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Background: Biallelic 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., Methods: A 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., Results: Disease 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., Conclusions: We 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., Competing Interests: Declaration of competing interest The authors declare no relevant conflicts of interest. Dr. Bernard is/was a consultant for Passage Bio Inc (2020-2021) and Ionis (2019). She is/was a site investigator for the Alexander's disease trial of Ionis (2021-present), Metachromatic leukodystrophy of Shire/Takeda (2020-2021), Krabbe and GM1 gene therapy trials of Passage Bio (2021-present), GM1 natural history study of Passage Bio (2021-present), and adrenoleukodystrophy/hematopoietic stem cell transplantation natural history study of Bluebird Bio (2019) and a site subinvestigator for the MPS II gene therapy trial of Regenxbio (2021-present) and the MPS II clinical trial of Denali (2022-present). She has received unrestricted educational grants from Takeda (2021-2022)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Salla disease (SD) is a rare lysosomal storage disorder characterised by intellectual disability ataxia, athetosis, nystagmus, and central nervous system demyelination. Although the neurological spectrum of SD's clinical phenotype is well defined, psychotic symptoms in SD remain unreported. We reviewed the presence of psychiatric symptoms in patients diagnosed with SD. Medical records of all SD patients at Oulu University Hospital during the years 1982-2015 were systematically reviewed to evaluate the presence of psychiatric symptoms. Psychiatric symptoms were frequently associated with SD (10/24, 42%), and two patients were described as developing psychosis as adolescents. We reported their clinical characteristics in detail and assessed the prevalence of psychiatric symptoms in a cohort of 24 patients. Other psychiatric factors associated with SD were sleeping disorders (8/24, 32%), aggressive behaviour disorders or restlessness (6/24, 25%), and off-label antipsychotic medication (4/24, 17%). This report expands the knowledge of the phenotypic spectrum of SD and demonstrates the importance of recognising the possibility of psychiatric symptoms, including psychosis, in persons with SD., (© 2022. The Author(s).)

Malfunction of the sialic acid transporter caused by various genetic mutations in the SLC17A5 gene encoding Sialin leads to a spectrum of neurodegenerative conditions called free sialic acid storage disorders. Unfortunately, how Sialin transports sialic acid/proton (H + ) and how pathogenic mutations impair its function are poorly defined. Here, we present the structure of human Sialin in an inward-facing partially open conformation determined by cryo-electron microscopy, representing the first high-resolution structure of any human SLC17 member. Our analysis reveals two unique features in Sialin: (i) The H + coupling/sensing requires two highly conserved Glu residues (E171 and E175) instead of one (E175) as implied in previous studies; and (ii) the normal function of Sialin requires the stabilization of a cytosolic helix, which has not been noticed in the literature. By mapping known pathogenic mutations, we provide mechanistic explanations for corresponding functional defects. We propose a structure-based mechanism for sialic acid transport mediated by Sialin.

Objectives: Urine free sialic acid (UFSA) is an important diagnostic biomarker for sialuria ( GNE variants) and infantile sialic acid storage disease/Salla disease ( SLC17A5 variants). Traditionally, UFSA has been measured using specific single-plex methodology in relatively small cohorts of patients with clinical symptoms suggestive of these disorders. The use of multiplex tandem mass spectrometry urine screening (UMSMS) has meant that UFSA can be measured semi-quantitatively in a much larger cohort of patients being investigated for suspected metabolic disorders. We hypothesised that the neuraminidase of Streptococcus pneumoniae may release free sialic acid from endogenous sialylated glycoconjugates and result in increased UFSA levels., Methods: We conducted a retrospective review of clinical records of patients who were identified as having S. pneumoniae infection and who also had UMSMS at the time of their acute infection., Results: We identified three cases of increased UFSA detected by UMSMS screening that were secondary to S. pneumoniae sepsis. Additional testing ruled out genetic causes of increased UFSA in the first patient. All three patients had overwhelming sepsis with multiorgan dysfunction which was fatal. Glycosylation abnormalities consistent with the removal of sialic acid were demonstrated in serum transferrin patterns in one patient., Conclusions: We have demonstrated in a retrospective cohort that elevation of UFSA levels have been observed in cases of S. pneumoniae sepsis. This expands our knowledge of UFSA as a biomarker in human disease. This research demonstrates that infection with organisms with neuraminidase activity should be considered in patients with unexplained increases in UFSA., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

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., (© 2021 Hasnain et al.; Published by Cold Spring Harbor Laboratory Press.)

