Balancing scientific interests and the rights of participants in designing a recall by genotype study

Recall by genotype (RbG) studies aim to better understand the phenotypes that correspond to genetic variants of interest, by recruiting carriers of such variants for further phenotyping. RbG approaches pose major ethical and legal challenges related to the disclosure of possibly unwanted genetic information. The Cooperative Health Research in South Tyrol (CHRIS) study is a longitudinal cohort study based in South Tyrol, Italy. Demand has grown for CHRIS study participants to be enrolled in RbG studies, thus making the design of a suitable ethical framework a pressing need. We here report upon the design of a pilot RbG study conducted with CHRIS study participants. By reviewing the literature and by consulting relevant stakeholders (CHRIS participants, clinical geneticists, ethics board, GPs), we identified key ethical issues in RbG approaches (e.g. complexity of the context, communication of genetic results, measures to further protect participants). The design of the pilot was based on a feasibility assessment, the selection of a suitable test case within the ProtectMove Research Unit on reduced penetrance of hereditary movement disorders, and the development of appropriate recruitment and communication strategies. An empirical study was embedded in the pilot study with the aim of understanding participants’ views on RbG. Our experience with the pilot study in CHRIS allowed us to contribute to the development of best practices and policies for RbG studies by drawing recommendations: addressing the possibility of RbG in the original consent, implementing tailored communication strategies, engaging stakeholders, designing embedded empirical studies, and sharing research experiences and methodology

