RESUMEN
The Drinking Water Directive (EU) 2020/2184 includes the parameter microcystin LR, a cyanotoxin, which drinking water producers need to analyze if the water source has potential for cyanobacterial blooms. In light of the increasing occurrences of cyanobacterial blooms worldwide and given that more than 50 percent of the drinking water in Sweden is produced from surface water, both fresh and brackish, the need for improved knowledge about cyanotoxin occurrence and cyanobacterial diversity has increased. In this study, a total of 98 cyanobacterial blooms were sampled in 2016-2017 and identified based on their toxin production and taxonomical compositions. The surface water samples from freshwater lakes throughout Sweden including brackish water from eight east coast locations along the Baltic Sea were analyzed for their toxin content with LC-MS/MS and taxonomic composition with 16S rRNA amplicon sequencing. Both the extracellular and the total toxin content were analyzed. Microcystin's prevalence was highest with presence in 82% of blooms, of which as a free toxin in 39% of blooms. Saxitoxins were found in 36% of blooms in which the congener decarbamoylsaxitoxin (dcSTX) was detected for the first time in Swedish surface waters at four sampling sites. Anatoxins were most rarely detected, followed by cylindrospermopsin, which were found in 6% and 10% of samples, respectively. As expected, nodularin was detected in samples collected from the Baltic Sea only. The cyanobacterial operational taxonomic units (OTUs) with the highest abundance and prevalence could be annotated to Aphanizomenon NIES-81 and the second most profuse cyanobacterial taxon to Microcystis PCC 7914. In addition, two correlations were found, one between Aphanizomenon NIES-81 and saxitoxins and another between Microcystis PCC 7914 and microcystins. This study is of value to drinking water management and scientists involved in recognizing and controlling toxic cyanobacteria blooms.
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Cianobacterias , Lagos , Toxinas Marinas , Microcistinas , Suecia , Cianobacterias/genética , Cianobacterias/aislamiento & purificación , Microcistinas/análisis , Lagos/microbiología , Toxinas Marinas/análisis , Saxitoxina/análisis , Monitoreo del Ambiente , ARN Ribosómico 16S/genética , Toxinas Bacterianas/análisis , Toxinas de Cianobacterias , Espectrometría de Masas en TándemRESUMEN
In recent years, several rational designed therapies have been developed for treatment of mucopolysaccharidoses (MPS), a group of inherited metabolic disorders in which glycosaminoglycans (GAGs) are accumulated in various tissues and organs. Thus, improved disease-specific biomarkers for diagnosis and monitoring treatment efficacy are of paramount importance. Specific non-reducing end GAG structures (GAG-NREs) have become promising biomarkers for MPS, as the compositions of the GAG-NREs depend on the nature of the lysosomal enzyme deficiency, thereby creating a specific pattern for each subgroup. However, there is yet no straightforward clinical laboratory platform which can assay all MPS-related GAG-NREs in one single analysis. Here, we developed and applied a GAG domain mapping approach for analyses of urine samples of ten MPS patients with various MPS diagnoses and corresponding aged-matched controls. We describe a nano-LC-MS/MS method of GAG-NRE profiling, utilizing 2-aminobenzamide reductive amination labeling to improve the sensitivity and the chromatographic resolution. Diagnostic urinary GAG-NREs were identified for MPS types IH/IS, II, IIIc, IVa and VI, corroborating GAG-NRE as biomarkers for these known enzyme deficiencies. Furthermore, a significant reduction of diagnostic urinary GAG-NREs in MPS IH (n = 2) and MPS VI (n = 1) patients under treatment was demonstrated. We argue that this straightforward glycomic workflow, designed for the clinical analysis of MPS-related GAG-NREs in one single analysis, will be of value for expanding the use of GAG-NREs as biomarkers for MPS diagnosis and treatment monitoring.