Lysosomal free sialic acid storage disorder (FSASD) is an extremely rare, autosomal recessive, neurodegenerative, multisystemic disorder caused by defects in the lysosomal sialic acid membrane exporter SLC17A5 (sialin). SLC17A5 defects cause free sialic acid and some other acidic hexoses to accumulate in lysosomes, resulting in enlarged lysosomes in some cell types and 10-100-fold increased urinary excretion of free sialic acid. Clinical features of FSASD include coarse facial features, organomegaly, and progressive neurodegenerative symptoms with cognitive impairment, cerebellar ataxia and muscular hypotonia. Central hypomyelination with cerebellar atrophy and thinning of the corpus callosum are also prominent disease features. Around 200 FSASD cases are reported worldwide, with the clinical spectrum ranging from a severe infantile onset form, often lethal in early childhood, to a mild, less severe form with subjects living into adulthood, also called Salla disease. The pathobiology of FSASD remains poorly understood and FSASD is likely underdiagnosed. Known patients have experienced a diagnostic delay due to the rarity of the disorder, absence of routine urine sialic acid testing, and non-specific clinical symptoms, including developmental delay, ataxia and infantile hypomyelination. There is no approved therapy for FSASD. We initiated a multidisciplinary collaborative effort involving worldwide academic clinical and scientific FSASD experts, the National Institutes of Health (USA), and the FSASD patient advocacy group (Salla Treatment and Research [S.T.A.R.] Foundation) to overcome the scientific, clinical and financial challenges facing the development of new treatments for FSASD. We aim to collect data that incentivize industry to further develop, obtain approval for, and commercialize FSASD treatments. This review summarizes current aspects of FSASD diagnosis, prevalence, etiology, and disease models, as well as challenges on the path to therapeutic approaches for FSASD., (Copyright © 2021. Published by Elsevier B.V.)

Disorders caused by defects in lysosomal membrane transporters form a distinct subgroup of lysosomal storage disorders (LSDs). To date, defects in only 10 lysosomal membrane transporters have been associated with inherited disorders. The clinical presentations of these diseases resemble the phenotypes of other LSDs; they are heterogeneous and often present in children with neurodegenerative manifestations. However, for pathomechanistic and therapeutic studies, lysosomal membrane transport defects should be distinguished from LSDs caused by defective hydrolytic enzymes. The involved proteins differ in function, localization, and lysosomal targeting, and the diseases themselves differ in their stored material and therapeutic approaches. We provide an overview of the small group of disorders of lysosomal membrane transporters, emphasizing discovery, pathomechanism, clinical features, diagnostic methods and therapeutic aspects. We discuss common aspects of lysosomal membrane transporter defects that can provide the basis for preclinical research into these disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Background: Sialuria is a rare inborn error of metabolism caused by excessive synthesis of sialic acid due to the mutation in the binding site of the cytidine monophosphate-sialic acid of UDPGlcNAc 2-Epimerase/ManNAc Kinase (GNE/MNK)., Objective: This is the first study investigating the molecular basis of neuronal disorders exhibiting sialuria in Pakistani children/adolescents., Methods: The current study genotyped GNE SNPs rs121908621, rs121908622 and rs121908623 by using PCR, RFLP, and DNA sequencing methods. Socioeconomic and clinical histories were also recorded., Results: Our data suggest that clinical symptoms and financial status play a significant role in conferring sialuria related Intellectual Disability (ID). SNP: rs121908623 showed G/A substitution (R263Q) in the GNE gene., Conclusion: We have identified one case study in Pakistan, so this makes our research a leap forward towards the identification of the 10th case study worldwide., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Nonimmune hydrops fetalis is the most severe clinical manifestation of lysosomal storage diseases (LSDs). Around 14 different LSDs have been accounted for as 1-15% of the cause of nonimmune hydrops fetalis. We report a Korean infant affected by an extremely rare but severe form of sialic acid storage disease. The patient presented with nonimmune hydrops fetalis, dysmorphic facial features, hepatosplenomegaly, and dysostosis multiplex and died at 39 days of age due to persistent pulmonary hypertension. LSD was suspected based on the presence of diffuse vacuolation of syncytiotrophoblast, villous stromal cells, and intermediate trophoblast in placental biopsy. Increased excretion of urinary free sialic acid was detected by liquid chromatography-tandem mass spectrometry. The patient was compound heterozygous of the c.908G>A (p.Trp303Ter) and the splicing mutation c.1259+5G>T (IVS9+5 G>T) in the SLC17A5 gene., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Likely 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 Description: This 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., Conclusion: We 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sialuria is a rare autosomal dominant disorder of mammalian metabolism, caused by defective feedback inhibition of the UDP-N-acetylglucosamine-2-epimerase N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. Sialuria is characterized by overproduction of free sialic acid in the cell cytoplasm. Patients exhibit vastly increased urinary excretion of sialic acid and show differently pronounced developmental delays. The physiopathology of sialuria is not well understood. Here we established a transgenic mouse line that expresses GNE containing the sialuria mutation R263L, in order to investigate the influence of an altered sialic acid concentration on the organism. The transgenic mice that expressed the mutated RNA excreted up to 400 times more N-acetylneuraminic acid than wild-type mice. Additionally, we found higher sialic acid concentration in the brain cytoplasm. Analyzing the (poly)sialylation of neural cell adhesion molecule (NCAM) revealed increased polysialylation in brains of transgenic mice compared to wild-type. However, we found only minor changes in membrane-bound sialylation in various organs but, surprisingly, a significant increase in surface sialylation on leukocytes. Our results suggest that the intracellular sialic acid concentration regulates polysialylation on NCAM in vivo; this could play a role in the manifestation of the developmental delays in sialuria patients., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sialic 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., Results: The 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., Conclusions: This 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.

