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The cytoplasmic peptide:N-glycanase (NGLY1) is ubiquitously expressed and functions as a de-N-glycosylating enzyme that degrades misfolded N-glycosylated proteins. NGLY1 deficiency due to biallelic loss-of-function NGLY1 variants is an ultrarare autosomal recessive deglycosylation disorder with multisystemic involvement; the neurological manifestations represent the main disease burden. Currently, there is no treatment for this disease. To develop a gene therapy, we first characterized a tamoxifen-inducible Ngly1-knockout (iNgly1) C57BL/6J mouse model, which exhibited symptoms recapitulating human disease, including elevation of the biomarker GlcNAc-Asn, motor deficits, kyphosis, Purkinje cell loss, and gait abnormalities. We packaged a codon-optimized human NGLY1 transgene cassette into 2 adeno-associated virus (AAV) capsids, AAV9 and AAV.PHPeB. Systemic administration of the AAV.PHPeB vector to symptomatic iNgly1 mice corrected multiple disease features at 8 weeks after treatment. Furthermore, another cohort of AAV.PHPeB-treated iNgly1 mice were monitored over a year and showed near-complete normalization of the neurological aspects of the disease phenotype, demonstrating the durability of gene therapy. Our data suggested that brain-directed NGLY1 gene replacement via systemic delivery is a promising therapeutic strategy for NGLY1 deficiency. Although the superior CNS tropism of AAV.PHPeB vector does not translate to primates, emerging AAV capsids with enhanced primate CNS tropism will enable future translational studies.

Cytosolic peptide: N-glycanase (PNGase/NGLY1 in mammals) is an amidase (EC:3.5.1.52) widely conserved in eukaryotes. It catalyzes the removal of N-glycans on glycoproteins, converting N-glycosylated Asn into Asp residues. This enzyme also plays a role in the quality control system for nascent glycoproteins. Since the identification of a patient with an autosomal recessive genetic disorder caused by NGLY1 gene dysfunction, known as NGLY1 deficiency or NGLY1 congenital disorder of deglycosylation (OMIM: 615273), in 2012, more than 100 cases have been reported worldwide. NGLY1 deficiency is characterized by a wide array of symptoms, such as global mental delay, intellectual disability, abnormal electroencephalography findings, seizure, movement disorder, hypolacrima or alacrima, and liver dysfunction. Unfortunately, no effective therapeutic treatments for this disease have been established. However, administration of adeno-associated virus 9 (AAV9) vector harboring human NGLY1 gene to an NGLY1-deficient rat model (Ngly1-/- rat) by intracerebroventricular injection was found to drastically improve motor function defects. This observation indicated that early therapeutic intervention could alleviate various symptoms originating from central nervous system dysfunction in this disease. Therefore, there is a keen interest in the development of facile diagnostic methods for NGLY1 deficiency. This review summarizes the history of assay development for PNGase/NGLY1 activity, as well as the recent progress in the development of novel plate-based assay systems for NGLY1, and also discusses future perspectives., (© The Author(s) 2024. Published by Oxford University Press.)

The conserved SKN-1A/Nrf1 transcription factor regulates the expression of proteasome subunit genes and is essential for maintenance of adequate proteasome function in animal development, aging, and stress responses. Unusual among transcription factors, SKN-1A/Nrf1 is a glycoprotein synthesized in the endoplasmic reticulum (ER). N-glycosylated SKN-1A/Nrf1 exits the ER and is deglycosylated in the cytosol by the PNG-1/NGLY1 peptide:N-glycanase. Deglycosylation edits the protein sequence of SKN-1A/Nrf1 by converting N-glycosylated asparagine residues to aspartate, which is necessary for SKN-1A/Nrf1 transcriptional activation of proteasome subunit genes. Homozygous loss-of-function mutations in the peptide:N-glycanase (NGLY1) gene cause NGLY1 deficiency, a congenital disorder of deglycosylation. There are no effective treatments for NGLY1 deficiency. Since SKN-1A/Nrf1 is a major client of NGLY1, the resulting proteasome deficit contributes to NGLY1 disease. We sought to identify targets for mitigation of proteasome dysfunction in NGLY1 deficiency that might indicate new avenues for treatment. We isolated mutations that suppress the sensitivity to proteasome inhibitors caused by inactivation of the NGLY1 ortholog PNG-1 in Caenorhabditis elegans. We identified multiple suppressor mutations affecting 3 conserved genes: rsks-1, tald-1, and ent-4. We show that the suppressors act through a SKN-1/Nrf-independent mechanism and confer proteostasis benefits consistent with amelioration of proteasome dysfunction. ent-4 encodes an intestinal nucleoside/nucleotide transporter, and we show that restriction of nucleotide availability is beneficial, whereas a nucleotide-rich diet exacerbates proteasome dysfunction in PNG-1/NGLY1-deficient C. elegans. Our findings suggest that dietary or pharmacological interventions altering nucleotide availability have the potential to mitigate proteasome insufficiency in NGLY1 deficiency and other diseases associated with proteasome dysfunction., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yanagi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 μg of crude extract, which is the equivalent of 5 × 10 3  cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

NGLY1 deficiency is a genetic disease caused by biallelic mutations of the Ngly1 gene. Although epileptic seizure is one of the most severe symptoms in patients with NGLY1 deficiency, preclinical studies have not been conducted due to the lack of animal models for epileptic seizures in NGLY1 deficiency. Here, we observed the behaviors of male and female Ngly1 -/- mice by video monitoring and found that these mice exhibit spontaneous seizure-like behaviors. Gene expression analyses and enzyme immunoassay revealed significant decreases in oxytocin, a well-known neuropeptide, in the hypothalamus of Ngly1 -/- mice. Seizure-like behaviors in Ngly1 -/- mice were transiently suppressed by a single intranasal administration of oxytocin. These findings suggest the therapeutic potential of oxytocin for epileptic seizure in patients with NGLY1 deficiency and contribute to the clarification of the disease mechanism., (© 2024. The Author(s).)

