Novel HEXB variant and first evidence of urinary Gb4 isoforms in Sandhoff disease: Biochemical and bioinformatic characterization in two Moroccan families

Sandhoff disease is a rare autosomal recessive lysosomal storage disorder caused by a deficiency of β-hexosaminidases A and B. These enzymes play a key role in the degradation of ganglioside GM2, GA2, globoside Gb4, and other glycolipids in neuronal and visceral tissues. Clinically, it is almost indistinguishable from Tay–Sachs disease, another disorder affecting hexosaminidase activity. We investigated two Moroccan families affected by Sandhoff disease through a multidisciplinary approach combining biochemical, chromatographic, bio-informatic, and genetic analyses. Urinary sphingolipids were characterized as potential biomarkers using thin-layer chromatography (TLC), high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS), and molecular networking. Enzyme activity of β-hexosaminidase A and total β-hexosaminidase were measured, and next-generation sequencing (NGS) was performed to identify disease-causing mutations in the HEXB gene. Lipidomic profiling of urinary extracts demonstrated accumulation of Gb4 and lactosylceramides. This is the first report showing that Gb4 isoforms are excreted in the urine of Sandhoff patients, highlighting their value as reliable, accessible and non-invasive biomarkers for resource-limited settings. Representative Gb4 isoforms were structurally assigned, and their exact molecular formulas were determined in patient urine. Enzyme essay revealed a marked reduction of β-hexosaminidase activity, consistent with Sandhoff disease. Molecular networking confirmed the abnormal sphingolipid signature. Genetic analysis identified a novel homozygous variant in the HEXB gene (c.1543A > C; p.Ser515Arg), associated with a severe clinical phenotype and early mortality. Our study expands the mutation spectrum of HEXB and provides first evidence of urinary Gb4 isoform excretion as potential biomarkers, demonstrating the utility of integrating urinary sphingolipid profiling with NGS.