S-Adenosylhomocysteine hydrolase deficiency (AHCYd) is an ultra-rare autosomal recessive disorder of methionine metabolism caused by biallelic pathogenic variants in the AHCY gene. First identified in Croatia in 2002, AHCYd disrupts cellular methylation, leading to progressive myopathy, developmental delay, and potentially life-threatening complications. Fewer than 20 cases have been reported worldwide. Although the core biochemistry is well described, largely through foundational work by the principal investigator of this proposal, muscle-specific disease mechanisms remain poorly understood, and no disease-modifying therapies are currently available. This project will establish the first patient-derived induced pluripotent stem cell (iPSC) model of AHCYd myopathy. Dermal fibroblasts from genetically confirmed patients will be reprogrammed using non-integrating Sendai virus vectors and differentiated into myoblasts. These models, together with CRISPR-corrected isogenic and healthy controls, will be analysed using an integrated multi-omics approach. For the first time in AHCYd, telomere-to-telomere long-read genome sequencing (T2T lrGS) will be applied to resolve structural variants and map native CpG methylation, complemented by RNA-seq and episignature analysis targeting myogenic regulatory elements. CRISPR-based base and prime editing will be used to correct pathogenic variants, followed by functional rescue studies. The project will (1) define methylation-driven epigenetic and transcriptomic mechanisms of AHCYd myopathy, (2) provide the first preclinical proof-of-concept for mutation-specific gene therapy, (3) generate a FAIR-compliant iPSC-myoblast platform and multi-omics dataset, and (4) develop recommendations for clinical implementation of T2T lrGS in rare diseases. By addressing a critical knowledge gap, the project will advance personalised medicine for AHCYd and establish a transferable framework for other methylation-related myopathies.