Higher precision, first tier newborn screening for metachromatic leukodystrophy using 16:1-OH-sulfatide.

Newborn screening (NBS) for metachromatic leukodystrophy (MLD) is based on first-tier measurement of sulfatides in dried blood spots (DBS) followed by second-tier measurement of arylsulfatase A in the same DBS. This approach is very precise with 0-1 false positives per ∼30,000 newborns tested. Recent data reported here shows that the sulfatide molecular species with an α-hydroxyl, 16­carbon, mono-unsaturated fatty acyl group (16:1-OH-sulfatide) is superior to the original biomarker 16:0-sulfatide in reducing the number of first-tier false positives. This result is consistent across 4 MLD NBS centers. By measuring 16:1-OH-sulfatide alone or together with 16:0-sulfatide, the estimated false positive rate is 0.048% and is reduced essentially to zero with second-tier arylsulfatase A activity assay. The false negative rate is predicted to be extremely low based on the demonstration that 40 out of 40 newborn DBS from clinically-confirmed MLD patients are detected with these methods. The work shows that NBS for MLD is extremely precise and ready for deployment. Furthermore, it can be multiplexed with several other inborn errors of metabolism already tested in NBS centers worldwide.

Arylsulfatase A pseudodeficiency in Mexico: Enzymatic activity and haplotype analysis.

BACKGROUND: Metachromatic Leukodystrophy (MLD, OMIM 250100) is a neurodegenerative disease caused by mutations in the ARSA gene (OMIM 607574) that lead to deficiency in Arylsulfatase A (ASA). ASA pseudodeficiency (PD-ASA) is a biochemical condition that substantially diminishes ASA activity but is not associated with clinical manifestations. PD-ASA is associated with the c.1055A>G (p.Asn352Ser) (rs2071421) and c.*96A>G (rs6151429) variants, which have an estimated frequency of 2% in the population. OBJECTIVE: To determine the activity of Arylsulfatase A and to identify variants and haplotypes in the ARSA gene in Mexican individuals with pseudodeficiency. METHODS: Two-hundred apparently healthy individuals were included to determine the enzymatic activity of ASA in leukocytes by spectrophotometric analysis, and identification of the PD-ASA alleles was performed by PCR-RFLP assays. Genotypes were confirmed by semi-automated Sanger sequencing. Haplotypes were constructed using Arlequin v.10.04, and linkage disequilibrium analysis was performed with Cube X. RESULTS: The enzymatic activity of ASA was determined to be 1.74-2.09 nmol/mg protein/min and later correlated with genotypes and haplotypes. For the (p.Asn352Ser) variant, we found 126 (0.63) individuals with the AA genotype, 62 with AG (0.31) and 12 with GG (0.06); the frequency of the polymorphic allele was 0.215 (86 alleles, 21.5%), and the variant was in HWE (p = .2484). The variant c.*96A>G was also in HWE (p = .2105): 185 individuals (0.925) with the AA genotype, 14 (0.07) with AG, and 1 (0.005) with (GG), with a frequency of 0.04 (4%) for the polymorphic allele. The inference of haplotypes resulted in 312 (0.78) AA, 72 (0.18) GA, and 16 (0.04) GG haplotypes. The AG haplotype was not found. The variants were found to be in linkage disequilibrium (D' = 1). Of the nine possible diplotypes, AA/AG, AA/GG, and AG/GG were not found, in concordance with the hypothesis that the G allele of c.*96A>G does not occur in the absence of the G allele of c.1055A>G. We found a slight correlation between ASA biochemical activity and variants, mainly due to the G allele of c.*96A>G in either genotypes or haplotypes. CONCLUSIONS: In Northwestern Mexico, the presence of PD-ASA alleles was biochemically and molecularly determined, and the frequencies were found to be in HWE. The frequency of PD-ASA for the North Western Mexican mestizo is 8%.

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.

Quantification of plasma sulfatides by mass spectrometry: Utility for metachromatic leukodystrophy.

Impaired sulfatide catabolism is the primary biochemical insult in patients with the inherited neurodegenerative disease, metachromatic leukodystrophy (MLD), and sulfatide elevation in body fluids is useful in the diagnostic setting. Here we used mass spectrometry to quantify fourteen species of sulfatide, in addition to the deacetylated derivative, lyso-sulfatide, using high pressure liquid chromatography-electrospray ionisation-tandem mass spectrometry in both positive and negative ion mode. A single phase extraction of 0.01 mL of MLD plasma identified all 14 sulfatide species in the positive ion mode but none in the negative ion mode. Interrogation of seven major and seven hydroxylated molecular species, as well as lyso-sulfatide, identified the C18 isoform as the most informative for MLD. The C18 produced a linear response and was below the limit of quantification (<10 pmol mL-1) in control plasma with concentrations in MLD plasma ranging from 12 to 196 pmol mL-1. Serial plasma samples from an MLD patient post-therapeutic bone marrow transplant proved similar to non-disease controls with C18 sulfatide concentrations below the limit of quantification, as did samples from three individuals with an arylsulfatase A pseudodeficiency - a population variant which appears deficient upon enzymatic assay, without manifestation of disease. These findings emphasise the utility of the C18 sulfatide species for the diagnosis of MLD and biochemical monitoring of MLD patients. Extension of this approach to a newborn screening card correctly identified an MLD patient at birth with elevated C18 sulfatide at levels almost double that present in the newborn card from his unaffected sibling, suggesting the methodology may have applicability for newborn screening.

