Circa 30 million people in Europe suffer from life-threatening rare diseases, which are poorly understood. This major societal burden forces patients into endless paths for a diagnosis often without cure. EU cooperation efforts between countries contribute to improve research and visibility of these diseases with a primary role of the ‘Ospedale pediatrico Bambino Gesù’ (OPBG, Italy). Here, via next generation sequencing technology, Dr. Tartaglia recently described new genetic variants, including some in the tubulin cofactors TBCE and TBCD which alter microtubules (MTs) dynamics (crucial for brain development and maintenance) and cause previously undescribed early onset neurodegeneration. Yet the underlying pathogenic mechanisms and impact on the brain physiology remain elusive. Given the difficulty to reproduce brain physiology in vitro and its general inaccessibility, a vertebrate model amenable to genetic and whole-brain in vivo investigation is necessary. With its rapid and transparent development and genetic and imaging tools improving continuously, the small teleost fish zebrafish is ideal. I will monitor live MTs behaviour and cellular processes (e.g. cell division, axons formation and innervation) in the precursor and mature neurons of zebrafish TBCE and TBCD mutants, focusing on motoneurons. To this aim, I will combine my expertise in fish neurobiology with the host’skills in human genetics (Dr. Tartaglia) and fish gene editing (Dr. Del Bene, France). In line with the current EU efforts, this work will advance our understanding on rare genetic variants causing neurodegeneration. Deciphering the role of MTs dynamics on brain physiology will be relevant also for common brain disorders and exome sequencing of undiagnosed patients will further contribute to a genotype-phenotype catalog. Via an intensive transfer of knowledge, the project will increase EU multi-disciplinary competence on rare disorders in general, providing new models for therapy development.

The current preclinical drug development pipeline is highly inefficient with 9 out of 10 drugs failing when first tested in humans. Of those failures, between 69% and 81% is due to a lack of efficacy (52-57%) or safety (17-24%), indicating that the predictivity of currently used cell culture and animal models is not high enough. While promising human cell culture models have arisen in the last decades (e.g. pluripotent stem cell models, spheroids, organoids), the environment of these models is too simplistic to reach their full potential and to translate certain responses to humans. One of the important aspects missing in current cell culture is kinetics: many processes in the human body have response times with resolutions of seconds, minutes or hours. In standard cell culture however, such temporal resolution is absent because cell culture medium remains unchanged for typically 1-3 days. So for kinetics, scientists turn to animal models with relatively low translational success and high costs. As an alternative, complex microfluidic setups have been used to enable perfusion in vitro and demonstrated a significant amount of evidence that kinetics can improve the relevance of cell culture models. Despite the evidence, typical cell culture biologists are not taking kinetics into account because the tools to control kinetics in cell culture are too complex. We solve this problem by starting from what cell culture biologists currently use: a standard well plate. We developed a µFluidic Adaptor that can be clamped on any 96 well plate and completely replaces the fluid in the well homogenously without disturbing the cell culture. Our goal is to develop a plug-and-play peripheral system that integrates automated fluidics and microscopic readout in a fully controlled environment: the δypha System. The system uses µFluidic Adaptors that can be designed for different applications or well plate formats.

Steroid-sensitive nephrotic syndrome (SSNS) is a rare, mostly childhood kidney disease defined by selective proteinuria, hypoalbuminaemia and minimal histological changes without Ig deposits. It has been postulated that SSNS is immune mediated and involves T-cell dysregulation. Association studies with HLA class-2 alleles are small or limited to an exome array on a South Asian population. Building on our GWAS in membranous nephropathy (MN), (Stanescu et al, NEJM 2011, 364: 616) and a first set of data obtained by GWAS and NGS in 425 and 96 SSNS patients, respectively, our objectives are to identify additional gene variants by GWAS, to sequence the GWAS-tagged loci by NGS, and to investigate the functional significance of the relevant gene variants. This project is very innovative because it combines multi-ethnic and family studies, full sequencing of GWAS-tagged loci, functional studies, and clinical correlations in deeply phenotyped cohorts, as well as a comparison with risk variants in MN. This study comprises 5 workpackages. WP 1: GWAS discovery of new SNPs associated with SSNS We have tripled the number of enrolled patients (non-Chinese available for the current study: 375 patients; Chinese: 500 patients; grand total, 1,400 patients including already studied patients) to discover new SNPs, particularly in non-HLA-D associated loci. WP 2: Deep genotyping of relevant loci by NGS: HLA-D and beyond Our first aim is to provide a fine mapping of loci tagged by GWAS within HLA-D locus and outside of it. Studies will include logistic regression analysis of top SNPs, and identification of ethnicity-specific SNPs which may explain part of the geographic heterogeneity of SSNS, as well as common, trans-ethnic SNPs, which may be associated with key pathways affecting the immune system or the podocyte. A second objective is to compare these data with those in MN (220 patients sequenced, unpublished results) because of shared characteristics with SSNS. WP 3: Family studies Our objective is to discover variants associated with 22 families of SSNS, which may reveal a strong effect of a rare variant of a gene involved as a common variant in sporadic cases. We aim to identify coding variants (mutations) by whole exome studies (WES) as well as non-coding and coding variants within HLA-D locus and outside of it, by targeted sequencing of candidate genes and loci. WP 4: Functional studies of relevant gene variants They will depend on the findings of genetic studies, which may provide clues to permeability factor(s) or to altered signaling pathways. Two lines of research can already be envisaged: • Characterization of regulatory and coding SNPs: We expect to find disease-associated non-coding SNPs within regulatory sequences as well as SNPs in the coding regions. Computational approaches will first guide the design of functional assays. We will use 41 glomerular transcriptomes from SSNS patients to correlate tag and regulatory SNPs with actual transcriptional changes in the glomerulus. • Identification of HLA-class 2 molecules: the road toward identification of T-cell epitopes: We will characterize risk allelotypes, haplotypes and amino acids on HLA class-2 molecules, and we will use the relevant anti-allelotype antibodies to immunoprecipitate the circulating HLA class-2 peptide complexes followed by mass spectometry analysis. WP 5: Clinical correlations Correlations will be established between clinical presentation (demography) and outcome, and genotype (alleles of top SNPs, risk allelotypes and amino acids). With our deeply phenotyped, multi-ethnic cohort also including adult patients (30% of the Spanish and 60% of the Chinese cohorts), we have the unique opportunity to establish correlations with response to treatment depending on age and ethnicity. These studies will contribute to SSNS pathogenesis and hopefully provide new disease biomarkers and drug targets.

