PARADIGM study, funded by the PNRR research grant, will focus on Eye Diseases (ED) and Neuro-Muscular Diseases (NMD) as groups of genetically heterogeneous diseases which are extensively studied by the Partners partecipating in the project; indeed ED and NMD are well clinically and molecularly characterized and approachable by drug-testing options already assessed and implemented by PARADIGM partners. ED and NMD represent good and compatible disease models as:

• both are genetically heterogeneous disorders where missing heritability is likely to be hidden in non-coding variants;
• many of the individual genes accountable for the ED and NMD cause autosomal recessive forms, increasing the chance of finding regulatory/splicing variants

UO1 (IRCCS Azienda Ospedaliero-Universitaria di Bologna, Coordinator) will coordinate the data collection and management protocols under fundamental ethical principles and relevant national, EU and international legislation; will set-up the bioinformatic data warehouse and deploy the workflows to analyze omics data analysis applying its own and published bioinformatic pipelines and will contribute to multi-omics data analysis.

UO2 (IRCCS Fondazione Istituto Neurologico Casimiro Mondino, Partner) will perform HiC/UMI-4C analyses and will contribute to the identification, validation and characterization of non-coding variants associated with RGDs, providing cellular and molecular genetic tools for the project to be tested in vitro or on patients' cell cultures from blood or skin. Only in specific cases (e.g. genes selectively expressed in the affected tissue), induced pluripotent stem cells (iPSCs) started from patients' fibroblasts will be used to generate 2D/3D cell models (e.g. retinal organoids) differentiated into the tissue of interes to dissect the molecular basis of unresolved RGDs.

UO3 (Azienda Ospedaliero-universitaria Luigi Vanvitelli, Partner) will carry out and integrate Genome Sequencing (GS) and transcriptome analysis on RNA from accessible tissues, e.g., cultured peripheral blood mononuclear cells (PBMCs), cultured fibroblasts (or fibroblast derived organoids), skeletal muscle (only when available in diagnostic routine).

UO4 (Università degli Studi di Napoli Federico II) will develop state-of-art gene therapy approaches to correct non-coding variants associated with RGDs, and test their efficiency in relevant retinal organoid models.

UO1, UO2, UO3 will be the clinical centers for recruitment of patients and collection of samples. This partnership gathers experts in the main omics, clinical geneticists, molecular biologists, and gene therapists, with massive experience in handling omics data, characterizing genetic variants for clinical purpose and developing therapeutics for genetic diseases up to the clinical stage.

Inclusion Criteria:

• patients/relatives of patients with clinical diagnosis of NMD/ED;
• patients/relatives of patients with inconclusive ES and aCGH data (no pathogenic/likely pathogenic variant) or finding of only a single hit (a pathogenic or likely pathogenic variant) in an autosomal recessive gene by ES (or aCGH) or no pathogenic or likely pathogenic variant but detection of a large region of genomic homozygosity surrounding a candidate gene;
• patients/relatives of patients with a finding of cryptic VUS (splicing/regulatory/noncoding CNVs) in ED/NMD genes or pathogenic cryptic variants in a selected number of representative cases.
• Signed informed consent to participate in the study.

Exclusion Criteria:

- Trios or nuclear families where both unaffected parents do not consent to participate will be excluded (similarly, a minimum number of 3 affected family members will be needed in multigenerational pedigrees).

The study population will include:

• patients previously in diagnostic routine, who have provided informed consent for storage and secondary use of their biological samples and for being recontacted; they will be informed about the current project and requested to provide a new specific informed consent;
• patients' parents and/or other relatives (e.g. siblings, auncle/aunt);
• age: from 0 years (newborns) with no upper age limit.