Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India.

BACKGROUND: Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India. RESULTS: 903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood. CONCLUSION: We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

Newborn Screening for 6 Lysosomal Storage Disorders in China.

Importance: Newborn screening (NBS) for lysosomal storage disorders (LSDs) is becoming an increasing concern in public health. However, the birth prevalence of these disorders is rarely reported in the Chinese population, and subclinical forms of diseases among patients identified by NBS have not been evaluated. Objective: To evaluate the birth prevalence of the 6 LSDs in the Shanghai population and determine subclinical forms based on clinical, biochemical, and genetic characteristics. Design, Setting, and Participants: This cohort study included 50 108 newborns recruited from 41 hospitals in Shanghai between January and December 2021 who were screened for 6 LSDs using tandem mass spectrometry (MS/MS). Participants with screen-positive results underwent molecular and biochemical tests and clinical assessments. Data were analyzed from January 2021 through October 2022. Exposures: All participants were screened for Gaucher, acid sphingomyelinase deficiency (ASMD), Krabbe, mucopolysaccharidosis type I, Fabry, and Pompe diseases using dried blood spots. Main Outcomes and Measures: Primary outcomes were the birth prevalence and subclinical forms of the 6 LSDs in the Shanghai population. Disease biomarker measurements, genetic testing, and clinical analysis were used to assess clinical forms of LSDs screened. Results: Among 50 108 newborns (26 036 male [52.0%]; mean [SD] gestational age, 38.8 [1.6] weeks), the mean (SD) birth weight was 3257 (487) g. The MS/MS-based NBS identified 353 newborns who were positive. Of these, 27 newborns (7.7%) were diagnosed with 1 of 6 LSDs screened, including 2 newborns with Gaucher, 5 newborns with ASMD, 9 newborns with Krabbe, 8 newborns with Fabry, and 3 newborns with Pompe disease. The combined birth prevalence of LSDs in Shanghai was 1 diagnosis in 1856 live births, with Krabbe disease the most common (1 diagnosis/5568 live births), followed by Fabry disease (1 diagnosis/6264 live births), and ASMD (1 diagnosis/10 022 live births). Biochemical, molecular, and clinical analysis showed that early-onset clinical forms accounted for 3 newborns with positive results (11.1%), while later-onset forms represented nearly 90% of diagnoses (24 newborns [88.9%]). Conclusions and Relevance: In this study, the combined birth prevalence of the 6 LSDs in Shanghai was remarkably high. MS/MS-based newborn screening, combined with biochemical and molecular genetic analysis, successfully identified and characterized newborns who were screen-positive, which may assist with parental counseling and management decisions.

Unraveling the link between neuropathy target esterase NTE/SWS, lysosomal storage diseases, inflammation, abnormal fatty acid metabolism, and leaky brain barrier.

Mutations in Drosophila Swiss cheese (SWS) gene or its vertebrate orthologue neuropathy target esterase (NTE) lead to progressive neuronal degeneration in flies and humans. Despite its enzymatic function as a phospholipase is well established, the molecular mechanism responsible for maintaining nervous system integrity remains unclear. In this study, we found that NTE/SWS is present in surface glia that forms the blood-brain barrier (BBB) and that NTE/SWS is important to maintain its structure and permeability. Importantly, BBB glia-specific expression of Drosophila NTE/SWS or human NTE in the sws mutant background fully rescues surface glial organization and partially restores BBB integrity, suggesting a conserved function of NTE/SWS. Interestingly, sws mutant glia showed abnormal organization of plasma membrane domains and tight junction rafts accompanied by the accumulation of lipid droplets, lysosomes, and multilamellar bodies. Since the observed cellular phenotypes closely resemble the characteristics described in a group of metabolic disorders known as lysosomal storage diseases (LSDs), our data established a novel connection between NTE/SWS and these conditions. We found that mutants with defective BBB exhibit elevated levels of fatty acids, which are precursors of eicosanoids and are involved in the inflammatory response. Also, as a consequence of a permeable BBB, several innate immunity factors are upregulated in an age-dependent manner, while BBB glia-specific expression of NTE/SWS normalizes inflammatory response. Treatment with anti-inflammatory agents prevents the abnormal architecture of the BBB, suggesting that inflammation contributes to the maintenance of a healthy brain barrier. Considering the link between a malfunctioning BBB and various neurodegenerative diseases, gaining a deeper understanding of the molecular mechanisms causing inflammation due to a defective BBB could help to promote the use of anti-inflammatory therapies for age-related neurodegeneration.

