A single nucleotide polymorphism that affects oligodendroglial lineage cells may augment the consequences of neurotoxicants.

Sulfatase-responsive phase-separating peptide coacervates target stress granules to reverse sorafenib resistance in hepatocellular carcinoma.

This study evaluated intravenous administration of allogeneic mesenchymal stem cells (MSCs) transduced with AAV9-ARSA to pigs. The MSCs were modified to overexpress human arylsulfatase A (ARSA). Thirty-five days after treatment, ARSA activity significantly increased in CNS tissues. No hepatotoxicity or systemic inflammation was observed. The results confirm the safety and efficacy of this MSC-based gene therapy for metachromatic leukodystrophy (MLD).

IntroductionSaposin B deficiency is an ultrarare lysosomal disorder caused by biallelic mutations in the PSAP gene. Although it clinically resembles classical arylsulfatase A (ARSA)-deficient metachromatic leukodystrophy (MLD), ARSA activity remains biochemically normal. Fewer than 30 genetically confirmed patients have been reported to date.Patient PresentationWe describe 2 pediatric patients with homozygous PSAP gene mutations. Both presented with progressive neurodegeneration, spastic quadriparesis, demyelinating peripheral neuropathy, and radiologic findings consistent with MLD. The first patient harbored a canonical splice-site variant (c.577-1G>T), whereas the second carried a recurrent missense mutation (p.Cys241Ser). Notably, the first patient also had a PMP22 duplication consistent with coexisting CMT1A. Despite preserved ARSA activity, both patients fulfilled clinical and imaging criteria for MLD. Abdominal imaging revealed gallbladder sludge in both patients, with additional biliary tract dilation in one. These hepatobiliary findings have not been previously reported in genetically confirmed saposin B deficiency, broadening the known phenotypic spectrum.ConclusionThese patients underscore the importance of PSAP gene sequencing in MLD-like presentations with normal ARSA activity and suggest that hepatobiliary involvement may be an underrecognized feature of saposin B-related MLD.

Encapsulated cells as an enzyme replacement therapy for metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal disorder affecting about 1 per 100,000 newborns. It is caused by biallelic variations in the arylsulfatase A (ARSA) gene, leading to deficiency of ARSA enzyme activity leading to elevation of sulfatides. Most affected individuals have the late-infantile or early-juvenile phenotype, associated with significant and progressive neurologic degeneration and death. For these phenotypes, treatment with a lentiviral gene therapy in infancy can improve survival and motor function. However, in the absence of screening or an affected older sibling, most cases are diagnosed much later. A two-tiered newborn screen, based on the presence of elevated sulfatides in dried-blood spots followed by finding low ARSA enzyme activity, can accurately identify newborns with the early-onset phenotypes of MLD for timely gene therapy. An MLD screening study with consent is ongoing in 8 hospitals in New York City and population-based newborn screening has been implemented in several regions in Europe. Although the false-positive rate is low, only one of these MLD newborn screening activities, in Hannover, Germany, has reported identifying cases. At least four newborn screening programs in the United States are in the process of implementing MLD screening.

Newborn screening for metachromatic leukodystrophy: Preparation of reagents and methodology for measurement of sulfatides and arylsulfatase A enzymatic activity in dried blood spots.

Metachromatic leukodystrophy (MLD) is an autosomal recessive neurodegenerative disorder caused by mutations in the arylsulfatase A (ARSA) gene, resulting in lower sulfatase activity and the toxic accumulation of sulfatides in the central and peripheral nervous system. Children account for 70% of cases and become progressively disabled, with death occurring within 10 years of disease onset. Gene therapy approaches to restore ARSA expression via adeno-associated virus (AAV) vectors have been promising but hampered by limited brain biodistribution. We report the development of an engineered capsid, AAV.GMU01, demonstrating superior biodistribution and transgene expression in the central nervous system of nonhuman primates (NHPs). Next, we show that AAV.GMU01-ARSA–treated MLD mice exhibit persistent, normal levels of sulfatase activity and a concomitant reduction in toxic sulfatides. Treated mice also show a reduction in MLD-associated pathology and auditory dysfunction. Lastly, we demonstrate that treatment with AAV.GMU01-ARSA in NHPs is well tolerated and results in potentially therapeutic ARSA expression in the brain. In summary, we propose AAV.GMU01-ARSA–mediated gene replacement as a clinically viable approach to achieve broad and therapeutic levels of ARSA.