Functional characterization of the p13 protein of HTLV-1

HTLV-1 is the causative agent of adult T-cell leukemia-lymphoma (ATLL), an aggressive neoplasm of mature CD4+ T cells refractory to current therapies, and of HAM/TSP, a neurodegenerative disease. HTLV-1 is a complex retrovirus whose genome encodes several regulatory and accessory proteins (Rex, Tax, HBZ, p12, p30, p13) in addition to the structural proteins (gag, pol, env) common to all retroviruses. Previous studies demonstrated that HTLV-1 p13, an 87-amino acid protein, is targeted to the inner membrane of mitochondria and induces mitochondrial fragmentation. In vitro studies carried out using the full-length synthetic protein and isolated mitochondria showed that p13 induces a potential-dependent potassium (K+) influx into the mitochondrial matrix, accompanied by a dose-dependent depolarization, and reduces the threshold for permeability transition pore (PTP) opening. This effect on K+ permeability is mediated by an amphipatic α-helical domain (amino acids 21-30), in which four positively charged arginines play a key role. p13 slows down cell proliferation, promotes apoptosis triggered by ceramide and Fas L, and interferes with tumor growth in experimental models. The studies described in the present thesis were aimed at testing the effects of p13 on mitochondria in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we demonstrated that p13 induces dose-dependent mitochondrial depolarization of HeLa cells. The p13RQ mutant, which is inactive on K+ permeability, had no effect on mitochondrial membrane potential (∆ψ). Similar results were obtained with Jurkat T-cells. As ∆ψ is a key factor controlling mitochondrial calcium (Ca2+) uptake, we analyzed the effect of p13 on Ca2+ homeostasis. To this end, we employed organelle-targeted aequorin and showed that p13 specifically reduced mitochondrial Ca2+ uptake, upon histamine stimulation, while it does not alter Ca2+ concentration in the cytoplasm or the endoplasmic reticulum (ER). This effect suggests that p13 might control key processes regulated through Ca2+ signalling, such as T-cell activation and death. The observation that p13's effect on ∆ψ was heterogeneous at intermediate levels of expression suggested the existence of mechanisms regulating p13 function. In the present study, we investigated the possible role of phosphorylation in the regulation of p13 function. In particular, we focused on serine 15 (S15), a putative protein kinase C (PKC) phosphorylation site located in the proximity of the α-helical domain. In vitro phosphorylation assays on p13[9-22] and p13S15A[9-22] synthetic peptides confirmed the phosphorylation of S15 by purified PKC and by cytosolic and mitochondrial lysates from Jurkat T-cells as a source of kinases. We also confirmed the in vitro phosphorylation of the full-length synthetic p13 by Jurkat T-cell lysates. Preliminary results obtained by 2D-electrophoresis analysis of Jurkat T-cells transiently transfected with p13 demonstrated that the protein is detected in two main forms differing in their pI. We then analyzed the functional effects of S15 phosphorylation by producing the phosphoablative (p13S15A) and phosphomimetic (p13S15D) mutants and studying their properties in HeLa cells. Results demonstrated that the presence of a negative charge on residue 15 enhances the effect of p13 on the induction of mitochondrial fragmentation. Interestingly, using TMRM, we found that the phosphomimetic mutant induces mitochondrial depolarization at much lower level of expression, compared to p13 wild-type. Taken together, these results suggest that phosphorylation on S15 might influence the biological properties of p13, and open the door to future studies of this and other cellular mechanisms regulating p13 function.Il virus T-linfotropico umano di tipo 1 (HTLV-1) è associato all'insorgenza della leucemia-linfoma a cellule T dell'adulto (ATLL), un'aggressiva neoplasia dei linfociti T CD4+ maturi, e della mielopatia associata ad HTLV-1/paraparesi spastica tropicale (HAM/TSP), una patologia degenerativa del sistema nervoso centrale. HTLV-1 è classificato come retrovirus complesso in quanto il suo genoma codifica, oltre a proteine strutturali (gag, env, pol), anche proteine regolatorie (Tax e Rex) ed accessorie (p30, p12, p13, HBZ), dalla funzione ancora non del tutto chiarita. Precedentamente è stato dimostrato che la proteina accessoria p13 (la cui sequenza è costituita da 87 aminoacidi) si localizza nella membrana mitocondriale interna ed induce marcata frammentazione di questi organelli. Mediante studi in vitro condotti con p13 sintetica e mitocondri isolati è stato dimostrato che p13 induce un influsso di potassio (K+) potenziale-dipendente all'interno della matrice mitocondriale. L'alterazione della permeabilità mitocondriale indotta da p13 è accompagnata da una depolarizzazione dose-dipendente e da una riduzione della soglia di apertura del poro di transizione della permeabilità mitocondriale (PTP). L'influsso di K+ è dovuto alla presenza di un dominio anfipatico ad α-elica (tra gli aminoacidi in posizione 21 e 30), in cui le quattro arginine in posizione 22, 25, 29 e 30 svolgono un ruolo fondamentale per la funzione di p13. p13 rallenta la proliferazione cellulare, promuove l'apoptosi mediata da ceramide e Fas L e interferisce con la crescita tumorale in modelli sperimentali in vivo. Gli studi descritti nella presente tesi sono stati mirati alla analisi degli effetti di p13 a livello mitocondriale in cellule vitali. Usando la sonda potenziale-dipendente tetrametil-rodamina-metil-estere (TMRM), è stato dimostrato che p13 induce una depolarizzazione mitocondriale dose-dipendente in cellule HeLa. Il mutante p13RQ, che non altera la permeabilità della membrana interna al K+, non ha evidenziato effetti a livello del potenziale di membrana mitocondriale (∆ψ). Risultati analoghi sono stati ottenuti utilizzando cellule T come modello cellulare. Dato che il ∆ψ è fondamentale nel controllo dell'ingresso di calcio (Ca2+) nel mitocondrio, abbiamo analizzati gli effetti di p13 nell'omeostasi del Ca2+. A tal fine abbiamo utilizzato la sonda Ca2+-sensibile equorina, specifica per dati compartimenti cellulari. I risultati di questa analisi hanno evidenziato che, in seguito a stimolazione con istamina, l'influsso mitocondriale di Ca2+ risulta ridotto in presenza di p13, che invece non induce alterazioni nella concentrazione di Ca2+ del citoplasma e del reticolo endoplasmatico. Questo effetto suggerisce che p13 potrebbe controllare fondamentali processi, regolati da Ca2+, come l'attivazione e la morte delle cellule T. L'osservazione dell' eterogeneità dell' effetto di p13 sul ∆ψ, a livelli intermedi di espressione, ha suggerito l'esistenza di meccanismi preposti alla regolazione della funzione della proteina. Abbiamo quindi analizzato il ruolo della fosforilazione come possibile evento regolativo della funzione di p13. In particolare, è stata individuata la serina in posizione 15 come possibile sito di fosforilazione da parte di protein-chinasi C (PKC). S15 potrebbe essere un sito chiave per la regolazione della funzione di p13 poiché è localizzata vicino al dominio funzionale ad α-elica, responsabile dell'influsso di K+ nella matrice mitocondriale. Saggi di fosforilazione in vitro condotti sui peptidi sintetici p13[9-22] e p13S15A[9-22] hanno confermato la fosforilazione di S15 da parte di lisati cellulari (mitocondriale e citosolico) di cellule T Jurkat, oltre che da parte di una miscela commerciale di PKC, come fonte di chinasi. La reazione di fosforilazione in vitro da parte di lisati cellulari è stata anche confermata sulla proteina sintetica p13[1-87]. Risultati preliminari ottenuti mediante esperimenti di elettroforesi bidimensionale di lisati di cellule T Jurkat trasfettate transientemente con p13 hanno dimostrato che la proteina, espressa in cellule T, esiste in due forme principali, che differiscono per punto isoelettrico (pI). Sono stati inoltre analizzati gli effetti funzionali della fosforilazione di S15 producendo dei mutanti fosfomimetico (p13S1D) e fosfoablativo (p13S15A) e studiando le loro proprietà in cellule HeLa. I risultati di questi studi hanno evidenziato che la presenza della carica negativa nel residuo 15 rafforza gli effetti di p13 nell'induzione della frammentazione mitocondriale. Mediante saggi di incubazione con TMRM è stato dimostrato che il mutante fosfomimetico induce depolarizzazione mitocondriale ad un livello di espressione inferiore rispetto alla proteina wild-type. Complessivamente, questi risultati suggeriscono che la fosforilazione in S15 possa influenzare le proprietà biologiche di p13. Questo studio apre la strada ad ulteriori indagini, finalizzate all'analisi di questo e di altri meccanismi alla base della regolazione della funzione di p13

