Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice.

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.

Brain cell type-specific endocytosis of arylsulfatase A identifies limitations of enzyme-based therapies for metachromatic leukodystrophy.

Enzyme replacement therapies, allogeneic bone marrow transplantation and gene therapies are treatment options for lysosomal storage diseases caused by inherited deficiencies of soluble lysosomal enzymes. Independent from the approach, the enzyme must be delivered to lysosomes of deficient patient cells. Little is known about the dissemination of enzyme within a tissue where cells compete for uptake via different receptor systems, binding affinities and endocytic rates. To evaluate dissemination and lysosomal targeting of a lysosomal enzyme in the CNS, we analysed receptor-mediated endocytosis of arylsulfatase A (ASA) by different types of brain-derived cell lines and primary murine brain cells. For ASA expressed by chinese hamster ovary cells for enzyme replacement therapy of metachromatic leukodystrophy, endocytic rates decline from microglia to neurons and astrocytes and to oligodendrocytes. Only immature oligodendrocytes endocytose significant amounts of enzyme. Uptake by non-microglial cells is due to mannose 6-phosphate receptors, whereas several receptor systems participate in endocytosis by microglial cells. Interestingly, ASA expressed by microglial cells cannot be taken up in a mannose 6-phosphate dependent manner. The resulting failure to correct non-microglial cells corroborates in vivo data and indicates that therapeutic effects of allogeneic bone marrow transplantation and hematopoietic stem cell gene therapy on metachromatic leukodystrophy are independent of metabolic cross-correction of neurons, astrocytes and oligodendrocytes by receptor-mediated endocytosis.

Optimization of Enzyme Essays to Enhance Reliability of Activity Measurements in Leukocyte Lysates for the Diagnosis of Metachromatic Leukodystrophy and Gangliosidoses.

(1) Lysosomal storage diseases are rare inherited disorders with no standardized or commercially available tests for biochemical diagnosis. We present factors influencing the quality of enzyme assays for metachromatic leukodystrophy (MLD) and gangliosidoses (GM1; GM2 variants B and 0) and validate the reliability and stability of testing in a retrospective analysis of 725 samples. (2) Patient leukocytes were isolated from ethylene-diamine-tetra-acetic acid (EDTA) blood and separated for subpopulation experiments using density gradient centrifugation or magnetic cell separation. Enzyme activities in whole leukocyte lysate and leukocyte subpopulations were determined. (3) The enzyme activities in leukocyte subpopulations differed significantly. Compared to lymphocytes, the respective enzyme activities were 2.31-4.57-fold higher in monocytes and 1.64-2.81-fold higher in granulocytes. During sample preparation, a considerable amount of the lysosomal enzymes was released from granulocytes. Nevertheless, with the sample preparation method used here, total leukocyte count proved to be more accurate than total protein amount as a reference unit for enzyme activities. Subsequent analysis of 725 individuals showed clear discrimination of enzyme activities in patient samples (48 MLD; 21 gangliosidoses), with a sensitivity of 100% and specificity of 98-99%.

Synthesis and structure-activity relationships of cerebroside analogues as substrates of cerebroside sulphotransferase and discovery of a competitive inhibitor.

Metachromatic leukodystrophy (MLD) is a rare genetic disease characterised by a dysfunction of the enzyme arylsulphatase A leading to the lysosomal accumulation of cerebroside sulphate (sulphatide) causing subsequent demyelination in patients. The enzyme galactosylceramide (cerebroside) sulphotransferase (CST) catalyses the transfer of a sulphate group from 3'-phosphoadenosine-5'-phosphosulphate (PAPS) to cerebrosides producing sulphatides. Substrate reduction therapy for arylsulphatase A by inhibition of CST was proposed as a promising therapeutic approach. To identify competitive CST inhibitors, we synthesised and investigated analogues of the substrate galactosylceramide with variations at the anomeric position, the acyl substituent and the carbohydrate moiety, and investigated their structure-activity relationships. While most of the compounds behaved as substrates, α-galactosylceramide 16 was identified as the first competitive CST inhibitor. Compound 16 can serve as a new lead structure for the development of drugs for the treatment of this devastating disease, MLD, for which small molecule therapeutics are currently not available.

