Free sialic acid storage disorder (FSASD) is caused by pathogenic biallelic variants in SLC17A5, which encodes the lysosomal sialic acid exporter, sialin. FSASD is characterized by the accumulation of lysosomal free sialic acid, leading to either a severe, childhood-lethal form or a more slowly progressive neurodegenerative disorder associated with the p.Arg39Cys (p.R39C) variant, i.e., Salla disease. While dysregulated glycosphingolipid (GSL) metabolism has been observed in cellular models of FSASD, this study provides the first in vivo biochemical dissection of GSL metabolism in a knock-in mouse model harboring the Slc17a5 p.R39C variant. We employed an integrated multi-modal approach, including sialic acid quantification, exploratory untargeted lipidomics, HPLC-based GSL profiling, bulk transcriptomics, and 4-MU-based lysosomal enzyme activity assays in brain and peripheral tissues (liver and kidney). Exploratory untargeted lipidomic screening revealed region-dependent lipid alterations, with more pronounced changes in the cerebellum than in the forebrain. Pathway-level analyses indicated enrichment of lipid classes related to sphingolipid and GSL metabolism. Targeted biochemical analyses demonstrated that several GSL species accumulate predominantly in the brain, with minimal changes in peripheral tissues, whereas glucosylceramide levels were significantly reduced in all brain regions analyzed. Transcriptomic profiling identified dysregulation of several genes involved in GSL and sialic acid metabolism. Enzyme activity assays corroborated the transcriptomic findings, demonstrating increased activity of several lysosomal glycohydrolases, including neuraminidase 1/3/4 and β-hexosaminidase. Collectively, these findings highlight dysregulated GSL metabolism as a prominent biochemical consequence of sialin deficiency in vivo and highlight its putative role in FSASD neuropathology.

Free sialic acid storage disorder (FSASD) is an autosomal recessive lysosomal storage disease caused by biallelic pathogenic variants in SLC17A5, which encodes the lysosomal sialic acid transporter, sialin. FSASD is characterized by excessive lysosomal free sialic acid accumulation, leading to either a severe, early-onset lethal phenotype or a progressive neurodegenerative course. To characterize biochemical alterations in FSASD models, we performed comprehensive profiling of glycosphingolipids (GSLs), including sialylated species (i.e., gangliosides), in mouse embryonic fibroblasts (MEFs) derived from Slc17a5-R39C/R39C and Slc17a5-KO/KO mouse models, as well as in human SLC17A5-deficient HEK-293 T cells generated via CRISPR-Cas9-mediated non-homologous end joining. HPLC-based analyses demonstrated GM3 ganglioside accumulation in MEFs and significant reductions in a-series GSLs-including GM2, GM1a, and GD1a-in SLC17A5-deficient HEK-293 T cells. Analysis of neuraminidase 1/3/4 activities revealed consistently elevated activity across all cell models, while cytosolic neuraminidase 2 showed only a modest increase in Slc17a5-R39C/R39C MEFs. Preliminary quantification showed elevated free sialic acid across all models, consistent with the characteristic biochemical defect observed in FSASD and supporting their relevance for mechanistic studies. These findings highlight that free sialic acid storage leads to changes in GSL homeostasis in FSASD mouse (MEFs) and human (SLC17A5-deficient HEK-293T) cellular models, underscoring their utility as models for studying FSASD pathogenesis.

