Sialic Acid Storage Disease Research Articles

Longitudinal Characterization of the Clinical Course of Intermediate-Severe Salla Disease

BackgroundBiallelic pathogenic variants in SLC17A5 cause three forms of free sialic acid storage disease categorized based on severity from least to most severe: Salla disease, intermediate-severe Salla disease, and infantile free sialic acid storage disease. Intermediate-severe Salla disease is the most recently described form. Here, we report a longitudinal characterization of intermediate-severe Salla disease progression in two sisters carrying the following biallelic variants in SLC17A5: c.406A>G (p.Lys136Glu) and c.819+1G>A. MethodsA retrospective review of medical records was performed. A developmental questionnaire was completed to obtain further clinical information. For functional characterization of the predicted splice site variant, RNA was extracted from patient blood samples and sequenced. ResultsDisease onset occurred within the first six months of life in both patients. Early childhood development was delayed with achievement of some milestones followed by a developmental plateau in late childhood. After this, both patients began a slow and progressive neurological regression in adolescence. Functional studies confirmed the pathogenicity of the c.819+1G>A variant, resulting in a frameshift and deletion of exon 6. ConclusionsWe present a detailed study describing the clinical course of intermediate-severe Salla disease with over 15 to 20 years of evolution and demonstrate the pathogenicity of the c.819+1G>A splice site variant.

Analysis of urinary oligosaccharide excretion patterns by UHPLC/HRAM mass spectrometry for screening of lysosomal storage disorders.

Oligosaccharidoses, sphingolipidoses and mucolipidoses are lysosomal storage disorders (LSDs) in which defective breakdown of glycan-side chains of glycosylated proteins and glycolipids leads to the accumulation of incompletely degraded oligosaccharides within lysosomes. In metabolic laboratories, these disorders are commonly diagnosed by thin-layer chromatography (TLC) but more recently also mass spectrometry-based approaches have been published. To expand the possibilities to screen for these diseases, we developed an ultra-high-performance liquid chromatography (UHPLC) with a high-resolution accurate mass (HRAM) mass spectrometry (MS) screening platform, together with an open-source iterative bioinformatics pipeline. This pipeline generates comprehensive biomarker profiles and allows for extensive quality control (QC) monitoring. Using this platform, we were able to identify α-mannosidosis, β-mannosidosis, α-N-acetylgalactosaminidase deficiency, sialidosis, galactosialidosis, fucosidosis, aspartylglucosaminuria, GM1 gangliosidosis, GM2 gangliosidosis (M. Sandhoff) and mucolipidosis II/III in patient samples. Aberrant urinary oligosaccharide excretions were also detected for other disorders, including NGLY1 congenital disorder of deglycosylation, sialic acid storage disease, MPS type IV B and GSD II (Pompe disease). For the latter disorder, we identified heptahexose (Hex7), as a potential urinary biomarker, in addition to glucose tetrasaccharide (Glc4), for the diagnosis and monitoring of young onset cases of Pompe disease. Occasionally, so-called "neonate" biomarker profiles were observed in young patients, which were probably due to nutrition. Our UHPLC/HRAM-MS screening platform can easily be adopted in biochemical laboratories and allows for simple and robust screening and straightforward interpretation of the screening results to detect disorders in which aberrant oligosaccharides accumulate.

A concerted action to explore therapies for free sialic acid storage disease

A concerted action to explore therapies for free sialic acid storage disease

A concerted action to explore therapies for free sialic acid storage disease (FSASD)

A concerted action to explore therapies for free sialic acid storage disease (FSASD)

Prenatal hydrops fetalis associated with infantile free sialic acid storage disease due to a novel homozygous deletion in the SLC17A5 gene

