Biochemical and molecular analysis of pediatric patients with metachromatic leukodystrophy in South China: functional characterization of five novel ARSA variants.

Metachromatic leukodystrophy (MLD) is a rare hereditary neurodegenerative disease caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). This study described the clinical and molecular characteristics of 24 Chinese children with MLD and investigated functional characterization of five novel ARSA variants. A retrospective analysis was performed in 24 patients diagnosed with MLD at Guangzhou Women and Children's Medical Center in South China. Five novel mutations were further characterized by transient expression studies. We recruited 17 late-infantile, 3 early-juvenile, 4 late-juvenile MLD patients. In late-infantile patients, motor developmental delay and gait disturbance were the most frequent symptoms at onset. In juvenile patients, cognitive regression and gait disturbance were the most frequent chief complaints. Overall, 25 different ARSA mutations were identified with 5 novel mutations.The most frequent alleles were p.W320* and p.G449Rfs. The mutation p.W320*, p.Q155=, p.P91L, p.G156D, p.H208Mfs*46 and p.G449Rfs may link to late-infantile type. The novel missense mutations were predicted damaging in silico. The bioinformatic structural analysis of the novel missense mutations showed that these amino acid replacements would cause severe impairment of protein structure and function. In vitro functional analysis of the six mutants, showing a low ARSA enzyme activity, clearly demonstrated their pathogenic nature. The mutation p.D413N linked to R alleles. In western blotting analysis of the ARSA protein, the examined mutations retained reduced amounts of ARSA protein compared to the wild type. This study expands the spectrum of genotype of MLD. It helps to the future studies of genotype-phenotype correlations to estimate prognosis and develop new therapeutic approach.

A systematic review on the birth prevalence of metachromatic leukodystrophy.

BACKGROUND: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by deficiency in arylsulfatase A (ASA) activity arising primarily from ASA gene (ARSA) variants. Late-infantile, juvenile and adult clinical subtypes are defined by symptom onset at ≤ 2.5, > 2.5 to < 16 and ≥ 16 years, respectively. Epidemiological data were sought to address knowledge gaps and to inform decisions regarding the clinical development of an investigational drug. METHODS: To synthesize all available estimates of MLD incidence and birth prevalence worldwide and in selected countries, Ovid MEDLINE and Embase were searched systematically (March 11, 2022) using a population, intervention, comparator, outcome, time and setting framework, complemented by pragmatic searching to reduce publication bias. Where possible, results were stratified by clinical subtype. Data were extracted from non-interventional studies (clinical trials, non-clinical studies and case reports were excluded; reviews were used for snowballing only). RESULTS: Of the 31 studies included, 14 reported birth prevalence (13 countries in Asia-Pacific, Europe, the Middle East, North America and South America), one reported prevalence and none reported incidence. Birth prevalence per 100,000 live births ranged from 0.16 (Japan) to 1.85 (Portugal). In the three European studies with estimates stratified by clinical subtypes, birth prevalence was highest for late-infantile cases (0.31-1.12 per 100,000 live births). The distribution of clinical subtypes reported in cases diagnosed over various time periods in 17 studies varied substantially, but late-infantile and juvenile MLD accounted for at least two-thirds of cases in most studies. CONCLUSIONS: This review provides a foundation for further analysis of the regional epidemiology of MLD. Data gaps indicate the need for better global coverage, increased use of epidemiological measures (e.g. prevalence estimates) and more stratification of outcomes by clinical and genetic disease subtype.

Complex genotypes in family with metachromatic leukodystrophy: Effect of trans and cis mutations distribution on the phenotype variability.

