere: Cohen's d = -0.530/-0.516) and smaller surface area (left/right hemisphere: Cohen's d = -0.251/-0.254), with the largest effect sizes for both in frontal and temporal lobe regions. Regional group differences in cortical thickness remained significant when statistically controlling for global cortical thickness, suggesting regional specificity. In contrast, effects for cortical surface area appear global. Case-control, negative, cortical thickness effect sizes were two to three times larger in individuals receiving antipsychotic medication relative to unmedicated individuals. Negative correlations between age and bilateral temporal pole thickness were stronger in individuals with schizophrenia than in healthy volunteers. Regional cortical thickness showed significant negative correlations with normalized medication dose, symptom severity, and duration of illness and positive correlations with age at onset. CONCLUSIONS: The findings indicate that the ENIGMA meta-analysis approach can achieve robust findings in clinical neuroscience studies; also, medication effects should be taken into account in future genetic association studies of cortical thickness in schizophrenia

The Sex-Specific Detrimental Effect of Diabetes and Gender-Related Factors on Pre-admission Medication Adherence Among Patients Hospitalized for Ischemic Heart Disease: Insights From EVA Study

Background: Sex and gender-related factors have been under-investigated as relevant determinants of health outcomes across non-communicable chronic diseases. Poor medication adherence results in adverse clinical outcomes and sex differences have been reported among patients at high cardiovascular risk, such as diabetics. The effect of diabetes and gender-related factors on medication adherence among women and men at high risk for ischemic heart disease (IHD) has not yet been fully investigated.Aim: To explore the role of sex, gender-related factors, and diabetes in pre-admission medication adherence among patients hospitalized for IHD.Materials and Methods: Data were obtained from the Endocrine Vascular disease Approach (EVA) (ClinicalTrials.gov Identifier: NCT02737982), a prospective cohort of patients admitted for IHD. We selected patients with baseline information regarding the presence of diabetes, cardiovascular risk factors, and gender-related variables (i.e., gender identity, gender role, gender relations, institutionalized gender). Our primary outcome was the proportion of pre-admission medication adherence defined through a self-reported questionnaire. We performed a sex-stratified analysis of clinical and gender-related factors associated with pre-admission medication adherence.Results: Two-hundred eighty patients admitted for IHD (35% women, mean age 70), were included. Around one-fourth of the patients were low-adherent to therapy before hospitalization, regardless of sex. Low-adherent patients were more likely diabetic (40%) and employed (40%). Sex-stratified analysis showed that low-adherent men were more likely to be employed (58 vs. 33%) and not primary earners (73 vs. 54%), with more masculine traits of personality, as compared with medium-high adherent men. Interestingly, women reporting medication low-adherence were similar for clinical and gender-related factors to those with medium-high adherence, except for diabetes (42 vs. 20%, p = 0.004). In a multivariate adjusted model only employed status was associated with poor medication adherence (OR 0.55, 95%CI 0.31–0.97). However, in the sex-stratified analysis, diabetes was independently associated with medication adherence only in women (OR 0.36; 95%CI 0.13–0.96), whereas a higher masculine BSRI was the only factor associated with medication adherence in men (OR 0.59, 95%CI 0.35–0.99).Conclusion: Pre-admission medication adherence is common in patients hospitalized for IHD, regardless of sex. However, patient-related factors such as diabetes, employment, and personality traits are associated with adherence in a sex-specific manner

Steroid therapy for a case of severe drug-induced cholestasis.

OBJECTIVE: To report a severe case of cholestatic liver disease successfully treated with corticosteroids following combined therapy with clarithromycin and nimesulide. CASE SUMMARY: A 15-year-old girl was admitted with cholestasis probably related to treatment with clarithromycin and nimesulide for an upper respiratory tract infection. Other causes of liver disease (infections, metabolic liver disorders, genetic cholestatic syndromes, autoimmune diseases, primary biliary tract disorders) were excluded. Liver biopsy showed a severe canalicular cholestasis with bile plugs in dilated bile canaliculi, giant cell transformation, and portal and lobular infiltrate. An objective causality assessment suggested that cholestasis was probably related to clarithromycin and/or nimesulide use. No benefit was derived from a course of ursodeoxycholic acid therapy. Since the patient experienced a progressive worsening in cholestasis, prednisone was started after 20 days. This therapy was promptly followed by improvement in clinical and laboratory test results. After 2 months of prednisone treatment, the patient became symptom-free with normal liver function tests. DISCUSSION: The manifestations of drug-induced hepatotoxicity are highly variable, ranging from asymptomatic hypertransaminemia to fulminant hepatic failure. No specific treatment for drug-induced hepatotoxicity exists. Early recognition and drug withdrawal are the keys to management of hepatotoxicity, but in some cases, liver disease may persist despite discontinuation of the drug. Possible advantages of corticosteroid therapy have not been well demonstrated. CONCLUSIONS: Application of the Naranjo probability scale indicates a probable relationship between cholestasis and nimesulide plus clarithromycin use. This case draws attention to a possible therapeutic option for some cases of drug-induced hepatotoxicity that show a severe course without any sign of improvement