Linking the impact of mutations of the extracellular domain of Myelin Protein Zero to the etiopathogenesis of Charcot-Marie-Tooth disease

Myelin Protein Zero (MPZ) is the primary adhesive protein of the peripheral nervous system (PNS) myelin sheath, where its extracellular domain (MPZex) mediates homophilic interactions essential for compacting myelin lamellae. Mutations in the MPZ gene are a leading cause of Charcot-Marie-Tooth (CMT) disease, the most common inherited neuropathy, but give rise to a remarkably heterogeneous spectrum of clinical phenotypes, from severe, early-onset demyelinating forms (CMT1B, DSS) to milder, late-onset axonal variants (CMT2I/J). This diversity is thought to reflect distinct molecular pathomechanisms, yet a quantitative framework directly linking the biophysical defects of a given mutation to its clinical outcome has been lacking. The primary objective of this thesis is to establish such a framework by systematically investigating the molecular consequences of pathogenic point mutations in MPZex, thereby decoding the complex genotype-phenotype correlations in MPZ-related neuropathies. The first part of this thesis provides a comprehensive biophysical characterization of the novel D134V mutation, identified in a patient with severe, childhood-onset CMT1B. Using a suite of spectroscopic, stability, and functional assays, we demonstrate that the D134V mutant, while retaining a globally folded β-sheet architecture, exhibits significant local conformational perturbations and appears markedly destabilized against thermal and acidic stress. This structural fragility predisposes the mutant to aggregation and translates into a failure of its biological function, with fluorescence-based assays revealing a dramatic (~80%) reduction in homophilic (MPZ-MPZ) binding affinity. This combination of structural instability, aggregation propensity, and severe loss of adhesive function could provide a direct molecular explanation for the severe demyelinating phenotype. The investigation was then expanded to a comparative analysis of a broader panel of eight pathogenic MPZex variants associated with a wide range of clinical severities. This analysis revealed that each mutation imparts a unique biophysical signature creating a spectrum of molecular defects in stability and aggregation kinetics. The homophilic adhesion assay proved to be a powerful functional 5 discriminator, revealing a clear dichotomy: one group of mutants displayed a severe impairment of self-association, whereas a second group retained near-wild-type binding capacity. These findings reveal a possible correlation, demonstrating that variants causing a profound loss of homophilic adhesion are predominantly associated with severe, early-onset demyelinating phenotypes, thus suggesting that the direct failure of myelin compaction is a primary driver of the most severe forms of the disease. Finally, the complex, multi-parameter dataset was integrated using Principal Component Analysis (PCA) to establish a quantitative, structure-based framework for decoding MPZ neuropathies. The PCA clustered the variants into distinct groups based on their integrated biophysical signatures, creating a "pathogenic landscape" where each mutant's position reflects its molecular defect profile and strongly correlates with its clinical outcome. Variants associated with severe, early-onset demyelinating phenotypes localized extreme positions in this landscape, reflecting profound instability and a catastrophic loss of adhesive function. Conversely, variants linked to milder, late-onset axonal neuropathies were located closer to the wild-type, consistent with more subtle defects. This work, therefore, establishes a robust framework that not only elucidates the molecular principles governing the clinical heterogeneity of MPZ-related neuropathies but also holds promise for predicting the severity of newly identified variants and for informing the development of tailored, mechanism-based therapeutic strategies for CMT.