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) catalyzes deglycosylation of N-glycans on glycoproteins. A genetic disorder caused by mutations in the NGLY1 gene leads to NGLY1 deficiency with symptoms including motor deficits and neurological problems. Effective therapies have not been established, though, a recent study used the administration of an adeno-associated viral vector expressing human NGLY1 to dramatically rescue motor functions in young Ngly1 -/- rats. Thus, early therapeutic intervention may improve symptoms arising from central nervous system dysfunction, and assay methods for measuring NGLY1 activity in biological samples are critical for early diagnostics. In this study, we established an assay system for plate-based detection of endogenous NGLY1 activity using a FRET-based probe. Using this method, we revealed significant changes in NGLY1 activity in rat brains during aging. This novel assay offers reliable disease diagnostics and provides valuable insights into the regulation of PNGase/NGLY1 activity in diverse organisms under different stress conditions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The cytosolic peptide:N-glycanase (PNGase; NGLY1 in human and PNG1 in budding yeast) is a deglycosylating enzyme widely conserved in eukaryotes. Initially, functional importance of this enzyme remained unknown as the png1Δ mutant in yeast did not exhibit any significant phenotypes. However, the discovery of NGLY1 deficiency, a rare genetic disorder with biallelic mutations in NGLY1 gene, prompted an intensification of research that has resulted in uncovering the significance of NGLY1 as well as the proteins under its influence that are involved in numerous cellular processes. A recent report by Rauscher et al. (Patient-derived gene and protein expression signatures of NGLY1 deficiency. J. Biochem. 2022; 171: 187-199) presented a comprehensive summary of transcriptome/proteome analyses of various cell types derived from NGLY1-deficient patients. The authors also provide a web application called 'NGLY1 browser', which will allow researchers to have access to a wealth of information on gene and protein expression signature for patients with NGLY1 deficiency., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: NGLY1-associated congenital disorder of deglycosylation (CDDG1: OMIM #615273) is a rare autosomal recessive disorder caused by a functional impairment of endoplasmic reticulum in degradation of glycoproteins. Neurocognitive dysfunctions have been documented in patients with CDDG1; however, deteriorating phenotypes of affected individuals remain elusive., Case Presentation: A Japanese boy with delayed psychomotor development showed ataxic movements from age 5 years and myoclonic seizures from age 12 years. Appetite loss, motor and cognitive decline became evident at age 12 years. Electrophysiological studies identified paroxysmal discharges on myoclonic seizure and a giant somatosensory evoked potential. Perampanel was effective for controlling myoclonic seizures. Exome sequencing revealed that the patient carried compound heterozygous variants in NGLY1, NM_018297.4: c.857G > A and c.-17_12del, which were inherited from mother and father, respectively. A literature review confirmed that myoclonic seizures were observed in 28.5% of patients with epilepsy. No other patients had progressive myoclonic epilepsy or cognitive decline in association with loss-of-function variations in NGLY1., Conclusion: Our data provides evidence that a group of patients with CDDG1 manifest slowly progressive myoclonic epilepsy and cognitive decline during the long-term clinical course., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest concerning the present study., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

NGLY1 deficiency is a rare congenital disorder of deglycosylation with a unique constellation of symptoms that include hypo- or alacrima, movement disorder, epilepsy, and severe intellectual disability (OMIM #615273). Here we report a patient with NGLY1 deficiency whose clinical presentation lacks many of the features associated with the disease and has a much milder intellectual disability than had been previously reported, expanding the phenotypic spectrum., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

NGLY1 deficiency, a rare disease with no effective treatment, is caused by autosomal recessive, loss-of-function mutations in the N-glycanase 1 (NGLY1) gene and is characterized by global developmental delay, hypotonia, alacrima, and seizures. We used a Drosophila model of NGLY1 deficiency to conduct an in vivo, unbiased, small molecule, repurposing screen of FDA-approved drugs to identify therapeutic compounds. Seventeen molecules partially rescued lethality in a patient-specific NGLY1 deficiency model, including multiple serotonin and dopamine modulators. Exclusive dNGLY1 expression in serotonin and dopamine neurons, in an otherwise dNGLY1 deficient fly, was sufficient to partially rescue lethality. Further, genetic modifier and transcriptomic data supports the importance of serotonin signaling in NGLY1 deficiency. Connectivity Map analysis identified glycogen synthase kinase 3 (GSK3) inhibition as a potential therapeutic mechanism for NGLY1 deficiency, which we experimentally validated with TWS119, lithium, and GSK3 knockdown. Strikingly, GSK3 inhibitors and a serotonin modulator rescued size defects in dNGLY1 deficient larvae upon proteasome inhibition, suggesting that these compounds act through NRF1, a transcription factor that is regulated by NGLY1 and regulates proteasome expression. This study reveals the importance of the serotonin pathway in NGLY1 deficiency, and serotonin modulators or GSK3 inhibitors may be effective therapeutics for this rare disease., Competing Interests: The authors have declared that no competing interests exist.