Clinical, biochemical and molecular characterization of prosaposin deficiency.

Prosaposin (PSAP) deficiency is an ultra-rare, fatal infantile lysosomal storage disorder (LSD) caused by variants in the PSAP gene, with seven subjects reported so far. Here, we provide the clinical, biochemical and molecular characterization of two additional PSAP deficiency cases. Lysoplex, a targeted resequencing approach was utilized to identify the variant in the first patient, while quantification of plasma lysosphingolipids (lysoSLs), assessed by liquid chromatography mass spectrometry (LC-MS/MS) and brain magnetic resonance imaging (MRI), followed by Sanger sequencing allowed to attain diagnosis in the second case. Functional studies were carried out on patients' fibroblast lines to explore the functional impact of variants. The two patients were homozygous for two different truncating PSAP mutations (c.895G>T, p.Glu299*; c.834_835delGA, p.Glu278Aspfs*27). Both variants led to a complete lack of processed transcript. LC-MS/MS and brain MRI analyses consistently provided a distinctive profile in the two children. Quantification of specific plasma lysoSLs revealed elevated levels of globotriaosylsphingosine (LysoGb3) and glucosylsphingosine (GlSph), and accumulation of autophagosomes, due to a decreased autophagic flux, was observed. This report documents the successful use of plasma lysoSLs profiling in the PSAP deficiency diagnosis, as a reliable and informative tool to obtain a preliminary information in infantile cases with complex traits displaying severe neurological signs and visceral involvement.

Sulfatide Analysis by Mass Spectrometry for Screening of Metachromatic Leukodystrophy in Dried Blood and Urine Samples.

BACKGROUND: Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by deficiency in arylsulfatase A activity, leading to accumulation of sulfatide substrates. Diagnostic and monitoring procedures include demonstration of reduced arylsulfatase A activity in peripheral blood leukocytes or detection of sulfatides in urine. However, the development of a screening test is challenging because of instability of the enzyme in dried blood spots (DBS), the widespread occurrence of pseudodeficiency alleles, and the lack of available urine samples from newborn screening programs. METHODS: We measured individual sulfatide profiles in DBS and dried urine spots (DUS) from MLD patients with LC-MS/MS to identify markers with the discriminatory power to differentiate affected individuals from controls. We also developed a method for converting all sulfatide molecular species into a single species, allowing quantification in positive-ion mode upon derivatization. RESULTS: In DBS from MLD patients, we found up to 23.2-fold and 5.1-fold differences in total sulfatide concentrations for early- and late-onset MLD, respectively, compared with controls and pseudodeficiencies. Corresponding DUS revealed up to 164-fold and 78-fold differences for early- and late-onset MLD patient samples compared with controls. The use of sulfatides converted to a single species simplified the analysis and increased detection sensitivity in positive-ion mode, providing a second option for sulfatide analysis. CONCLUSIONS: This study of sulfatides in DBS and DUS suggests the feasibility of the mass spectrometry method for newborn screening of MLD and sets the stage for a larger-scale newborn screening pilot study.

Quantification of sulfatides and lysosulfatides in tissues and body fluids by liquid chromatography-tandem mass spectrometry.

Sulfatides are found in brain as components of myelin, oligodendrocytes, and neurons but are also present in various visceral tissues. Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A, leading to severe white matter disease due to the accumulation of sulfatides and lysosulfatides. To study the physiological role of sulfatides, accessible and sensitive quantitative methods are required. We developed a sensitive LC/MS/MS method to quantify total sulfatide and lysosulfatide content as well as individual molecular species in urine and plasma from MLD patients and plasma and tissues from an MLD mouse model. Our results demonstrate that the method can quantify a wide range of sulfatide concentrations and can be used to quantify total sulfatide content and levels of individual molecular species of sulfatides in tissues, cells, and body fluids. Even though plasma sulfatides and lysosulfatides would seem attractive candidate biomarkers that could possibly correlate with the severity of MLD and be of use to monitor the effects of therapeutic intervention, our results indicate that it is unlikely that the determination of these storage products in plasma will be useful in this respect.

Hematopoietic SCT: a useful treatment for late metachromatic leukodystrophy.