Nonimmune hydrops fetalis, the excessive accumulation of serous fluid in the subcutaneous tissues and serous cavities of the fetus, has many possible etiologies, providing a diagnostic challenge for the physician. Lysosomal storage diseases have been reported in up to 5%–16% of nonimmune hydrops fetalis pregnancies. Infantile free sialic acid storage disease (ISSD) (OMIM #269920) is a severe form of autosomal recessive sialic acid storage disease. ISSD is caused by mutations in SLC17A5 (OMIM #604322), which encodes sialin, a lysosomal-membrane sialic acid transporter. We describe a case of fetal hydrops due to a novel homozygous deletion in the SLC17A5 gene. Prenatal single-nucleotide polymorphism (SNP) array analysis was performed on amniocytes after the discovery of fetal hydrops at 24 wk gestation revealing no copy-number variants. The SNP array, however, reported several regions of homozygosity (ROHs) including one on Chromosome 6 encompassing the SLC17A5 gene. High levels of urine sialic acid in the newborn were detected. SLC17A5 gene sequencing was initiated with no sequence variants identified; however, the assay failed to amplify exons 8 and 9, prompting an exon-level copy-number analysis that revealed a novel homozygous deletion of exons 8 and 9, inherited from heterozygous carrier parents. ISSD should be considered in the workup of patients with nonimmune hydrops fetalis, and analysis for SLC17A5 deletions should be carried out when variants are not detected by gene sequencing.

Lysosomal storage disorders as an etiology of nonimmune hydrops fetalis: A systematic review

We performed a systematic review of the literature to evaluate the incidence and types of lysosomal storage disorders (LSD) in case series of nonimmune hydrops fetalis (NIHF). PubMed and Ovid were reviewed for case series evaluating the workup of NIHF diagnosed in utero or in the neonatal period in human subjects from 1979 to August 2020. Retrospective case series with at least five cases of fetal and/or neonatal NIHF with its workup mentioned were identified. Idiopathic NIHF was defined as NIHF without an apparent cause after initial standard-of-care workup. In total, twenty-two case series with 2,678 total cases of NIHF were identified. The overall incidence of LSD was 6.6% (177/2,663) in NIHF cases that were tested for any LSD, and 8.2% (177/2,151) in idiopathic NIHF cases. The most common LSD identified in cases of NIHF were mucopolysaccharidosis type VII, galactosialidosis, infantile sialic acid storage disease, Gaucher disease, GM1 gangliosidosis and sialidosis. More than 40% of the most common LSD causes of NIHF have a potential postnatal treatment. LSD testing for NIHF allows for early diagnosis, better counseling and appropriate management, planning for possible early treatment, and counseling for recurrence risk.

779 The spectrum of lysosomal storage diseases in non-immune hydrops fetalis and potential treatments

We performed a systematic literature review to determine the spectrum of lysosomal storage diseases (LSD) that result in non-immune hydrops fetalis (NIHF) and evaluate the existing therapies available. PubMed and Ovid were reviewed for case series evaluating the workup of NIHF diagnosed in utero or in the neonatal period in human subjects. Search terms were ‘non-immune hydrops,’ ‘non immune hydrops,’ ‘metabolic genetic disorders,’ ‘lysosomal storage disorders,’ ‘inborn errors of metabolism,’ and ‘inherited metabolic diseases.’ The time period searched was 1979 – August 2020. Retrospective case series with at least five cases of fetal and/or neonatal NIHF with reported workup were included. MOOSE guidelines were followed. Twenty-two case series with 2,678 total cases of NIHF were identified. The overall incidence of LSD was 7.2% (194/2,678) in NIHF cases that tested for any LSD. Fifteen different LSD were identified as potential causes of NIHF, with the five most common LSD causes being mucopolysaccharidosis type VII (45/194, 23.2%), galactosialidosis (26/194, 13.4%), infantile sialic acid storage disease (25/194, 12.9%), Gaucher disease (23/194, 11.9%), and GM1-gangliosidosis (22/194,11.3%). Of the fifteen different LSD identified as a cause of NIHF, six have approved postnatal therapies (Table 1) and several have clinical trials investigating potential therapies (Table 2). 43% of LSD identified as causes of NIHF in our review have available approved therapies. Given that 7% of NIHF cases are caused by LSD, and more than 40% of NIHF cases caused by LSD have available therapies, LSD testing should be considered if the standard workup for NIHF is negative.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Lysosomal storage disease spectrum in nonimmune hydrops fetalis: a retrospective case control study.