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Glicosaminoglicanos , Mucopolisacaridosis , Humanos , Anciano , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Glicómica , Flujo de Trabajo , Mucopolisacaridosis/diagnóstico , Mucopolisacaridosis/orina , BiomarcadoresRESUMEN
BACKGROUND: Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system. The glycosphingolipid sulfatide, a lipid particularly enriched in the myelin sheath, has been shown to be involved the maintenance of this specific membrane structure. Sulfatide in cerebrospinal fluid (CSF) may reflect demyelination, a dominating feature of MS. We investigated the diagnostic utility of CSF sulfatide isoform levels to separate different courses or phenotypes of MS disease. MATERIAL AND METHODS: This was a mono-center, cross-sectional study of relapsing-remitting MS (RRMS) (n = 45) and progressive MS (PMS) (n = 42) patients (consisting of primary PMS (n = 17) and secondary PMS (n = 25)) and healthy controls (n = 19). In total, 20 sulfatide isoforms were measured in CSF by liquid chromatography-mass spectrometry. RESULTS: CSF total sulfatide concentrations, as well as CSF sulfatide isoform distribution, did not differ across the study groups, and their levels were independent of disease course/phenotype, disease duration, time to conversion to secondary PMS, age, and disability in MS patients. CONCLUSION: CSF sulfatide isoforms lack diagnostic and prognostic utility as a biomarker for progressive MS.
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Esclerosis Múltiple Crónica Progresiva , Esclerosis Múltiple Recurrente-Remitente , Esclerosis Múltiple , Humanos , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Esclerosis Múltiple/diagnóstico , Sulfoglicoesfingolípidos/líquido cefalorraquídeo , Sulfoglicoesfingolípidos/química , Estudios Transversales , Esclerosis Múltiple Crónica Progresiva/diagnóstico , Esclerosis Múltiple Crónica Progresiva/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Isoformas de ProteínasRESUMEN
Here, we present the first two Swedish cases of Conserved Oligomeric Golgi complex subunit 6-congenital disorders of glycosylation (COG6-CDG). Their clinical symptoms include intellectual disability, Attention Deficit/Hyperactivity Disorder (ADHD), delayed brain myelinization, progressive microcephaly, joint laxity, hyperkeratosis, frequent infections, and enamel hypoplasia. In one family, compound heterozygous variants in COG6 were identified, where one (c.785A>G; p.Tyr262Cys) has previously been described in patients of Moroccan descent, whereas the other (c.238G>A; p.Glu80Lys) is undescribed. On the other hand, a previously undescribed homozygous duplication (c.1793_1795dup) was deemed the cause of the disease. To confirm the pathogenicity of the variants, we treated patient and control fibroblasts with the ER-Golgi transport inhibitor Brefeldin-A and show that patient cells manifest a significantly slower anterograde and retrograde ER-Golgi transport.
RESUMEN
Fabry disease (FD) is a rare genetic lysosomal storage disorder, resulting from partial or complete lack of alpha-galactosidase A (α-GAL) enzyme, leading to systemic accumulation of substrate glycosphingolipids with a broad range of tissue damage. Current in vivo models are laborious, expensive, and fail to adequately mirror the complex FD physiopathology. To address these issues, we developed an innovative FD model in zebrafish. Zebrafish GLA gene encoding α-GAL enzyme presents a high (>70%) homology with its human counterpart, and the corresponding protein has a similar tissue distribution, as evaluated by immunohistochemistry. Moreover, a similar enzymatic activity in different life stages could be demonstrated. By using CRISPR/Cas9 technology, we generated a mutant zebrafish with decreased GLA gene expression, and decreased expression of the specific gene product in the kidney. Mutant animals showed higher plasma creatinine levels and proteinuria. Transmission electron microscopy (TEM) studies documented an increased podocyte foot process width (FPW) in mutant, as compared to wild type zebrafish. This zebrafish model reliably mirrors distinct features of human FD and could be advantageously used for the identification of novel biomarkers and for an effective screening of innovative therapeutic approaches.
