Amyotrophic Lateral Sclerosis (ALS)

The study has two components, one in the field of expology and the other in epidemiology. The first part will involve instantaneous atmospheric measurement of organophosphorus additives in oils, namely TCP isomers, TXP isomers (commercially available) and TBP, during any fume event, using a portable device developed for this type of event (pre-positioned on all aircraft), over a short time span (around 1 h). An identical measurement campaign will be systematically carried out on a "control" flight free of fume event, within a week. The aim of this experiment is to sample all fume event (around 100) occurring over a period of 12 months and as many fume event free flights. At the same time, carbon monoxide will be measured along with organophosphates during the event. In parallel with these measurements, a "flight self-questionnaire" will be compiled on flight characteristics and cabin environmental conditions will be completed by the flight's in-charge. The second epidemiological phase will take place in two stages. Each measurement, whether or not it concerns event or not, will be coupled with a systematic self-questionnaire collection of symptoms experienced during the flight, reported by exposed and non-exposed flight crews. (T0), no later than 72 hours after the end of the flight. In addition, in order to identify acute and delayed neurological impairment and to assess cognitive functions, each subject included (exposed and unexposed) will perform : * A self-assessment within 72 hours (Phase 1) and 3 months later (Phase 2) of their neurocognitive performance using the CANTAB (Cambridge Neuropsychological Test Automated Battery) coupled with a self-assessment of fatigue and anxiety levels using self-questionnaires. * A medical consultation 3 months later (Phase 2), including a standardized medical check-up with neuromuscular and neurosensory tests, self-questionnaires and neurocognitive evaluation identical to those used in Phase 1. Phase 2 is carried out three months after exposure, in order to investigate effects with a latency up to 5 weeks after exposure.

This trial aims to evaluate the effects (i.e. safety and uptake) of a new radiotracer molecule. Study participants will take part in the study by attending two to three study visits over a period of up to 3 months (from the screening visit up to the last study visit). The study consists of three parts in which a total of up to 45 participants may be included: Part 1 may include in total up to 15 participants: * up to 5 healthy controls (HCs) * up to 5 symptomatic progranulin gene (GRN) and up to 5 symptomatic chromosome 9 open reading frame 72 (C9orf72) mutation carriers with FTD (including prodromal) either with or without motor neuron disease (MND) characteristics. If the safety and dosimetry are satisfactory in the first subjects and sufficient data are obtained from this part, Part 2 may be initiated. Part 2 may include in total up to 30 participants including: * up to 25 patients with TDP-43 proteinopathies: up to 10 additional mutation carriers (including other mutations than GRN and C9orf72, and/or asymptomatic carriers) with FTD (including prodromal) either with or without MND characteristics; up to 10 sporadic or genetic (excluding mutations with known absence of TDP-43 pathology, e.g. Superoxide Dismutase 1 (SOD1) or fused in sarcoma protein gene (FUS)) ALS; up to 10 suspected TDP-43 related sporadic FTD or FTD-MND; up to 5 patients with other neurodegenerative diseases, e.g. AD or suspected Limbic-Predominant Age-related TDP-43 Encephalopathy (LATE) pathology * up to 5 additional HVs may also be imaged, if necessary, to enable a better distinction of brain binding in this population compared to the population of subjects with TDP-43 proteinopathies Part 3 aims to assess test-retest reliability. Up to 5 participants from Part 1 and/or Part 2 will have an additional scan within 1 month after their first scan to determine test-retest reliability.

In the field of diagnosing brain neurodegenerative diseases, it is now a well-established practice to inject positron-emitting tracers into the human body. These tracers bind to specific target proteins, allowing their distribution to be visualized via PET imaging. Currently, several research groups worldwide are engaged in developing and clinically validating their own tau imaging agents. This clinical research project aims to visualize abnormal tau pathology in the living human brain using \[18F\]NIDF PET imaging. \[18F\]NIDF is a 2-arene-azaindole-based tracer that offers stronger binding affinity to tau neurofibrillary tangles and reduced non-specific/off-target binding compared to existing tau-PET imaging agents. The study primarily focuses on evaluating the safety and diagnostic efficacy of \[18F\]NIDF PET imaging in human subjects.