BackgroundMetachromatic leukodystrophy (MLD) is an ultrarare, severe lysosomal storage disorder caused by a deficiency of arylsulfatase A (ARSA).MethodsWe treated patients who had MLD with atidarsagene autotemcel (arsa-cel), a hematopoietic stem-cell–based gene therapy, in two prospective open-label clinical studies and expanded-access programs. We compared their outcomes with those of untreated patients (natural history cohort). The primary end point was survival free from severe motor impairment (the time from birth to the first occurrence of loss of locomotion and of sitting without support or death from any cause).ResultsA total of 39 treated patients and 49 untreated patients were included. The median follow-up was 6.76 years (range, 0.64 to 12.19). Arsa-cel resulted in a significantly lower risk of severe motor impairment or death than no treatment among patients with presymptomatic late-infantile MLD (P<0.001), those with presymptomatic early-juvenile MLD (P=0.04), and those with early-symptomatic early-juvenile MLD (P<0.001). The estimated percentage of patients surviving without severe motor impairment at 6 years of age was 0% (95% confidence interval [CI], not evaluable) among untreated patients with late-infantile MLD and 100% (95% CI, 100 to 100) among treated patients with presymptomatic late-infantile MLD. The estimated percentage of patients surviving without severe motor impairment at 10 years of age was 11.2% (95% CI, 0.9 to 36.4) among untreated patients with early-juvenile MLD and 87.5% (95% CI, 38.7 to 98.1) and 80.0% (95% CI, 40.9 to 94.6) among treated patients with presymptomatic and early-symptomatic early-juvenile MLD, respectively. No evidence of insertional oncogenesis was found. The most common grade 3 or higher adverse event was febrile neutropenia. Anti-ARSA antibodies were detected transiently in 6 of 39 patients (15%). Three deaths occurred, all of which were considered by the investigators to be unrelated to arsa-cel.ConclusionsAmong patients with presymptomatic late-infantile or early-juvenile MLD and those with early-symptomatic early-juvenile MLD, the risk of severe motor impairment or death was significantly lower among those who received treatment with arsa-cel than in a natural history cohort that did not receive treatment. (Funded by Orchard Therapeutics and others; ClinicalTrials.gov numbers, NCT01560182 and NCT03392987.)

Metachromatic leukodystrophy (MLD) is a rare inherited disorder of lysosomal storage, caused by a deficiency in the arylsulfatase A (ARSA) enzyme, leading to toxic accumulation of sulfatides, which progressively impair motor and cognitive function. MLD is a candidate for inclusion in newborn screening (NBS) programs, due to the narrow pre-symptomatic window for effective therapeutic intervention. We set up a prospective pilot NBS program for MLD in Tuscany, based on a two-step approach. The first-tier test quantified four sulfatides; if levels exceeded the cut-off, we performed the second-tier test by measuring ARSA activity on the same neonatal dried blood spot (DBS). We performed the first-tier test on 42,262 newborns over two years and the second-tier test on residual neonatal DBS from 90 of them (0.21%). We recalled 10 newborns (0.02%) for an additional DBS, due to insufficient residual material for a second-tier test (n = 4) or to low ARSA activity (n = 6). We found normal ARSA activity in all new DBS and identified no new cases of MLD. Retrospective analysis of eight neonatal and fifteen non-neonatal DBS from patients with genetically confirmed MLD showed that the algorithm accurately identified MLD patients. This diagnostic algorithm proved feasible and accurate for early detection of MLD in prospective NBS.

The myelin is responsible for providing stability to the axons of the nerve cells, but above all, to improve transmission speed of the nerve impulse in vertebrates. Over 70% of the myelin sheath is composed of lipids and the remaining portion by approximately 2,000 proteins. The myelin sheath has been constantly evolving, and it is known that unusually high concentrations of galactosylceramide (GalCer) and its sulfated form play a major role in the biophysical properties of the myelin. To gain insights of the evolutionary role of GalCer, we have studied two lysosomal enzymes involved in GalCer degradation, arylsulfatase A (ARSA) and galactocerebrosidase (GALC). Deficiency of ARSA or GALC causes demyelinating disorders. We conducted phylogenetic analyses of 105 ARSA and 110 GALC orthologs representing more than 600 million years ago of evolution. We examined i) low values of the ratio of nonsynonymous to synonymous nucleotide-substitution rates (dN/dS) indicating purifying selection and ii) negative selection of amino acids located in the active site preventing pathogenic mutations. Gene structure analyses showed evidence of rearrangement with gain and loss of exons while there were conserved regions mainly located around the active site. We also found a limited number of sites under positive selection pressure that do not cause alterations to the overall protein structure. Our results indicate that ARSA and GALC have been highly conserved during the evolutionary process to maintain the metabolism of GalCer, which is essential for the integrity of the white matter in vertebrate species.