Regulation of Human Adenovirus Alternative RNA Splicing by the Adenoviral L4-33K and L4-22K Proteins

Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced viral mRNAs. Studies aimed at characterizing the interactions between the virus and the host cell RNA splicing machinery have identified three viral proteins of special significance for the control of late viral gene expression: L4-33K, L4-22K, and E4-ORF4. L4-33K is a viral alternative RNA splicing factor that controls L1 alternative splicing via an interaction with the cellular protein kinases Protein Kinase A (PKA) and DNA-dependent protein kinase (DNA-PK). L4-22K is a viral transcription factor that also has been implicated in the splicing of a subset of late viral mRNAs. E4-ORF4 is a viral protein that binds the cellular protein phosphatase IIA (PP2A) and controls Serine/Arginine (SR)-rich protein activity by inducing SR protein dephosphorylation. The L4-33K, and most likely also the L4-22K protein, are highly phosphorylated in vivo. Here we will review the function of these viral proteins in the post-transcriptional control of adenoviral gene expression and further discuss the significance of potential protein kinases phosphorylating the L4-33K and/or L4-22K proteins

The dynamic consent of the Cooperative Health Research in South Tyrol (CHRIS) study : broad aim within specific oversight and communication

In biobanking and genomics research, data and samples are stored for long time and used in further studies, which may not be sufficiently specified or foreseen at the time of the initial consent. The dynamic consent of the CHRIS study integrates broad research aims, specific oversight and governance mechanisms, and continuous communication with participants, and allows nuanced choices to be changed over time. With this paper, we describe the CHRIS dynamic consent, and illustrate, by discussing data sharing and ongoing consent in the CHRIS study, how dynamic consent can actualize an informed consent model that is suitable for biobanking and genomic research

Italians locked down : people's responses to early COVID-19 pandemic public health measures

At the beginning of 2020, the widespread diffusion of SARS-CoV-2 rapidly became a worldwide priority. In Italy, the government implemented a lockdown for more than two months (March 9–May 18). Aware of the uniqueness of such an experience, we designed an online qualitative study focused on three main dimensions: daily life during the lockdown, relationships with others, and public health issues. The aim was to gain insights into people’s experiences of, and attitudes toward, the changes caused by public health measures implemented as a response to the COVID-19 pandemic. We conducted 18 semi-structured interviews with Italian residents. The interviewees were recruited through mediators using purposive sampling to obtain a balanced sample with respect to age, gender, education, and geographical residence. Interviews were analyzed through qualitative content analysis. The lockdown affected a variety of aspects of people’s life, resulting in a significant re-shaping of daily activities and relationships. These changes, which entailed both positive and negative aspects, were met with resilience. Even though public health measures were generally considered acceptable and adequate, they were also perceived to generate uncertainty and stress as well as to reveal tensions within the public health system. When tasked with imagining a scenario with saturated intensive care units and the need for selection criteria, respondents showed a tendency to dodge the question and struggled to formulate criteria. Media and news were found to be confusing, leading to a renewed critical attitude toward information. The findings shed some light on the impact of the lockdown on people’s daily life and its effects on relationships with others. Furthermore, the study contributes to an understanding of people’s reasons for, and capacity to respond to, emergency public health measures

Ethical, legal and social/societal implications (ELSI) of recall-by-genotype (RbG) and genotype-driven-research (GDR) approaches : a scoping review

Recall by Genotype (RbG), Genotype-driven-recall (GDR), and Genotype-based-recall (GBR) strategies are increasingly used to conduct genomic or biobanking sub-studies that single out participants as eligible because of their specific individual genotypic information. However, existing regulatory and governance frameworks do not apply to all aspects of genotype-driven research approaches. The recall strategies disclose or withhold personal genotypic information with uncertain clinical utility. Accordingly, this scoping review aims to identify peculiar, explicit and implicit ethical, legal, and societal/social implications (ELSI) of RbG study designs. We conducted a systematic literature search of three electronic databases from November 2020 to February 2021. We investigated qualitative and quantitative research methods used to report ELSI aspects in RbG research. Congruent with other research findings, we identified a lack of qualitative research investigating the particular ELSI challenges with RbG. We included and analysed the content of twenty-five publications. We found a consensus on RbG posing significant ethical issues, dilemmas, barriers, concerns and societal challenges. However, we found that the approaches to disclosure and study-specific recall and communication strategies employed consent models and Return of Research Results (RoRR) policies varied considerably. Furthermore, we identified a high heterogeneity in perspectives of participants and experts about ELSI of study-specific RbG policies. Therefore, further fine-mapping through qualitative and empirical research is needed to draw conclusions and re-fine ELSI frameworks