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Sulfatide (3-O-sulfogalactosylceramide, SM4) is a glycosphingolipid, highly multifunctional and particularly enriched in the myelin sheath of neurons. The role of sulfatide has been implicated in various biological fields such as the nervous system, immune system, host-pathogen recognition and infection, beta cell function and haemostasis/thrombosis. Thus, alterations in sulfatide metabolism and production are associated with several human diseases such as neurological and immunological disorders and cancers. The unique lipid-rich composition of myelin reflects the importance of lipids in this specific membrane structure. Sulfatide has been shown to be involved in the regulation of oligodendrocyte differentiation and in the maintenance of the myelin sheath by influencing membrane dynamics involving sorting and lateral assembly of myelin proteins as well as ion channels. Sulfatide is furthermore essential for proper formation of the axo-glial junctions at the paranode together with axonal glycosphingolipids. Alterations in sulfatide metabolism are suggested to contribute to myelin deterioration as well as synaptic dysfunction, neurological decline and inflammation observed in different conditions associated with myelin pathology (mouse models and human disorders). Body fluid biomarkers are of importance for clinical diagnostics as well as for patient stratification in clinical trials and treatment monitoring. Cerebrospinal fluid (CSF) is commonly used as an indirect measure of brain metabolism and analysis of CSF sulfatide might provide information regarding whether the lipid disruption observed in neurodegenerative disorders is reflected in this body fluid. In this review, we evaluate the diagnostic utility of CSF sulfatide as a biomarker for neurodegenerative disorders associated with dysmyelination/demyelination by summarising the current literature on this topic. We can conclude that neither CSF sulfatide levels nor individual sulfatide species consistently reflect the lipid disruption observed in many of the demyelinating disorders. One exception is the lysosomal storage disorder metachromatic leukodystrophy, possibly due to the genetically determined accumulation of non-metabolised sulfatide. We also discuss possible explanations as to why myelin pathology in brain tissue is poorly reflected by the CSF sulfatide concentration. The previous suggestion that CSF sulfatide is a marker of myelin damage has thereby been challenged by more recent studies using more sophisticated laboratory techniques for sulfatide analysis as well as improved sample selection criteria due to increased knowledge on disease pathology.
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Vaina de Mielina , Sulfoglicoesfingolípidos , Animales , Axones , Biomarcadores , Humanos , Ratones , NeuroglíaRESUMEN
BACKGROUND: Sulfatides (STs) in cerebrospinal fluid (CSF), as well as magnetic resonance imaging (MRI)-detected white matter hyperintensities (WMHs), may reflect demyelination. Here, we investigated the diagnostic utility of CSF ST levels in the subcortical small vessel type of dementia (SSVD), which is characterized by the presence of brain WMHs. OBJECTIVE: To study the diagnostic utility of CSF ST levels in SSVD. METHODS: This was a mono-center, cross-sectional study of SSVD (nâ=â16), Alzheimer's disease (nâ=â40), mixed dementia (nâ=â27), and healthy controls (nâ=â33). Totally, 20 ST species were measured in CSF by liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: CSF total ST levels, as well as CSF levels of hydroxylated and nonhydroxylated ST species, did not differ across the study groups. In contrast, CSF neurofilament light chain (NFL) levels separated the patient groups from the controls. CSF total ST level correlated with CSF/serum albumin ratio in the total study population (râ=â0.64, pâ<â0.001) and in all individual study groups. Furthermore, CSF total ST level correlated positively with MRI-estimated WMH volume in the total study population (râ=â0.30, pâ<â0.05), but it did not correlate with CSF NFL level. CONCLUSION: Although there was some relation between CSF total ST level and WMH volume, CSF ST levels were unaltered in all dementia groups compared to the controls. This suggests that CSF total ST level is a poor biomarker of demyelination in SSVD. Further studies are needed to investigate the mechanisms underlying the marked correlation between CSF total ST level and CSF/serum albumin ratio.
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Enfermedad de Alzheimer , Demencia Vascular , Enfermedades Desmielinizantes , Imagen por Resonancia Magnética/métodos , Sulfoglicoesfingolípidos/líquido cefalorraquídeo , Sustancia Blanca/diagnóstico por imagen , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Cromatografía Liquida/métodos , Estudios Transversales , Demencia Vascular/líquido cefalorraquídeo , Demencia Vascular/diagnóstico , Demencia Vascular/fisiopatología , Enfermedades Desmielinizantes/líquido cefalorraquídeo , Enfermedades Desmielinizantes/diagnóstico , Diagnóstico Diferencial , Técnicas de Diagnóstico Neurológico , Femenino , Humanos , Masculino , Proteínas de Neurofilamentos/líquido cefalorraquídeo , Utilización de Procedimientos y Técnicas , Reproducibilidad de los Resultados , Sustancia Blanca/patologíaRESUMEN
Lysosomal storage diseases, including mucopolysaccharidoses, result from genetic defects that impair lysosomal catabolism. Here, we describe two patients from two independent families presenting with progressive psychomotor regression, delayed myelination, brain atrophy, neutropenia, skeletal abnormalities, and mucopolysaccharidosis-like dysmorphic features. Both patients were homozygous for the same intronic variant in VPS16, a gene encoding a subunit of the HOPS and CORVET complexes. The variant impaired normal mRNA splicing and led to an ~85% reduction in VPS16 protein levels in patient-derived fibroblasts. Levels of other HOPS/CORVET subunits, including VPS33A, were similarly reduced, but restored upon re-expression of VPS16. Patient-derived fibroblasts showed defects in the uptake and endosomal trafficking of transferrin as well as accumulation of autophagosomes and lysosomal compartments. Re-expression of VPS16 rescued the cellular phenotypes. Zebrafish with disrupted vps16 expression showed impaired development, reduced myelination, and a similar accumulation of lysosomes and autophagosomes in the brain, particularly in glia cells. This disorder resembles previously reported patients with mutations in VPS33A, thus expanding the family of mucopolysaccharidosis-like diseases that result from mutations in HOPS/CORVET subunits.