Nonimmune hydrops fetalis (NIHF) accounts for 90% of hydrops fetalis cases. About 15% to 29% of unexplained NIHF cases are caused by lysosomal storage diseases (LSD). We review the spectrum of LSD and associated clinical findings in NIHF in a cohort of patients referred to our institution. We present a retrospective case-control study of cases with NIHF referred for LSD biochemical testing at a single center. Cases diagnosed with LSD were matched to controls with NIHF and negative LSD testing and analyzed according to the STROBE criteria to the extent the retrospective nature of this study allowed. Between January 2006 and December 2018, 28 patients with NIHF were diagnosed with a LSD. Eight types of LSD were diagnosed: galactosialidosis 8/28 (28.6%), sialic acid storage disease (SASD) 5/28 (17.9%), mucopolysaccharidosis VII 5/28 (17.9%), Gaucher 4/28 (14.3%), sialidosis 2/28 (7.1%), GM1 gangliosidosis 2/28 (7.1%), Niemann-Pick disease type C 1/28 (3.6%), and mucolipidosis II/III 1/28 (3.6%). Associated clinical features were hepatomegaly 16/21 (76.2%) vs 22/65 (33.8%), P < .05, splenomegaly 12/20 (60.0%) vs 14/58 (24.1%), P < .05, and hepatosplenomegaly 10/20 (50.0%) vs 13/58 (22.4%) P < .05. The most common LSD in NIHF were galactosialidosis, SASD, mucopolysaccharidosis VII, and Gaucher disease. LSD should be considered in unexplained NIHF cases, particularly if hepatomegaly, splenomegaly, or hepatosplenomegaly is visualized on prenatal ultrasound.

A cross-sectional quantitative analysis of the natural history of free sialic acid storage disease—an ultra-orphan multisystemic lysosomal storage disorder

PurposeQuantitative definition of the natural history of free sialic acid storage disease (SASD, OMIM 604369), an orphan disorder due to the deficiency of the proton-driven carrier SLC17A5. MethodsAnalysis of published cases with SASD (N = 116) respecting STROBE criteria. Main outcome parameters: survival and diagnostic delay. Phenotype, phenotype–biomarker associations, and geographical patient distribution were explored. ResultsMedian age at disease onset was 0.17 years. Median age at diagnosis was 3 years with a median diagnostic delay of 2.5 years. Median survival was 11 years. The biochemical phenotype clearly predicted the disease course: patients with a urinary free sialic acid excretion below 6.37-fold or an intracellular free sialic acid storage in fibroblasts below 7.37-fold of the mean of normal survived longer than patients with biochemical values above these thresholds. Cluster analysis of disease features suggested a continuous phenotypic spectrum. Patient distribution was panethnic. ConclusionCombination of neurologic symptoms, visceromegaly, and dysmorphic features and/or nonimmune hydrops fetalis should prompt specific tests for SASD, reducing diagnostic delay. The present quantitative data inform clinical studies and may stimulate and accelerate development of specific therapies. Biomarker–phenotype association is particularly important for both counseling parents and study design.

Effects of the Cell Cycle on Vacuole Size in S. cerevisiae yeast

Malfunctions in a cell's ability to degrade unnecessary chemicals or proteins may lead to one of fifty distinctive lysosomal storage disorders in humans. Lysosomal storage diseases such as Tay‐Sach's, Salla, or Sialic Acid Storage diseases often lead to premature death in infants. Lysosomes are vital for degrading subcellular proteins no longer needed by the cell. Our research uses a functional homologue of lysosomes, the vacuole in S. cerevisiae yeast as a model system, which also functions in protein degradation.We study vacuoles in S. cerevisiae yeast to understand protein degradation. We investigate whether changes in vacuole size and degradative function are influenced by the progression of the cell cycle. We hypothesize that changes in vacuolar size and specific phases of the cell cycle play an important role in degradative function. To address this, we designed experiments to approach this by linking vacuole size changes to the progression of the cell cycle. We halted the cells at each cell cycle stage to track vacuolar changes as the cell cycle progresses. We did this by incubating the cells with 4 cell cycle inhibition drugs: Tributyltin (G1 phase), Hydroxyurea (S phase), Epothilone B (G2 phase), and Nocodazole (M phase). Vacuoles were fluorescently labelled with VPH1‐GFP, a vacuolar surface protein. We imaged the samples using Spinning Disk Confocal Microscopy. We then analyzed the images with ImageJ and MatLab algorithms to track, compare, and characterize vacuolar change. Thus far, we have found that the vacuoles change in terms of copy number (the number of vacuolar lobes) as the cell cycle progresses. This preliminary evidence suggests that there is a correlation between changes in vacuolar size along with the progression of the cell cycle. Because of this, we hypothesize that vacuole size is influenced by the progression of the cell cycle.In addition, decreases in degradative ability are correlated with enlarged vacuolar phenotypes, hence a logical future direction would be to test whether degradative ability may also be synchronized with the progression of the cell cycle. Characterizing these changes would allow us to predict degradative efficiency based on the phase of the cell cycle. Further research could determine whether protein degradation and vacuolar morphology depend on one another. Overall, our research is significant because it will provide molecular insight on how vacuolar morphology and degradation are regulated, which may lead to new treatments to help those who suffer from lethal lysosomal storage disorders, and those who struggle with inefficiencies in degradative ability.Support or Funding InformationNSF‐Center for Cellular Construction GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A New Patient With Intermediate Severe Salla Disease With Hypomyelination: A Literature Review for Salla Disease

