Cellular Functional Analyses of ARX Variants Reveal New Insights Into Genotype-Phenotype Correlations in...

Neurodevelopmental disorders (NDDs) encompass a wide range of conditions often linked to genetic causes, with mutations in the X-linked ARX gene representing a recurrent contributor. ARX encodes a transcription factor critical for GABAergic neuron development and functioning, regulating the expression of key neurodevelopmental target genes. Variants in ARX result in a wide clinical spectrum, ranging from asymptomatic female carriers to severe developmental syndromes in both sexes. This study investigates the functional impact of 16 ARX variants, including known pathogenic, likely pathogenic, and novel de novo variants, some associated with atypical presentations such as sudden infant death. Using transient expression in N2a neuroblastoma cells, we employed a comprehensive functional approach-including luciferase reporter assays, Western blotting, immunofluorescence, and RT-qPCR-to assess protein expression levels, subcellular localization, their interaction with known corepressors (TLE1 and CtBP1), and their transcriptional activity on selected ARX-known targets. Our results demonstrate that all tested variants disrupt normal ARX transcriptional function, with several also altering protein localization or expression. Remarkably, a subset of variants exhibited dominant-negative effects, offering a compelling explanation for unexpectedly severe phenotypes in female patients, likely exacerbated by skewed X-inactivation and other modifying factors. By integrating experimental data with a literature-based review of published ARX variants, we provide refined genotype-phenotype correlations, highlighting the importance of mutation type, positional context within key ARX functional domains, and patient sex. Altogether, this study advances our understanding of the molecular mechanisms driving ARX-related NDDs, emphasizing the importance of functional testing for accurate variant interpretation and paving the way toward informed genetic counseling and potential therapeutic development.