In metachromatic leukodystrophy (MLD), the deficiency of the lysosomal enzyme arylsulfatase A (ARSA) leads to demyelination in the central and peripheral nervous system and ultimately to death. Allogeneic hematopoietic SCT (HSCT) is currently the only treatment for adult and late-onset juvenile MLD, although it is still in question because of insufficient follow-up. We wanted to determine whether HSCT could halt the progression of adult and late-onset juvenile MLD. Four treated unrelated patients and three untreated siblings were included in the study, and followed regularly for up to 18 years after transplantation. The patients were assessed from clinical examination, ARSA enzyme levels, magnetic resonance imaging of the brain and neuropsychological and neurophysiological tests. In the treated patients, ARSA levels were normal up to 18 years after transplantation. The parameters evaluated stabilized and remained stable after a latency period of 12-24 months. Two patients live normal lives, partially in a protected environment. The other two patients stabilized at a low cognitive and functional level. One of the controls is demented, one is in a vegetative state and one died. We conclude that, in comparison with their untreated siblings, HSCT halted the progression of the disease in our treated patients.

Leukodystrophies underlying cryptic spastic paraparesis: frequency and phenotype in 76 patients.

BACKGROUND AND PURPOSE: In chronic progressive spasticity of the legs many rare causes have to be considered, including leukodystrophies due to neurometabolic disorders. To determine the frequency of leukodystrophies and the phenotypic spectrum patients with cryptic spasticity of the legs were screened for underlying neurometabolic abnormalities. METHODS: Seventy-six index patients presenting with adult-onset lower limb spasticity of unknown cause consistent with autosomal recessive inheritance were included in this study. Screening included serum levels of very long chain fatty acids for X-linked adrenoleukodystrophy/adrenomyeloneuropathy and lysosomal enzyme activities in leukocytes for metachromatic leukodystrophy, GM1-gangliosidosis, Tay-Sachs, Sandhoff and Krabbe disease. If clinical evidence was indicative of other types of leukodystrophies, additional genetic testing was conducted. Clinical characterization included neurological and psychiatric features and magnetic resonance imaging. RESULTS: Basic screening detected one index patient with metachromatic leukodystrophy, two patients with Krabbe disease and four patients with adrenoleukodystrophy/adrenomyeloneuropathy. Additional genetic testing revealed one patient with vanishing white matter disease. These patients accounted for an overall share of 11% of leukodystrophies. One patient with Krabbe disease and three patients with adrenoleukodystrophy/adrenomyeloneuropathy presented with pure spasticity of the lower limbs, whilst one patient each with Krabbe disease, metachromatic leukodystrophy and adrenoleukodystrophy/adrenomyeloneuropathy showed additional complicating symptoms. CONCLUSIONS: Adult patients presenting with cryptic spasticity of the legs should be screened for underlying X-linked adrenoleukodystrophy/adrenomyeloneuropathy and lysosomal disorders, irrespective of the presence of additional complicating symptoms. Leukodystrophies may manifest as late as the sixth decade and hyperintensity of cerebral white matter on magnetic resonance FLAIR images is not obligatory.

Ultra-performance liquid chromatography/tandem mass spectrometry for determination of sulfatides in dried blood spots from patients with metachromatic leukodystrophy.

RATIONALE: Metachromatic leukodystrophy (MLD) is a genetic autosomal recessive disease caused by a deficiency in arylsulfatase A. Accumulated sulfatides can be detected in the urine and detection of sulfatiduria is a useful test for diagnosis and monitoring. To our knowledge, no studies have explored the accumulation of sulfatides in dried blood spots (DBSs). We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for measuring sulfatides in DBSs from patients with MLD. METHODS: DBSs were eluted with internal standard. After mixing and centrifugation, the organic layer was transferred to a 96-well microplate and dried, then resuspended in methanol/propanol solution. Samples were analyzed on an UPLC system. Total running time was 4 min. Quantification was achieved by multiple reaction monitoring using a tandem mass spectrometer. We evaluated the precision, linearity, and ion suppression of the method and analyzed sulfatide concentrations in DBS specimens from MLD patients (n = 9), pseudodeficiency (PD) patient (n = 1), obligate heterozygotes (OH) (n = 2) and normal controls (n = 124). RESULTS: In negative-ion mode, sulfatides species subjected to collision-induced dissociation readily fragment to produce an intense ion at m/z 96.8 (HSO4(-)). The precisions of low and high concentration controls ranged from 5.4 to 19.9%. The sulfatides produced linear responses. Molecular species of sulfatides were barely detected in DBSs from normal individuals and the PD-OH group [mean (range), 0.07 (<0.05-0.34) and 0.13 (<0.05-0.22) µg/mL, respectively]. In contrast, the DBSs from MLD patients showed a marked increase in several molecular species of sulfatide [mean (range), 2.02 (1.18-3.89) µg/mL]. CONCLUSIONS: Simultaneous detection for sulfatides using UPLC/MS/MS can be successfully applied to DBS analysis. This method provides a fast and effective screening and monitoring tool for the diagnosis and treatment of MLD.