Metachromatic leukodystrophy (MLD) is a rare childhood disease arises by arylsulfatase A (ARSA) deficiency. Storage of sulfatides causes dysmyelination in the central nervous system resulting clinically neurodegenerative process. Ichthyosis can be seen in multiple sulfatase deficiency (MSD) and steroid sulfatase deficiency but ichthyosis with arylsulfatase deficiency is not defined before. Herein we present an individual diagnosed late infantile metachromatic leukodystrophy with ichthyosis and ARSA gene analysis revealed homozygote mutation (c.619G&gt;C). To our knowledge ichthysosis with ARSA deficiency was not reported previously.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ARSA). Accumulation of sulfatide, substrate of ARSA, in the central and peripheral nervous system causes neurodegeneration, which leads to neurologic and psychiatric symptoms. Adult-onset MLD is the least frequent type of MLD and shows both genetic and clinical heterogeneity. The clinical presentation differs according to pathogenic variants in the ARSA gene. Therefore, establishing genotype-phenotype correlation is crucial for the diagnosis and management of patients with adult-onset MLD. A family of multiple individuals with adult-onset psychomotor deterioration was assessed. The patients had behavioral disturbances initially, and neurological deficits had developed in the later stages of the disease. The family was analyzed using karyotype analysis, Sanger sequencing, custom next-generation sequencing (NGS) panel of the genes related to dementia, and whole exome sequencing (WES). Karyotype analysis and NGS dementia panel showed no pathogenic aberration. However, WES analysis revealed heterozygous variants in the ARSA gene: c.542T>G (p.Ile181Ser) and c.661T>A (p.Phe221Ile). Segregation analysis, performed by Sanger sequencing, showed that all individuals with the same clinical findings were compound heterozygous for c.542T>G and c.661T>A substitutions. The compound heterozygosity of c.542T>G and c.661T>A variants of the ARSA gene cause adult-onset MLD. The genotype detected in the patients was not reported before in the literature. Moreover, the clinical course of the patients followed a similar pattern with dominantly psychiatric symptoms at the initial stage of the disease, indicating a distinct phenotype caused by the two pathogenic variants of the ARSA gene.

Effective gene therapy for metachromatic leukodystrophy achieved with minimal lentiviral genomic integrations.

Continued advances in variant effect prediction are necessary to demonstrate the ability of machine learning methods to accurately determine the clinical impact of variants of unknown significance (VUS). Towards this goal, the ARSA Critical Assessment of Genome Interpretation (CAGI) challenge was designed to characterize progress by utilizing 219 experimentally assayed missense VUS in the Arylsulfatase A (ARSA) gene to assess the performance of community-submitted predictions of variant functional effects. The challenge involved 15 teams, and evaluated additional predictions from established and recently released models. Notably, a model developed by participants of a genetics and coding bootcamp, trained with standard machine-learning tools in Python, demonstrated superior performance among submissions. Furthermore, the study observed that state-of-the-art deep learning methods provided small but statistically significant improvement in predictive performance compared to less elaborate techniques. These findings underscore the utility of variant effect prediction, and the potential for models trained with modest resources to accurately classify VUS in genetic and clinical research.

Sulfatides are a class of sphingolipids which are abundant in the myelin sheet and oligodendrocytes, therefore they play a crucial role in the nervous system. Abnormal sulfatide excretion has been linked to several neurodegenerative disorders including metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD). In MLD and MSD, sulfatide catabolism is impaired due to the reduced lysosomal arylsulfatase A (ARSA) activity resulting in an accumulation of sulfatides, which can be useful in a diagnostic setting. The current study aims to develop a method for semi-quantitation of urine sulfatides as a diagnostic tool for MLD and MSD. We developed a sensitive and accurate method for identifying 48 urinary molecular sulfatide species by UHPLC-Orbitrap-HRMS analysis. Newborns were classified according to their gestational age. The proportion of sulfatides bearing saturated fatty acids attached to d18:1 and d18:0 sulfatide backbone was higher in newborns and increased with prematurity. The 5 most abundant sulfatide species in all samples (controls, MLD and MSD) were C22:0, C24:0, C22:0-OH, C24:0-OH and C24:1-OH fatty acid attached to d18:1 sulfatide backbone. The top discriminant feature between MLD patients and controls was d18:1/C26:1-OH. Total semi-quantitation of 6 sulfatide species (5 most abundant sulfatides+d18:1/C26:1-OH) shows that overall excretion gradually decreases with age and all MLD patients were successfully discriminated from their age-matched controls. While sulfatide excretion was increased in the severe MSD patients (n=2), it was normal in the attenuated MSD patients, who had high residual ARSA activity. This study proves the feasibility of diagnosing MLD and severe MSD based on sulfatide excretion in urine. We established (gestational) age-specific cut-offs of the total sulfatide excretion and composition. Interpretation of the composition (e.g. by calculation the ratio (d18:1/C22:0+d18:1/C24:0)/(d18:1/C22:0-OH+d18:1/C24:0-OH)) may reduce false positives, especially when sampling at young age.

