Phosphomannomutase deficiency (PMM2-CDG), the most common congenital disorder of glycosylation (CDG), is characterized by multisystem involvement and a lack of disease-modifying therapies. While previous transcriptomic studies have uncovered disrupted cellular pathways, the functional consequences of these alterations remain poorly understood. To further investigate PMM2-CDG pathophysiology, we integrated proteomic and metabolomic profiling of patient-derived fibroblasts with previously published transcriptomic data. Proteomic analysis was performed using Tandem Mass Tag-based mass spectrometry, while metabolomics was conducted via Nuclear Magnetic Resonance spectroscopy. Multi-omics integration was performed using principal component analysis-based dimensionality reduction, incorporating clinical metadata as supplementary variables. Proteomic analysis identified 43 significantly altered proteins, with enrichment in the retinoic acid synthesis pathway, wound healing, and cytoskeletal organization. Metabolomic profiling revealed altered amino acid levels and elevated concentrations of UDP-GlcNAc, consistent with perturbation of the hexosamine biosynthesis pathway, and increased levels of myo-inositol. Notably, myo-inositol levels showed a strong association with disease severity in the integrated analysis. RT-qPCR confirmed the upregulation of GFPT2. This integrative multi-omics study identifies consistent alterations in the retinoic acid synthesis pathway and hexosamine biosynthesis in PMM2-CDG patient-derived fibroblasts and reveals an association between intracellular myo-inositol levels and disease severity. These findings provide new insights into PMM2-CDG-associated molecular alterations and illustrate the value of multi-omics integration for hypothesis generation in rare diseases.

Reliable detection of structural variants (SVs) and copy number variations (CNVs) is crucial in the contemporary diagnostics of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, limitations of commonly used conventional and molecular cytogenetic methods may hinder the accurate genetic characterization of patients. Optical genome mapping (OGM) offers a reliable alternative by enabling high-resolution, genome-wide detection of CNVs and SVs. Chromosomal aberrations were screened using OGM in 51 children with B-ALL. The results were compared with those of karyotyping, fluorescence in situ hybridization (FISH), digital multiplex ligation-dependent probe amplification (digitalMLPA), and targeted RNA sequencing (RNA-seq). OGM data showed high congruency with karyotyping and FISH findings, detecting clinically relevant variants beyond G-banding results and unraveling a complex KMT2A fusion undetected by FISH. Gene fusions involved in complex ETV6::RUNX1 translocations, but not detected by RNA-seq, were confirmed using FISH. Normalization of OGM copy number values with DNA-index-improved concordance with FISH-derived copy numbers in near-tri/tetraploid cases. In the peripheral regions of OGM variants (fringe-zones), a novel evaluation strategy called 'FriZone' was applied, which significantly improved the concordance between OGM and digitalMLPA. In addition, a co-segregation analysis revealed strong associations between ETV6::RUNX1 fusion and deletions of ETV6, RAG2, and NR3C2. OGM uncovered complex rearrangements undetected by widely used methods in 15% of cases, improving genetic classification and risk stratification in 10% of the patients. The FriZone analysis and normalization by DNA-index provide a refined, more accurate approach to OGM variant interpretation, facilitating the efficient application of OGM in clinical diagnostics. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.