Patient preferences in genetic newborn screening for rare diseases: study protocol.

INTRODUCTION: Rare diseases (RDs) collectively impact over 30 million people in Europe. Most individual conditions have a low prevalence which has resulted in a lack of research and expertise in this field, especially regarding genetic newborn screening (gNBS). There is increasing recognition of the importance of incorporating patients' needs and general public perspectives into the shared decision-making process regarding gNBS. This study is part of the Innovative Medicine Initiative project Screen4Care which aims at shortening the diagnostic journey for RDs by accelerating diagnosis for patients living with RDs through gNBS and the use of digital technologies, such as artificial intelligence and machine learning. Our objective will be to assess expecting parent's perspectives, attitudes and preferences regarding gNBS for RDs in Italy and Germany. METHODS AND ANALYSIS: A mixed method approach will assess perspectives, attitudes and preferences of (1) expecting parents seeking genetic consultation and (2) 'healthy' expecting parents from the general population in two countries (Germany and Italy). Focus groups and interviews using the nominal group technique and ranking exercises will be performed (qualitative phase). The results will inform the treatment of attributes to be assessed via a survey and a discrete choice experiment (DCE). The total recruitment sample will be 2084 participants (approximatively 1000 participants in each country for the online survey). A combination of thematic qualitative and logit-based quantitative approaches will be used to analyse the results of the study. ETHICS AND DISSEMINATION: This study has been approved by the Erlangen University Ethics Committee (22-246_1-B), the Freiburg University Ethics Committee (23-1005 S1-AV) and clinical centres in Italy (University of FerraraCE: 357/2023/Oss/AOUFe and Hospedale Bambino Gesu: No.2997 of 2 November 2023, Prot. No. _902) and approved for data storage and handling at the Uppsala University (2022-05806-01). The dissemination of the results will be ensured via scientific journal publication (open access).

Genetic newborn screening and digital technologies: A project protocol based on a dual approach to shorten the rare diseases diagnostic path in Europe.

Since 72% of rare diseases are genetic in origin and mostly paediatrics, genetic newborn screening represents a diagnostic "window of opportunity". Therefore, many gNBS initiatives started in different European countries. Screen4Care is a research project, which resulted of a joint effort between the European Union Commission and the European Federation of Pharmaceutical Industries and Associations. It focuses on genetic newborn screening and artificial intelligence-based tools which will be applied to a large European population of about 25.000 infants. The neonatal screening strategy will be based on targeted sequencing, while whole genome sequencing will be offered to all enrolled infants who may show early symptoms but have resulted negative at the targeted sequencing-based newborn screening. We will leverage artificial intelligence-based algorithms to identify patients using Electronic Health Records (EHR) and to build a repository "symptom checkers" for patients and healthcare providers. S4C will design an equitable, ethical, and sustainable framework for genetic newborn screening and new digital tools, corroborated by a large workout where legal, ethical, and social complexities will be addressed with the intent of making the framework highly and flexibly translatable into the diverse European health systems.

The mycotoxin beauvericin impairs development, fertility and life span in the nematode Caenorhabditis elegans accompanied by increased germ cell apoptosis and lipofuscin accumulation.

Beauvericin is an ubiquitous mycotoxin with relevant occurrence in food and feed. It causes a high toxicity in several cell lines, but its general mechanism of action is not fully understood and only limited in vivo studies have been performed. We used Caenorhabditis elegans as a model organism to investigate effects of beauvericin. The mycotoxin displays a moderate acute toxicity at 100 µM; at this concentration also reproductive toxicity occurred (reduction of total progeny to 32.1 %), developmental toxicity was detectable at 250 µM. However, even lower concentrations were capable to reduce stress resistance and life span of the nematode: A significant reduction was detected at 10 µM beauvericin (decrease in mean survival time of 4.3 % and reduction in life span of 12.9 %). An increase in lipofuscin fluorescence was demonstrated starting at 10 µM suggesting oxidative stress as a mechanism of beauvericin toxicity. Beauvericin (100 µM) increases the number of apoptotic germ cells comparable to the positive control UV-C (400 J/m2). Conclusion: Low concentrations of beauvericin are capable to cause adverse effects in C. elegans, which may be relevant for hazard identification of this compound.

Agrimonia procera Wallr. Extract Increases Stress Resistance and Prolongs Life Span in Caenorhabditis elegans via Transcription Factor DAF-16 (FoxO Orthologue).

Agrimonia procera is a pharmacologically interesting plant which is proposed to protect against various diseases due to its high amount of phytochemicals, e.g., polyphenols. However, in spite of the amount of postulated health benefits, studies concerning the mechanistic effects of Agrimonia procera are limited. Using the nematode Caenorhabditis elegans, we were able to show that an ethanol extract of Agrimonia procera herba (eAE) mediates strong antioxidative effects in the nematode: Beside a strong radical-scavenging activity, eAE reduces accumulation of reactive oxygen species (ROS) accumulation and protects against paraquat-induced oxidative stress. The extract does not protect against amyloid-ß-mediated toxicity, but efficiently increases the life span (up to 12.7%), as well as the resistance to thermal stress (prolongation of survival up to 22%), of this model organism. Using nematodes deficient in the forkhead box O (FoxO)-orthologue DAF-16, we were able to demonstrate that beneficial effects of eAE on stress resistance and life span were mediated via this transcription factor. We showed antioxidative, stress-reducing, and life-prolonging effects of eAE in vivo and were able to demonstrate a molecular mechanism of this extract. These results may be important for identifying further molecular targets of eAE in humans.

Polygonum multiflorum Extract Exerts Antioxidative Effects and Increases Life Span and Stress Resistance in the Model Organism Caenorhabditis elegans via DAF-16 and SIR-2.1.

Extracts of the Chinese plant Polygonum multiflorum (PME) are used for medicinal purposes as well as food supplement due to anti-aging effects. Despite of the common use of these food supplements, experimental data on physiological effects of PME and its underlying molecular mechanisms in vivo are limited. We used the model organism Caenorhabditis elegans to analyze anti-aging-effects of PME in vivo (life span, lipofuscin accumulation, oxidative stress resistance, thermal stress resistance) as well as the molecular signaling pathways involved. The effects of PME were examined in wildtype animals and mutants defective in the sirtuin-homologue SIR-2.1 (VC199) and the FOXO-homologue DAF-16 (CF1038). PME possesses antioxidative effects in vivo and increases oxidative stress resistance of the nematodes. While the accumulation of lipofuscin is only slightly decreased, PME causes a significant elongation (18.6%) of mean life span. DAF-16 is essential for the reduction of thermally induced ROS accumulation, while the resistance against paraquat-induced oxidative stress is dependent on SIR-2.1. For the extension of the life span, both DAF-16 and SIR-2.1 are needed. We demonstrate that PME exerts protective effects in C. elegans via modulation of distinct intracellular pathways.