Metachromatic leukodystrophy (MLD) is a severe metabolic disorder caused by the deficient activity of arylsulfatase A due to ARSA gene mutations. According to the age of onset, MLD is classified into three forms: infantile, juvenile, and adult. In our study, we aimed to perform a genetic analysis for two siblings with juvenile MLD for a better characterization of the molecular mechanisms behind the disease. A consanguineous family including two MLD patients (PII.1 and PII.2) was enrolled in our study. The diagnosis was made based on the clinical and neuroimaging investigations. The sequencing of ARSA gene was performed followed by in silico analysis. Besides, the cis/trans distribution of the variants was verified through a PCR-RFLP. The ARSA gene sequencing revealed three known variants, two exonic c.1055A > G and c.1178C > G and an intronic one (c.1524 + 95A > G) in the 3'UTR region. All variants were present at heterozygous state in the two siblings and their mother. The assessment of the cis/trans distribution showed the presence of these variants in cis within the mother, while PII.2 and PII.2 present the c.1055A > G/c.1524 + 95A > G and the c.1178C > G in trans. Additionally, PII.1 harbored a de novo novel missense variant c.1119G > T, whose pathogenicity was supported by our predictive results. Our genetic findings, supported by a clinical examination, confirmed the affection of the mother by the adult MLD. Our results proved the implication of the variable distribution of the found variants in the age of MLD onset. Besides, we described a variable severity between the two siblings due to the de novo pathogenic variant. In conclusion, we identified a complex genotype of ARSA variants within two MLD siblings with a variable severity due to a de novo variant present in one of them. Our results allowed the establishment of an adult MLD diagnosis and highlighted the importance of an assessment of the trans/cis distribution in the cases of complex genotypes.

Engineered arylsulfatase A with increased activity, stability and brain delivery for therapy of metachromatic leukodystrophy.

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.

Evaluating meaningful changes in physical functioning and cognitive declines in metachromatic leukodystrophy: a caregiver interview study.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a rare lysosomal storage disease caused by deficient activity of arylsulfatase A (ASA). Treatment options for patients are limited; gene therapy based on haematopoietic stem cell transplantation is the only approved treatment for some subtypes of MLD. Any therapeutic benefit of treatments must be meaningful for patients and their families. We evaluated the clinical meaningfulness of slowing the decline in gross motor function as measured by the Gross Motor Function Classification in MLD (GMFC-MLD) from the caregiver perspective via semi-structured telephone interviews with caregivers of children with late-infantile MLD. We also evaluated the perceived significance of declines in communication abilities measured by the Expressive Language Function Classification in MLD (ELFC-MLD). This work could help to inform the endpoints of a phase 2 clinical trial (NCT03771898) assessing the efficacy of intrathecal recombinant human ASA in MLD. RESULTS: Twelve caregivers were recruited, reporting on 12 children with MLD. Children had a mean age of 6.1 years; mean age at symptom onset was 17.6 months. Most children (10/12) progressed from walking without support (categories 0-1) to a loss of locomotion (categories 5-6) in ≤ 2 years. Caregivers felt that GMFC-MLD and ELFC-MLD accurately described motor and language declines in their children, respectively. Most caregivers (10/12) reported that the idea of delaying disease progression would be meaningful. Further, a slowing of motor function decline in GMFC-MLD, from category 1 to category 3 or from category 2 to category 4 over 2 years, was seen as meaningful by all caregivers asked; however, only 3/12 caregivers reported that delayed decline would be meaningful if baseline category was ≥ 3. Caregivers also reported that delaying expressive language decline at any level that did not indicate a complete loss of expressive language (indicated by categories 1-3) would be meaningful. CONCLUSIONS: Caregivers of children with MLD felt that a delayed decline in gross motor function, as assessed by the GMFC-MLD, would be meaningful, supporting the selection of primary and secondary endpoints for the phase 2 clinical trial. Communication abilities were another area of significance for consideration in future clinical trial design.

Safety and Efficacy of Intravenous and Intrathecal Delivery of AAV9-Mediated ARSA in Minipigs.

Metachromatic leukodystrophy (MLD) is a hereditary neurodegenerative disease characterized by demyelination and motor and cognitive impairments due to deficiencies of the lysosomal enzyme arylsulfatase A (ARSA) or the saposin B activator protein (SapB). Current treatments are limited; however, gene therapy using adeno-associated virus (AAV) vectors for ARSA delivery has shown promising results. The main challenges for MLD gene therapy include optimizing the AAV dosage, selecting the most effective serotype, and determining the best route of administration for ARSA delivery into the central nervous system. This study aims to evaluate the safety and efficacy of AAV serotype 9 encoding ARSA (AAV9-ARSA) gene therapy when administered intravenously or intrathecally in minipigs, a large animal model with anatomical and physiological similarities to humans. By comparing these two administration methods, this study contributes to the understanding of how to improve the effectiveness of MLD gene therapy and offers valuable insights for future clinical applications.