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Mucopolisacaridosis , Pez Cebra , Animales , Endosomas , Humanos , Lisosomas , Proteínas de Transporte Vesicular/genéticaRESUMEN
ß-Mannosidosis is a lysosomal storage disorder characterized by accumulation of disaccharides due to deficiency of the lysosomal enzyme ß-mannosidase. The disease is caused by mutations in MANBA and is extremely rare in humans. Although the clinical presentation is heterogeneous, common symptoms include various degrees of developmental delay, behavioral disturbances, hearing loss, and frequent infections. We report a 15-yr-old girl presenting with mild intellectual disability, sensorineural hearing loss, severe behavioral disturbances, dysmorphic traits, and evolving angiokeratomas. Copy-number variation analysis of next-generation sequencing (NGS) data indicated increased coverage in exons 8-11 of MANBA Low ß-mannosidase activity (1 µkatal/kg protein, refv 25-40) established the diagnosis of ß-mannosidosis. Whole-genome sequencing (WGS) and cDNA analysis revealed a novel homozygous intragenic inverted duplication in MANBA, where a 13.1-kb region between introns 7 and 11 was duplicated and inserted in an inverted orientation, creating a 67-base nonduplicated gap at the insertion point. Both junctions showed microhomology regions. The inverted duplication resulted in exon skipping of exons 8-9 or 8-10. Our report highlights the importance of copy-number variation analysis of data from NGS and in particular the power of WGS in the identification and characterization of copy-number variants.
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Angioqueratoma/genética , Variaciones en el Número de Copia de ADN , Manosidasas/genética , beta-Manosidosis/genética , Adolescente , Angioqueratoma/diagnóstico , Angioqueratoma/patología , ADN Complementario/genética , Exones/genética , Femenino , Duplicación de Gen , Pérdida Auditiva/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Discapacidad Intelectual/genética , Mutación , Fenotipo , Análisis de Secuencia de ADN , Secuenciación Completa del Genoma , beta-Manosidosis/diagnóstico , beta-Manosidosis/patologíaRESUMEN
The pathological cascade leading from primary storage to neural cell dysfunction and death in metachromatic leukodystrophy (MLD) has been poorly elucidated in human-derived neural cell systems. In the present study, we have modeled the progression of pathological events during the differentiation of patient-specific iPSCs to neuroepithelial progenitor cells (iPSC-NPCs) and mature neurons, astrocytes, and oligodendrocytes at the morphological, molecular, and biochemical level. We showed significant sulfatide accumulation and altered sulfatide composition during the differentiation of MLD iPSC-NPCs into neuronal and glial cells. Changes in sulfatide levels and composition were accompanied by the expansion of the lysosomal compartment, oxidative stress, and apoptosis. The neuronal and glial differentiation capacity of MLD iPSC-NPCs was significantly impaired. We showed delayed appearance and/or reduced levels of oligodendroglial and astroglial markers as well as reduced number of neurons and disorganized neuronal network. Restoration of a functional Arylsulfatase A (ARSA) enzyme in MLD cells using lentiviral-mediated gene transfer normalized sulfatide levels and composition, globally rescuing the pathological phenotype. Our study points to MLD iPSC-derived neural progeny as a useful in vitro model to assess the impact of ARSA deficiency along NPC differentiation into neurons and glial cells. In addition, iPSC-derived neural cultures allowed testing the impact of ARSA reconstitution/overexpression on disease correction and, importantly, on the biology and functional features of human NPCs, with important therapeutic implications.
