Glycosphingolipids comprise a hydrophobic ceramide backbone, consisting of a long-chain base (sphingosine) and a fatty acid, conjugated with a hydrophilic oligosaccharide moiety. These amphipathic molecules are integral constituents of cellular membranes, playing pivotal roles in modulating membrane protein functionality and receptor-mediated signaling. Among glycosphingolipids, gangliosides, defined by their inclusion of sialic acid residues, are abundantly enriched in the central nervous system. Notably, four predominant species, GM1, GD1a, GD1b, and GT1b, constitute the majority of gangliosides in the mammalian brain and are indispensable for neuronal development, synaptic architecture, and signal transduction. These gangliosides are critically involved in neurogenesis, differentiation, membrane stability, and the modulation of receptor function, ion channel activity, and immunological signaling within the nervous system. The biosynthesis of these gangliosides is orchestrated by key enzymes, including GM3 synthase (ST3GAL5) and GM2/GD2 synthase (B4GALNT1) catalyzing the formation of downstream intermediates. Pathogenic variants in ST3GAL5 result in GM3 synthase deficiency (GM3SD), an autosomal recessive disorder characterized by infantile-onset epileptic encephalopathy and profound developmental regression. In contrast, biallelic mutations in B4GALNT1 cause a complex form of hereditary spastic paraplegia (SPG26), marked by progressive spasticity and intellectual impairment. ST3GAL3, another α2,3-sialyltransferase, contributes to the synthesis of GD1a and GT1b, as well as to glycoprotein sialylation. Mutations in this gene underlie neurodevelopmental disorders, including developmental and epileptic encephalopathy type 15 (DEE15). In this review, we summarize the current understanding of the molecular pathogenesis of congenital ganglioside biosynthesis disorders, integrating data from genetically engineered mouse models and affected individuals.

Background: GM3 Synthase Deficiency (GM3SD) is a rare autosomal recessive neurodevelopmental disease characterized by recurrent seizures and neurological deficits. The disorder stems from mutations in the ST3GAL5 gene, encoding GM3 synthase (GM3S), a key enzyme in ganglioside biosynthesis. While enzyme deficiencies affecting ganglioside catabolism are well-documented, the consequences of impaired ganglioside biosynthesis remain less explored. Methods: To investigate GM3SD, we used a Human Embryonic Kidney 293-T (HEK293-T) knockout (KO) cell model generated via CRISPR/Cas9 technology. Lipid composition was assessed via high-performance thin-layer chromatography (HPTLC); glycohydrolase activity in lysosomal and plasma membrane (PM) fractions was enzymatically analyzed. Lysosomal homeostasis was evaluated through protein content analysis and immunofluorescence, and cellular bioenergetics was measured using a luminescence-based assay. Results: Lipidome profiling revealed a significant accumulation of lactosylceramide (LacCer), the substrate of GM3S, along with increased levels of monosialyl-globoside Gb5 (MSGb5), indicating a metabolic shift in glycosphingolipid biosynthesis. Lipid raft analysis revealed elevated cholesterol levels, which may impair microdomain fluidity and signal transduction. Furthermore, altered activity of lysosomal and plasma membrane (PM)-associated glycohydrolases suggests secondary deregulation of glycosphingolipid metabolism, potentially contributing to abnormal lipid patterns. In addition, we observed increased lysosomal mass, indicating potential lysosomal homeostasis dysregulation. Finally, decreased adenosine triphosphate (ATP) levels point to impaired cellular bioenergetics, emphasizing the metabolic consequences of GM3SD. Conclusions: Together, these findings provide novel insights into the molecular alterations associated with GM3SD and establish the HEK293-T KO model as a promising platform for evaluating potential therapeutic strategies.

GM3 synthase deficiency (GM3SD) is an autosomal recessive disorder resulting from mutations in the ST3GAL5 gene. It is characterized by intellectual disability, microcephaly, psychomotor and developmental delay, hearing and visual impairments, and changes in skin pigmentation. This study aims to broaden the genetic mutation spectrum of GM3SD through the report of a de novo mutation and a comprehensive summary of GM3SD phenotype to aid in genetic counseling and prenatal diagnosis. Compound heterozygous variants in ST3GAL5 (NM_003896.4: c.1000delC, p.Arg334Glufs*15 and c.207-1G > T, p.Cys70Glufs*81) were identified via trio-whole exome sequencing (trio-WES) and confirmed pathogenic through functional experiments. Notably, c.207-1G > T was a newly discovered variant. Additionally, previously reported GM3SD mutations were classified into R288X and non-R288X, revealing that R288X mutations were more likely to manifest developmental, emotional abnormalities, and severe feeding difficulties. This study reveals a novel mutation in ST3GAL5 and provides a comprehensive overview of GM3SD phenotype, aiding in the diagnosis and genetic counseling of GM3SD in clinical practice.

Ganglioside GM3 synthase is a key enzyme involved in the biosynthesis of gangliosides. GM3 synthase deficiency (GM3SD) causes an absence of GM3 and all downstream biosynthetic derivatives, including all the a-, b-, c-series gangliosides, commonly found in neural tissues. The affected individuals manifest with severe irritability, intractable seizures, hearing loss, blindness, and profound intellectual disability. It has been reported that oral ganglioside supplementation has achieved some significant improvements in clinical symptoms, growth parameters, and developmental and cognitive scores in GM3SD patients. To gain insight into the molecular mechanisms of this supplementation, we performed supplementation of oral bovine milk gangliosides to GM3 synthase-deficient mice from early weaning periods. The oral milk ganglioside preparations were dominated by GM3 and GD3 gangliosides. Oral milk ganglioside supplementation improved the decreased cognitive function observed in GM3 synthase-deficient mice. The improvement in cognitive function was accompanied by increased ganglioside levels and neurogenesis in the hippocampus in the supplemented animals.

GM3 synthase deficiency (GM3SD) is caused by biallelic variants in the ST3GAL5 gene. Early clinical features of GM3SD include infantile onset of severe irritability and feeding difficulties, early intractable seizures, growth failure, hypotonia, sensorineural hearing impairment. We describe the generation and characterization the human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a 13-year-old girl with GM3 synthase deficiency resulted compound heterozygous for two new variants in the ST3GAL5 gene, c.1166A > G (p.His389Arg) and the c.1024G > A (p.Gly342Ser). The generated hiPSC line shows a normal karyotype, expresses pluripotency markers, and is able to differentiate into the three germ layers.