RESUMEN
Mutations in the X-linked methyl-CpG-binding 2 (MECP2) gene lead to Rett Syndrome (RTT; OMIM 312750), a devasting neurodevelopmental disorder. RTT clinical manifestations are complex and with different degrees of severity, going from autistic-like behavior to loss of acquired speech, motor skills and cardiac problems. Furthermore, the correlation between the type of MECP2 mutation and the clinical phenotype is still not fully understood. Contextually, different genotypes can differently affect the patient's phenotype and omics methodologies such as proteomics could be an important tool for a molecular characterization of genotype/phenotype correlation. The aim of our study was focused on evaluating RTT oxidative stress (OS) responses related to specific MECP2 gene mutations by using proteomics and bioinformatics approaches. Primary fibroblasts isolated from patients affected by R133C and R255× mutations were compared to healthy controls (HC). After clustering primary dermal fibroblasts based on their specific MECP2 mutations, fibroblast-derived protein samples were qualitative and quantitative analyzed, using a label free quantification (LFQ) analysis by mass spectrometry (MS), achieving a preliminary correlation for RTT genotype/phenotype. Among the identified proteins involved in redox regulation pathways, NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) was found to be absent in R255× cells, while it was present in R133C and in HC fibroblasts. Moreover, NQO1 aberrant gene regulation was also confirmed when cells were challenged with 100 µM hydrogen peroxide (H2O2). In conclusion, by employing a multidisciplinary approach encompassing proteomics and bioinformatics analyses, as well as molecular biology assays, the study uncovered phenotypic responses linked to specific MECP2 gene mutations. These findings contribute to a better understanding of the complexity of RTT molecular pathways, confirming the high heterogeneity among the patients.