The GENESIS study is a multicenter, prospective, non-interventional, clinical study with a target of 12,000 subjects and an anticipated total duration of 36 months. The aim of study GENESIS is to provide a pilot map of HLA genetic variation in the Greek population in order to be used in medical research and for possible clinical applications (evaluation of possible correlations with selected underlying diseases). During the study, each subject will conduct one visit to the participating cite, in which they will provide: 1. Demographic information \[i.e. date of birth, gender, race, ancestry (including information about the subject's grandparents' birthplace), height, weight\], 2. Other information about smoking/vaping, alcohol consumption, arterial blood pressure, diagnosed diseases (if any), current treatments (if any), and 3. Recent (up to 12 months prior to sample collection) results if/when are available from clinical lab tests such as blood count (Hct, Hb, RBC, WBC, PLT count), including a metabolic panel, liver enzymes and biochemical parameters (Glu, HbA1c, TC, TG, LDL-C, HDL-C, ALT, AST, ALP, γGT, bilirubin, LDH, insulin, C-peptide). Upon completion of the data registry, two buccal swabs will be collected per subject and they will be stored at ALTP premises until their shipment to Galatea.Bio. All buccal swab samples will be subjected to genetic material (DNA) extraction. The DNA samples will be further proceeded for HLA genotyping analysis. A follow up analysis will be performed in selected DNA samples via full low-pass whole genome sequencing (LP-WGS), which aims to further investigate the association between the HLA region and autoimmune diseases. Upon completion of the analysis, an individualized ancestry report will be securely made available to all study subjects which they can access, as and if they elect to.

2\. INTERVENTION The first phase of the intervention will last four weeks and will be done in the hospital. After this intervention, if the patients meet the inclusion criteria, they will be enrolled in the second phase, which will be done at the patient's home for two additional weeks. Three assessments will be performed during the study: The first one ("Day 0") will be assessed before starting the treatment, the second one will be done ("Week 4") directly after the intervention at a hospital setting, and the last one ("Week 6"), after the home intervention. The intervention will be conducted in 1-hour sessions three times a week for four weeks, performing different exercises using an augmented reality platform accessed through RGSweb. If the patients continue the intervention at home, a three-session training for two weeks will be conducted in the presence of a patient's relative, who will accept to be the training partner. 3\. EQUIPMENT AND TECHNICAL SPECIFICATIONS. RGSweb can be used with any computer or device with a camera. However, our target users are patients who might have visibility issues due to old age. Therefore, bigger screens will be used to enhance the patients'; visibility. Additional screen support will be required to hold the monitor. There are three protocols in RGSweb: Twister Buddy: It has been developed for balance training, which involves a series of levels intended to strengthen the muscles crucial for maintaining upright posture, notably those in the legs. The aim is to improve stability and help prevent falls. Levels The user will have to shift their weight from one leg to the other while doing different exercises, such as touching targets with their hands and feet or taking steps. All levels will start with a calibration phase to ensure the user is in the correct position. Level 1 -Balance: In this level, two colored columns will be displayed on the screen, while user motion trackers will also be reflected on the screen. The goal is to align the trackers with the same color column. Level 2 - Balance + Hands: In addition to the balance movement from Level 1, a target will be displayed on the screen. The user must maintain balance on the highlighted column while reaching the target with their hand. Level 3 - Side Steps: To initiate, the user must have their feet on a square-like target at the bottom center of the screen. After that, a target will appear adjacent to the square. The user's task is to adjust their foot position so that both the toe and heel are inside this target area. Once achieved, the user must return their foot to the original position within the square, prompting the appearance of the next target for the opposite foot. Level 4 - Front steps: Similar to level 4, the user begins by positioning both feet on the square target. Then, an arrow will appear above one of the user's feet, indicating the direction of the step (either forward or backward). Once the user moves the designated foot past a predefined threshold value, currently indicated by a slider, the arrow will disappear, prompting the user to return both feet to the square. Subsequently, an arrow will appear above the other foot, indicating the direction for the next step. The direction of the steps is randomized for each interaction. Level 5 -- Balance + Hands + Feet: This is the next step from level 4. It starts with Balance, then the target for the hand, and finally, the target for the opposite foot. Like level 2, the user must maintain only the bottom part (hip and under) inside the column while reaching the target with their hand and foot. Costume Party: This protocol's main focus is sit-to-stand. The levels of this exercise follow different steps to stand up from a chair, starting with leaning forward. Levels All levels will start with a calibration phase to ensure the user is in the correct position. The objective is to dress up for a costume party using a reference shown at the bottom right of the screen. Various costume parts will appear on a bar and move across the screen. The user needs to catch the right parts for their costume and put them on. Once the costume is complete, there will be a few seconds to admire it before the process starts over with a new costume reference. Level 1-Lean Forward: In this level, the costume elements move from one side of the screen to another at the user's eye level. To grab the element, they must lean forward enough to surpass a threshold value defined by a slider. Level 2-Flex Knees: The user starts the motion of standing up by flexing the knees and rising slightly from the chair. The elements will appear close to the user's head, and leaning forward is unnecessary. Level 3-Stand Up: This level is similar to level 2, but the elements are positioned higher, requiring the user to stand up to reach them. Gentle Giant: This exercise targets less-impaired patients who can perform sit-to-stand and balance training exercises. Levels: The aim is to allow the elements to move from one platform to another following color coding. To do so, the user will act as a bridge between the different floors. All levels will start with a calibration phase to ensure the user is in the correct position. Level 1-Squats: In this level, the user must move the elements from one platform to another by moving the bridge up and down, that is, by squatting to go down and standing again to come up. Level 2-Tip-Toe: In this level, one platform will be higher than the user's height. The aim is similar, though: The user must stand in a tip-toe position to move the bridge higher and connect the platforms.