Atidarsagene autotemcel for metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a rare autosomal recessive disorder of sphingolipid metabolism, due to a deficiency of the enzyme arylsulfatase A (ARSA). The main clinical signs of the disease are secondary to central and peripheral nervous system demyelination. MLD is subdivided into early- and late-onset subtypes based upon the onset of neurological disease. The early-onset subtype is associated with a more rapid progression of the disease that leads to death within the first decade of life. Until recently, no effective treatment was available for MLD. The blood-brain barrier (BBB) prevents systemically administered enzyme replacement therapy from reaching target cells in MLD. The evidence for the efficacy of hematopoietic stem cell transplantation is limited to the late-onset MLD subtype. Here, we review the preclinical and clinical studies that facilitated the approval of the ex vivo gene therapy atidarsagene autotemcel for early-onset MLD by the European Medicines Agency (EMA) in December 2020. This approach was studied in an animal model first and then in a clinical trial, eventually proving its efficacy in preventing disease manifestations in presymptomatic patients and stabilizing its progression in paucisymptomatic subjects. This new therapeutic consists of patients' CD34+ hematopoietic stem/progenitor cells (HSPCs) transduced with a lentiviral vector encoding functional ARSA cDNA. The gene-corrected cells get reinfused into the patients after a cycle of chemotherapy conditioning.

Case report: A compound heterozygous mutations in ARSA associated with adult-onset metachromatic leukodystrophy.

Metachromatic Leukodystrophy (MLD) is a rare autosomal recessive disease, which is caused by mutations in the arylsulfatase A (ARSA) gene. The ARSA gene is located on chromosome 22q13, containing eight exons. According to the age of onset, MLD can be divided into late infantile type, juvenile type, and adult type. Adult MLD has an insidious onset after the age of 16 years. Additionally, intellectual as well as behavioral changes, such as memory deficits or emotional instability, are commonly the first presenting symptoms. There is a study that reported an adult-onset MLD manifested cognitive impairment progressively due to compound heterozygous mutations of NM_000487: c.[185_186dupCA], p.(Asp63GlnfsTer18), and NM_000487: c.[154G>T], p.(Gly172Cys), rs74315271 in the ARSA gene, finding that the c.[154G>T], p.(Gly172Cys) is a novel missense mutation. Brain magnetic resonance imaging (MRI) revealed symmetrical demyelination of white matter. The activity of ARSA enzymatic in leukocytes decreased. Nerve conduction studies displayed that evidence of polyneuropathy was superimposed upon diffuse, uniform demyelinating, and sensorimotor polyneuropathy. Family genes revealed that each family member carried one of two heterozygous mutant genes. She has been discharged and is currently being followed up. This study found a compound heterozygous mutation in the ARSA gene associated with MLD and identified a novel missense mutation NM_000487: c.[154G>T], p.(Gly172Cys), rs74315271. This will provide a critical clue for prenatal diagnosis of the offspring in this family, and expand the mutation spectrum of MLD-related ARSA.

Long-Term Consequences of Increased Activity of Urine Enzymes After Cardiac Surgery - A Prospective Observational Study.

Introduction: Cardiac surgery associated AKI (CSA-AKI) complicates recovery and may be associated with a greater risk of developing chronic kidney disease and mortality. The aim of this study was to assess long-term clinical consequences of transient increased activity of urinary enzymes after cardiac surgery (CS). Methods: An observational study was conducted in a group of 88 adult patients undergoing planned coronary artery bypass grafting (CABG), but all samples were obtained from 79 patients. The activity of urinary enzymes: N-acetyl-beta-glucosaminidase (NAG), arylsulfatase A (ASA) and beta-glucuronidase was evaluated in sequential urine samples. A comparative analysis of biochemical parameters was performed regarding the occurrence of acute kidney injury (AKI) defined by KIDGO at 24 hours, at day 30 and 5-years after the operation. Results: During the first 24 hours after CS AKI was diagnosed in 13 patients. A comparison of the activity of urinary enzymes in pre-defined time-points showed significant differences for ASA and NAG (post OP-sample p < 0.028 and p < 0.022; POD 1 sample p < 0.004 and p < 0.001 respectively). No patient had any biochemical or clinical features of kidney failure at day 30. In the AKI group kidney failure was diagnosed in 36% of patients within 5 years of follow-up as opposed to 5% in the no AKI group. The activities of tubular enzymes in urine reflect a general injury of kidney tubules during and after the operation. However, they are not ideal biomarkers for prediction of the degree of kidney injury and further poor prognosis of CS-AKI.

