Molecular and Structural Characterization of Five Novel GLA Gene Variants in Fabry Disease

Abstract

Fabry disease is an X-linked lysosomal storage disorder caused by pathogenic variants in the GLA gene, encoding α-galactosidase A. Enzyme deficiency leads to progressive globotriaosylceramide (Gb3) accumulation and multisystemic involvement. Here, we characterize five previously undescribed GLA variants (p.D109N, p.N215T, p.N192H, p.L166P, and p.F248S) through an integrated approach combining biomolecular and computational analyses to investigate their effects on enzyme structure, catalytic activity, and dimerization. The identified substitutions affect residues located in regions critical for protein folding and active-site integrity. The p.F248S variant may destabilize the hydrophobic core and reduce thermodynamic stability, whereas p.D109N and p.N192H may disrupt hydrogen-bond networks required for proper catalytic geometry. The p.N215T substitution is associated with impaired glycosylation, while p.L166P may induce local conformational changes that compromise correct folding. Biochemical analyses of all samples carrying these variants revealed reduced α-galactosidase A activity and increased the Gb3 levels, but also of uncertain significance (VUS). Overall, these findings support an effect of the five variants on α-galactosidase A structure and function and highlight the value of integrating clinical, genetic, biochemical, and computational data for variant interpretation. Molecular characterization of novel variants may improve genetic diagnosis, inform therapeutic decisions, and support the development of targeted treatment strategies in Fabry disease.