The effectiveness of a multidomain lifestyle intervention on the prevention of cognitive decline and dementia have not been studied in Asian elderly at high risk of dementia conversion. Dementia is caused by both nonmodifiable genetic variables, and modifiable lifestyle risk factors. While neuroimaging biomarkers have been well documented in the neurophysiology of ageing and age-associated cognitive decline, their role as surrogate endpoints and intermediate variables between multi-domain lifestyle intervention and cognitive benefits has not been studied. The current study aims to understand brain functional and structural changes that may result from a multi-domain lifestyle intervention and whether the changes correlate with improvement in cognitive function. At risk elderly aged 60-80 years will be randomly allocated to either the control arm (self-guided management) or the intervention (multi-domain lifestyle) arm, which consists of nutritional guidance, physical exercise, cognitive training and the monitoring and management of vascular and metabolic risk factors. We hypothesize that the multi-domain lifestyle intervention will promote favorable changes in cognitive function. Moreover, such intervention will slow down the progression of cerebrovascular disease and neurodegeneration in participants in the intervention arm. Findings from the present study will shed light on the biological mechanisms of age-related cognitive decline and neurodegenerative disease. Insight obtained from the study could be translated into new targets of nonpharmacological interventions which aim at the potential causal molecular pathways implicated in ageing and age-related cognitive decline. Adaption and implementation of our findings into clinical and public health practice will further promote healthy and confident ageing among Chinese elderly, to eventually expand their health span.

The use of deep brain stimulation (DBS) has expanded to include multiple conditions in children including dystonia, epilepsy, Tourette syndrome and mood disorders. Despite its growing application, DBS remains a low-volume procedure in most pediatric centers, which limits opportunities for large-scale research studies. To overcome this challenge, an international data-sharing platform is essential for advancing knowledge about DBS in pediatric patients, particularly concerning surgical techniques and patient outcomes across various conditions. This study aims to establish a multicenter pediatric DBS registry. With limited data on pediatric DBS outcomes and a small number of cases at individual centers, there is a need for a comprehensive registry to enable large-scale, well-powered analyses of DBS safety and effectiveness. The primary goals of this study are to: * Establish and implement a multi-center pediatric DBS registry * Facilitate large-scale analyses of DBS safety and effectiveness in children * Refine DBS as a treatment option for dystonia and other hyperkinetic movement disorders in children. Secondary objectives include: * Identifying which patients benefit most from DBS * Determining clinical variables that influence DBS responsiveness * Identifying optimal implant sites for specific conditions * Understanding the long-term effects of DBS in children * Assessing the impact of DBS on the quality of life in pediatric patients The study will involve both prospective and retrospective data collection from pediatric DBS patients.

This is a retrospective, multicenter cohort study using anonymized electronic health record data collected in a prospective study (PORC -EDGE 2364) performed at UZA and an identical prospective data collection performed by collegues at Department of Sleep Laboratory and Sleep surgery at Heim Pal National Pediatric Institute at Budapest.Data are collected from otherwise healthy children and children with varying comorbidities undergoing (adeno)-tonsillecty for Obstructive Sleepapnea (OSAS). Per-postoperative management was carried out according to a predefined treatment protocol. As this study uses existing data, no informed consent was required. The aim of the current project is to answer the following questions. * What is the prevalence of Postoperative Respiratory Complications (PORC) in children undergoing surgical treatment for OSA and is the prevalence different according to the underlying comorbid condition (i.e obesity, craniofacial malformation, syndromes Down syndrome, neurological conditions affecting upper airway muscle tone)? * Which factors are associated with the occurrence of PORC (Polysomnogram (PSG) related factors such as obstructive Apnea-Hypopnea Index (oAHI), minimum oxygen saturation during PSG, and patient related factors such as age at time of surgery, presence of a comorbidity)? * Is it possible to develop a management algorithm for postoperative management based upon the risk factors for PORC above.