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Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Leucodistrofia Metacromática/patología , Modelos Biológicos , Células-Madre Neurales/patología , Neuroglía/patología , Neuronas/patología , Apoptosis , Glicoesfingolípidos/biosíntesis , Humanos , Lisosomas/metabolismo , Degeneración Nerviosa/patología , Células-Madre Neurales/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Sulfoglicoesfingolípidos/metabolismoRESUMEN
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Several biomarkers including proteins and lipids have been reported in MS cerebrospinal fluid (CSF), reflecting different aspects of the pathophysiology particularly of relapsing-remitting MS (RRMS). Sulfatide, abundant in the myelin sheath and a proposed target for autoimmune attack in MS, has been reported altered in MS CSF. Here, we investigated the potential of CSF sulfatide and its isoforms as biomarkers in MS. A highly sensitive and quantitative mass spectrometry method was employed to determine levels of sulfatide isoforms in CSF from RRMS and progressive MS (PMS) patients, and healthy donors (HD). We demonstrate that levels of total CSF sulfatide and C24:1, C26:1, and C26:1-OH isoforms were significantly increased in PMS compared with RRMS patients and HD, while C23:0-OH was significantly decreased in CSF from PMS patients compared to the other two groups. Multivariate discriminant analysis showed that CSF sulfatide isoform pattern in PMS patients was distinct and non-overlapping with that of RRMS patients and HD. Sulfatide levels did not correlate with tested biomarkers or clinical parameters. The results suggest that CSF sulfatide isoform levels may be used to discriminate the phenotype of MS and might play a role in the progression of the disease.
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Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/diagnóstico , Sulfoglicoesfingolípidos/líquido cefalorraquídeo , Adulto , Estudios de Casos y Controles , Citocinas/líquido cefalorraquídeo , Progresión de la Enfermedad , Femenino , Proteína Ácida Fibrilar de la Glía/líquido cefalorraquídeo , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Proteínas de Neurofilamentos/líquido cefalorraquídeo , Análisis de Componente Principal , Isoformas de Proteínas/líquido cefalorraquídeo , Curva ROC , Sulfoglicoesfingolípidos/química , Linfocitos T/metabolismo , Adulto JovenRESUMEN
BACKGROUND: The peroxisome biogenesis disorders, which are caused by mutations in any of 13 different PEX genes, include the Zellweger spectrum disorders. Severe defects in one of these PEX genes result in the absence of functional peroxisomes which is seen in classical Zellweger syndrome. These patients present with hypotonia and seizures shortly after birth. Other typical symptoms are dysmorphic features, liver disease, retinal degeneration, sensorineural deafness, polycystic kidneys, and the patient does not reach any developmental milestones. CASE PRESENTATION: We report a case of a patient with Zellweger spectrum disorder due to a novel mutation in the PEX10 gene, presenting with a mild late-onset neurological phenotype. The patient, an Assyrian girl originating from Iraq, presented with sensorineural hearing impairment at the age of 5 followed by sensorimotor polyneuropathy, cognitive delay, impaired gross and fine motor skills, and tremor and muscle weakness in her teens. Analyses of biochemical markers for peroxisomal disease suggested a mild peroxisomal defect and functional studies in fibroblasts confirmed the existence of a peroxisome biogenesis disorder. Diagnosis was confirmed by next generation sequencing analysis, which showed a novel homozygous mutation (c.530 T > G (p.Leu177Arg) (NM_153818.1)) in the PEX10 gene predicted to be pathogenic. CONCLUSIONS: This case highlights the importance of performing biochemical, functional, and genetic peroxisomal screening in patients with clinical presentations milder than those usually observed in Zellweger spectrum disorders.