Leukocyte and Dried Blood Spot Arylsulfatase A Assay by Tandem Mass Spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.

Gene-Based Approaches to Inherited Neurometabolic Diseases.

In the last decade, the gene therapy (GT) field experienced a renaissance, thanks to crucial understandings and innovations in vector design, stem cell manipulation, conditioning protocols, and cell/vector delivery. These efforts were successfully coupled with unprecedented clinical results of the trials employing the newly developed technology and with the novel establishment of academic-industrial partnerships. A renewed and strengthened interest is rising in the development of gene-based approaches for inherited neurometabolic disorders with severe neurological involvement. Inherited metabolic disorders are monogenetic diseases caused by enzymatic or structural deficiencies affecting the lysosomal or peroxisomal metabolic activity. The metabolic defect can primarily affect the central nervous system, leading to neuronal death, microglial activation, inflammatory demyelination, and axonal degeneration. This review provides an overview of the GT strategies currently under clinical investigation for neurometabolic lysosomal and peroxisomal storage diseases, such as adrenoleukodystrophy and metachromatic leukodystrophy, as well as novel emerging indications such as mucopolysaccharidoses, gangliosidoses, and neuronal ceroid lipofuscinoses, with a comprehensive elucidation of the main features and mechanisms at the basis of a successful GT approach for these devastating diseases.

Measurement of recombinant human arylsulfatase A and leukocyte sulfatase activities by analytical isotachophoresis.

Metachromatic Leukodystrophy (MLD) and Multiple Sulfatase Deficiency (MSD) are rare and ultra-rare lysosomal storage diseases. Due to enzyme defects, patients are unable to split the sulfategroup from the respective substrates. In MSD all sulfatases are affected due to a defect of the Sulfatase Modifying Factor 1 (SUMF1) gene coding for the formylglycine generating enzyme (FGE) necessary for the modification of the active site of sulfatases. In MLD mutations in the arylsulfatase A (ARSA) gene cause ARSA deficiency with subsequent accumulation of 3-sulfogalactocerebroside especially in oligodendrocytes. The clinical consequence is demyelination and a devastating neurological disease. Enzyme replacement therapy (ERT) with recombinant human arylsulfatase A (rhARSA), gene therapy, and stem cell transplantation are suggested as new therapeutic options. The aim of our study was to characterize rhARSA concerning its substrate specificity using analytical isotachophoresis (ITP). Substrate specificity could be demonstrated by sulfate splitting from the natural substrates 3-sulfogalactocerebroside and ascorbyl-2-sulfate and the artificial substrate p-nitrocatecholsulfate, whereas galactose-6-sulfate, a substrate of galactose-6­sulfurylase, was totally resistant. In contrast, leukocyte extracts of healthy donors were able to split sulfate also from galactose-6-sulfate. The ITP method allows therefore a rapid and simple differentiation between samples of MLD and MSD patients and healthy donors. Therefore, the isotachophoretic diagnostic assay from leukocyte extracts described here provides a fast and efficient way for the diagnosis of MLD and MSD patients and an elegant system to differentiate between these diseases in one assay.

Enzymatic characterization of novel arylsulfatase A variants using human arylsulfatase A-deficient immortalized mesenchymal stromal cells.