In a single surgical procedure electrodes will be inserted into muscles of the trunk and hip musculature. The electrode leads are connected to a stimulator/telemeter located in the abdomen. After a typical post-operative period of two to five days, neuroprosthesis recipients will be discharged to home for two to six weeks of restricted activity to promote healing of all surgical incisions. After a period of exercise and training, functional use of the neuroprosthesis will begin. Laboratory assessments of strength, balance, and functional abilities with and without the system, as well as the technical performance of the implanted components will be evaluated.

Specific aim 1 To perform an unbiased genetic screening and to measure biochemical and neuroradiological biomarkers in a deep-phenotyped cohort of ALS patients. In particular, the activities will be divided into four WPs: WP 1. The investigators will recruit 200 incident patients with a diagnosis of ALS according to the revised El Escorial Criteria and Gold Coast Criteria from the UO1, UO2, and UO4; patients will be classified as classic, bulbar, flail arm, flail leg, and prevalent upper motor neuron ALS. The research group will consider neurophysiological parameters. Given the clinical continuum of ALS with FTD, the cognitive assessment will be evaluated through a complete neuropsychological test battery according to the consensus criteria for the diagnosis of frontotemporal cognitive and behavioral syndromes in ALS. Every patient will be followed up every three months, performing the MRC scale for muscle strength, ALS-FRS-R score for functional status, and King¿s and MITOS staging systems. WP 2. Genetics The investigators will perform NGS analysis on recruited patients to identify genetic variants associated with ALS susceptibility and phenotypic variability. Annotated variants will be subdivided according to the ACMG criteria and pathogenic, likely pathogenic, and variants of unknown significance (VUS) will be selected for further validation in Aim 3. To prioritize identified variants for analysis, researchers will focus on: 1) genes associated with monogenic types of ALS; 2) genes associated with ALS susceptibility; 3) genes associated with other degenerative diseases overlapping with ALS. WP 3. Imaging Brain Magnetic Resonance Imaging (MRI) phenotyping will be acquired by UO1, UO2, and UO4 with 3T magnets. The imaging protocol will include: 3D T1-weighted images (gradient-echo sequence Inversion Recovery prepared Fast Sypoiled Gradient Recalled-echo) for voxel-based morphometry (VBM) processing; 3D multi-echo gradient echo sequence for quantitative susceptibility mapping (QSM) analysis; FLuid-Attenuated Inversion Recovery (FLAIR) sequence; gradient-echo (GRE) echo-planar-imaging (EPI) sequence for whole-brain diffusion tensor imaging (DTI); repeated gradient-echo echoplanar imaging T2\*-weighted sequence for resting-state functional MRI (RS-fMRI) analysis. Brain fluorodeoxyglucose(FDG)-positron emission tomography (PET) will be performed at UO1 and UO4. WP 4. Biomarker analysis in biofluids. The investigators will measure a panel of protein biomarkers in plasma and CSF to evaluate the relationships between different biomarkers and between each biomarker and genotypic-phenotypic features, and their longitudinal trajectories (at baseline, after 6 and 12 months). The researchers selected established biomarkers mirroring fundamental pathophysiological processes in ALS such as neuroaxonal degeneration (NFL, tau), alterations in protein homeostasis (UCHL1, PPIA), TDP-43 pathology (TDP-43), and neuroinflammation (MCP-1, GFAP, MMP-9, PPIA). All biomarkers will be measured by means of advanced and automated immunoassay workstations that allow measuring proteins in biological fluids with the highest accuracy and sensitivity. Biomarkers will be measured in the plasma of all patients longitudinally (baseline, 6 and 12 months from first evaluation) and in CSF of selected patients. The investigators will analyze EVs isolated from plasma and CSF samples of all patients recruited in the project as markers of alterations in cell-cell communication and protein homeostasis. They will measure biophysical parameters and protein cargos in EVs by an established procedure as described (PMID 34376243). Specific aim2 To use Artificial Intelligence (AI) approaches for integrating the different data and correlate genetic and biomarker data with phenotypic traits. The investigators will develop AI approaches through machine-learning algorithms for integrating the different phenotypical, genetic, and neurobiological data to provide predictive models of disease phenotype, disease risk, and progression. In particular, they aim at deriving novel ALS subtype re-categorizations to better investigate the heterogeneity of ALS, enlightening novel genetic, neurochemical, and/or molecular divergences or overlaps between subsets of patients through data-driven approaches, and at identifying