Variants of uncertain significance (VUS) represent variants that lack sufficient evidence to be confidently associated with a disease, thus posing a challenge in the interpretation of genetic testing results. Here we report an improved method for predicting the VUS of Arylsulfatase A (ARSA) gene as part of the Critical Assessment of Genome Interpretation challenge (CAGI6). Our method uses a transfer learning approach that leverages a pre-trained protein language model to predict the impact of mutations on the activity of the ARSA enzyme, whose deficiency is known to cause a rare genetic disorder, metachromatic leukodystrophy. Our innovative framework combines zero-shot log odds scores and embeddings from the ESM, an evolutionary scale model as features for training a supervised model on gene variants functionally related to the ARSA gene. The zero-shot log odds score feature captures the generic properties of the proteins learned due to its pre-training on millions of sequences in the UniProt data, while the ESM embeddings for the proteins in the ARSA family capture features specific to the family. We also tested our approach on another enzyme, N-acetyl-glucosaminidase (NAGLU), that belongs to the same superfamily as ARSA. Our results demonstrate that the performance of our family models (augmented ESM models) is either comparable or better than the ESM models. The ARSA model compares favorably with the majority of state-of-the-art predictors on area under precision and recall curve (AUPRC) performance metric. However, the NAGLU model outperforms all pathogenicity predictors evaluated in this study on AUPRC metric. The improved AUPRC has relevance in a diagnostic setting where variant prioritization generally entails identifying a small number of pathogenic variants from a larger number of benign variants. Our results also indicate that genes that have sparse or no experimental variant impact data, the family variant data can serve as a proxy training data for making accurate predictions. Attention analysis of active sites and binding sites in ARSA and NAGLU proteins shed light on probable mechanisms of pathogenicity for positions that are highly attended.

Very few cases have been described so far with co-occurrence of Phelan-McDermid syndrome (PMS) and metachromatic leukodystrophy (MLD). Those patients harbour a chromosome (chr) 22q13deletion encompassing at least the SHANK3 and ARSA genes and a pathogenic variant in the arylsulfatase A (ARSA) gene residing on the other allele. The deletion in chr22q13 results in PMS phenotype and the presence of pathogenic variation on the other intact copy of ARSA gene, leads to MLD phenotype due to biallelic loss of ARSA function. We describe a male infant, born to a third-degree consanguineous couple, who exhibited neuroregression at the age of 8 months, presented with developmental delay, hypotonia which rapidly progressed to feeding difficulties, axial hypotonia, ptosis, sleep disturbance, dystonia, spasticity, and abnormal eye movements. Brain MRI showed T2 hyperintensities consistent with MLD. Biochemical workup showed deficiency of ARSA enzyme activity. Genetic investigations revealed heterozygous deletion of size 2.5 Mb on chr 22q13.3 encompassing the entire ARSA gene and a pathogenic variant in the other copy of ARSA gene. Parents and sibling were tested and informed about the disease management and genetic testing in extended family members to understand the risk and preventive measures. Few case reports have proposed screening for urine sulfatides levels at the time of PMS diagnosis to identify pre-symptomatic or asymptomatic MLD patients to facilitate management and our case supports this proposal.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by mutations in the arylsulfatase A (ARSA) gene. Few studies have assessed the spectrum of ARSA mutations among Iranian patients. Here, we report eight Iranian patients with clinical features of MLD. Whole exome sequencing led to identification of the underlying mutation in ARSA gene in these patients. Among identified mutations was the recurrent c.938G > C (p.R313P) mutation in exon 5 of this gene, showing its relatively high frequency among Iranians. The results of this study helps in design of population-specific panels for screening purposes in order to decrease the burden of MLD.

Metachromatic leukodystrophy (MLD) is an autosomal recessive hereditary neurodegenerative disease caused by a deficiency in arylsulfatase A (ARSA) activity and belongs to the group of lysosomal storage diseases. A biochemical diagnosis of MLD is based on determining the residual ARSA activity in leukocytes, skin fibroblasts, and urine. This study documents our biochemical experience and estimates the relative frequency of MLD over 21 years (2001-2022). This study analyzed 4357 suspected cases of MLD in Egypt. The ARSA activity was spectrophotometrically determined in leukocytes in all the referred cases. Of these 4357 referred cases, 577 (13%) possessed decreased ARSA activity, less than 10% of the low normal range (50-200 micromole/gram protein/hour (μmol/g protein/h), and 104 cases were diagnosed as having a pseudodeficiency in enzyme activity (<20-50% of low-normal ARSA activity). The prevalence of MLD was 1.6/100,000. A diagnosis of MLD in Egypt is based on enzyme activity levels and clinical suspicion; molecular analysis was performed in a few cases.