Metachromatic leukodystrophy (MLD) is an autosomal-recessive lysosomal storage disease caused by mutations in the ARSA gene leading to arylsulfatase A (ARSA) deficiency and causing sulfatide accumulation. Main symptoms of the disease are progressive demyelination, neurological dysfunction, and reduced life expectancy. To date, more than 200 different ARSA variants have been reported in MLD patients. Here, we report the biochemical characterization of seven novel pathogenic variants (c.98T > C, c.195delC, c.229G > C, c.545C > G, c.674A > G, c.852T > A, and c.1274A > G), which were found when sequencing a cohort of 31 German MLD families. For that purpose, the ARSA cDNAs carrying the respective mutations inserted by site-directed mutagenesis were cloned into a MigR1 (MSCV, IRES, GFP, retrovirus-1) vector. The constructs were overexpressed using retroviral gene transfer in immortalized, human multipotent mesenchymal stromal cells prepared from a patient deficient in ARSA activity (late infantile MLD). In this novel ARSA-/- cell system, the seven ARSA mutants showed ARSA activity of less than 10% when compared with wild type, which is evidence for the pathogenicity of all seven variants. In conclusion, the system of ARSA-/- -immortalized MSC turned out to be a helpful novel tool for the biochemical characterization of ARSA variants.

Generation of Human Induced Pluripotent Stem Cell-Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy.

Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells Translational Medicine 2017;6:352-368.

Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases.

Enzyme replacement in the CSF to treat metachromatic leukodystrophy in mouse model using single intracerebroventricular injection of self-complementary AAV1 vector.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a functional deficiency in human arylsulfatase A (hASA). We recently reported that ependymal cells and the choroid plexus are selectively transduced by intracerebroventricular (ICV) injection of adeno-associated virus serotype 1 (AAV1) vector and serve as a biological reservoir for the secretion of lysosomal enzymes into the cerebrospinal fluid (CSF). In the present study, we examined the feasibility of this AAV-mediated gene therapy to treat MLD model mice. Preliminary experiments showed that the hASA level in the CSF after ICV injection of self-complementary (sc) AAV1 was much higher than in mice injected with single-stranded AAV1 or scAAV9. However, when 18-week-old MLD mice were treated with ICV injection of scAAV1, the concentration of hASA in the CSF gradually decreased and was not detectable at 12 weeks after injection, probably due to the development of anti-hASA antibodies. As a result, the sulfatide levels in brain tissues of treated MLD mice were only slightly reduced compared with those of untreated MLD mice. These results suggest that this approach is potentially promising for treating MLD, but that controlling the immune response appears to be crucial for long-term expression of therapeutic proteins in the CSF.

A 5-year-old male child with late infantile metachromatic leukodystrophy: a case report.

Metachromatic leukodystrophy is a rare disorder of myelin metabolism. This degenerative disorder results from the accumulation of cerebroside sulfatide within the myelin sheath of central and peripheral nervous system, due to deficiency of aryl sulfatase A enzyme. We report a 5-year-old male child, who presented with regression of milestones, recurrent seizures and spasticity from second year of life. Initially neurodegenerative disorder was considered and the case was investigated with neuroimaging and enzyme levels. Computed tomography (CT) of the brain showed hypodensities in the corpus callosum and bilateral periventricular and deep cerebral white matter suggestive of neurodegenerative disorder. Subsequently, magnetic resonance imaging (MRI) of the brain was done, which showed symmetrical hyperintensities in the periventricular white matter with classical sparing of subcortical "U" fibers. The ß-galactosidase enzyme activity was normal; however, the activity of aryl sulfatase A enzyme was undetectable, confirming the diagnosis of late infantile variant of metachromatic leukodystrophy.

[Analysis of phenotype and genotype in a family with late infantile metachromatic leukodystrophy].

OBJECTIVE: To study genotype-phenotype correlation of a family with late infantile metachromatic leukodystrophy(MLD). METHODS: Clinical data were collected and ARSA gene was tested by PCR and sequencing in a pedigree. RESULTS: The male proband onset with walking dysfunction at 19 months, arylsulfatase A activity of leucocyte from his peripheral blood was 20.2 nmol/mg.17h, and his cranial MRI showed wildly symmetrical demyelination. Homozygosis for novel c.622delC (p.His208Metfs46X) in exon 3 of ARSA gene was identified in proband, and heterozygous for the same mutation in parents and grandma of the proband. CONCLUSION: Late infantile metachromatic leukodystrophy is characterized by rapid and progressive regression of neuropsychiatric and motor development. There is a significant correlation between the mutation of c.622delC(p.His208Metfs*46) in the ARSA gene and the phenotype presenting as O/O patients.