different patients¿ subtypes and novel ALS-related variants that require more specific therapies for improved treatment. In addition, researches will apply supervised and semi-supervised models for phenotype/disease progression prediction and relevant prognostic factors identification. Specific aim 3 To evaluate the biological significance of the identified phenotypes in human ALS motor neurons differentiated from iPSC (induced pluripotent stem cells). According to the genetic data obtained in Aim 1 and to the AI results from Aim 2, a set of pathogenetic/risk variants will be selected to be modeled in vitro. A preliminary functional screening will be performed using human neuroblastoma cells and the more appropriate assay will be chosen based on the type/nature of the identified gene variants. Selected ALS patients' cells (PBMC or fibroblasts) will be then reprogrammed into iPSC using the non-integrating Sendai virus expressing the Yamanaka¿s factors. The obtained iPSC will be fully characterized and differentiated according to well-established protocols to assess their capacity to generate functional motor neurons. Several morphological and viability parameters will be monitored to assess possible differences with wild-type control iPSC already available for the project. Experimental design aim1: WP 1. Patients ALS patients (n=200) will be enrolled at diagnosis, following written informed consent. Their motor phenotype will be classified as: classic, bulbar, flail arm, flail leg, and prevalent upper motor neuron ALS according to a published algorithm (PMID 21402743). The cognitive axis will be investigated during the diagnostic work-up through a full neuropsychological assessment, encompassing executive function, social cognition, language, memory, behavior, and neuropsychiatric symptoms, in agreement with the ALS-FTD revised diagnostic criteria (PMID 28054827). Deep family history will be collected, focusing on ALS and FTD, other neurodegenerative diseases, psychiatric disorders, suicide, glaucoma, and Paget's disease of bone. At baseline and in three-month follow-up visits, muscle strength will be assessed using the MRC scale and the ALSFRS-R will be collected. King¿s and MITOS stages at every visit will be extrapolated from the ALSFRS-R score (PMID 24720420, 24336810). WP 2. Genetics Genomic DNA will be extracted from whole blood. All samples will be pre-screened for C9orf72 hexanucleotide expansion (GGGGCC) and ATXN2 trinucleotide expansion (CAG) according to already established protocols. Whole-genome sequencing (WGS) will be performed on a HiSeq or NovaSeq platform (Illumina) with an expected coverage yield of\>30x. Read alignment, variant calling, and quality control will be performed using the BWA/GATK pipeline. ExpansionHunter will be used to determine the presence of large expansion of short tandem repeats (STRs) within genes of interest. Rare variant burden association analysis will be performed using PLINK/SEQ and RVtests, prioritizing variants according to the ACMG guidelines and genes involved in neurodegeneration. Validation of candidate variants associated with ALS susceptibility and phenotypic traits will be performed in independent cohorts, exploiting both internally generated NGS data, as well as public disease-specific databases (Project MinE). WP 3. Imaging MRI images will be acquired with 3T magnets. Within two weeks from clinical and neuropsychological assessments, eligible patients will undergo MRI sessions of about 30 minutes, acquiring: a high-resolution 3D T1 sequence (gradient-echo sequence Inversion Recovery prepared Fast Spoiled Gradient Recalled-echo) for voxel-based morphometry (VBM) analysis; T2-fluid attenuation inversion recovery (FLAIR) sequence, to exclude severe cerebrovascular disease according to standard clinical neuroradiological criteria; 3D multi-echo gradient echo sequence for quantitative susceptibility mapping (QSM) analysis; spin-echo (SE) EPI sequence (b value=2,000 s/mm2, 64 isotropically distributed gradients, frequency encoding RL) to perform whole-brain Tract-based Spatial Statistics (TBSS) DTI analysis; gradient-echo echo-planar imaging (GRE-EPI) sequence generating 320 T2\*-weighted volumes of 44 axial slices to perform RS-fMRI. MRI scan will be repeated after 6 and 12 months from baseline. Brain fluorodeoxyglucose (FDG)-positron emission tomography (PET) will be performed at diagnosis. WP 4. Biomarker analysis in biofluids. A panel of protein biomarkers was selected because of previous evidence of a correlation with clinical variables (PMID: 32385188; 16567701; 35263489; 32515902; 34972208; 28011744; 31742901; 34376243). In particular, NFL, tau, UCHL1, PPIA, TDP-43, MCP-1, GFAP, MMP-9 will be measured in plasma and CSF samples of ALS patients. The levels of plasma biomarkers will be measured in 200 incident patients during disease progression, at three-time points: baseline, 6, and 12 months from the first evaluation, while the levels of CSF biomarkers will be