Advances in Immune Tolerance Induction in Enzyme Replacement Therapy.

Inborn errors of metabolism (IEMs) are a group of genetic diseases that occur due to the either deficiency of an enzyme involved in a metabolic/biochemical pathway or other disturbances in the metabolic pathway including transport protein or activator protein deficiencies, cofactor deficiencies, organelle biogenesis, maturation or trafficking problems. These disorders are collectively significant due to their substantial impact on both the well-being and survival of affected individuals. In the quest for effective treatments, enzyme replacement therapy (ERT) has emerged as a viable strategy for patients with many of the lysosomal storage disorders (LSD) and enzyme substitution therapy in the rare form of the other inborn errors of metabolism including phenylketonuria and hypophosphatasia. However, a major challenge associated with enzyme infusion in patients with these disorders, mainly LSD, is the development of high antibody titres. Strategies focusing on immunomodulation have shown promise in inducing immune tolerance to ERT, leading to improved overall survival rates. The implementation of immunomodulation concurrent with ERT administration has also resulted in a decreased occurrence of IgG antibody development compared with cases treated solely with ERT. By incorporating the knowledge gained from current approaches and analysing the outcomes of immune tolerance induction (ITI) modalities from clinical and preclinical trials have demonstrated significant improvement in the efficacy of ERT. In this comprehensive review, the progress in ITI modalities is assessed, drawing insights from both clinical and preclinical trials. The focus is on evaluating the advancements in ITI within the context of IEM, specifically addressing LSDs managed through ERT.

Clinical investigator perspectives on patient outcomes in children with neuronopathic mucopolysaccharidosis II during intrathecal idursulfase-IT treatment.

BACKGROUND: Mucopolysaccharidosis II (MPS II) is a rare lysosomal storage disease characterized by iduronate-2-sulfatase gene (IDS) deficiency and downstream glycosaminoglycan accumulation. Two-thirds of patients present with neuronopathic disease and evaluating cognitive function in these patients is challenging owing to limitations of currently available tests. During the clinical development of intrathecal idursulfase (idursulfase-IT), regulatory authorities requested qualitative data to further understand the neurocognitive changes observed by the investigators through the clinical trials. RESULTS: This qualitative study consisted of semi-structured interviews with all nine of the principal investigators who participated in the idursulfase-IT phase 2/3 (NCT02055118) and extension (NCT02412787) trials. These investigators enrolled the 56 patients with neuronopathic MPS II who qualified for the extension phase of the trial. The investigators were asked to rate the disease status of their patients. Of the 56 patients, 49 (88%) were rated as having disease that was improved/improving, stabilized or slowing progression compared with the expected outcomes with no treatment. Three patients were rated as worsening, while the remaining four patients were considered to have slowing progression or worsening disease. Similar results were demonstrated for patients aged from 3 to under 6 years at baseline, with 33 of 39 patients (85%) rated as having disease that was improved/improving, stabilized or slowing progression. Of the seven patients rated with slowing progression/worsening or worsening disease, five of them had an IDS variant other than missense, while two had a missense class variant. All the assigned improved/improving ratings were in patients receiving idursulfase-IT from the start of the phase 2/3 trial. Moreover, patients under 3 years of age at baseline were all rated as either improved/improving or stabilized disease. In a blinded review of patient profiles, investigators were requested to assign a disease status rating to 18 patients with large IDS deletions; 67% of these patients were rated as improved/improving or stabilized disease. CONCLUSIONS: This qualitative analysis provides a snapshot of clinicians' considerations when evaluating treatment in patients with neuronopathic MPS II, compared with the expected decline in cognitive function in the absence of treatment. The results highlight the importance of robust assessment tools in treatment evaluation.

Alterations in Proteostasis Mechanisms in Niemann-Pick Type C Disease.