We delineated the phenotypic spectrum of epilepsy in individuals with NGLY1 deficiency from an international cohort. We collected detailed clinical and electroencephalographic data from 29 individuals with bi-allelic (likely) pathogenic variants in NGLY1 as part of an ongoing prospective natural history study. Participants were evaluated in-person at a single center and/or remotely. Historical medical records were reviewed. Published cases were included for comprehensive phenotyping. Of 29 individuals (mean 11.4 years, range 3-27 years), 17 (58.6%) participants had a history of epilepsy. Seizure onset was in early childhood (mean 43 months, range 2 months to 19 years). The most common seizure types were myoclonic and atonic. Epilepsy course was variable, but 35.2% (6/17) of participants with epilepsy achieved seizure freedom. The most common medications included levetiracetam, valproate, lamotrigine, and clobazam. Electroencephalogram (EEGs) were abnormal in 80% (12/15) of participants with or without epilepsy, although encephalopathy was uncommon. There was a trend in neurodevelopmental outcomes that participants with epilepsy had more developmental delays. In summary, epilepsy is common in NGLY1 deficiency. Over half of the participants had a history of epilepsy and nearly all had EEG abnormalities indicating an increased risk of epilepsy. This work expands the electroclinical phenotype of NGLY1 deficiency and supports a high clinical suspicion for seizures. Some of the more common seizure types (epileptic spasms, myoclonic, and atonic seizures) can be subtle and require counseling to ensure early recognition and treatment to ensure the best possible outcomes. Despite transient liver enzyme abnormalities in this disorder, hepatically metabolized medications were well tolerated., (© 2022 SSIEM.)

The cytosolic PNGase (peptide:N-glycanase), also known as peptide-N4-(N-acetyl-β-glucosaminyl)-asparagine amidase, is a well-conserved deglycosylation enzyme (EC 3.5.1.52) which catalyzes the non-lysosomal hydrolysis of an N(4)-(acetyl-β-d-glucosaminyl) asparagine residue (Asn, N) into a N-acetyl-β-d-glucosaminyl-amine and a peptide containing an aspartate residue (Asp, D). This enzyme (NGLY1) plays an essential role in the clearance of misfolded or unassembled glycoproteins through a process named ER-associated degradation (ERAD). Accumulating evidence also points out that NGLY1 deficiency can cause an autosomal recessive (AR) human genetic disorder associated with abnormal development and congenital disorder of deglycosylation. In addition, the loss of NGLY1 can affect multiple cellular pathways, including but not limited to NFE2L1 pathway, Creb1/Atf1-AQP pathway, BMP pathway, AMPK pathway, and SLC12A2 ion transporter, which might be the underlying reasons for a constellation of clinical phenotypes of NGLY1 deficiency. The current comprehensive review uncovers the NGLY1'ssdetailed structure and its important roles for participation in ERAD, involvement in CDDG and potential treatment for NGLY1 deficiency.

N -Glycanase 1 (NGLY1) is a cytosolic enzyme involved in removing N -linked glycans of misfolded N -glycoproteins and is considered to be a component of endoplasmic reticulum-associated degradation (ERAD). The 2012 identification of recessive NGLY1 mutations in a rare multisystem disorder has led to intense research efforts on the roles of NGLY1 in animal development and physiology, as well as the pathophysiology of NGLY1 deficiency. Here, we present a review of the NGLY1-deficient patient phenotypes, along with insights into the function of this gene from studies in rodent and invertebrate animal models, as well as cell culture and biochemical experiments. We will discuss critical processes affected by the loss of NGLY1, including proteasome bounce-back response, mitochondrial function and homeostasis, and bone morphogenetic protein (BMP) signaling. We will also cover the biologically relevant targets of NGLY1 and the genetic modifiers of NGLY1 deficiency phenotypes in animal models. Together, these discoveries and disease models have provided a number of avenues for preclinical testing of potential therapeutic approaches for this disease.

Substrate-derived biomarkers are necessary in slowly progressing monogenetic diseases caused by single-enzyme deficiencies to identify affected patients and serve as surrogate markers for therapy response. N-glycanase 1 (NGLY1) deficiency is an ultra-rare autosomal recessive disorder characterized by developmental delay, peripheral neuropathy, elevated liver transaminases, hyperkinetic movement disorder and (hypo)-alacrima. We demonstrate that N-acetylglucosamine-asparagine (GlcNAc-Asn; GNA), is the analyte most closely associated with NGLY1 deficiency, showing consistent separation in levels between patients and controls. GNA accumulation is directly linked to the absence of functional NGLY1, presenting strong potential for its use as a biomarker. In agreement, a quantitative liquid chromatography with tandem mass spectrometry assay, developed to assess GNA from 3 to 3000 ng/ml, showed that it is conserved as a marker for loss of NGLY1 function in NGLY1-deficient cell lines, rodents (urine, cerebrospinal fluid, plasma and tissues) and patients (plasma and urine). Elevated GNA levels differentiate patients from controls, are stable over time and correlate with changes in NGLY1 activity. GNA as a biomarker has the potential to identify and validate patients with NGLY1 deficiency, act as a direct pharmacodynamic marker and serve as a potential surrogate endpoint in clinical trials., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society.)