Extremely low arylsulfatase A enzyme activity does not necessarily cause symptoms: A long-term follow-up and review of the literature.

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by deficiency of arylsulfatase A (ARSA). Heterozygous carriers of disease-causing variants and individuals harbouring pseudodeficiency alleles in the ARSA gene exhibit reduced ARSA activity. In the context of these genotypes, low ARSA activity has been suggested to lead to an atypical form of MLD or other neurological abnormalities, but data are limited. The aim of our study was to analyse the impact of low ARSA activity in two subjects who are heterozygous for the ARSA pseudodeficiency allele and a disease-causing variant. Biochemical testing included ARSA activity measurements and urinary sulfatide analysis. Biochemical data of a large cohort of MLD patients, heterozygotes, pseudodeficient individuals and healthy controls were analysed. MRI was performed at 3T using T1- and T2-weighted sequences and MR spectroscopy. We present two long-term follow-ups who are heterozygous for the ARSA pseudodeficiency allele and a disease-causing variant in the ARSA gene in cis. The two related index cases exhibit markedly reduced ARSA activity compared to controls and heterozygous carriers. The neurological evaluation and MRI do not reveal any abnormalities. Our data underline that extremely low enzyme activity due to a pseudodeficiency allele and a disease-causing variant in the ARSA gene even in cis does not lead to clinical symptoms or pre-symptomatic MRI changes suspicious for MLD. The review of literature corroborates that any association of low ARSA activity with disease features remains questionable. It seems important to combine the measurement of ARSA activity with elevated sulfatide as well as genetic testing, as done in current newborn screening approaches. Heterozygosity for metachromatic leukodystrophy and an arylsulfatase A pseudodeficiency allele does not cause neurological or neuropsychiatric features.

Plasma arylsulfatase A levels are associated with cognitive function in Parkinson's disease.

BACKGROUND: Arylsulfatase A (ARSA), a lysosomal enzyme, has been shown to inhibit the aggregation and propagation of α-synuclein (α-syn) through its molecular chaperone function. The relationship between ARSA levels and Parkinson's disease (PD) in the Chinese Han population remains controversial, and few quantitative research studies have investigated the relationship between plasma ARSA levels and PD. OBJECTIVES: The purpose of this study was to investigate the relationships between ARSA levels and cognitive function in PD patients and to evaluate the association of ARSA and α-syn levels with nonmotor symptoms. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to measure the plasma ARSA and α-syn levels in 50 healthy controls, 120 PD patients (61 PD patients with no cognitive impairment (PD-NCI) and 59 PD patients with cognitive impairment (PD-CI)). Motor symptoms and nonmotor symptoms (cognitive function, Unified Parkinson's Disease Rating Scale (UPDRS) score, depression, anxiety, constipation, olfactory dysfunction, sleep disruption, and other symptoms) were assessed with the relevant scales. The Kruskal-Wallis H test was used for comparison between groups, and Pearson/Spearman analysis was used for correlation analysis. RESULTS: The plasma ARSA concentrations were lower in the PD-CI group than in the PD-NCI group. The plasma α-syn levels in the PD-CI group were higher than those in the healthy control group, and the plasma ARSA levels were correlated with the Mini-Mental State Examination (MMSE scores) and Hoehn and Yahr (H-Y) stage. CONCLUSION: We used a quantitative assessment method to show that low plasma ARSA levels and high α-syn levels are related to cognitive impairment in PD patients. Plasma ARSA levels gradually decrease with PD progression.