A new analytical bench assay for the determination of arylsulfatase a activity toward galactosyl-3-sulfate ceramide: implication for metachromatic leukodystrophy diagnosis.

Here, we present the design and validation of a new assay for the diagnosis of metachromatic leukodystrophy. The method is highly specific, simple, reproducible, and straightforward. In our spectrophotometric method, the determination of arylsulfatase A (ARSA) activity toward the natural substrate, galactosyl-3-sulfate ceramide (or sulfatide), is performed using neat sulfatide without chemical modification. This confers to the assay high analytical specificity. The hydrolyzed sulfatide is monitored upon inclusion of the colorimetric reagent Azure A. The nonhydrolyzed sulfatide-Azure A is recovered and measured at a wavelength of λ = 650 nm. Thus, ARSA activity toward the sulfatide is obtained by subtracting the nonhydrolyzed sulfatide from the total sulfatide used in the enzyme reaction (sulfatide-Azure A present in a parallel assay performed in the absence of ARSA). Within a clinical context, our method definitely discriminated between healthy subject samples and metachromatic leukodystrophy patient samples, and, therefore, it is suitable for diagnostic applications and for monitoring the efficacy of therapeutic treatments in patients or animal models.

Lysosomal leukodystrophies: Krabbe disease and metachromatic leukodystrophy.

Genetic deficiencies of lysosomal catabolic pathways lead to storage disorders with multiple organ abnormalities or to degeneration of purely nervous structures. Krabbe disease and metachromatic leukodystrophy are caused by metabolic errors concerning lipids of neural membranes. They are characterized by demyelination of the central nervous system and, variably, the peripheral nerves. Their clinical presentation is a relentlessly progressive motor and mental deterioration starting at any age between infancy and adolescence. MRI demonstrates characteristic lesions of brain white matter. In Krabbe disease, deficient galactocerebroside ß-galactosidase activity causes accumulation of lipids in "globoid" macrophages and of psychosine, which is toxic to oligodendrocytes. Diagnosis depends on demonstration of the enzyme deficiency. Experimental treatment is limited to hematopoietic stem cell transplantation, which can favorably alter the course of disease in certain situations. In metachromatic leukodystrophy, deficient activity of arylsulfatase A, or lack of a cofactor, causes accumulation of sulfatide in various tissues and diffuse demyelination. Symptoms are neurological, but gallbladder dysfunction may be present. Diagnosis depends on demonstrating the enzyme deficiency and elevated urinary sulfatide. In a rare variant, multiple sulfatases are deficient. Stem cell transplantation may prevent disease progression in selected cases. Enzyme replacement is being evaluated, and gene therapies are being developed.

Correction of brain oligodendrocytes by AAVrh.10 intracerebral gene therapy in metachromatic leukodystrophy mice.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in glial cells and neurons, the result of an inherited deficiency of arylsulfatase A (ARSA; EC 3.1.6.8) and myelin degeneration in the central and peripheral nervous systems. No effective treatment is currently available for the most frequent late infantile (LI) form of MLD, which results in rapid neurological degradation and early death after the onset of clinical manifestations. To potentially arrest or reverse disease progression, ARSA enzyme must be rapidly delivered to brain oligodendrocytes of patients with LI MLD. We previously showed that brain gene therapy with adeno-associated virus serotype 5 (AAV5) driving the expression of human ARSA cDNA under the control of the murine phosphoglycerate kinase (PGK) promoter alleviated most long-term disease manifestations in MLD mice. Herein, we evaluated the short-term effects of AAVrh.10 driving the expression of human ARSA cDNA under the control of the cytomegalovirus/ß-actin hybrid (CAG/cu) promoter in 8-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 2 months, and in contrast to results with the AAV5-PGK-ARSA vector, a single intrastriatal injection of AAVrh.10cuARSA resulted in correction of brain sulfatide storage, accumulation of specific sulfatide species in oligodendrocytes, and associated brain pathology in the injected hemisphere. Better potency of the AAVrh.10cuARSA vector was mediated by higher neuronal and oligodendrocyte transduction, axonal transport of the AAVrh.10 vector and ARSA enzyme, as well as higher CAG/cu promoter driven expression of ARSA enzyme. These results strongly support the use of AAVrh.10cuARSA vector for intracerebral gene therapy in rapidly progressing early-onset forms of MLD.