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Cytosolic peptide:N-glycanase (NGLY1 in mammals), a highly conserved enzyme in eukaryotes, catalyses the deglycosylation of N-glycans that are attached to glycopeptide/glycoproteins. In 2012, an autosomal recessive disorder related to the NGLY1 gene, which was referred to as NGLY1 deficiency, was reported. Since then, more than 100 patients have been identified. Patients with this disease exhibit various symptoms, including various motor deficits and other neurological problems. Effective therapeutic treatments for this disease, however, have not been established. Most recently, it was demonstrated that the intracerebroventricular administration of an adeno-associated virus 9 vector expressing human NGLY1 during the weaning period allowed some motor functions to be recovered in Ngly1-/- rats. This observation led us to hypothesize that a therapeutic intervention for improving these motor deficits or other neurological symptoms found in the patients might be possible. To achieve this, it is critical to establish robust and facile methods for assaying NGLY1 activity in biological samples, for the early diagnosis and evaluation of the therapeutic efficacy for the treatment of NGLY1 deficiency. In this mini review, we summarize progress made in the development of various assay methods for NGLY1 activity, as well as a recent progress in the identification of NGLY1 deficiency-specific biomarkers., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

NGLY1 deficiency is a rare recessive genetic disease caused by mutations in the NGLY1 gene which codes for N-glycanase 1 (NGLY1). Here, we report the generation of two gene corrected iPSC lines using a patient-derived iPSC line (NCATS-CL6103) that carried a homozygous p.R401X mutation in the NGLY1 gene. These lines contain either one (NCATS-CL6104) or two (NCATS-CL6105) CRISPR/Cas9 corrected alleles of NGLY1. This pair of NGLY1 mutation corrected iPSC lines can be used as a control for the NCATS-CL6103 which serves as a cell-based NGLY1 disease model for the study of the disease pathophysiology and evaluation of therapeutics under development., (Published by Elsevier B.V.)

N-glycanase 1 (NGLY1) deficiency is a rare inherited disorder characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, motor deficits, and other neurological symptoms. The underlying mechanisms of the NGLY1 phenotype are poorly understood, and no effective therapy is currently available. Similar to human patients, the rat model of NGLY1 deficiency, Ngly1-/-, shows developmental delay, movement disorder, somatosensory impairment, scoliosis, and learning disability. Here we show that single intracerebroventricular administration of AAV9 expressing human NGLY1 cDNA (AAV9-hNGLY1) to Ngly1-/- rats during the weaning period restored NGLY1 expression in the brain and spinal cord, concomitant with increased enzymatic activity of NGLY1 in the brain. hNGLY1 protein expressed by AAV9 was found predominantly in mature neurons, but not in glial cells, of Ngly1-/- rats. Strikingly, intracerebroventricular administration of AAV9-hNGLY1 normalized the motor phenotypes of Ngly1-/- rats assessed by the rota-rod test and gait analysis. The reversibility of motor deficits in Ngly1-/- rats by central nervous system (CNS)-restricted gene delivery suggests that the CNS is the primary therapeutic target organs for NGLY1 deficiency, and that the Ngly1-/- rat model may be useful for evaluating therapeutic treatments in pre-clinical studies.

NGLY1 deficiency is a recently described autosomal recessive disorder, involved in deglycosylation of proteins, and for that reason grouped as the congenital disorders of deglycosylation together with the lysosomal storage disorders. The typical phenotype is characterized by intellectual disability, liver malfunctioning, muscular hypotonia, involuntary movements, and decreased or absent tear production. Liver biopsy demonstrates vacuolar amorphous cytoplasmic storage material. NGLY1 deficiency is caused by bi-allelic variants in NGLY1 which catalyzes protein deglycosylation. We describe five patients from two families with NGLY1 deficiency due to homozygosity for two novel NGLY1 variants, and compare their findings to those of earlier reported patients. The typical features of the disorder are present in a limited way, and there is intra-familial variability. In addition in one of the families the muscle atrophy and posture abnormalities are marked. These can be explained either as variability of the phenotype or as sign of slowly progression of features as the present affected individuals are older than earlier reported patients., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

N-Glycanase 1 (NGLY1) deficiency is a congenital disorder caused by mutations in the NGLY1 gene. Because systemic Ngly1 -/- mice with a C57BL/6 (B6) background are embryonically lethal, studies on the mechanism of NGLY1 deficiency using mice have been problematic. In this study, B6-Ngly1 -/+ mice were crossed with Japanese wild mice-originated Japanese fancy mouse 1 (JF1) mice to produce viable F 2 Ngly1 -/- mice from (JF1×B6)F 1 Ngly1 -/+ mice. Systemic Ngly1 -/- mice with a JF1 mouse background were also embryonically lethal. Hybrid F1 Ngly1 -/- (JF1/B6F1) mice, however, showed developmental delay and motor dysfunction, similar to that in human patients. JF1/B6F1 Ngly1 -/- mice showed increased levels of plasma and urinary aspartylglycosamine, a potential biomarker for NGLY1 deficiency. JF1/B6F1 Ngly1 -/- mice are a useful isogenic animal model for the preclinical testing of therapeutic options and understanding the precise pathogenic mechanisms responsible for NGLY1 deficiency.

N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0 to 100% lethality. Association analysis on the lethality phenotype, as well as an evolutionary rate covariation analysis, generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2 ), a conserved ion transporter. Analyses in NGLY1 -/- mouse cells demonstrated that NKCC1 has an altered average molecular weight and reduced function. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function in NGLY1 deficiency patients., Competing Interests: DT, KO, EC, GM, JP, RP, KH, AZ, NC, CL, AR, CC No competing interests declared, (© 2020, Talsness et al.)

Mutations in human N-glycanase 1 (NGLY1) cause the first known congenital disorder of deglycosylation (CDDG). Patients with this rare disease, which is also known as NGLY1 deficiency, exhibit global developmental delay and other phenotypes including neuropathy, movement disorder, and constipation. NGLY1 is known to regulate proteasomal and mitophagy gene expression through activation of a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1). Loss of NGLY1 has also been shown to impair energy metabolism, but the molecular basis for this phenotype and its in vivo consequences are not well understood. Using a combination of genetic studies, imaging, and biochemical assays, here we report that loss of NGLY1 in the visceral muscle of the Drosophila larval intestine results in a severe reduction in the level of AMP-activated protein kinase α (AMPKα), leading to energy metabolism defects, impaired gut peristalsis, failure to empty the gut, and animal lethality. Ngly1-/- mouse embryonic fibroblasts and NGLY1 deficiency patient fibroblasts also show reduced AMPKα levels. Moreover, pharmacological activation of AMPK signaling significantly suppressed the energy metabolism defects in these cells. Importantly, the reduced AMPKα level and impaired energy metabolism observed in NGLY1 deficiency models are not caused by the loss of NFE2L1 activity. Taken together, these observations identify reduced AMPK signaling as a conserved mediator of energy metabolism defects in NGLY1 deficiency and suggest AMPK signaling as a therapeutic target in this disease., Competing Interests: The authors have declared that no competing interests exist.