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Mutación Missense/genética , Peroxinas/genética , Receptores Citoplasmáticos y Nucleares/genética , Síndrome de Zellweger/diagnóstico , Adolescente , Femenino , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Linaje , Síndrome de Zellweger/genética , Síndrome de Zellweger/fisiopatologíaRESUMEN
Sulfatides (STs) are a group of glycosphingolipids that are highly expressed in brain. Due to their importance for normal brain function and their potential involvement in neurological diseases, development of accurate and sensitive methods for their determination is needed. Here we describe a high-throughput oriented and quantitative method for the determination of STs in cerebrospinal fluid (CSF). The STs were extracted using a fully automated liquid/liquid extraction method and quantified using ultra-performance liquid chromatography coupled to tandem mass spectrometry. With the high sensitivity of the developed method, quantification of 20 ST species from only 100 µl of CSF was performed. Validation of the method showed that the STs were extracted with high recovery (90%) and could be determined with low inter- and intra-day variation. Our method was applied to a patient cohort of subjects with an Alzheimer's disease biomarker profile. Although the total ST levels were unaltered compared with an age-matched control group, we show that the ratio of hydroxylated/nonhydroxylated STs was increased in the patient cohort. In conclusion, we believe that the fast, sensitive, and accurate method described in this study is a powerful new tool for the determination of STs in clinical as well as preclinical settings.
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Fraccionamiento Químico/métodos , Cromatografía Líquida de Alta Presión/métodos , Pruebas de Química Clínica/métodos , Sulfoglicoesfingolípidos/líquido cefalorraquídeo , Sulfoglicoesfingolípidos/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos , Adulto , Anciano , Automatización , Humanos , Persona de Mediana EdadRESUMEN
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.
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Intrones/genética , Transportadores de Anión Orgánico/genética , Enfermedad por Almacenamiento de Ácido Siálico/genética , Simportadores/genética , Elementos Transponibles de ADN/genética , Exones/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Masculino , Ácido N-Acetilneuramínico/metabolismo , Reacción en Cadena de la Polimerasa , Piel/citología , Secuenciación del Exoma/métodosRESUMEN
BACKGROUND: With the advent new sequencing technologies, we now have the tools to understand the phenotypic diversity and the common occurrence of phenocopies. We used these techniques to investigate two Norwegian families with an autosomal recessive cerebellar ataxia with cataracts and mental retardation. METHODS AND RESULTS: Single nucleotide polymorphism (SNP) chip analysis followed by Exome sequencing identified a 2 bp homozygous deletion in GBA2 in both families, c.1528_1529del [p.Met510Valfs*17]. Furthermore, we report the biochemical characterization of GBA2 in these patients. Our studies show that a reduced activity of GBA2 is sufficient to elevate the levels of glucosylceramide to similar levels as seen in Gaucher disease. Furthermore, leucocytes seem to be the proper enzyme source for in vitro analysis of GBA2 activity. CONCLUSIONS: We report GBA2 mutations causing a Marinesco-Sjögren-like syndrome in two Norwegian families. One of the families was originally diagnosed with Marinesco-Sjögren syndrome based on an autosomal recessive cerebellar ataxia with cataracts and mental retardation. Our findings highlight the phenotypic variability associated with GBA2 mutations, and suggest that patients with Marinesco-Sjögren-like syndromes should be tested for mutations in this gene.
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Mutación/genética , Degeneraciones Espinocerebelosas/genética , beta-Glucosidasa/genética , Anciano , Niño , Preescolar , Femenino , Glucosilceramidasa , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , LinajeRESUMEN
Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells.
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Glicoesfingolípidos Acídicos/metabolismo , Diferenciación Celular , Gangliósidos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Glicoesfingolípidos Acídicos/química , Glicoesfingolípidos Acídicos/inmunología , Biomarcadores/metabolismo , Secuencia de Carbohidratos , Línea Celular , Regulación hacia Abajo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epítopos/inmunología , Citometría de Flujo , Gangliósidos/química , Gangliósidos/inmunología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Espectrometría de MasasAsunto(s)
1-Desoxinojirimicina/análogos & derivados , Inhibidores Enzimáticos/uso terapéutico , Enfermedad de Niemann-Pick Tipo C/diagnóstico , Trastornos Psicóticos/etiología , 1-Desoxinojirimicina/uso terapéutico , Adulto , Proteínas Portadoras/genética , Diagnóstico Tardío , Fibroblastos/metabolismo , Filipina/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Glicoproteínas de Membrana/genética , Enfermedades del Sistema Nervioso/etiología , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/complicaciones , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedades Raras/complicaciones , Enfermedades Raras/diagnóstico , Enfermedades Raras/tratamiento farmacológico , Coloración y Etiquetado/métodos , Resultado del TratamientoRESUMEN
Natural killer T (NKT) lymphocytes are implicated in the early response to microbial infection. Further, sulfatide, a myelin self-glycosphingolipid, activates a type II NKT cell subset and can modulate disease in murine models. We examined the role of NKT cells and the effect of sulfatide treatment in a murine model of Staphylococcus aureus sepsis. The lack of CD1d-restricted NKT cells did not alter survival after a lethal inoculum of S. aureus. In contrast, sulfatide treatment significantly improved the survival rate of mice with S. aureus sepsis, accompanied by decreased levels of tumor necrosis factor alpha and interleukin-6 in the blood. The protective effect of sulfatide treatment depended on CD1d but not on type I NKT cells, suggesting that activation of type II NKT cells by sulfatide has beneficial effects on the outcome of S. aureus sepsis in this model.