Brain MRI and biological diagnosis in five Tunisians MLD patients.

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is characterized by an accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). we studied 5/200 cases of MLD and clearly distinguished three clinical forms. One of them presented the juvenile form; two presented the late infantile form; and two other presented the adult form. The Magnetic Resonance Imaging (MRI) of these patients showed a diffuse, bilateral and symmetrical demyelination. The biochemical diagnosis of MLD patients evidencing the low activity of ASA and sulfatide accumulation. PATIENTS AND METHODS: We studied 5/200 MLD patients addressed to us for behavioral abnormalities and progressive mental deterioration. All of them were diagnosed at first by brain MRI evidencing a bilateral demyelination, then the measurement of ASA activity using P-nitrocathecol sulfate as substrate, finally the sulfatiduria was performed using thin-layer chromatography using alpha-naphtol reagent. RESULTS: In this study, from 200 patients presenting behavioral abnormalities and a progressive mental deterioration, we reported just 2 patients were diagnosed as late-infantile form of MLD. Only1 case presented as the juvenile form; and 2 patients with the adult-type of MLD. The brain magnetic resonance imaging (MRI) of all patients showed characteristic lesions of MLD with extensive demyelination. Biochemical investigations of these patients detected a low level of ASA activity at 0°C and 37°C; the excess of sulfatide in sulfatiduria. CONCLUSION: MRI is required to orient the diagnosis of MLD patients; the latter must be confirmed by the biochemical investigations which is based on the measurement of ASA activity and the excess of sulfatide showed in the sulfatiduria. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1791578262610232.

Developing treatment options for metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) represents a devastating lysosomal storage disease characterized by intralysosomal accumulation of the sphingolipid sulfatide in various tissues. Three types of the disease are currently distinguished: the late-infantile, which is the most commonly observed, the juvenile and the adult type. Demyelination represents the main histopathological feature of the disorder, leading to neurological impairment with no curative treatment currently available. Nevertheless, the increased scientific interest on the disease has led to the experimental use of innovative therapeutic approaches in animal models, aiming to provide an effective therapeutic regimen for human patients, as well. This paper provides an overview of developing treatment options among patients with MLD. Apart from hematopoietic stem cell transplantation, already in use for decades, other recent data discussed includes umbilical cord blood and stem cell transplantation, enzyme replacement therapy, gene therapy and autologous hematopoietic transplantation of genetically modified stem cells. Gene therapy with oligodedroglial, neural progenitor, embryonic and microencapsulated recombinant cells represents add-on treatment options still on experimental level.

[Diagnostic strategy of metachromatic leukodystrophy in Tunisia]. / La leucodystrophie métachromatique : stratégie diagnostique en Tunisie.

We recruited a 44-year-old woman who had a dementia with behavioral and personality troubles. A biochemical analysis which includes a qualitative study of urinary sulfatides by thin layer chromatography followed by the determination of the enzymatic activity of arylsulfatase A (ARSA) was performed. The Molecular analysis concerned the research of the most frequent mutations (459 +1 G> A, p.P426L, p.I179S). The profile that has revealed the presence of 3-O-sulfogalactosylceramide fraction was in favor of metachromatic leukodystrophy. The activity of arylsulfatase A was collapsed in the index case which confirmed the phenotype of the adult form of the diagnosed MLD. The molecular study showed the presence of the mutation p.I179S in the homozygous state in the index case.