Objective: To screen high risk children for lysosomal storage diseases (LSD) in southern China and analyze the spectrum characteristics of LSD in this region. Methods: A cross-sectional study was conducted. A total of 7 435 children at high risk of LSD were screened at Guangzhou Women and Children's Medical Center, Guangzhou Medical University from January 2009 to December 2024. The activities of 22 lysosomal enzymes from peripheral blood leukocytes or plasma were measured by fluorescence or colorimetric assays with synthetic substrates to screen for 24 LSD subtypes. Results: Among the 7 435 high risk children, 759 children were diagnosed with LSD (10.2%). The diagnosed cases included 506 males and 253 females, with an age at diagnosis of 3.0 (2.5, 5.5) years. The common disease types were mucopolysaccharidosis (MPS) (390 cases (51.4%)), sphingolipidoses (269 cases (35.4%)), glycogen storage disease (62 cases (8.2%)), and mucolipidosis types Ⅱ and Ⅲ (29 cases (3.8%)). Among the positive cases, 21 disease subtypes were identified. The 5 frequent subtypes, in descending order, were MPS type Ⅱ (197 cases (26.0%)), Gaucher disease (111 cases (14.6%)), MPS type ⅣA (87 cases (11.5%)), glycogen storage disease type Ⅱ (62 cases (8.2%)), and metachromatic leukodystrophy (MLD) (49 cases (6.5%)). The rarest subtypes were mannosidosis, multiple sulfatase deficiency and Wolman disease, each with 1 case (0.1%). Conclusions: Enzyme activity screening is essential for diagnosing high risk children with LSD. In Southern China, the most common LSD subtypes are MPS Ⅱ, Gaucher disease, MPS ⅣA, glycogen storage disease type Ⅱ, and MLD, while mannosidosis, multiple sulfatase deficiency and Wolman disease are the rarest. 目的： 筛查中国南部地区溶酶体贮积症（LSD）高危患儿，分析该地区LSD疾病谱特征。 方法： 横断面研究。对2009年1月至2024年12月在广州医科大学附属妇女儿童医疗中心就诊的7 435例LSD高危患儿进行筛查，利用人工合成底物荧光法或比色分析法检测外周血白细胞或血浆中22种溶酶体酶的活性，筛查24种LSD亚型。 结果： 7 435例高危患儿中共确诊LSD患儿759例（10.2%），其中男506例、女253例，确诊年龄3.0（2.5，5.5）岁；常见的疾病类型为黏多糖贮积症（MPS）［390例（51.4%）］、鞘脂贮积病［269例（35.4%）］、糖原贮积病［62例（8.2%）］及黏脂贮积症Ⅱ和Ⅲ型［29例（3.8%）］。阳性病例中共发现21种疾病亚型，常见的5种依次为MPS Ⅱ型［197例（26.0%）］、戈谢病［111例（14.6%）］、MPS ⅣA型［87例（11.5%）］、糖原贮积病Ⅱ型［62例（8.2%）］和异染性脑白质营养不良［49例（6.5%）］，罕见的是甘露糖贮积症、多种硫酸酯酶缺乏症和Wolman病［各1例（0.1%）］。 结论： LSD高危患儿需要通过酶活性筛查进行确诊，中国南部地区常见的LSD亚型依次为 MPS Ⅱ型、戈谢病、MPS ⅣA型、糖原贮积病Ⅱ型及异染性脑白质营养不良，罕见的是甘露糖贮积症、多种硫酸酯酶缺乏症和Wolman病。.