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Antígenos CD1d/metabolismo , Factores Inmunológicos/administración & dosificación , Células Asesinas Naturales/inmunología , Sepsis/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/inmunología , Sulfoglicoesfingolípidos/administración & dosificación , Animales , Femenino , Interleucina-6/sangre , Ratones , Ratones Endogámicos C57BL , Sepsis/inmunología , Sepsis/microbiología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidad , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/sangreRESUMEN
BACKGROUND: Chlamydia psittaci is an intracellular bacterium primarily causing respiratory diseases in birds but may also be transmitted to other animals, including humans. The prevalence of the pathogen in wild birds in Sweden is largely unknown. METHODS: DNA was extracted from cloacae swabs and screened for C. psittaci by using a 23S rRNA gene PCR assay. Partial 16S rRNA and ompA gene fragments were sequence determined and phylogenies were analysed by the neighbour-joining method. RESULTS AND CONCLUSION: The C. psittaci prevalence was 1.3% in 319 Peregrine Falcons and White-tailed Sea Eagles, vulnerable top-predators in Sweden. 16S rRNA and ompA gene analysis showed that novel Chlamydia species, as well as novel C. psittaci strains, are to be found among wild birds.
RESUMEN
Niemann-Pick type C (NPC) is a progressive neurodegenerative lysosomal disease with altered cellular lipid trafficking. The metabolism of amyloid-ß (Aß) - previously mainly studied in Alzheimer's disease - has been suggested to be altered in NPC. Here we aimed to perform a detailed characterization of metabolic products from the amyloid precursor protein (APP) in NPC models and patients. We used multiple analytical technologies, including immunoassays and immunoprecipitation followed by mass spectrometry (IP-MS) to characterize Aß peptides and soluble APP fragments (sAPP-α/ß) in cell media from pharmacologically (U18666A) and genetically (NPC1 ( -/- ) ) induced NPC cell models, and cerebrospinal fluid (CSF) from NPC cats and human patients. The pattern of Aß peptides and sAPP-α/ß fragments in cell media was differently affected by NPC-phenotype induced by U18666A treatment and by NPC1 ( -/- ) genotype. U18666A treatment increased the secreted media levels of sAPP-α, AßX-40 and AßX-42 and reduced the levels of sAPP-ß, Aß1-40 and Aß1-42, while IP-MS showed increased relative levels of Aß5-38 and Aß5-40 in response to treatment. NPC1 ( -/- ) cells had reduced media levels of sAPP-α and Aß1-16, and increased levels of sAPP-ß. NPC cats had altered CSF distribution of Aß peptides compared with normal cats. Cats treated with the potential disease-modifying compound 2-hydroxypropyl-ß-cyclodextrin had increased relative levels of short Aß peptides including Aß1-16 compared with untreated cats. NPC patients receiving ß-cyclodextrin had reduced levels over time of CSF Aß1-42, AßX-38, AßX-40, AßX-42 and sAPP-ß, as well as reduced levels of the axonal damage markers tau and phosphorylated tau. We conclude that NPC models have altered Aß metabolism, but with differences across experimental systems, suggesting that NPC1-loss of function, such as in NPC1 ( -/- ) cells, or NPC1-dysfunction, seen in NPC patients and cats as well as in U18666A-treated cells, may cause subtle but different effects on APP degradation pathways. The preliminary findings from NPC cats suggest that treatment with cyclodextrin may have an impact on APP processing pathways. CSF Aß, sAPP and tau biomarkers were dynamically altered over time in human NPC patients.