Identification of neurodegeneration indicators and disease progression in metachromatic leukodystrophy using quantitative NMR-based urinary metabolomics.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency of the arylsulfatase A (ARSA). ARSA deficiency leads to an accumulation of sulfatides primarily in the nervous system ultimately causing demyelination. With evolving therapeutic options, there is an increasing need for indicators to evaluate disease progression. Here, we report targeted metabolic urine profiling of 56 MLD patients including longitudinal sampling, using 1H (proton) nuclear magnetic resonance (NMR) spectroscopy. 1H-NMR urine spectra of 119 MLD samples and 323 healthy controls were analyzed by an in vitro diagnostics research (IVDr) tool, covering up to 50 endogenous and 100 disease-related metabolites on a 600-MHz IVDr NMR spectrometer. Quantitative data reports were analyzed regarding age of onset, clinical course, and therapeutic intervention. The NMR data reveal metabolome changes consistent with a multiorgan affection in MLD patients in comparison to controls. In the MLD cohort, N-acetylaspartate (NAA) excretion in urine is elevated. Early onset MLD forms show a different metabolic profile suggesting a metabolic shift toward ketogenesis in comparison to late onset MLD and controls. In samples of juvenile MLD patients who stabilize clinically after hematopoietic stem cell transplantation (HSCT), the macrophage activation marker neopterin is elevated. We were able to identify different metabolic patterns reflecting variable organ disturbances in MLD, including brain and energy metabolism and inflammatory processes. We suggest NAA in urine as a quantitative biomarker for neurodegeneration. Intriguingly, elevated neopterin after HSCT supports the hypothesis that competent donor macrophages are crucial for favorable outcome.

Measurement of sulfatides in the amniotic fluid supernatant: A useful tool in the prenatal diagnosis of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal disorder caused by deficiency of arylsulfatase A (ARSA), leading to an accumulation of sulfatides. Sulfatides have been quantified in urine, dried blood spots (DBS), and tissues of patients with MLD. Newborn screening (NBS) for MLD has already been proposed based on a two-tier approach with the quantification of sulfatides in DBS followed by the quantification of ARSA by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Prenatal screening for MLD is also crucial, and sulfatide quantification in amniotic fluid (AF) can aid diagnosis. The prenatal study was initiated due to a family history of MLD at 19 weeks of gestation. ARSA was quantified in cultured amniocytes. C16:0 sulfatide was quantified by LC-MS/MS in the supernatant of AF. Molecular analysis of the ARSA gene was performed in cultured amniocytes. ARSA was deficient in fetal cells, and C16:0 sulfatides were significantly elevated in comparison to age-matched controls (3-fold higher). Genetic studies identified the c.465+1G>A variant in homozygosis in the ARSA gene. Our study shows that sulfatides can be quantified in the supernatant of AF of MLD fetuses, and it could potentially aid in a faster and more accurate diagnosis of MLD patients.

Acute-onset paralytic strabismus in toddlers is important to consider as a potential early sign of late-infantile Metachromatic Leukodystrophy.

OBJECTIVES: Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease characterized by progressive demyelination within the central and peripheral nervous system. Rapid diagnosis is crucial in view of evolving therapeutic options. Strabismus has anecdotally been described as a feature in children with MLD. Our first aim was to examine the prevalence of strabismus as an early or even presenting sign of MLD in two nationwide cohorts. Second, we aimed to investigate the temporal relation between the onset of strabismus and gross motor deterioration, other early onset eye movement disorders and brain white matter abnormalities. METHODS: Clinical records of 204 MLD patients at the University Children's Hospital Tubingen and Amsterdam University Medical Center were reviewed on the presence of strabismus and other eye movement disorders. Gross motor deterioration and white matter abnormalities on brain MRI were evaluated by using the Gross Motor Function Classification in MLD and MLD LOES score, respectively. RESULTS: We identified strabismus as an early sign in MLD patients with the late-infantile form, with a prevalence of 27% (N = 17). The onset of strabismus preceded gross motor symptoms and brain white matter abnormalities in 71% and 46% respectively of the cases. Important characteristics were an acute-onset paralytic esotropia, partly accompanied by other eye movement abnormalities, and gadolinium enhancement of the cranial nerves. CONCLUSIONS: Acute-onset paralytic strabismus in toddlers should be considered a potential early sign of late-infantile MLD and might result from early cranial nerve involvement. Brain MRI with gadolinium contrast may facilitate early diagnosis.