Despite their importance, rare diseases' impact on patients and families is understudied. This is particularly true for ultrarare disorders, such as multiple sulfatase deficiency (MSD), a pediatric neurodegenerative disorder. To address this gap, we captured caregiver perspectives on how multiple sulfatase deficiency affects their child, themselves, and their families regarding adaptive behaviors and health-related quality of life.Overall, 19 multiple sulfatase deficiency caregivers participated in assessments capturing health outcomes related to daily functional abilities (Vineland Adaptive Behavior Scale-Third Edition [VABS-3]: n = 19), child health-related quality of life (Caregiver Priorities and Child Health Index of Life with Disabilities: n = 12; Pediatric Quality of Life Inventory-generic core scales: n = 13), and caregiver health-related quality of life (Pediatric Quality of Life Inventory-family impact module: n = 12; Traumatic Brain Injury Caregiver Quality of Life: n = 15). The Pediatric Quality of Life Inventory-family impact module results were compared to a data set from metachromatic leukodystrophy (n = 30), a rare disease with an overlapping sulfatase deficiency.The Vineland Adaptive Behavior Scale-Third Edition captured global impairment across domains in multiple sulfatase deficiency. Despite these functional limitations, the Caregiver Priorities and Child Health Index of Life with Disabilities and Pediatric Quality of Life Inventory-generic core scales captured relative preservation of health-related quality of life, especially related to emotional well-being. Compared with the Pediatric Quality of Life Inventory-generic core scales, the Caregiver Priorities and Child Health Index of Life with Disabilities captured a broader spectrum of health-related quality of life across all domains and caregivers' top priorities in disease management and care coordination. The health-related quality of life of caregivers was severely impacted, with caregivers reporting profound feelings of grief and entrapment. Additionally, there was a similar caregiver burden between multiple sulfatase deficiency and metachromatic leukodystrophy.Our results will help inform psychosocial outcome measures for rare disease families and patient-centered endpoints in impending multiple sulfatase deficiency clinical trials.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplantion (HSCT) with lentiviral (LV)-based gene therapy has great potential to treat MLD. Achieving the optimal balance between high enzyme production for therapeutic efficacy and maintaining a low vector copy number (VCN) is crucial. Insufficient enzyme levels can lead to the progression of motor symptoms, undermining treatment goals. Conversely, elevated VCN increases the risk of genotoxicity, which poses safety concerns, and contributes to higher production costs, making the therapy less accessible. Striking this balance is essential to maximize clinical benefit while minimizing risks and costs. To address this need, we increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human hematopoietic stem cells (HSCs) with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4× more ARSA activity than the currently US and European Union (EU)-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa-knockout (KO) MLD mice transplanted with Arsa-KO bone marrow (BM) cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching wild-type (WT) mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating MLD patients.

Glycosaminoglycans (GAGs) accumulate in patients with mucopolysaccharidoses (MPS), multiple sulfatase deficiency, and mucolipidoses; measurement of total GAGs and the specific excretion pattern by fractionation can aid in their diagnosis. Since 1993, the College of American Pathologists with the American College of Medical Genetics and Genomics has offered proficiency testing (PT) for urine GAG analysis accessible to laboratories worldwide. Data from PT surveys administered from 2016 to 2022 were assessed for trends in participation and methodological platforms used, as well as analytical performance and diagnostic accuracy by method and disease. The number of participating laboratories declined from 43 in 2016 to 28 in 2022. Fourteen urine samples with clinical vignettes were distributed; the median of correct diagnoses reported was 91.5% (range: 74%-100%). The best performing methodologies for total GAG analysis and fractionation were dimethylmethylene blue-dye-binding spectrophotometric assay and liquid chromatography-tandem mass spectrometry, respectively. MPS IV samples posed the greatest diagnostic challenge, whereas the overall false-positive rate was low. Based on the data reviewed, best patient care for those at risk of an MPS is achieved by a combination of total GAG analysis and GAG fractionation. Development of liquid-chromatography-tandem-mass-spectrometry-based methods for quantitative, differentiated GAG analysis or disease-specific GAG-derived nonreducing end oligosaccharide fragments in combination with multiplexed lysosomal enzyme assays will likely improve diagnostic accuracy. The decline of laboratories participating in PT is concerning because MPS are increasingly included in newborn screening programs and urinary GAGs can be used to monitor the effectiveness of new therapies.

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.