Lysosomal free sialic acid storage disorder (FSASD) is a rare neurodegenerative disease caused by biallelic mutations in SLC17A5, encoding the lysosomal sialic acid exporter, SLC17A5 (sialin). While the involvement of oligodendroglia in FSASD pathogenesis is established, the roles of other neural cell types remain elusive. In this study, we utilized radial glial cells (iRGCs), immature and mature astrocytes (iIAs and iMAs, respectively), and cortical neurons (iCNs) differentiated from induced pluripotent stem cells (iPSCs) derived from two individuals with FSASD, alongside two independent healthy donors for comparison. We employed a multifaceted profiling approach, including the assessment of cellular glycosphingolipids (GSLs), transcriptomics focused on GSL metabolism genes, and 4-methylumbelliferone-based lysosomal enzyme activity measurements. Our findings revealed significant elevations in free sialic acid levels across all FSASD cell types, indicating that iPSCs and derived iRGCs, iIAs, iMAs and iCNs may be used to model FSASD in vitro. We observed significant alterations in the abundance of specific GSL species, predominantly in mature astrocytes, with fewer changes in other cell types. Transcriptomic analyses uncovered differential expression of genes involved in GSL catabolism, including those encoding glycohydrolases. Enzyme assays corroborated the transcriptomic findings, showing heightened glycohydrolase activities, particularly in mature astrocytes. Collectively, these data may help refine our understanding of neural cell phenotypes and potential contributors to selective vulnerability in FSASD. Free sialic acid storage disorder (FSASD) is a spectrum of neurodegenerative phenotypes resulting from increased lysosomal storage of free sialic acid. Less severe FSASD (historically called Salla disease) is characterized by normal appearance and absence of neurologic findings at birth, followed by slowly progressive neurologic deterioration resulting in mild-to-moderate psychomotor delays, spasticity, athetosis, and epileptic seizures. Severe FSASD (historically referred to as infantile free sialic acid storage disease, or ISSD) is characterized by severe developmental delay, coarse facial features, hepatosplenomegaly, and cardiomegaly; death usually occurs in early childhood. The diagnosis of FSASD is established in a proband by identification of biallelic pathogenic variants in SLC17A5 by molecular genetic testing. Treatment of manifestations: Management is symptomatic and supportive: standard treatment of seizures; developmental and educational support; rehabilitation to optimize mobility; supplementation of calcium and vitamin D for low bone density; feeding therapy and provision of adequate nutrition; treatment of ophthalmologic manifestations per ophthalmologist with low vision services as needed; treatment of cardiomegaly per cardiologist; treatment of nephropathy / nephrotic syndrome per nephrologist; surgical treatment of hernia as needed; family and social support. Surveillance: Assessment of seizures, other neurologic manifestations, development, mobility, growth, nutrition, feeding, respiratory status, and family needs at each visit. Annual ophthalmology exam in those with intermediate or severe FSASD. Annual EKG and echocardiography to assess for cardiomegaly. Annual urinalysis for proteinuria. FSASD is inherited in an autosomal recessive manner. If both parents are known to be heterozygous for an SLC17A5 pathogenic variant, each sib of an affected individual has at conception a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Once the SLC17A5 pathogenic variants have been identified in an affected family member, molecular genetic carrier testing and prenatal/preimplantation genetic testing are possible.

Lysosomal free sialic acid storage disorder (FSASD) is a rare, multisystem neurodegenerative disease caused by biallelic pathogenic variants in SLC17A5, encoding sialin. FSASD is characterized by aberrant accumulation of unconjugated "free" sialic acid (Neu5Ac) within lysosomes. Depending on the specific genetic variants, affected individuals may present with either a rapidly fatal disease or progressive neurodegeneration. While skin fibroblasts have traditionally been used for diagnosis and research, the use of leukocytes in FSASD remains underexplored. This study examined Neu5Ac levels in leukocytes from three individuals with FSASD carrying distinct SLC17A5 variants. The levels in affected individuals were compared to three different groups: (1) the unaffected biological parents of each case; (2) subjects for whom 14 distinct lysosomal storage disorders (LSDs) were excluded based on enzyme analysis (n = 11); and (3) participants with a confirmed LSD diagnosis, as determined by enzyme analysis (n = 9). Individuals with FSASD exhibited significantly higher levels of free Neu5Ac compared to their unaffected biological parents (36-fold), LSD-negative subjects (22-fold), and individuals with other LSDs (49-fold). Although total Neu5Ac levels showed a non-significant trend toward an increase in FSASD (1.3-fold), this was primarily due to elevated free Neu5Ac, as bound Neu5Ac was slightly decreased in the leukocytes of FSASD cases relative to their unaffected parents. Overall, these findings highlight leukocytes as a valuable, minimally invasive cellular model for FSASD, offering an alternative, reliable diagnostic tool and a potential platform for monitoring therapeutic responses in future intervention trials.