Neurofilament light chain and glial fibrillary acidic protein levels in metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lethal metabolic leukodystrophy, with emerging treatments for early disease stages. Biomarkers to measure disease activity are required for clinical assessment and treatment follow-up. This retrospective study compared neurofilament light chain and glial fibrillary acidic protein (GFAP) levels in CSF (n = 11) and blood (n = 92) samples of 40 patients with metachromatic leukodystrophy (aged 0-42 years) with 38 neurologically healthy children (aged 0-17 years) and 38 healthy adults (aged 18-45 years), and analysed the associations between these levels with clinical phenotype and disease evolution in untreated and transplanted patients. Metachromatic leukodystrophy subtype was determined based on the (expected) age of symptom onset. Disease activity was assessed by measuring gross motor function deterioration and brain MRI. Longitudinal analyses with measurements up to 23 years after diagnosis were performed using linear mixed models. CSF and blood neurofilament light chain and GFAP levels in paediatric controls were negatively associated with age (all P < 0.001). Blood neurofilament light chain level at diagnosis (median, interquartile range; picograms per millilitre) was significantly increased in both presymptomatic (14.7, 10.6-56.7) and symptomatic patients (136, 40.8-445) compared to controls (5.6, 4.5-7.1), and highest among patients with late-infantile (456, 201-854) or early-juvenile metachromatic leukodystrophy (291.0, 104-445) and those ineligible for treatment based on best practice (291, 57.4-472). GFAP level (median, interquartile range; picogram per millilitre) was only increased in symptomatic patients (591, 224-1150) compared to controls (119, 78.2-338) and not significantly associated with treatment eligibility (P = 0.093). Higher blood neurofilament light chain and GFAP levels at diagnosis were associated with rapid disease progression in late-infantile (P = 0.006 and P = 0.051, respectively) and early-juvenile patients (P = 0.048 and P = 0.039, respectively). Finally, blood neurofilament light chain and GFAP levels decreased during follow-up in untreated and transplanted patients but remained elevated compared with controls. Only neurofilament light chain levels were associated with MRI deterioration (P < 0.001). This study indicates that both proteins may be considered as non-invasive biomarkers for clinical phenotype and disease stage at clinical assessment, and that neurofilament light chain might enable neurologists to make better informed treatment decisions. In addition, neurofilament light chain holds promise assessing treatment response. Importantly, both biomarkers require paediatric reference values, given that their levels first decrease before increasing with advancing age.

Accumulation of Seminolipid in Sertoli Cells Is Associated with Increased Levels of Reactive Oxygen Species and Male Subfertility: Studies in Aging Arsa Null Male Mice.

Seminolipid (also known as sulfogalactosylglycerolipid-SGG), present selectively in male germ cells, plays important roles in spermatogenesis and sperm-egg interaction. The proper degradation of SGG in apoptotic germ cells is also as important. Sertoli cells first phagocytose apoptotic germ cells, then Sertoli lysosomal arylsulfatase A (ARSA) desulfates SGG, the first step of SGG degradation. We have reported that aging male Arsa-/- mice become subfertile with SGG accumulation in Sertoli cell lysosomes, typical of a lysosomal storage disorder (LSD). Since reactive oxygen species (ROS) levels are increased in other glycolipid-accumulated LSDs, we quantified ROS in Arsa-/- Sertoli cells. Our analyses indicated increases in superoxide and H2O2 in Arsa-/- Sertoli cells with elevated apoptosis rates, relative to WT counterparts. Excess H2O2 from Arsa-/- Sertoli cells could travel into testicular germ cells (TGCs) to induce ROS production. Our results indeed indicated higher superoxide levels in Arsa-/- TGCs, compared with WT TGCs. Increased ROS levels in Arsa-/- Sertoli cells and TGCs likely caused the decrease in spermatogenesis and increased the abnormal sperm population in aging Arsa-/- mice, including the 50% decrease in sperm SGG with egg binding ability. In summary, our study indicated that increased ROS production was the mechanism through which subfertility manifested following SGG accumulation in Sertoli cells.

Long-term disease course of two patients with multiple sulfatase deficiency differs from metachromatic leukodystrophy in a broad cohort.

