Short communication

The assay of arylsulphatases A and B in human urine

Metachromatic leukodystrophy (MLD) is a devastating rare neurodegenerative disease. Typically, loss of motor and cognitive skills precedes early death. The disease is characterised by deficient lysosomal arylsulphatase A (ARSA) activity and an accumulation of undegraded sulphatide due to pathogenic variants in the ARSA gene. Atidarsagene autotemcel (arsa-cel), an ex vivo haematopoietic stem cell gene therapy was approved for use in the UK in 2021 to treat early-onset forms of pre- or early-symptomatic MLD. Optimal outcomes require early diagnosis, but in the absence of family history this is difficult to achieve without newborn screening (NBS). A pre-pilot MLD NBS study was conducted as a feasibility study in Manchester UK using a two-tiered screening test algorithm. Pre-established cutoff values (COV) for the first-tier C16:0 sulphatide (C16:0-S) and the second-tier ARSA tests were evaluated. Before the pre-pilot study, initial test validation using non‑neonatal diagnostic bloodspots demonstrated ARSA pseudodeficiency status was associated with normal C16:0-S results for age (n = 43) and hence not expected to cause false positive results in this first-tier test. Instability of ARSA in bloodspot required transfer of NBS bloodspots from ambient temperature to −20°C storage within 7–8 days after heel prick, the earliest possible in this UK pre-pilot study. Eleven of 3687 de-identified NBS samples in the pre-pilot were positive for C16:0-S based on the pre-established COV of ≥170 nmol/l or ≥ 1.8 multiples of median (MoM). All 11 samples were subsequently tested negative determined by the ARSA COV of <20% mean of negative controls. However, two of 20 NBS samples from MLD patients would be missed by this C16:0-S COV. A further suspected false negative case that displayed 4% mean ARSA activity by single ARSA analysis for the initial test validation was confirmed by genotyping of this NBS bloodspot, a severe late infantile MLD phenotype was predicted. This led to urgent assessment of this child by authority approval and timely commencement of arsa-cel gene therapy at 11 months old. Secondary C16:0-S analysis of this NBS bloodspot was 150 nmol/l or 1.67 MoM. This was the lowest result reported thus far, a new COV of 1.65 MoM is recommended for future pilot studies. Furthermore, preliminary data of this study showed C16:1-OH sulphatide is more specific for MLD than C16:0-S. In conclusion, this pre-pilot study adds to the international evidence that recommends newborn screening for MLD, making it possible for patients to benefit fully from treatment through early diagnosis.

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

Bisphenol A is recognized as an endocrine disruptor that can affect several biological processes in marine species. Consequently, its use has been restricted and it has been replaced with other similar compounds named bisphenol A analogues (BPA analogues). BPA analogues are speculatively considered safer compounds than BPA and their usage is increasing with a consequent higher environmental release. In this study, specimens of the clam Ruditapes philippinarum were exposed to three main BPA analogues, namely BPAF, BPF, BPS and their mixture at an environmentally relevant concentration of 300 ng/L for 7 and 14 days. Effects on biomarkers indicative of cytotoxicity, oxidative stress and damage and neurotoxicity were evaluated. In addition, bioaccumulation of the compound tested was analysed in clam soft tissues. Results showed that BPA analogues at an environment concentration affected cellular parameters and antioxidant system causing also oxidative damage, suggesting that BPA analogues can be harmful compounds for clams.

A deficiency of human arylsulfatase A (hASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disease characterized by sulfatide accumulation and central nervous system (CNS) demyelination. Efficacy of enzyme replacement therapy (ERT) is increased by genetic engineering of hASA to elevate its activity and transfer across the blood-brain barrier (BBB), respectively. To further improve the enzyme’s bioavailability in the CNS, we mutated a cathepsin cleavage hot spot and obtained hASAs with substantially increased half-lives. We then combined the superstabilizing exchange E424A with the activity-promoting triple substitution M202V/T286L/R291N and the ApoEII-tag for BBB transfer in a trimodal modified neoenzyme called SuPerTurbo-ASA. Compared with wild-type hASA, half-life, activity, and M6P-independent uptake were increased more than 7-fold, about 3-fold, and more than 100-fold, respectively. ERT of an MLD-mouse model with immune tolerance to wild-type hASA did not induce antibody formation, indicating absence of novel epitopes. Compared with wild-type hASA, SuPerTurbo-ASA was 8- and 12-fold more efficient in diminishing sulfatide storage of brain and spinal cord. In both tissues, storage was reduced by ∼60%, roughly doubling clearance achieved with a 65-fold higher cumulative dose of wild-type hASA previously. Due to its enhanced therapeutic potential, SuPerTurbo-ASA might be a decisive advancement for ERT and gene therapy of MLD.

The majority of lysosomal storage diseases affect the brain. Treatment of the brain with intravenous enzyme replacement therapy is not successful, because the recombinant lysosomal enzymes do not cross the blood–brain barrier (BBB). Biologic drugs, including lysosomal enzymes, can be re-engineered for BBB delivery as IgG-enzyme fusion proteins. The IgG domain of the fusion protein is a monoclonal antibody directed against an endogenous receptor-mediated transporter at the BBB, such as the insulin receptor or the transferrin receptor. This receptor transports the IgG across the BBB, in parallel with the endogenous receptor ligand, and the IgG acts as a molecular Trojan horse to ferry into brain the lysosomal enzyme genetically fused to the IgG. The IgG-enzyme fusion protein is bi-functional and retains both high affinity binding for the BBB receptor, and high lysosomal enzyme activity. IgG-lysosomal enzymes are presently in clinical trials for treatment of the brain in Mucopolysaccharidosis.

Metachromatic leukodystrophy (MLD) is a rare autosomal recessive lysosomal storage disease caused by a deficiency of the enzyme arylsulfatase A (ARSA), which is encoded by the ARSA gene. Mutations discovered in the ARSA gene continuously improve our knowledge and understanding of this rare disease. We identified a Chinese family with 15 members. Two of them were diagnosed as MLD. We used Sanger sequencing to analyze eight exons of the ARSA gene and measured the ARSA activity in leukocyte or amniotic cells with p-nitrocatechol sulfate as the substrate. The potential pathological functions of these variants were predicted through SIFT, Polyphen2, and Mutation Taster. Two family members with a diagnosis of MLD were a two-year-old boy and a fetus. Both had a complex heterozygous mutation: c.413C > T (p.Pro138Leu) in exon 2 and c.1344dupC (p.G449fs) in exon 8. This complex heterozygous mutation is the first case of concurrent missense and code shift mutations reported in MLD in China. In these two family members, ARSA activity (20 nmol/17 h/mg protein and 9.8 nmol/17 h/mg protein) is only 10% compared to the normal population.