analyzed in selected patients (n=80) at baseline. EVs will be isolated from plasma samples and characterized for their biophysical properties and protein cargos (PPIA and HSP90). Multidimensional data for each patient will be provided to UO4 for patient characterization and classification. Experimental design aim 2 The investigators will develop AI approaches through machine-learning algorithms for deriving novel ALS subtype re-categorizations to better investigate the heterogeneity of ALS, and for identifying different patients' subtypes and novel ALS-related variants that require more specific therapies for personalized treatments. The research group will apply both unsupervised models (e.g., from cluster analysis) and supervised and semi-supervised models for phenotype/disease progression prediction and relevant prognostic factor identification. After data processing the investigators will generate several multi-dimensional 'views' for each patient data instance and cluster analysis will be performed to identify robust clusters of patients. They will essentially compare two schemes of data integration: intermediate integration, whereby features from different views are combined prior to clustering, and late integration, whereby a consensus approach will be applied to validate the different clusters of patients emerging from the different views. A linear discriminant analysis will be applied to assess the level of differentiation achieved at the clustering stage with the new groupings of patients. Moreover, among supervised models for relevant factor identification, the research group will apply the XGBoost algorithm, which trains a non-linear model from a dataset with labels and several features available and then uses the trained model to predict the labels on a new dataset's features. The advantage of the XGBoost algorithm is two-fold: first, together with the model, it provides a ranking of importance among the features (and views); second, it is robust to the correlation among features and the unbalance of the groups. Next, to investigate multimodal patterns potentially able to predict disease progression and patient prognosis, the investigators will apply non-parametric techniques to transform patients' survival data into hazard ranks. These models, including, e.g. the GuanRank model, as well as a Random forest analysis, will be used to identify brain MRI/FDG-PET and molecular variables that best predict the cognitive and behavioral scores and physical disability (i.e. phenotypic heterogeneity) and disease prognosis. Further, researchers will apply imbalance-aware hyperSMURF hyper-ensembles (ensemble of the ensemble) handling high class-imbalance by distributional-aware over and under-sampling. These ensembles may achieve state-of-the-art results in prioritizing pathogenic variants in Mendelian genetic diseases and will be applied to recognize the ALS-related variants from those extracted, to support the discovery of pathogenic variants associated with ALS. The investigators will generate patient-derived iPSC to conduct a functional characterization of the ALS-associated gene variants identified by the WGS analyses in Aim 1 and further defined by the AI-based approach. Human iPSCs have the great advantage of maintaining the patient's genetic background and of being potentially differentiated into different types of neuro-glial cells, including motoneurons. A subset of gene variants identified in our ALS cohort will be prioritized according to biological parameters, cell pathways involved and AI-derived prediction scores to be modeled in vitro. Blood cells (PBMC) or primary fibroblasts from selected ALS patients will be reprogrammed into iPSC using the non-integrating Sendai virus system as already published by UO2 (PMID: 32125773). The obtained iPSC lines will be fully characterized for the expression of stemness markers, their pluripotency state, and their capacity to spontaneously differentiate into the three embryonic germ layers, and for the absence of gross chromosomal rearrangements after reprogramming. In order to obtain human motoneurons, patient-derived iPSC will be differentiated according to already established protocols which include the formation of embryoid bodies and subsequent maturation of motoneuronal cells for 14-30 days. As a first functional parameter, the capacity of the generated iPSC to properly differentiate into motoneurons will be tested by image analysis of specific neuronal/motoneuronal markers and morphometric parameters by ImageJ software together with cell viability assays. According to the nature of the selected genes, the investigators will design and conduct ad hoc functional assays to further assess the biological effects of the predicted variants. The establishment of ALS patient-derived iPSC and motoneurons will allow a personalized in vitro modeling of the disease which represents an important step towards the understanding of the clinical heterogeneity defined in Aim 1.