Niemann-Pick Type C (NPC) represents an autosomal recessive disorder with an incidence rate of 1 in 150,000 live births, classified within lysosomal storage diseases (LSDs). The abnormal accumulation of unesterified cholesterol characterizes the pathophysiology of NPC. This phenomenon is not unique to NPC, as analogous accumulations have also been observed in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. Interestingly, disturbances in the folding of the mutant protein NPC1 I1061T are accompanied by the aggregation of proteins such as hyperphosphorylated tau, α-synuclein, TDP-43, and ß-amyloid peptide. These accumulations suggest potential disruptions in proteostasis, a regulatory process encompassing four principal mechanisms: synthesis, folding, maintenance of folding, and protein degradation. The dysregulation of these processes leads to excessive accumulation of abnormal proteins that impair cell function and trigger cytotoxicity. This comprehensive review delineates reported alterations across proteostasis mechanisms in NPC, encompassing changes in processes from synthesis to degradation. Additionally, it discusses therapeutic interventions targeting pharmacological facets of proteostasis in NPC. Noteworthy among these interventions is valproic acid, a histone deacetylase inhibitor (HDACi) that modulates acetylation during NPC1 synthesis. In addition, various therapeutic options addressing protein folding modulation, such as abiraterone acetate, DHBP, calnexin, and arimoclomol, are examined. Additionally, treatments impeding NPC1 degradation, exemplified by bortezomib and MG132, are explored as potential strategies. This review consolidates current knowledge on proteostasis dysregulation in NPC and underscores the therapeutic landscape targeting diverse facets of this intricate process.

Rapid and long-lasting efficacy of high-dose ambroxol therapy for neuronopathic Gaucher disease: A case report and literature review.

Gaucher disease (GD) is a lysosomal storage disorder caused by a deficiency in the GBA1-encoded enzyme, ß-glucocerebrosidase. Enzyme replacement therapy is ineffective for neuronopathic Gaucher disease (nGD). High-dose ambroxol has been administered as an alternative treatment for a group of patients with nGD. However, little is known about the clinical indication and the long-term outcome of patients after ambroxol therapy. We herein report a case of a female patient who presented with a progressive disease of GD type 2 from 11 months of age and had the pathogenic variants of p.L483P (formerly defined as p.L444P) and p.R502H (p.R463H) in GBA1. A combined treatment of imiglucerase with ambroxol started improving the patient's motor activity in 1 week, while it kept the long-lasting effect of preventing the deteriorating phenotype for 30 months. A literature review identified 40 patients with nGD, who had received high-dose ambroxol therapy. More than 65% of these patients favorably responded to the molecular chaperone therapy, irrespective of p.L483P homozygous, heterozygous or the other genotypes. These results highlight the long-lasting effect of ambroxol-based chaperone therapy for patients with an expanding spectrum of mutations in GBA1.

Modeling Lysosomal Storage Disorders in an Innovative Way: Establishment and Characterization of Stem Cell Lines from Human Exfoliated Deciduous Teeth of Mucopolysaccharidosis Type II Patients.

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.

Lack of SPNS1 results in accumulation of lysolipids and lysosomal storage disease in mouse models.

Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1-KO cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global KO of Spns1 caused embryonic lethality between E12.5 and E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC), and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1-KO mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K/AKT signaling pathway. Furthermore, we identified 3 human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.

Drug Repurposing and Lysosomal Storage Disorders: A Trick to Treat.

Rare diseases, or orphan diseases, are defined as diseases affecting a small number of people compared to the general population. Among these, we find lysosomal storage disorders (LSDs), a cluster of rare metabolic diseases characterized by enzyme mutations causing abnormal glycolipid storage. Drug repositioning involves repurposing existing approved drugs for new therapeutic applications, offering advantages in cost, time savings, and a lower risk of failure. We present a comprehensive analysis of existing drugs, their repurposing potential, and their clinical implications in the context of LSDs, highlighting the necessity of mutation-specific approaches. Our review systematically explores the landscape of drug repositioning as a means to enhance LSDs therapies. The findings advocate for the strategic repositioning of drugs, accentuating its role in expediting the discovery of effective treatments. We conclude that drug repurposing represents a viable pathway for accelerating therapeutic discovery for LSDs, emphasizing the need for the careful evaluation of drug efficacy and toxicity in disease-specific contexts.

