Genotype-phenotype correlations of GFAP variants in type I Alexander disease subtypes

Introduction: Alexander disease (AxD) is a rare and progressive leukodystrophy caused by variants in the Glial Fibrillary Acidic Protein (GFAP) gene. AxD presents as Type I and Type II, based on clinical and neuroradiological features. However, Type I patients variable disease progression has led to sub-classify them into subtypes Ia-Ib-Ic-Id. Our study investigated genotype-phenotype correlations in Type I AxD across these subtypes. Methods: A cohort of 74 genetically confirmed Type I AxD patients was analysed, with variants classified according to the clinical subtypes Ia, Ib, Ic, and Id. Genotypic–phenotypic correlations were explored both across all subtype groups and in stratified analyses based on patients' ability to achieve independent ambulation versus those who never acquired this milestone. To investigate the functional consequences of a representative variant, in vitro assays were performed. Selected mutant GFAP protein was expressed and their ability to assemble into intermediate filament networks was assessed. Results: The study revealed a progressive decrease in GFAP allelic heterogeneity across disease subtypes from Ia to Id. Additionally, GFAP pathogenic variants in exon 1 were more frequent in milder forms, whereas mutations in exon 4 were predominantly observed in severe phenotypes. In addition, changes at p.R239 and p.R79 residues showed an opposite trend, with variants affecting p.R239 never detected in Id subtype, while variants affecting p.R79 never detected in Ia. Specific aminoacidic changes at the p.R79 residue were able to stratify patients based on ability to acquire independent ambulation or not. Furthermore, the distribution of Combined Annotation Dependent Depletion (CADD) scores showed an inverse correlation with the acquisition of autonomous ambulation. Conclusions: These results suggest that specific GFAP variants could be potential predictors for disease progression in Type I AxD, supporting patients' subclassification and underscoring the importance of considering both genetic and clinical factors in AxD management, particularly as therapeutic interventions are being developed.