BackgroundLikely pathogenic variants in SLC17A5 results in allelic disorders of free sialic acid metabolism including (1) infantile free sialic acid storage disease with severe global developmental delay, coarse facial features, hepatosplenomegaly, and cardiomegaly; (2) intermediate severe Salla disease with moderate to severe global developmental delay, hypotonia, and hypomyelination with or without coarse facial features, and (3) Salla disease with normal appearance, mild cognitive dysfunction, and spasticity. Patient DescriptionThis five-year-old girl presented with infantile-onset severe global developmental delay, truncal hypotonia, and generalized dystonia following normal development during her first six months of life. Brain magnetic resonance imaging showed marked hypomyelination and a thin corpus callosum at age 19 months, both unchanged on follow-up at age 28 months. Urine free sialic acid was moderately elevated. Cerebrospinal fluid free sialic acid was marginally elevated. Sequencing of SLC17A5 revealed compound heterozygous likely pathogenic variants, namely, a known missense (c.291G>A) variant and a novel truncating (c.819+1G>A) variant, confirming the diagnosis of Salla disease at age 3.5 years. ConclusionWe report a new patient with intermediate severe Salla disease. Normal or marginally elevated urine or cerebrospinal fluid free sialic acid levels cannot exclude Salla disease. In patients with progressive global developmental delay and hypomyelination on brain magnetic resonance imaging, Salla disease should be included into the differential diagnosis.

Identification of a large intronic transposal insertion in SLC17A5 causing sialic acid storage disease

BackgroundSialic acid storage diseases are neurodegenerative disorders characterized by accumulation of sialic acid in the lysosome. These disorders are caused by mutations in SLC17A5, the gene encoding sialin, a sialic acid transporter located in the lysosomal membrane. The most common form of sialic acid storage disease is the slowly progressive Salla disease, presenting with hypotonia, ataxia, epilepsy, nystagmus and findings of cerebral and cerebellar atrophy. Hypomyelination and corpus callosum hypoplasia are typical as well. We report a 16 year-old boy with an atypically mild clinical phenotype of sialic acid storage disease characterized by psychomotor retardation and a mixture of spasticity and rigidity but no ataxia, and only weak features of hypomyelination and thinning of corpus callosum on MRI of the brain.ResultsThe thiobarbituric acid method showed elevated levels of free sialic acid in urine and fibroblasts, indicating sialic acid storage disease. Initial Sanger sequencing of SLC17A5 coding regions did not show any pathogenic variants, although exon 9 could not be sequenced. Whole exome sequencing followed by RNA and genomic DNA analysis identified a homozygous 6040 bp insertion in intron 9 of SLC17A5 corresponding to a long interspersed element-1 retrotransposon (KF425758.1). This insertion adds two splice sites, both resulting in a frameshift which in turn creates a premature stop codon 4 bp into intron 9.ConclusionsThis study describes a novel pathogenic variant in SLC17A5, namely an intronic transposal insertion, in a patient with mild biochemical and clinical phenotypes. The presence of a small fraction of normal transcript may explain the mild phenotype. This case illustrates the importance of including lysosomal sialic acid storage disease in the differential diagnosis of developmental delay with postnatal onset and hypomyelination, as well as intronic regions in the genetic investigation of inborn errors of metabolism.