Leukodystrophy with Macrocephaly, Refractory Epilepsy, and Severe Hyponatremia-The Neonatal Type of Alexander Disease.

INTRODUCTION: Alexander disease (AxD) is a rare neurodegenerative condition that represents the group of leukodystrophies. The disease is caused by GFAP mutation. Symptoms usually occur in the infantile age with macrocephaly, developmental deterioration, progressive quadriparesis, and seizures as the most characteristic features. In this case report, we provide a detailed clinical description of the neonatal type of AxD. METHOD: Next-Generation Sequencing (NGS), including a panel of 49 genes related to Early Infantile Epileptic Encephalopathy (EIEE), was carried out, and then Whole Exome Sequencing (WES) was performed on the proband's DNA extracted from blood. CASE DESCRIPTION: In the first weeks of life, the child presented with signs of increased intracranial pressure, which led to ventriculoperitoneal shunt implementation. Recurrent focal-onset motor seizures with secondary generalization occurred despite phenobarbital treatment. Therapy was modified with multiple anti-seizure medications. In MRI contrast-enhanced lesions in basal ganglia, midbrain and cortico-spinal tracts were observed. During the diagnostic process, GLUT-1 deficiency, lysosomal storage disorders, organic acidurias, and fatty acid oxidation defects were excluded. The NGS panel of EIEE revealed no abnormalities. In WES analysis, GFAP missense heterozygous variant NM_002055.5: c.1187C>T, p.(Thr396Ile) was detected, confirming the diagnosis of AxD. CONCLUSION: AxD should be considered in the differential diagnosis in all neonates with progressive, intractable seizures accompanied by macrocephaly.

SNX8 enables lysosome reformation and reverses lysosomal storage disorder.

Lysosomal Storage Disorders (LSDs), which share common phenotypes, including enlarged lysosomes and defective lysosomal storage, are caused by mutations in lysosome-related genes. Although gene therapies and enzyme replacement therapies have been explored, there are currently no effective routine therapies against LSDs. During lysosome reformation, which occurs when the functional lysosome pool is reduced, lysosomal lipids and proteins are recycled to restore lysosome functions. Here we report that the sorting nexin protein SNX8 promotes lysosome tubulation, a process that is required for lysosome reformation, and that loss of SNX8 leads to phenotypes characteristic of LSDs in human cells. SNX8 overexpression rescued features of LSDs in cells, and AAV-based delivery of SNX8 to the brain rescued LSD phenotypes in mice. Importantly, by screening a natural compound library, we identified three small molecules that enhanced SNX8-lysosome binding and reversed LSD phenotypes in human cells and in mice. Altogether, our results provide a potential solution for the treatment of LSDs.

What should rheumatologists know about Gaucher disease and Fabry disease? Connecting the dots for an overview.

Gaucher and Fabry diseases are lysosomal storage disorders in which deficient enzyme activity leads to pathological accumulation of sphingolipids. These diseases have a broad phenotypic presentation. Musculoskeletal symptoms and pain complaints are frequently reported by patients. Thus, rheumatologists can be contacted by these patients, contributing to the correct diagnosis, earlier indication of appropriate treatment and improvement of their prognosis. This review describes important concepts about Gaucher and Fabry diseases that rheumatologists should understand to improve patients' quality of life and change the natural history of these diseases.

Investigation on lysosomal accumulation by a quantitative analysis of 2D phase-maps in digital holography microscopy.