The medical sector is striving to enhance treatment options, decrease serious adverse events and increase patient quality of life. The area of surgery is continuously developing. Minimal invasive surgery (MIS) has become an important aspect of surgery in adults. In paediatric surgery, new surgical techniques are also being developed. However, there is a large knowledge gap between the newly developed surgical techniques and its use within Europe and even within the rest of the world. In order to tackle skills gaps and mismatches, the focus of this TEACHER project will be on the rare disease Esophageal Atresia (EA) and tracheal malformations in newborns. For this disease, only few medical centres in Europe have the expertise to perform innovative paediatric surgical techniques (both open surgery and via MIS). The level of education on innovative paediatric surgery is very low and an educational programme on EA and tracheal malformations does not exist. Also, newly discovered surgical techniques are not well shared among medical centres within Europe.TEACHER counteracts the fragmented and internally focused medical centres within Europe, by setting up this network between Italy, Sweden, the UK and The Netherlands. Creating an inclusive higher medical education programme will be the start of a strong strategic network of medical centres to exchange innovative techniques, thereby exponentially increasing knowledge and skills. The TEACHER partners have therefore formulated three objectives:-To define a competency profile for paediatric surgical EA and tracheal malformations (IO1)-To develop an online training programme for junior paediatric surgeons (IO2)-To develop a blended teach the teacher programme for senior paediatric surgeons (IO3)The TEACHER partnership consists of 4 medical centres and two industry partners. The medical partners are worldwide renowned centres for paediatric surgery and have specific expertise in the field of EA and tracheal malformations. The two industry partners Elevate and Incision bring in their expertise on online education. Elevate brings in knowledge and expertise regarding the development of distance-learning online curricula. Incision will bring in their knowledge about online course material containing step-by-step best practice protocols for surgical procedures.The activities are divided in three IO’s:•IO1: The first IO focuses on the development of a competency profile for paediatric surgeons with clinical and research tasks. This forms the basis for the education of paediatric surgeons. The competency profile will be based on literature review, questionnaires and focus groups.•IO2: A training programme for junior paediatric surgeons of 30 ECTS will be developed. This will be a blended learning programme based on online education combined with hands-on education by senior paediatric surgeons. The training programme will be based on the four-component instructional design model (4C/ID model). Pilot course runs will be carried out to evaluate the training programme. •IO3: The blended teach the teacher programme for senior paediatric surgeons of 20 ECTS will start with describing the intended learning outcomes. Based on the learning outcomes, the learning tools and instructional design models or theories will be defined in order to reach the learning outcomes. Next step is to create a blended learning course outline and syllabus.TEACHER will deliver three IOs. The work for each IO is coordinated by a lead partner who will be responsible for setting up, managing, monitoring, evaluating and finalizing the activities and for producing the deliverables. In distributing and dividing the work in the project, the partners have taken background, knowledge and expertise of each of the partners as starting point. UMC Utrecht as project coordinator, is responsible for the overall management of the project.TEACHER will increase the knowledge level of the paediatric surgeons with a focus on EA and tracheal malformations. Junior and senior paediatric surgeons will develop new skills, not only related to the medical skills but also related to multiple roles healthcare professionals need to play today, like setting up treatment plans and acquiring necessary research skills. It will lead to more uniformity in working methods and procedures. As such it will also improve the quality of research. TEACHER will build a knowledge structure of benefit for European medical professionals and researchers. Throughout Europe several centres of expertise will develop and strengthen their position. From a healthcare perspective this project contributes to an increased quality of life of patients and equal opportunities for patients in all member states of the EU.