Unlabelled: Sialuria, a rare inborn error of metabolism, was diagnosed in a healthy 12-year-old boy through whole exome sequencing. The patient had experienced mild delays of speech and motor development, as well as persistent hepatomegaly. Identification of the 8th individual with this disorder, prompted follow-up of the mother-son pair of patients diagnosed over 15years ago. Hepatomegaly was confirmed in the now 19-year-old son, but in the 46-year-old mother a clinically silent liver tumor was detected by ultrasound and MRI. The tumor was characterized as an intrahepatic cholangiocarcinoma (IHCC) and DNA analysis of both tumor and normal liver tissue confirmed the original GNE mutation. As the maternal grandmother in the latter family died at age 49years of a liver tumor, a retrospective study of the remaining pathology slides was conducted and confirmed it to have been an IHCC as well. The overall observation generated the hypothesis that sialuria may predispose to development of this form of liver cancer. As proof of sialuria in the grandmother could not be obtained, an alternate cause of IHCC cannot be ruled out. In a series of 102 patients with IHCC, not a single instance was found with the allosteric site mutation in the GNE gene. This confirms that sialuria is rare even in a selected group of patients, but does not invalidate the concern that sialuria may be a risk factor for IHCC., Synopsis: Sialuria is a rare inborn error of metabolism characterized by excessive synthesis and urinary excretion of free sialic acid with only minimal clinical morbidity in early childhood, but may be a risk factor for intrahepatic cholangiocarcinoma in adulthood., (Copyright © 2016 Elsevier Inc. All rights reserved.)

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme of sialic acid biosynthesis in vertebrates. It catalyzes the first two steps of the cytosolic formation of CMP-N-acetylneuraminic acid from UDP-N-acetylglucosamine. In this review we give an overview of structure, biochemistry, and genetics of the bifunctional enzyme and its complex regulation. Furthermore, we will focus on diseases related to UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.

We describe here a 34 months child, practically asymptomatic which presented with high levels of free sialic acid in urine by biochemical detection in second-tier tests newborn screening and with two disease causing mutations in SLC17A5 gene. SLC17A5 mutation analysis showed p.Tyr306* previously described and the novel mutation p.Leu167Pro. This early onset diagnosis allowed us to perform a fast and accurate genetic counseling to the family, helped to better understanding the natural history of this rare disease and probably it could promote cost reduction in future diagnostic tests in the hypothetic case of starting symptoms without diagnosis established. Moreover, an early diagnosis could save family from a long period of time until achieving a definitive diagnostic and to develop an early symptomatic and supportive management of patient to attenuate, as much as possible, disease complications. But, above all, this case illustrates the huge ethical dilemma which arises from any secondary finding (second tier) in newborn screening., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Infection of Spodoptera frugiperda (Sf9) cells by baculovirus (BV) is well established for transgene expression of soluble proteins, but few correctly folded transmembrane proteins have been so produced. We here report the use of the BV/Sf9 (BVES) method for the expression and transfer, via microvesicles, of the exclusive lysosomal exporters for cystine and sialic acid, human cystinosin and sialin. These proteins and their mRNA are released into the culture medium as very low-density microvesicles (~1.05 g/ml), which do not label for lysobisphosphatidic acid. The presence of the human transgene proteins in the vesicles was confirmed by western blotting and confirmed and quantified by mass spectrometry. Addition of vesicles to cultures of human fibroblast lines deficient in either cystinosin or sialin produced a progressive depletion of stored lysosomal cystine or sialic acid, respectively. The depletion effect was slow (T1/2 ~48 h), saturable (down to ~40% of initial after 4 days) and stable (>one week). Surprisingly, BV infection of Spodoptera appeared to induce expression and release into microvesicles of the insect orthologue of cystinosin, but not of sialin. We conclude that BVES is an effective method to express and transfer functional transmembrane proteins so as to study their properties in mammalian cells, and has a generic potential for transport protein replacement therapy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ ManNAc kinase (GNE/MNK), encoded by the GNE gene, catalyzes the first two committed, rate-limiting steps in the biosynthesis of N-acetylneuraminic acid (sialic acid). GNE/MNK is feedback inhibited by binding of the downstream product, CMP-sialic acid in its allosteric site. GNE mutations can result in two human disorders, hereditary inclusion body myopathy (HIBM) or sialuria. So far, no active site geometry predictions or conformational transitions involved with function are available for mammalian GNE/MNK. The N-terminal GNE domain is homologous to various prokaryotic 2-epimerases, some of which have solved crystallographic structures. The C-terminal MNK domain belongs to the sugar kinases superfamily; its crystallographic structure is solved at 2.84 A and three-dimensional structures have also been reported for several other kinases. In this work, we employed available structural data of GNE/MNK homologs to model the active sites of human GNE/MNK and identify critical amino acid residues responsible for interactions with substrates. In addition, we modeled effects of GNE/MNK missense mutations associated with HIBM or sialuria on helix arrangement, substrate binding, and enzyme action. We found that all reported mutations are associated with the active sites or secondary structure interfaces of GNE/MNK. The Persian-Jewish HIBM founder mutation p.M712T is located at the interface alpha4alpha10 and likely affects GlcNAc, Mg2+, and ATP binding. This work contributes to further understanding of GNE/MNK function and ligand binding, which may assist future studies for therapeutic options that target misfolded GNE/MNK in HIBM and/or sialuria.