Lysosomes are the terminal end of catabolic pathways in the cell, as well as signaling centers performing important functions such as the recycling of macromolecules, organelles, and nutrient adaptation. The importance of lysosomes in human health is supported by the fact that the deficiency of most lysosomal genes causes monogenic diseases called as a group Lysosomal Storage Diseases (LSDs). A common phenotypic hallmark of LSDs is the expansion of the lysosomal compartment that can be detected by using conventional imaging methods based on immunofluorescence protocols or overexpression of tagged lysosomal proteins. These methods require the alteration of the cellular architecture (i.e., due to fixation methods), can alter the behavior of cells (i.e., by the overexpression of proteins), and require sample preparation and the accurate selection of compatible fluorescent markers in relation to the type of analysis, therefore limiting the possibility of characterizing cellular status with simplicity. Therefore, a quantitative and label-free methodology, such as Quantitative Phase Imaging through Digital Holographic (QPI-DH), for the microscopic imaging of lysosomes in health and disease conditions may represent an important advance to study and effectively diagnose the presence of lysosomal storage in human disease. Here we proof the effectiveness of the QPI-DH method in accomplishing the detection of the lysosomal compartment using mouse embryonic fibroblasts (MEFs) derived from a Mucopolysaccharidosis type III-A (MSP-IIIA) mouse model, and comparing them with wild-type (WT) MEFs. We found that it is possible to identify label-free biomarkers able to supply a first pre-screening of the two populations, thus showing that QPI-DH can be a suitable candidate to surpass fluorescent drawbacks in the detection of lysosomes dysfunction. An appropriate numerical procedure was developed for detecting and evaluate such cellular substructures from in vitro cells cultures. Results reported in this study are encouraging about the further development of the proposed QPI-DH approach for such type of investigations about LSDs.

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.

Prenatal Testing for Variants in Genes Associated with Hereditary Cancer Risk: Laboratory Experience and Considerations.

Prenatal molecular genetic testing for familial variants that cause inherited disorders has been performed for decades and is accepted as standard of care. However, the spectrum of genes considered for prenatal testing is expanding because of genetic testing for hereditary cancer risk (HCR) and inclusion of conditions with associated cancer risk in carrier screening panels. A few of these disorders, such as ataxia telangiectasia and Bloom syndrome, include increased cancer risk as part of the phenotype, already meet professional guidelines for prenatal testing, and may be associated with increased cancer risk in heterozygous carriers. In addition, recent studies implicate heterozygosity for variants in lysosomal storage disease genes in HCR etiology. Currently, there is no specific professional guidance regarding prenatal testing for HCR. To determine the prevalence of such testing, we reviewed 1345 consecutive prenatal specimens received in our laboratory for familial variant-specific testing and identified 65 (4.8%) with a known or likely HCR component, plus 210 (15.6%) for lysosomal storage disease. These specimens were classified into five distinct categories for clarity and to enable evaluation. Our experience assessing prenatal specimens for variants associated with HCR, with or without a constitutional phenotype, provides metrics for and contributes to the points to consider in prenatal testing for HCR.

Innate immune sensing of lysosomal dysfunction drives multiple lysosomal storage disorders.

Lysosomal storage disorders (LSDs), which are characterized by genetic and metabolic lysosomal dysfunctions, constitute over 60 degenerative diseases with considerable health and economic burdens. However, the mechanisms driving the progressive death of functional cells due to lysosomal defects remain incompletely understood, and broad-spectrum therapeutics against LSDs are lacking. Here, we found that various gene abnormalities that cause LSDs, including Hexb, Gla, Npc1, Ctsd and Gba, all shared mutual properties to robustly autoactivate neuron-intrinsic cGAS-STING signalling, driving neuronal death and disease progression. This signalling was triggered by excessive cytoplasmic congregation of the dsDNA and DNA sensor cGAS in neurons. Genetic ablation of cGAS or STING, digestion of neuronal cytosolic dsDNA by DNase, and repair of neuronal lysosomal dysfunction alleviated symptoms of Sandhoff disease, Fabry disease and Niemann-Pick disease, with substantially reduced neuronal loss. We therefore identify a ubiquitous mechanism mediating the pathogenesis of a variety of LSDs, unveil an inherent connection between lysosomal defects and innate immunity, and suggest a uniform strategy for curing LSDs.

Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease.