N-glycanase 1 (NGLY1) deficiency, an autosomal recessive disease caused by mutations in the NGLY1 gene, is characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, movement disorders and other neurological phenotypes. Because of few animal models that recapitulate these clinical signatures, the mechanisms of the onset of the disease and its progression are poorly understood, and the development of therapies is hindered. In this study, we generated the systemic Ngly1-deficient rodent model, Ngly1-/- rats, which showed developmental delay, movement disorder, somatosensory impairment and scoliosis. These phenotypes in Ngly1-/- rats are consistent with symptoms in human patients. In accordance with the pivotal role played by NGLY1 in endoplasmic reticulum-associated degradation processes, cleaving N-glycans from misfolded glycoproteins in the cytosol before they can be degraded by the proteasome, loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins in the neurons of the central nervous system of Ngly1-/- rats. Histological analysis identified prominent pathological abnormalities, including necrotic lesions, mineralization, intra- and extracellular eosinophilic bodies, astrogliosis, microgliosis and significant loss of mature neurons in the thalamic lateral and the medial parts of the ventral posterior nucleus and ventral lateral nucleus of Ngly1-/- rats. Axonal degradation in the sciatic nerves was also observed, as in human subjects. Ngly1-/- rats, which mimic the symptoms of human patients, will be a useful animal model for preclinical testing of therapeutic options and understanding the detailed mechanisms of NGLY1 deficiency., (© The Author(s) 2020. Published by Oxford University Press.)

Background: NGLY1-related congenital disorder of deglycosylation (NGLY1-CDDG) is a multisystemic neurodevelopmental disorder in which affected individuals show developmental delay, epilepsy, intellectual disability, abnormal liver function, and poor growth. This study presents a 10-month-old female infant with elevated liver transaminases, developmental delay, epilepsy (subclinical seizures), and constipation who possesses two compound heterozygous mutations in NGLY1., Case Presentation: The proband was admitted to the Department of Gastroenterology, Children's Hospital of Soochow University, with elevated liver transaminases. She had a history of intrauterine growth retardation and exhibited elevated transaminases, global developmental delay, seizures and light constipation during early infancy. Whole-exome sequencing (WES) and Sanger sequencing revealed two compound heterozygous mutations in NGLY1 that had been inherited in an autosomal recessive manner from her parents. One was a termination mutation, c.1168C > T (p.R390*), and the other was a missense mutation, c.1156G > T (p.D386Y). NGLY1-CDDG is a rare disorder, with a few dozen cases. The two mutations of this proband has not been previously identified., Conclusions: This study investigated a Chinese proband with NGLY1-CDDG born from healthy parents who was studied using WES and Sanger sequencing to identify the causative mutations. We identified two novel compound heterozygous mutations in NGLY1, c.1168C > T (p.R390*)/c.1156G > T (p.D386Y), which are probably causative of disease.

NGLY1 deficiency is the first and only autosomal recessive congenital disorder of N-linked deglycosylation (NGLY1-CDDG). To date, no patients with NGLY1 deficiency has been reported from mainland China or East Asia in English literature. Here, we present six patients with a diagnosis of NGLY1-CDDG on the basis of clinical phenotype, genetic testing, and functional studies. We retrospectively analyzed clinical phenotypes and NGLY1 genotypes of six cases from four families. Informed consent was obtained for diagnosis and treatment. In-silico tools and in vitro enzyme activity assays were used to determine pathogenicity of NGLY1 varaints. All patients had typical features of NGLY1-CDDG, including global developmental delay, microcephaly, hypotonia, hypertransaminasemia, alacrimia, and feeding difficulty. Dysmorphic features found in our patients include flat nasal bridge, loose and hollow cheeks, short stature, malnutrition, and ptosis. Pachylosis could be a novel cutaneous feature that may be explained by lack of sweat. We found three novel variants, including one missense (c.982C > G/p.Arg328Gly), one splice site (c.1003+3A > G), and one frame-shift (c.1637-1652delCATCTTTTGCTTATAT/p.Ser546PhefsTer) variant. All mutations were predicted to be disease causing with in-silico prediction tools, and affected at least one feature of gene splicing. Protein modeling showed missense variants may affect covalent bonding within the protein structure, or interrupt active/binding amino-acid residues. In vitro studies indicated that proteins carrying missense variants (p.Arg328Gly and p.Tyr342Cys) lost the enzyme activity. We expanded clinical phenotype and genetic mutation spectrum of NGLY1-CDDG by reporting six cases, three novel variants, and novel clinical features from mainland China.

