Immunopathology in PMM2-CDG: Defective glycosylation impact in the TNFα -TNFR1 signalling pathway.

Introduction: Glycosylation is a post-translational modification that plays a crucial role in immune system activity. Phosphomannomutase 2-Congenital Disorder of Glycosylation (PMM2-CDG) is a rare genetic disease affecting glycosylation with a multi-systemic impact. PMM2-CDG patients commonly show immune disfunction and elevated pro-inflammatory cytokine levels that may link to other symptoms. However, the underlying immune mechanisms remain unclear. Given Tumour Necrosis Factor (TNF)'s key role in inflammation, this study proposes that defective glycosylation of its receptors disrupts intracellular signalling, leading to changes in the immune response of PMM2-CDG patients. Methods: To address this, we applied an integrative approach, combining transcriptomics, glycomics, and immune-related assays to investigate the impact of TNF-a stimulation via TNF receptor 1 (TNFR1) in a cohort of PMM2-CDG patients' skin fibroblasts. Results: Our results reveal a multifaceted disruption of TNF-a signalling in PMM2-CDG fibroblasts. We observed structural abnormalities in TNFR1, including altered receptor shedding. PMM2-CDG cells also showed an altered N-glycosylation profile, affecting particularly, high mannose N-glycans. At transcriptional level, PMM2-CDG cells, especially those bearing the R141H heterozygous variant, exhibited a distinct gene expression profile, after stimulation, characterized by dysregulation of immune and signalling pathways. Functionally, these molecular alterations translated into a diminished secretion of key inflammation and infection mediators, such as interleukin-6 (IL-6) and C-C Motif chemokine ligand 5 (CCL5) upon TNF-a stimulation. Similarly, essential signalling kinases including extracellular-signal-regulated kinase (ERK) 1/2, p38 and c-Jun N- terminal kinase (JNK) 2 showed reduced expression in PMM2-CDG cells, and their expression did not alter following TNF-a stimulation, unlike control cells. Conslusion: Our findings point to TNFR1 signalling dysregulation as a key contributor to immune dysfunction in PMM2-CDG. Importantly, our study identifies TNFR1 as a promising therapeutic target, suggesting that strategies aimed at modulating TNFR1 activity or restoring glycosylation homeostasis could provide new approaches for treatment development. This work advances our understanding of PMM2 -CDG immunopathology and opens opportunities for targeted therapeutics.