Objective: To investigate body fluids of patients with undiagnosed leukodystrophies using in vitro (1)H-NMR spectroscopy (H-NMRS)., Methods: We conducted a cross-sectional study using high-resolution in vitro H-NMRS on CSF and urine samples., Results: We found a significant increase of free sialic acid in CSF or urine in 6 of 41 patients presenting with hypomyelination of unknown etiology. Molecular genetic testing revealed pathogenic mutations in the SLC17A5 gene in all 6 patients. H-NMRS revealed an increase of N-acetylaspartylglutamate in the CSF of all patients with SLC17A5 mutation (range 13-114 micromol/L, reference <12 micromol/L)., Conclusion: In patients with undiagnosed leukodystrophies, increased free sialic acid in CSF or urine is a marker for free sialic acid storage disorder and facilitates the identification of the underlying genetic defect. Because increase of N-acetylaspartylglutamate in CSF has been observed in other hypomyelinating disorders, it can be viewed as a marker of a subgroup of hypomyelinating disorders.

Aspartate, an excitatory amino acid, is known to be stored in synaptic vesicles and exocytosed from some neurons to perform aspartergic neurotransmission. Through in vitro reconstitution, we found that sialin, a lysosomal sialic acid exporter, is responsible for the vesicular storage of aspartate in hippocampal neurons and pinealocytes. Mutations found in Salla disease cause decreased aspartate transport activity without affecting sialic acid transport. Thus, sialin is a multifunctional transporter. It is possible that people with Salla disease lose the ability of aspartergic neurotransmission, and this could explain why Salla disease involves severe neurological defects.

We performed high-resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid concentration was increased in cerebrospinal fluid of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.

Dominant disease alleles are attractive therapeutic targets for allele-specific gene silencing by small interfering RNA (siRNA). Sialuria is a dominant disorder caused by missense mutations in the allosteric site of GNE, coding for the rate-limiting enzyme of sialic acid biosynthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase. The resultant loss of feedback inhibition of GNE-epimerase activity by CMP-sialic acid causes excessive production of free sialic acid. For this study we employed synthetic siRNAs specifically targeting the dominant GNE mutation c.797G>A (p.R266Q) in sialuria fibroblasts. We demonstrated successful siRNA-mediated down-regulation of the mutant allele by allele-specific real-time PCR. Importantly, mutant allele-specific silencing resulted in a significant decrease of free sialic acid, to within the normal range. Feedback inhibition of GNE-epimerase activity by CMP-sialic acid recovered after silencing demonstrating specificity of this effect. These findings indicate that allele-specific silencing of a mutated allele is a viable therapeutic strategy for autosomal dominant diseases, including sialuria.

Background Information: Free sialic acid storage diseases are caused by mutations of a lysosomal sialic acid transporter called sialin. We showed recently that the milder clinical form, Salla disease, and a related non-Finish case, are characterized by residual transport, whereas sialin mutants found in lethal infantile cases are inactive. In the present study, we have characterized the molecular effects of a putative polymorphism (M316I) and of four pathogenic mutations associated with either infantile (G127E and R57C) or Salla-like (G409E) phenotypes, or both (G328E). The transport activity of human sialin was analysed using a novel assay that was based on a construct without the functional lysosomal sorting motif, which is expressed at the plasma membrane., Results: The lysosomal localization of human sialin was not (M316I and G328E) or only partially (R57C, G127E and G409E) affected by the missense mutations. In contrast, all pathogenic mutations abolished transport, whereas the putative M316I polymorphism induced an approx. 5-fold decrease of sialic acid transport., Conclusions: The molecular effects of the R57C and G127E mutations strengthen the conclusion that the infantile phenotype is caused by loss-of-function mutations. On the other hand, the milder severity of the heterozygous G409E patient may reflect an incomplete expression of the splicing mutation present on the second allele. In the case of the G328E mutation, found in the homozygous state in a clinically heterogeneous family, the apparent severity of the transport phenotype suggests that the genetic or environmental factors underlying this clinical heterogeneity might be protective.