The cytoplasmic peptide:N-glycanase (Ngly1) is a de-N-glycosylating enzyme that cleaves N-glycans from misfolded glycoproteins and is involved in endoplasmic reticulum-associated degradation. The recent discovery of NGLY1-deficiency, which causes severe systemic symptoms, drew attention to the physiological function of Ngly1 in mammals. While several studies have been carried out to reveal the physiological necessity of Ngly1, the semi-lethal nature of Ngly1-deficient animals made it difficult to analyze its function in adults. In this study, we focus on the physiological function of Ngly1 in liver (hepatocyte)-specific Ngly1-deficient mice generated using the cre-loxP system. We found that hepatocyte-specific Ngly1-deficient mice showed abnormal hepatocyte nuclear size/morphology with aging but did not show other notable defects in unstressed conditions. This nuclear phenotype did not appear to be related to the function of the only gene currently reported to rescue Ngly1-deficient murine lethality so far, endo-β-N-acetylglucosaminidase. We also found that under a high fructose diet induced stress, the hepatocyte-specific Ngly1-deletion resulted in liver transaminases elevation and increased lipid droplet accumulation. We showed that the processing and localization of the transcription factor, nuclear factor erythroid 2-like 1 (Nfe2l1), was impaired in the Ngly1-deficient hepatocytes. Therefore, Nfe2l1, at least partially, contributes to the phenotypes observed in hepatocyte-specific Ngly1-deficient mice. Our results indicate that Ngly1 plays important roles in the adult liver impacting nuclear morphology and lipid metabolism. Hepatocyte-specific Ngly1-deficient mice could thus serve as a valuable animal model for assessing in vivo efficacy of drugs and/or treatment for NGLY1-deficiency., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Together with the lysosomal storage diseases, N-glycanase 1 deficiency is a congenital disorder of deglycosylation, which has been diagnosed in 27 patients, including two of them from Poland. The pathogenesis remains unknown, however, the main role is attributed to the disturbed endoplasmic reticulum-associated protein degradation process. The most characteristic symptoms include global developmental disability, hyperkinetic movement disorder, hypo-/alacrimia, and elevated serum transaminases. Identification of pathogenic variants in the NGLY1 gene is required to confirm the diagnosis.

N-glycanase 1 deficiency is a congenital disorder of deglycosylation, which has been diagnosed in 27 patients, including 2 of them from Poland. The most characteristic symptoms include global developmental disability, hyperkinetic movement disorder, hypo-/alacrimia, and elevated serum transaminases. We reported on a patient in whom the liver biopsy done at the age of 3 years revealed the presence of steatosis, fibrosis, and an amorphous periodic acid-Schiff staining positive diastases-digested material in the cytoplasm.

Hypothesis generation is a critical step in research and a cornerstone in the rare disease field. Research is most efficient when those hypotheses are based on the entirety of knowledge known to date. Systematic review articles are commonly used in biomedicine to summarize existing knowledge and contextualize experimental data. But the information contained within review articles is typically only expressed as free-text, which is difficult to use computationally. Researchers struggle to navigate, collect and remix prior knowledge as it is scattered in several silos without seamless integration and access. This lack of a structured information framework hinders research by both experimental and computational scientists. To better organize knowledge and data, we built a structured review article that is specifically focused on NGLY1 Deficiency, an ultra-rare genetic disease first reported in 2012. We represented this structured review as a knowledge graph and then stored this knowledge graph in a Neo4j database to simplify dissemination, querying and visualization of the network. Relative to free-text, this structured review better promotes the principles of findability, accessibility, interoperability and reusability (FAIR). In collaboration with domain experts in NGLY1 Deficiency, we demonstrate how this resource can improve the efficiency and comprehensiveness of hypothesis generation. We also developed a read-write interface that allows domain experts to contribute FAIR structured knowledge to this community resource. In contrast to traditional free-text review articles, this structured review exists as a living knowledge graph that is curated by humans and accessible to computational analyses. Finally, we have generalized this workflow into modular and repurposable components that can be applied to other domain areas. This NGLY1 Deficiency-focused network is publicly available at http://ngly1graph.org/., Availability and Implementation: Database URL: http://ngly1graph.org/. Network data files are at: https://github.com/SuLab/ngly1-graph and source code at: https://github.com/SuLab/bioknowledge-reviewer., Contact: asu@scripps.edu., (© The Author(s) 2020. Published by Oxford University Press.)

Patients with pathogenic mutations in NGLY1 cannot make tears and have global developmental delay and liver dysfunction. Traditionally, NGLY1 cleaves intact N-glycans from misfolded, retrotranslocated glycoproteins before proteasomal degradation. We demonstrate that Ngly1-null mouse embryonic fibroblasts, NGLY1 knockout human cells, and patient fibroblasts are resistant to hypotonic lysis. Ngly1-deficient mouse embryonic fibroblasts swell slower and have reduced aquaporin1 mRNA and protein expression. Ngly1 knockdown and overexpression confirms that Ngly1 regulates aquaporin1 and hypotonic cell lysis. Patient fibroblasts and NGLY1 knockout cells show reduced aquaporin11 mRNA, supporting NGLY1 as regulating expression of multiple aquaporins across species. Complementing Ngly1-deficient cells with catalytically inactive NGLY1 (p.Cys309Ala) restores normal hypotonic lysis and aquaporin1 protein. We show that transcription factors Atf1/Creb1 regulate aquaporin1 and that the Atf1/Creb1 signaling pathway is disrupted in Ngly1-deficient mouse embryonic fibroblasts. These results identify a non-enzymatic, regulatory function of NGLY1 in aquaporin transcription, possibly related to alacrima and neurological symptoms., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