The ceroid lipofuscinosis neuronal 1 (CLN1) disease, formerly called infantile neuronal ceroid lipofuscinosis, is a fatal hereditary neurodegenerative lysosomal storage disorder. This disease is caused by loss-of-function mutations in the CLN1 gene, encoding palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes depalmitoylation of S-palmitoylated proteins for degradation and clearance by lysosomal hydrolases. Numerous proteins, especially in the brain, require dynamic S-palmitoylation (palmitoylation-depalmitoylation cycles) for endosomal trafficking to their destination. While 23 palmitoyl-acyl transferases in the mammalian genome catalyze S-palmitoylation, depalmitoylation is catalyzed by thioesterases such as PPT1. Despite these discoveries, the pathogenic mechanism of CLN1 disease has remained elusive. Here, we report that in the brain of Cln1-/- mice, which mimic CLN1 disease, the mechanistic target of rapamycin complex-1 (mTORC1) kinase is hyperactivated. The activation of mTORC1 by nutrients requires its anchorage to lysosomal limiting membrane by Rag GTPases and Ragulator complex. These proteins form the lysosomal nutrient sensing scaffold to which mTORC1 must attach to activate. We found that in Cln1-/- mice, two constituent proteins of the Ragulator complex (vacuolar (H+)-ATPase and Lamtor1) require dynamic S-palmitoylation for endosomal trafficking to the lysosomal limiting membrane. Intriguingly, Ppt1 deficiency in Cln1-/- mice misrouted these proteins to the plasma membrane disrupting the lysosomal nutrient sensing scaffold. Despite this defect, mTORC1 was hyperactivated via the IGF1/PI3K/Akt-signaling pathway, which suppressed autophagy contributing to neuropathology. Importantly, pharmacological inhibition of PI3K/Akt suppressed mTORC1 activation, restored autophagy, and ameliorated neurodegeneration in Cln1-/- mice. Our findings reveal a previously unrecognized role of Cln1/Ppt1 in regulating mTORC1 activation and suggest that IGF1/PI3K/Akt may be a targetable pathway for CLN1 disease.

Impact of COVID-19 pandemic on healthcare delivery for lysosomal storage disorders at a tertiary care public hospital in Mumbai.

Introduction: Management of lysosomal storage disorders (LSDs) requires periodic visits for medical surveillance and hospitalizations. Management of LSDs may have been adversely impacted during the COVID-19 pandemic. Objective: To identify the factors impacting health care for patients with LSDs during the COVID-19 pandemic. Methods: An observational study was conducted in Mumbai comparing infusion practices and reasons for missed infusions for 15 months before March 2020 versus two phases during the pandemic (April 2020-March 2021 and April 2021-March 2022) in patients receiving intravenous enzyme replacement therapy (ERT) and on oral substrate reduction therapy (SRT). Results: Fifteen patients with LSDs were enrolled. Before the pandemic, 6/13 (46%) were receiving ERT at the study site, 4/13 (31%) at a local hospital, and 3/13 (23%) at home; two were on SRT. The median distance traveled for receiving ERT was 37 km, and 4.4 infusions/patient were missed. From April 2020 to March 2021, two more patients opted for home ERT infusions. The median distance traveled for receiving ERT was 37 km, and 11.6 infusions/patient were missed. From April 2021 to March 2022, one more patient opted for home ERT infusions. The median distance traveled for receiving ERT was 7 km, and 5.6 infusions/patient were missed. The pandemic also affected SRT compliance adversely. For all patients, the cause of disrupted treatment was travel curbs (69%) and fear of getting COVID-19 infection (38%). Conclusions: Treatment of LSDs was disrupted during the pandemic, with an increase in missed ERT infusions and SRT doses.

Update on leukodystrophies and developing trials.

Leukodystrophies are a heterogeneous group of rare genetic disorders primarily affecting the white matter of the central nervous system. These conditions can present a diagnostic challenge, requiring a comprehensive approach that combines clinical evaluation, neuroimaging, metabolic testing, and genetic testing. While MRI is the main tool for diagnosis, advances in molecular diagnostics, particularly whole-exome sequencing, have significantly improved the diagnostic yield. Timely and accurate diagnosis is crucial to guide symptomatic treatment and assess eligibility to participate in clinical trials. Despite no specific cure being available for most leukodystrophies, gene therapy is emerging as a potential treatment avenue, rapidly advancing the therapeutic prospects in leukodystrophies. This review will explore diagnostic and therapeutic strategies for leukodystrophies, with particular emphasis on new trials.