Quantification of sulfatides in dried blood and urine spots from metachromatic leukodystrophy patients by liquid chromatography/electrospray tandem mass spectrometry.

BACKGROUND: Treatments are being developed for metachromatic leukodystrophy (MLD), suggesting the need for eventual newborn screening. Previous studies have shown that sulfatide molecular species are increased in the urine of MLD patients compared to samples from non-MLD individuals, but there is no data using dried blood spots (DBS), the most common sample available for newborn screening laboratories. METHODS: We used ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) to quantify sulfatides in DBS and dried urine spots from 14 MLD patients and 50 non-MLD individuals. RESULTS: Several sulfatide molecular species were increased in dried urine samples from all MLD samples compared to non-MLD samples. Sulfatides, especially low molecular species, were increased in DBS from MLD patients, but the sulfatide levels were relatively low. There was good separation in sulfatide levels between MLD and non-MLD samples when dried urine spots were used, but not with DBS, because DBS from non-MLD individuals have measurable levels of sulfatides. CONCLUSION: Sulfatide accumulation studies in urine, but not in DBS, emerges as the method of choice if newborn screening is to be proposed for MLD.

Reduced intensity conditioning haematopoietic stem cell transplantation with mesenchymal stromal cells infusion for the treatment of metachromatic leukodystrophy: a case report.

We report the case of a 23-year-old woman who presented with an adult form of metachromatic leukodystrophy (MLD) evolving over one year with a progressive neurological deterioration. A non-myeloablative matched related haematopoietic stem cell transplantation (HSCT) with concomitant mesenchymal stromal cells (MSCs) infusion was performed. Engraftment occurred rapidly with no significant toxicity or side effects following the MSC infusion. At a follow up of 40 months, the patient had a stabilisation of all neurological manifestations of her disease. This case report suggests the feasibility and the potential efficacy of reduced intensity conditioning (RIC) allogeneic HSCT combined with MSC infusion for patients with the adult form of MLD.

[Differentiation between arylsulfatase A deficiency and pseudo-deficiency]. / Dyferentsiatsiia spravzhn'oho defitsytu ta psevdodefitsytu arylsul'fatazy A.

Metachromatic leukodystrophy (MLD)--lysosomal storage disease caused arylsulfatase A (ARSA) deficiency. Biochemical diagnostic of MLD is complicated by arylsulfatase A pseudodeficiency. There is possibility of mistake in MLD diagnoses in case of pseudodeficiency ARSA and non-MLD neurological disease combination. We suggest the new modification of arylsulfatase A activity detection method which allows to identify the arylsulfatase A pseudodeficiency without molecular genetic methods.

MRI appearances of metachromatic leukodystrophy.

BACKGROUND: The leukodystrophies constitute a wide spectrum of cerebral disorders of varying etiology. The imaging appearances on CT and MRI are recognizable as abnormalities of white matter; however, it may be impossible to arrive at the correct diagnosis based on imaging studies alone. PATIENTS AND METHODS: Three patients of varying age and clinical symptomatology diagnosed with metachromatic leukodystrophy (MLD) had remarkably similar MRI appearances. A "tigroid" or "leopard-skin" appearance was demonstrated within deep white matter in each case. RESULTS: All of the patients had biochemical confirmation of MLD. CONCLUSION: Although the "tigroid" pattern previously was considered to be pathognomonic of Pelizaeus-Merzbacher disease, the diagnosis of MLD must now be considered when these MRI appearances are encountered.

Arylsulfatase A (ASA) defect and psychiatric illness. A review.

The detection of homozygous (disease state) and heterozygous (carrier) forms of metachromatic leukodystrophy (MLD) and their prevalence among psychiatric individuals are reviewed. Levels of Arylsulfatase A (ASA) activity in peripheral leukocytes, mixed white cell populations, and lymphocytes are compared in normal and psychiatric patients. The prevalence of low levels of enzyme activity in psychiatric patients, and the implications of such levels with regard to the metabolic disease states and associated psychiatric illnesses are discussed. In addition, the use and reliability of leukocyte enzyme assay systems as a criteria for determining and distinguishing between the homozygous and heterozygous conditions are evaluated.

Metachromatic leukodystrophy in Greece: observations on 4 cases.

We report our findings in four cases of metachromatic leukodystrophy diagnosed in Greece during the last 4 years. The age of onset and the clinical symptoms were those described for the late infantile form of the disease. However, one patient retained his speech and mental abilities despite his pronounced motor regression and neurological involvement. This was combined with high residual arylsulphatase A activity in white blood cell homogenates even in the 0 degrees C incubation assay.