N -glycanase 1 ( NGLY1 ) deficiency is an ultra-rare and complex monogenic glycosylation disorder that affects fewer than 40 patients globally. NGLY1 deficiency has been studied in model organisms such as yeast, worms, flies and mice. Proteasomal and mitochondrial homeostasis gene networks are controlled by the evolutionarily conserved transcriptional regulator NRF1, whose activity requires deglycosylation by NGLY1. Hypersensitivity to the proteasome inhibitor bortezomib is a common phenotype observed in whole-animal and cellular models of NGLY1 deficiency. Here, we describe unbiased phenotypic drug screens to identify FDA-approved drugs that are generally recognized as safe natural products, and novel chemical entities, that rescue growth and development of NGLY1 -deficient worm and fly larvae treated with a toxic dose of bortezomib. We used image-based larval size and number assays for use in screens of a 2560-member drug-repurposing library and a 20,240-member lead-discovery library. A total of 91 validated hit compounds from primary invertebrate screens were tested in a human cell line in an NRF2 activity assay. NRF2 is a transcriptional regulator that regulates cellular redox homeostasis, and it can compensate for loss of NRF1. Plant-based polyphenols make up the largest class of hit compounds and NRF2 inducers. Catecholamines and catecholamine receptor activators make up the second largest class of hits. Steroidal and non-steroidal anti-inflammatory drugs make up the third largest class. Only one compound was active in all assays and species: the atypical antipsychotic and dopamine receptor agonist aripiprazole. Worm and fly models of NGLY1 deficiency validate therapeutic rationales for activation of NRF2 and anti-inflammatory pathways based on results in mice and human cell models, and suggest a novel therapeutic rationale for boosting catecholamine levels and/or signaling in the brain., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

NGLY1 deficiency is a rare inherited disorder caused by mutations in the NGLY1 gene encoding N-glycanase 1 that is a hydrolase for N-linked glycosylated proteins. An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 16-year-old patient with homozygous mutation of p.R401X (c.1201 A>T) in the NGLY1 gene. Our iPSC model offers a useful resource to study the disease pathophysiology and to develop therapeutics for treatment of NGLY1 patients., (Published by Elsevier B.V.)

Background: NGLY1-CDDG is a congenital disorder of deglycosylation caused by a defective peptide:N-glycanase (PNG). To date, all but one of the reported patients have been diagnosed through whole-exome or whole-genome sequencing, as no biochemical marker was available to identify this disease in patients. Recently, a potential urinary biomarker was reported, but the data presented suggest that this marker may be excreted intermittently., Methods: In this study, we performed untargeted direct-infusion high-resolution mass spectrometry metabolomics in seven dried blood spots (DBS) from four recently diagnosed NGLY1-CDDG patients, to test for small-molecule biomarkers, in order to identify a potential diagnostic marker. Results were compared to 125 DBS of healthy controls and to 238 DBS of patients with other diseases., Results: We identified aspartylglycosamine as the only significantly increased compound with a median Z-score of 4.8 (range: 3.8-8.5) in DBS of NGLY1-CDDG patients, compared to a median Z-score of -0.1 (range: -2.1-4.0) in DBS of healthy controls and patients with other diseases., Discussion: The increase of aspartylglycosamine can be explained by lack of function of PNG. PNG catalyzes the cleavage of the proximal N-acetylglucosamine residue of an N-glycan from the asparagine residue of a protein, a step in the degradation of misfolded glycoproteins. PNG deficiency results in a single N-acetylglucosamine residue left attached to the asparagine residue which results in free aspartylglycosamine when the glycoprotein is degraded. Thus, we here identified aspartylglycosamine as the first potential small-molecule biomarker in DBS for NGLY1-CDDG, making a biochemical diagnosis for NGLY1-CDDG potentially feasible., (Copyright © 2019 Elsevier Inc. All rights reserved.)

ALG3-CDG is one of the very rare types of congenital disorder of glycosylation (CDG) caused by variants in the ER-mannosyltransferase ALG3. Here, we summarize the clinical, biochemical, and genetic data of four new ALG3-CDG patients, who were identified by a type I pattern of serum transferrin and the accumulation of Man 5 GlcNAc 2 -PP-dolichol in LLO analysis. Additional clinical symptoms observed in our patients comprise sensorineural hearing loss, right-descending aorta, obstructive cardiomyopathy, macroglossia, and muscular hypertonia. We add four new biochemically confirmed variants to the list of ALG3-CDG inducing variants: c.350G>C (p.R117P), c.1263G>A (p.W421*), c.1037A>G (p.N346S), and the intron variant c.296+4A>G. Furthermore, in Patient 1 an additional open-reading frame of 141 bp (AAGRP) in the coding region of ALG3 was identified. Additionally, we show that control cells synthesize, to a minor degree, a hybrid protein composed of the polypeptide AAGRP and ALG3 (AAGRP-ALG3), while in Patient 1 expression of this hybrid protein is significantly increased due to the homozygous variant c.160_196del (g.165C>T). By reviewing the literature and combining our findings with previously published data, we further expand the knowledge of this rare glycosylation defect., (© 2019 Wiley Periodicals, Inc.)