In contrast to lysosomal hydrolytic enzymes, the lysosomal membrane remains poorly characterized. In particular, although the genetic study of cystinosis and sialic acid storage disorders led to the identification of two lysosomal transporters for cystine and sialic acids, respectively, ten years ago, most transporters responsible for exporting lysosomal hydrolysis products to the cytosol are still unknown at the molecular level. However, two lines of investigation recently started to fill this gap in the knowledge of lysosomal biology. First, novel proteomic approaches are now able to provide a reliable inventory of lysosomal membrane proteins. On the other hand, a novel functional approach based on intracellular trafficking mechanisms allows direct transport measurement in whole cells by redirecting recombinant lysosomal transporters to the cell surface. After surveying the current state of knowledge in this field, the review focuses on the sialic acid transporter sialin and shows how recent functional data using the above whole-cell approach shed new light on the pathogenesis of sialic acid storage disorders by revealing the existence of a residual transport activity associated with Salla disease.

Two disease-associated missense mutations in the sialin gene (G328E and G409E) have recently been identified in patients with lysosomal free sialic acid storage disease. We have assessed the effect of these mutations and find complete loss of measurable transport activity with both and impaired trafficking of the G409E protein. These results suggest that the two residues are important for proper function of sialin and confirm the association of loss of transport with disease causative mutations.

Free sialic acid storage disorders, Salla disease (SD) and Infantile sialic acid storage disease (ISSD), are lysosomal storage diseases due to impaired function of a sialic acid transporter, sialin, at the lysosomal membrane. Several mutations of the sialin gene, SLC17A5, are known, leading either to the severe neonatal/infantile disease or to the milder, adult-type developmental disorder, Salla disease. Free sialic acid accumulation in lysosomes causes increased tissue concentration and consequently elevated urinary excretion. Prenatal diagnosis of SASD is possible either by determination of free sialic acid concentration or by mutation analysis of the SLC17A5 gene in fetal specimen, in chorionic villus biopsy particularly. Both techniques have been successfully applied in several cases, sialic acid assay more often in ISSD cases but mutation analysis preferentially in SD. Sialic acid assay of amniotic fluid supernatant or cultured amniotic fluid cells may give erroneous results and should not be used for prenatal diagnosis of these disorders. The present comments are mainly based on our experience of prenatal diagnosis of SD in Finnish families. A founder mutation in SLC17A5 gene, 115C-> T, represents 95% of the disease alleles in the Finnish SD patients, which provides a unique possibility to apply mutation analysis. Therefore, molecular studies have successfully been used in 17 families since the identification of the gene and the characterization of the SD mutations. Earlier, eight prenatal studies were performed by measuring the free sialic acid concentration in chorionic villus samples.

Sialic acid (SA), a terminal monosaccharide of glycoconjugates, has a central role in human biological function. Various point mutations result in the malmetabolism of SA and inherited disorders: Defective SA synthesis causes sialuria and defective SA catabolism causes sialidosis and sialic acid storage disease (SASD). These inborn errors of metabolism are characterised by increased urinary free SA. This article reviews biochemical and clinical features that are distinct to each disorder. In view of recent evidence indicating a wide underestimation in the prevalence of sialic acid disorders, laboratory methods for determining urinary free SA and its implications for screening and prenatal diagnosis are evaluated.

Lysosomal free sialic acid storage diseases are recessively inherited allelic neurodegenerative disorders that include Salla disease (SD) and infantile sialic acid storage disease (ISSD) caused by mutations in the SLC17A5 gene encoding for a lysosomal membrane protein, sialin, transporting sialic acid from lysosomes. The classical form of SD, enriched in the Finnish population, is related to the p.R39C designed Salla(FIN) founder mutation. A more severe phenotype is due both to compound heterozygosity for the p.R39C mutation and to different mutations. The p.R39C has not been reported in ISSD. We identified the first case of SD caused by the homozygosity for p.K136E (c.406A>G) mutation, showing a severe clinical picture, as demonstrated by the early age at onset, the degree of motor retardation, the occurrence of peripheral nerve involvement, as well as cerebral hypomyelination. Recently, in vitro functional studies have shown that the p.K136E mutant produces a mislocalization and a reduced activity of the intracellular sialin. We discuss the in vivo phenotypic consequence of the p.K136E in relation to the results obtained by the in vitro functional characterization of the p.K136E mutant. The severity of the clinical picture, in comparison with the classical SD, may be explained by the fact that the p.K136E mutation mislocalizes the protein to a greater degree than p.R39C. On the other hand, the presence of a residual transport activity may account for the absence of hepatosplenomegaly, dysostosis multiplex, and early lethality typical of ISSD and related to the abolished transport activity found in this latter form.