Multiple sulfatase deficiency (MSD) is a lysosomal storage disease caused by a deficiency of formylglycine-generating enzyme due to SUMF1 defects. MSD may be misdiagnosed as metachromatic leukodystrophy (MLD), as neurological and neuroimaging findings are similar, and arylsulfatase A (ARSA) deficiency and enhanced urinary sulfatide excretion may also occur. While ARSA deficiency seems a cause for neurological symptoms and later neurodegenerative disease course, deficiency of other sulfatases results in clinical features such as dysmorphism, dysostosis, or ichthyosis. We report on a girl and a boy of the same origin presenting with severe ARSA deficiency and neurological and neuroimaging features compatible with MLD. However, exome sequencing revealed not yet described homozygosity of the missense variant c.529G > C, p.Ala177Pro in SUMF1. We asked whether dynamics of disease course differs between MSD and MLD. Comparison to a cohort of 59 MLD patients revealed different disease course concerning onset and disease progression in both MSD patients. The MSD patients showed first gross motor symptoms earlier than most patients with juvenile MLD (<10th percentile of Gross-Motor-Function in MLD [GMFC-MLD] 1). However, subsequent motor decline was more protracted (75th and 90th percentile of GMFC-MLD 2 (loss of independent walking) and 75th percentile of GMFC-MLD 5 (loss of any locomotion)). Language decline started clearly after 50th percentile of juvenile MLD and progressed rapidly. Thus, dynamics of disease course may be a further clue for the characterization of MSD. These data may contribute to knowledge of natural course of ultra-rare MSD and be relevant for counseling and therapy.

Co-occurrence of Metachromatic Leukodystrophy in Phelan-McDermid Syndrome.

Phelan-McDermid syndrome or 22q13.3 deletion syndrome is a rare neurodevelopmental disorder characterized by neonatal hypotonia, severe speech delay, moderate to profound intellectual disability, and minor dysmorphic features. Regression of developmental milestones is often recognized as characteristic of this syndrome. We report a 6-year-old patient with Phelan-McDermid syndrome who presented with rapid neurologic deterioration secondary to metachromatic leukodystrophy due to a mutation of the arylsulfatase A gene (ARSA) on the other allele of 22q13.3. Metachromatic leukodystrophy was diagnosed later after clinical deterioration. Currently, there are no guidelines for screening Phelan-McDermid syndrome patients for metachromatic leukodystrophy. We propose screening for urine sulfatides at the time of Phelan-McDermid syndrome diagnosis to identify patients with pre-symptomatic or early symptomatic metachromatic leukodystrophy as it is important to facilitate discussion of treatment options and prognosis and provide medical surveillance for associated complications.

Chinese Cases of Metachromatic Leukodystrophy with the Novel Missense Mutations in ARSA Gene.

Metachromatic leukodystrophy(MLD) is an autosomal recessive hereditary neurodegenerative lysosomal storage disorder caused by the mutations in arylsulfatase A gene (ARSA), which results in the deficiency of ARSA enzyme. The common clinical characteristics of MLD are abnormal gait, and then gradually appears ataxia, spastic quadriplegia, optic atrophy, cortical blindness, and dementia. We describe two patients in China who were diagnosed with MLD and find that the four ARSA gene mutations (c.1115G>A, c.302G>T, c.893 G> T, and c.302G>T) are associated with MLD, in which c.893 G>T and c.302G>T are novel mutations by gene sequence and clinical manifestations, to further understand the relationship between MLD and ARSA gene.

Clinical, Biochemical, and Molecular Characterization of Metachromatic Leukodystrophy Among Egyptian Pediatric Patients: Expansion of the ARSA Mutational Spectrum.

Metachromatic leukodystrophy (MLD) is a neurodegenerative disorder characterized by progressive demyelination due to deficiency of the enzyme arylsulfatase A (ARSA) in leukocytes, and consequently leads to impaired degradation and accumulation of cerebroside-3-sulfate (sulfatide). This study aimed to sequence the ARSA gene in a total of 43 patients with metachromatic leukodystrophy descendant from 40 Egyptian families. In addition, four carrier parents from two families with children who had died from MLD came to the clinic for genetic analysis. Prenatal diagnosis was performed for four families with molecularly diagnosed MLD sibs. Different mutations were characterized in our cohort, including missense, nonsense, splice, and deletion. Overall, 21 different mutations in the ARSA gene were detected, with 12 novel mutations, i.e. p.Arg60Pro, p.Tyr65*, p.Val112Asp, p.Arg116*, p.Gly124Asp, p.Pro193Ser, p.Gln238*, p.Gln456*, p.Thr276Lys, and p.Gly311Arg, in addition to two new acceptor splice-site mutations 685-1G > A and c.954_956 delCTT. The amniotic fluid samples revealed two carrier fetuses with heterozygous monoallelic mutations, and two affected fetuses had the homozygous biallelic mutations. In conclusion, the current study sheds light on the underlying ARSA gene defect, with an expansion of the mutation spectrum. To our knowledge, this is the first molecular study of MLD among the Egyptian population.