Progressive leukoencephalopathy impairs neurobehavioral development in sialin-deficient mice

Slc17a5−/− mice represent an animal model for the infantile form of sialic acid storage disease (SASD). We analyzed genetic and histological time-course expression of myelin and oligodendrocyte (OL) lineage markers in different parts of the CNS, and related this to postnatal neurobehavioral development in these mice. Sialin-deficient mice display a distinct spatiotemporal pattern of sialic acid storage, CNS hypomyelination and leukoencephalopathy. Whereas few genes are differentially expressed in the perinatal stage (p0), microarray analysis revealed increased differential gene expression in later postnatal stages (p10–p18). This included progressive upregulation of neuroinflammatory genes, as well as continuous down-regulation of genes that encode myelin constituents and typical OL lineage markers. Age-related histopathological analysis indicates that initial myelination occurs normally in hindbrain regions, but progression to more frontal areas is affected in Slc17a5−/− mice. This course of progressive leukoencephalopathy and CNS hypomyelination delays neurobehavioral development in sialin-deficient mice. Slc17a5−/− mice successfully achieve early neurobehavioral milestones, but exhibit progressive delay of later-stage sensory and motor milestones. The present findings may contribute to further understanding of the processes of CNS myelination as well as help to develop therapeutic strategies for SASD and other myelination disorders.

A study of circulating sialic acid levels in pregnancy

Background: The sialic acid level is significantly higher in full term cord firbrinogen than in controls and higher in premature than term samples and degree of hypersialation of fetal fibrinogen is a function of gestational age,prenatal diagnosis and confirmation of infantile sialic acid storage disease have been carried out by amniocentesis. Aims &amp; Objectives: To correlate the physiological stress occurring during different trimesters of pregnancy with circulating level of serum sialic acid. Methodology: In this study 30 blood samples were collected from healthy pregnant women in each trimester of pregnancy of age group between 18-35 years along with age and sex matched non pregnant control group. The blood samples was collected from all participants and analyzed for Serum sialic acid ,total protein, albumin and Blood sugar. Results: The mean concentration of serum sialic acid (Micmol/dl) in non pregnant control was estimated to be 119.1±4.68 as compared to 138.8± 4.57,155.76± 5.1, 171.18± 6.7 in first, second and third trimester of pregnancy respectively. Conclusion: The level of total sialic acid is found to be elevated as the pregnancy advances. This increase in maternal circulating serum TSA levels can be attributed to the immunological and metabolic adjustments that the pregnant women undergoes to bring about successful pregnancy.

Diagnostic challenges in Salla disease

Sialic acid storage disease (Salla disease) is an autosomal recessive disorder caused by mutations in a lysosomal sialic acid export protein, SLC17A5 (OMIM #604369). This disorder was initially described in Northern Finland but more recently has been reported in patients of other ethnicities. We describe the clinical presentation and the neuroimaging findings of two non-Finnish children where a diagnosis of Salla disease was suspected on the basis of brain magnetic resonance imaging. The biochemical confirmation of this diagnosis posed a challenge as both patients had elevated percent free urine sialic acid but biochemical analyses in fibroblasts were not conclusive; therefore, molecular testing was necessary for confirmation of the diagnosis. The described encounters demonstrate the importance of pursuing confirmatory molecular diagnostic testing when a sialic acid storage disorder is suspected.

Successful Prediction of Substrate-binding Pocket in SLC17 Transporter Sialin

Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.

In response to “Prenatal screening of sialic acid storage disease and confirmation in cultured fibroblasts by LC-MS/MS” by van den Bosch et al.

With great interest we have read the recent paper by Van den Bosch et al. (2011) who reported reference values of free sialic acid (FSA) in amniotic fluid supernatant (AFS). Reference values in AFS were lower (0–8.2 μmol/L) at 15–25 weeks of gestation compared to 26–38 weeks of gestation (3.2–12.0 μmol/L), indicating that FSA in AFS tends to increase with advancing gestation. Recently, we developed a method for quantification of sialic acid (SA) in cerebrospinal fluid (CSF) (van der Ham et al. 2010) since brain specific disorders of SA metabolism are recognized nowadays. When establishing reference values, we did not take into account the impact of gestational age (GA). The study of Van den Bosch et al. (2011) suggested SA metabolism to be dependent on GA. We therefore performed a study to evaluate CSF SA values in newborn infants (both preterm and full term born infants). This knowledge is essential for the interpretation of SA concentrations in CSF in these populations. The study included 27 CSF samples from preterm and 42 CSF samples from full term newborn infants. CSF was obtained for different clinical reasons. Prematurity was defined as a post menstrual age of less than 37 weeks at the time of CSF withdrawal. Post menstrual ages of preterm and full term infants were between 28 and 37 weeks and between 37 and 54 weeks, respectively. SA in CSF was quantified by HPLC-MS/MS (van der Ham et al. 2010). FSA concentrations were 72.2 ± 21.3 μmol/L and 49.3 ± 20.6 μmol/L in preterm and full term newborn infants, respectively (p < 0.0005). There was a significant negative correlation between FSA and post menstrual age (r = −0.664) with a 3.0 μmol/L decrease in FSA for each week of advancing post menstrual age (p < 0.0005). No difference was observed between conjugated sialic acid from preterm and full term newborn infants (80.0 ± 19.1 μmol/L versus 73.6 ± 28.0 μmol/L (p = 0.303)). In conclusion, with increasing gestational age, there was a considerable decrease in the amount of FSA in CSF. These results demonstrate that proper interpretation of metabolic investigations in preterm newborns depends on group-specific reference ranges, as concentrations of metabolites may differ significantly between full term and preterm born infants.