An Old Order Mennonite child was evaluated for gross motor delay, truncal ataxia, and slow linear growth. The diagnostic evaluation, which included sub-specialty consultations, neuroimaging, and metabolic testing, was long, costly, and did not yield a diagnosis. Recognition of a similarly affected second cousin prompted a genome-wide homozygosity mapping study using high-density single nucleotide polymorphism (SNP) arrays. SNP genotypes from two affected individuals and their parents were used to localize the disease locus to a 14.9 Mb region on chromosome 6. This region contained 55 genes, including SLC17A5, the gene encoding the lysosomal N-acetylneuraminic acid transport protein. Direct sequencing of SLC17A5 in the proband revealed homozygosity for the 115C --> T (R39C) sequence variant, the common cause of Salla disease in Finland. Three additional affected Mennonite individuals, ages 8 months to 50 years, were subsequently identified by directed molecular genetic testing. This small-scale mapping study was rapid, inexpensive, and analytically simple. In families with shared genetic heritage, genome-wide SNP arrays with relatively high marker density allow disease gene mapping studies to be incorporated into routine diagnostic evaluations.

The objective of this article is to describe the clinical, radiographic, and molecular genetic features of a new intermediate form of free sialic storage disease. Free sialic storage disease is a rare autosomal recessive lysosomal disorder that results from mutations in SLC17A5, a gene that codes for sialin, a lysosomal membrane sialic acid transporting protein. Infantile sialic acid storage disease has a severe phenotype, and Salla disease (Finnish variant) is generally milder in phenotype; intermediate forms have also been described. There have been few reports of magnetic resonance imaging (MRI) in the sialic acid storage disorders; leukodystrophy has been the characteristic finding, along with hypoplasia of the corpus callosum. An 8-month-old non-Finnish child presented with hypotonia and global developmental delay. Serial MRIs with magnetic resonance spectroscopy at 9 and 16 months revealed severe hypomyelination and hypogenesis of the corpus callosum. There was mild elevation of urinary sialic acid (4.5 times above normal). Electron microscopy of a skin biopsy showed lysosomal enlargement with oligosaccharide storage, and confirmatory molecular genetic testing revealed compound heterozygosity for two new SLC17A5 mutations. Free sialic storage disease of the intermediate type is an important part of the differential diagnosis of a hypotonic, delayed child with abnormal white matter on MRI. Intermediate types of free sialic acid overlap in phenotype with infantile sialic acid storage disease and the milder Salla disease and thus might be more difficult to identify clinically; the lack of Finnish ethnicity should not preclude testing for this probably under-recognized disorder. White-matter abnormalities appear to be characteristic of the entire phenotypic spectrum.

Sialic acids are expressed as terminal sugars in many glycoconjugates and play an important role during development and regeneration, as they are involved as polysialic acid in a variety of cell-cell interactions mediated by the neural cell adhesion molecule NCAM. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the consequences after expression of a sialuria-mutated GNE. Expression of the sialuria-mutated GNE leads to a dramatic increase of both cellular sialic acid and polysialic acid on NCAM. This could also be achieved by application of the sialic acid precursor N-acetylmannosamine. Our data suggest that biosynthesis of sialic acid regulates and limits the synthesis of polysialic acid.

Previous studies have reported that insect cell lines lack the capacity to generate endogenously the nucleotide sugar, CMP-Neu5Ac, required for sialylation of glycoconjugates. In this study, the biosynthesis of this activated form of sialic acid completely from endogenous metabolites is demonstrated for the first time in insect cells by expressing the mammalian genes required for the multistep conversion of endogenous UDP-GlcNAc to CMP-Neu5Ac. The genes for UDP-GlcNAc-2-epimerase/ManNAc kinase (EK), sialic acid 9-phosphate synthase (SAS), and CMP-sialic acid synthetase (CSAS) were coexpressed in insect cells using baculovirus expression vectors, but the CMP-Neu5Ac and precursor Neu5Ac levels synthesized were found to be lower than those achieved with ManNAc supplementation due to feedback inhibition of the EK enzyme by CMP-Neu5Ac. When sialuria-like mutant EK genes, in which the site for feedback regulation has been mutated, were used, CMP-Neu5Ac was synthesized at levels more than 4 times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAc feeding. Addition of N-acetylglucosamine (GlcNAc), a precursor for UDP-GlcNAc, to the media increased the levels of CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAc supplementation, creating a bottleneck in the conversion of Neu5Ac to CMP-Neu5Ac at higher levels of UDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance the levels of the sialylation donor molecule, CMP-Neu5Ac, a critical limiting substrate for the generation of complex glycoproteins in insect cells and other cell culture systems.