Control of cell death pathways by HTLV-1 proteins

Individuals infected with HTLV-1 harbor the virus mainly in CD4+ memory T-cells as a lifelong infection that remains subclinical in the majority of cases. However, about 3-5% of HTLV-1-infected individuals develop an aggressive T-cell neoplasia (ATLL) or a neurodegenerative disease (TSP/HAM) after a latency period ranging from years to decades. This review summarizes the current knowledge of the effects of the HTLV-1 proteins Tax, p13 and p12 on cell death and survival pathways. Tax, the major oncogenic determinant of HTLV-1, enhances cell survival through its effects on the NF-kappaB, CREB and AKT pathways and on the tumor suppressors p53 and Rb. p13 is targeted to the inner mitochondrial membrane and sensitizes cells to the Fas/ceramide apoptotic pathway and reactive oxygen species-mediated cell death. p12 enhances release of calcium from the endoplasmic reticulum and therefore may influence calcium-dependent apoptotic signals, including opening of the mitochondrial permeability transition pore. The long-term fate of HTLV-1-infected cells (apoptosis, survival, transformation) may therefore depend on the balance of the effects of Tax, p13 and p12 on cell death pathways

Municipal wastewater affects adipose deposition in male mice and increases 3T3-L1 cell differentiation

Trace concentration of EDs (endocrine disrupting compounds) in water bodies caused by wastewater treatment plant effluents is a recognized problem for the health of aquatic organisms and their potential to affect human health. In this paper we show that continuous exposure of male mice from early development to the adult life (140days) to unrestricted drinking of wastewater collected from a municipal sewage treatment plant, is associated with an increased adipose deposition and weight gain during adulthood because of altered body homeostasis. In parallel, bisphenol A (BPA) at the administration dose of 5μg/kg/body weight, shows an increasing effect on total body weight and fat mass. In vitro, a solid phase extract (SPE) of the wastewater (eTW), caused stimulation of 3T3-L1 adipocyte differentiation at dilutions of 0.4 and 1% in the final culture medium which contained a concentration of BPA of 40nM and 90nM respectively. Pure BPA also promoted adipocytes differentiation at the concentration of 50 and 80μM. BPA effect in 3T3-L1 cells was associated to the specific activation of the estrogen receptor alpha (ERα) in undifferentiated cells and the estrogen receptor beta (ERβ) in differentiated cells. BPA also activated the Peroxisome Proliferator Activated Receptor gamma (PPARγ) upregulating a minimal 3XPPARE luciferase reporter and the PPARγ-target promoter of the aP2 gene in adipose cells, while it was not effective in preadipocytes. The pure estrogen receptor agonist diethylstilbestrol (DES) played an opposite action to that of BPA inhibiting PPARγ activity in adipocytes, preventing cell differentiation, activating ERα in preadipocytes and inhibiting ERα and ERβ regulation in adipocytes. The results of this work show that the drinking of chemically-contaminated wastewater promotes fat deposition in male mice and that EDs present in sewage are likely responsible for this effect through a nuclear receptor-mediated mechanism

Ten years of dynamic consent in the CHRIS study : informed consent as a dynamic process

The Cooperative Health Research in South Tyrol (CHRIS) is a longitudinal study in Northern Italy, using dynamic consent since its inception in 2011. The CHRIS study collects health data and biosamples for research, and foresees regular follow-ups over time. We describe the experience with the CHRIS study dynamic consent, providing an overview of its conceptualization and implementation, and of the participant-centered strategies used to assess and improve the process, directly linked to participation and communication. In order to comply with high ethical standards and to allow broadness in the areas of research, CHRIS dynamic consent was conceived as an interactive process: based on a strong governance and an ongoing tailored communication with participants, it aims to promote autonomy and to develop a trust-based engaged relationship with participants, also relevant for retention. Built within an online platform, the consent allows granular choices, which can be changed over time. In a process of co-production, participants views have been investigated and kept into account in policy development. Participants showed a high degree of participation, thus enabling the consolidation of the CHRIS resources. Even though a low change rate was reported in the baseline, participants valued the possibility of changing their informed consent choices. Communication (language-tailored, ongoing, multimedia) was important for participants, and for participation and retention. In our experience, dynamic consent was proven to be a flexible consent model, which allowed to meet ethical and legal standards for participation in research, and to accommodate participants' and researchers' needs