Functional characterization of vesicular excitatory amino acid transport by human sialin

Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Δψ)-driven aspartate and glutamate transport into synaptic vesicles in addition to H+/sialic acid co-transport in lysosomes. Rodent sialin mutants harboring the mutations associated with Salla disease in humans did not transport aspartate and glutamate whereas H+/sialic acid co-transport activity was about one-third of the wild-type protein. In this study, we investigate the effects of various mutations on the transport activities of human sialin. Proteoliposomes containing purified heterologously expressed human sialin exhibited both Δψ-driven aspartate and glutamate transport activity and H+/sialic acid co-transport activity. Aspartate and glutamate transport was not detected in the R39C and K136E mutant forms of SLC17A5 protein associated with Salla disease, whereas H+/sialic acid co-transport activity corresponded to 30-50% of the recombinant wild-type protein. In contrast, SLC17A5 protein harboring the mutations associated with infantile sialic acid storage disease, H183R and Δ268SSLRN272 still showed normal levels of Δψ-driven aspartate and glutamate transport even though H+/sialic acid co-transport activity was absent. Human sialin carrying the G328E mutation that causes both phenotypes, and P334R and G378V mutations that cause infantile sialic acid storage disease showed no transport activity. These results support the idea that people suffering from Salla disease have been defective in aspartergic and glutamatergic neurotransmissions.

Prenatal screening of sialic acid storage disease and confirmation in cultured fibroblasts by LC‐MS/MS

Sialic acid storage disease (SASD) is an inborn error resulting from defects in the lysosomal membrane protein sialin. The SASD phenotypical spectrum ranges from a severe presentation, infantile sialic acid storage disease (ISSD) which may present as hydrops fetalis, to a relatively mild form, Salla disease. Screening for SASD is performed by determination of free sialic acid (FSA) in urine or amniotic fluid supernatant (AFS). Subsequent diagnosis of SASD is performed by quantification of FSA in cultured fibroblasts and by mutation analysis of the sialin gene, SLC17A5. We describe simple quantitative procedures to determine FSA as well as conjugated sialic acid in AFS, and FSA in cultured fibroblasts, using isotope dilution (13C3-sialic acid) and multiple reaction monitoring LC-ESI-MS/MS. The whole procedure can be performed in 2–4 h. Reference values in AFS were 0–8.2 μmol/L for 15–25 weeks of gestation and 3.2-12.0 μmol/L for 26–38 weeks of gestation. In AFS samples from five fetuses affected with ISSD FSA was 23.9-58.9 μmol/L demonstrating that this method is able to discriminate ISSD pregnancies from normal ones. The method was also validated for determination of FSA in fibroblast homogenates. FSA in SASD fibroblasts (ISSD; 20–154 nmol/mg protein, intermediate SASD; 12.9-15.1 nmol/mg, Salla disease; 5.9-7.4 nmol/mg) was clearly elevated compared to normal controls (0.3-2.2 nmol/mg). In conclusion, we report simple quantitative procedures to determine FSA in AFS and cultured fibroblasts improving both prenatal diagnostic efficacy for ISSD as well as confirmatory testing in cultured fibroblasts following initial screening in urine or AFS.

Sf9 cell vesicle-mediated correction of cystine storage in cystinotic fibroblasts and sialic acid storage in infantile sialic acid storage disease fibroblasts

Sf9 cell vesicle-mediated correction of cystine storage in cystinotic fibroblasts and sialic acid storage in infantile sialic acid storage disease fibroblasts