Salla disease and infantile sialic acid storage disorder are autosomal recessive neurodegenerative diseases characterized by loss of a lysosomal sialic acid transport activity and the resultant accumulation of free sialic acid in lysosomes. Genetic analysis of these diseases has identified several unique mutations in a single gene encoding a protein designated sialin (Verheijen, F. W., Verbeek, E., Aula, N., Beerens, C. E., Havelaar, A. C., Joosse, M., Peltonen, L., Aula, P., Galjaard, H., van der Spek, P. J., and Mancini, G. M. (1999) Nat. Genet. 23, 462-465; Aula, N., Salomaki, P., Timonen, R., Verheijen, F., Mancini, G., Mansson, J. E., Aula, P., and Peltonen, L. (2000) Am. J. Hum. Genet. 67, 832-840). From the biochemical phenotype of the diseases and the predicted polytopic structure of the protein, it has been suggested that sialin functions as a lysosomal sialic acid transporter. Here we directly demonstrate that this activity is mediated by sialin and that the recombinant protein has functional characteristics similar to the native lysosomal sialic acid transport system. Furthermore, we describe the effect of disease-causing mutations on the protein. We find that the majority of the mutations are associated with a complete loss of activity, while the mutations associated with the milder forms of the disease lead to reduced, but residual, function. Thus, there is a direct correlation between sialin function and the disease state. In addition, we find with one mutation that the protein is retained in the endoplasmic reticulum, indicating that altered trafficking of sialin is also associated with disease. This analysis of the molecular mechanism of sialic acid storage disorders is a further step in identifying therapeutic approaches to these diseases.

The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes and several diseases are caused by defective sialic acid metabolism. Sialic acids cleaved off from degraded sialoglycoconjugates are exported from lysosomes by a membrane transporter, named sialin, which is defective in two allelic inherited diseases: infantile sialic acid storage disease (ISSD) and Salla disease. To develop a functional assay of human sialin, we redirected the protein to the plasma membrane by mutating a dileucine-based internalization motif. Cells expressing the plasmalemmal construct accumulated neuraminic acid at acidic pH by a process equivalent to lysosomal efflux. The assay was used to determine how pathogenic mutations affect transport. Interestingly, while two missense mutations and one small, in-frame deletion associated with ISSD abolished transport, the mutation causing Salla disease (R39C) slowed down, but did not stop, the transport cycle, thus explaining why the latter disorder is less severe. Since neurological symptoms predominate in Salla disease, our results suggest that sialin is rate-limiting to specific sialic acid-dependent processes of the nervous system.

The allelic autosomal recessive lysosomal storage disorders Salla disease and infantile free sialic acid storage disease (ISSD) result from mutations in SLC17A5. This gene codes for sialin, a lysosomal membrane protein that transports the charged sugar, N-acetylneuraminic acid (sialic acid), out of lysosomes. ISSD has a severe phenotype with infantile onset, while the Finnish variant, Salla disease, has a milder phenotype with later onset. Both disorders cause developmental delay, and ISSD is generally fatal in early childhood. We describe a 30-month old non-Finnish, Caucasian child with global developmental delay of postnatal onset, language, and motor skills stagnant at a 3-4 month level, hypotonia, and mild but progressive coarsening of facial features. Urinary excretion of free sialic acid was elevated 4.5 times above control. EM of a skin biopsy revealed enlarged secondary lysosomes consistent with oligosaccharide storage. Free sialic acid in fibroblasts was 3.8+/-0.9 nmol/mg protein (concurrent normal controls, 0.5+/-0.1); differential centrifugation indicated a lysosomal location. Genomic analysis revealed compound heterozygosity for two new SLC17A5 mutations. This child's clinical manifestations of a lysosomal free sialic acid storage disease are consistent with her sialin mutations and biochemical findings. The differential diagnosis of postnatal developmental delay should include free sialic acid storage disorders such as ISSD and Salla disease.

Four members of an extended consanguineous Bedouin family presented with different phenotypic variants of an autosomal recessive lysosomal free sialic acid storage disease. One affected individual had congenital ascites followed by rapid clinical deterioration and death, a presentation concordant with the clinical course of infantile free sialic acid storage disorder. His three first cousins had a more slowly progressive neurodegenerative disease, in line with the clinical phenotype of the milder form (Salla type) of this lysosomal disorder. Diagnosis of free sialic acid storage disease was based on clinical findings, histology, and biochemical assays of sialic acid. Molecular studies showed that all four affected individuals were homozygous for the same novel 983G > A mutation in exon 8 of the SLC17A5 gene, replacing glycine with glutamic acid at position 328 of the sialin protein. This family demonstrates the significant phenotypic variability of the disease in affected members of a single inbred kindred with precisely the same mutation, suggesting a role for modifier genes or environmental factors. It also highlights the need to consider this rare disorder in the differential diagnosis of congenital ascites and of unexplained psychomotor retardation, ataxia, and hypomyelination in infancy.

SLC17A5 encodes a lysosomal membrane protein, sialin, which transports sialic acid from lysosomes. Mutations in sialin result in neurodegenerative sialic acid storage disorders, Salla disease (SD) and infantile sialic acid storage disease (ISSD). Here we analyzed sialin in mouse central nervous system (CNS) and primary cortical and hippocampal neurons and glia. In the CNS, sialin was predominantly expressed in neurons, especially in the proliferative zone of the prospective neocortex and the hippocampus in developing brain. In nonneuronal cells and primary glial cell cultures, mouse sialin was localized into lysosomes but interestingly, in primary neuronal cultures sialin was not targeted into lysosomes but rather revealed a punctate staining along the neuronal processes and was also seen in the plasma membrane. These data demonstrate a nonlysosomal localization of sialin in neurons and would imply a role for sialin in the secretory processes of neuronal cells.

The differential diagnosis of developmental delays and growth retardation in early childhood includes the allelic lysosomal sialic acid storage disorders, Salla disease and infantile free sialic acid storage disease (ISSD). These diseases, due to defective free sialic acid transport out of lysosomes, derive from mutations in the SLC17A5 gene coding for the protein sialin. We present two patients with clinical, biochemical, and molecular data indicative of lysosomal free sialic acid storage disorders. One patient, with a severe clinical course typical of ISSD, had 86-fold elevated levels of fibroblast free sialic acid, with 62% in the lysosomal fraction. His SLC17A5 mutations include a 148-bp deletion of exon 9, due to a G >A splice site mutation in position 1 of intron 9, and a 15-bp deletion (del 801-815) in exon 6. Another patient, with "intermediate severe" Salla disease, had 9-fold elevated levels of free sialic acid in cultured fibroblasts, of which 87% resided in the lysosomal fraction. This girl is compound heterozygous for the SLC17A5 mutation commonly found in Finnish Salla disease patients (R39C) and a 15-bp deletion found in ISSD patients (del 801-815). These observations emphasize the importance of considering free sialic acid disorders in infants with developmental delays and growth retardation, regardless of whether they are of Finnish ancestry., (Copyright 2003 Wiley-Liss, Inc.)

Salla disease, one of three disease phenotypes that manifest increased urinary excretion of unconjugated sialic acid, is an autosomal recessive condition caused by a mutation in SLC17A5. This gene encodes sialin, a lysosomal membrane transporter for sialic acid. Salla disease is rare outside of individuals of Finnish ancestry. In this report we describe the disorder in non-Finnish monozygous twin siblings, the first reported American cases of Salla disease., (Copyright 2003 Wiley-Liss, Inc.)

This paper gives an overview of the two sialic acid storage disorders, Salla disease and infantile sialic acid storage disease, and the related disorders cystinosis, sialuria, sialidosis, and galactosialidosis. Sialic acid storage disease and cystinosis are models for a deficient lysosomal transport of monosaccharides and amino acids, respectively. Several gene mutations leading to the production of the faulty membrane proteins sialin and cystinosin have been identified in recent years. Knowledge of the underlying pathophysiology is a prerequisite for future research projects, which will focus on the expression of the disease genes in living systems and the physical characterization of these proteins by X-ray crystallography and nuclear magnetic resonance spectroscopy.

We report on a boy who presented at birth with gastroschisis and thereafter developed the characteristic clinical symptoms of infantile sialic acid storage disease within the first two months of life. Measurements of free sialic acid excretion (tenfold increase) in the urine and a 15-fold elevation of free sialic acid in cultured fibroblasts proved the diagnosis. The clinical course was complicated by hypertrophic cardiomyopathy, recurrent infections, hypothyroidism, and intestinal protein losses, which had never been described before in an infantile sialic acid storage disease patient. The child died at the age of 10 months. Clinical and laboratory findings are discussed and compared with other cases described in the literature.

It has recently become clear that lysosomes have more complex functions than simply being the end-point on a degradative pathway. Similarly, it is now emerging that there are interesting functions for the limiting membranes around these organelles and their associated proteins. Although it has been known for several decades that the lysosomal membrane contains several highly N-glycosylated proteins, including the lysosome-associated membrane proteins LAMP-1 and LAMP-2 and lysosomal integral membrane protein-2/lysosomal membrane glycoprotein-85 (LIMP-2/LGP85), specific functions of these proteins have only recently begun to be recognized. Although the normal functions of LAMP-1 can be substituted by the structurally related LAMP-2, LAMP-2 itself has more specific tasks. Knockout of LAMP-2 in mice has revealed roles for LAMP-2 in lysosomal enzyme targeting, autophagy and lysosomal biogenesis. LAMP-2 deficiency in humans leads to Danon disease, a fatal cardiomyopathy and myopathy. Furthermore, there is evidence that LAMP-2 functions in chaperone-mediated autophagy. LIMP-2/LGP85 also seems to have specific functions in maintaining endosomal transport and lysosomal biogenesis. The pivotal function of lysosomal membrane proteins is also highlighted by the recent identification of disease-causing mutations in cystine and sialic acid transporter proteins, leading to nephropathic cystinosis and Salla disease.