Background: NGLY1 deficiency is a rare autosomal recessive disorder caused by loss in enzymatic function of NGLY1, a peptide N-glycanase that has been shown to play a role in endoplasmic reticulum associated degradation (ERAD). ERAD dysfunction has been implicated in other well-described proteinopathies, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. The classical clinical tetrad includes developmental delay, hypolacrima, transiently elevated transaminases, and hyperkinetic movement disorders. The musculoskeletal system is also commonly affected, but the orthopaedic phenotype has been incompletely characterized. Best practices for orthopaedic clinical care have not been elucidated and considerable variability has resulted from this lack of evidence base. Our study surveyed patients enrolled in an international registry for NGLY1 deficiency in order to characterize the orthopaedic manifestations, sequelae, and management., Results: Our findings, encompassing the largest cohort for NGLY1 deficiency to date, detail levels of motor milestone achievement; physical exam findings; fracture rates/distribution; frequency of motor skill regression; non-pharmacologic and non-procedural interventions; pharmacologic therapies; and procedural interventions experienced by 29 participants. Regarding the orthopaedic phenotype, at time of survey response, we found that over 40% of patients experienced motor skill regression from their peak. Over 80% of patients had at least one orthopaedic diagnosis, and nearly two-thirds of the total had two or more. More than half of patients older than 6 years had sustained a fracture. Related to orthopaedic non-medical management, we found that 93 and 79% of patients had utilized physical therapy and non-operative orthoses, respectively. In turn, the vast majority took at least one medication (including for bone health and antispasmodic therapy). Finally, nearly half of patients had undergone an invasive procedure. Of those older than 6 years, two-thirds had one or more procedures. Stratification of these analyses by sex revealed distinctive differences in disease natural history and clinical management course., Conclusions: These findings describing the orthopaedic natural history and standard of care in patients with NGLY1 deficiency can facilitate diagnosis, inform prognosis, and guide treatment recommendations in an evidence-based manner. Furthermore, the methodology is notable for its partnership with a disease-specific advocacy organization and may be generalizable to other rare disease populations. This study fills a void in the existing literature for this population and this methodology offers a precedent upon which future studies for rare diseases can build.

Mutations in the NGLY1 (N-glycanase 1) gene, encoding an evolutionarily conserved deglycosylation enzyme, are associated with a rare congenital disorder leading to global developmental delay and neurological abnormalities. The molecular mechanism of the NGLY1 disease and its function in tissue and immune homeostasis remain unknown. Here, we find that NGLY1 -deficient human and mouse cells chronically activate cytosolic nucleic acid-sensing pathways, leading to elevated interferon gene signature. We also find that cellular clearance of damaged mitochondria by mitophagy is impaired in the absence of NGLY1, resulting in severely fragmented mitochondria and activation of cGAS-STING as well as MDA5-MAVS pathways. Furthermore, we show that NGLY1 regulates mitochondrial homeostasis through transcriptional factor NRF1. Remarkably, pharmacological activation of a homologous but nonglycosylated transcriptional factor NRF2 restores mitochondrial homeostasis and suppresses immune gene activation in NGLY1 -deficient cells. Together, our findings reveal novel functions of the NGLY1-NRF1 pathway in mitochondrial homeostasis and inflammation and uncover an unexpected therapeutic strategy using pharmacological activators of NRF2 for treating mitochondrial and immune dysregulation., (© 2018 Yang et al.)

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.

N-glycanase 1 (NGLY1) Deficiency is a rare monogenic multi-system disorder first described in 2014. NGLY1 is evolutionarily conserved in model organisms. Here we conducted a natural history study and chemical-modifier screen on the Drosophila melanogaster NGLY1 homolog, Pngl We generated a new fly model of NGLY1 Deficiency, engineered with a nonsense mutation in Pngl at codon 420 that results in a truncation of the C-terminal carbohydrate-binding PAW domain. Homozygous mutant animals exhibit global development delay, pupal lethality and small body size as adults. We developed a 96-well-plate, image-based, quantitative assay of Drosophila larval size for use in a screen of the 2,650-member Microsource Spectrum compound library of FDA approved drugs, bioactive tool compounds, and natural products. We found that the cholesterol-derived ecdysteroid molting hormone 20-hydroxyecdysone (20E) partially rescued the global developmental delay in mutant homozygotes. Targeted expression of a human NGLY1 transgene to tissues involved in ecdysteroidogenesis, e.g. , prothoracic gland, also partially rescues global developmental delay in mutant homozygotes. Finally, the proteasome inhibitor bortezomib is a potent enhancer of global developmental delay in our fly model, evidence of a defective proteasome "bounce-back" response that is also observed in nematode and cellular models of NGLY1 Deficiency. Together, these results demonstrate the therapeutic relevance of a new fly model of NGLY1 Deficiency for drug discovery and gene modifier screens., (Copyright © 2018 Rodriguez et al.)

N-glycanase deficiency (NGLY1 deficiency, NGLY1-CDDG), the first autosomal recessive congenital disorder of N-linked deglycosylation (CDDG), is caused by pathogenic variants in NGLY1. The majority of affected individuals have been identified using exome or genome sequencing. To date, no reliable, clinically available biomarkers have been identified. Urine oligosaccharide analysis was included as part of a routine evaluation for possible biomarkers in patients with confirmed NGLY1-CDDG. During the qualitative review of oligosaccharide profiles by an experienced laboratory director an abnormal analyte with a proposed structure of Neu5Ac1Hex1GlcNAc1-Asn was identified in NGLY1-CDDG patient urine samples. The same species has been observed in profiles from individuals affected with aspartylglucosaminuria, although the complete spectra are not identical. Additional studies using tandem mass spectrometry confirmed the analyte's structure. In addition to the known NGLY1-CDDG patients identified by this analysis, a single case was identified in a population referred for clinical testing who subsequently had a diagnosis of NGLY1-CDDG confirmed by molecular testing. Urine oligosaccharide screening by MALDI-TOF MS can identify individuals with NGLY1-CDDG. In addition, this potential biomarker might also be used to monitor the effectiveness of therapeutic options as they become available., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Autosomal recessive loss-of-function mutations in N-glycanase 1 (NGLY1) cause NGLY1 deficiency, the only known human disease of deglycosylation. Patients present with developmental delay, movement disorder, seizures, liver dysfunction and alacrima. NGLY1 is a conserved cytoplasmic component of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway. ERAD clears misfolded proteins from the ER lumen. However, it is unclear how loss of NGLY1 function impacts ERAD and other cellular processes and results in the constellation of problems associated with NGLY1 deficiency. To understand how loss of NGLY1 contributes to disease, we developed a Drosophila model of NGLY1 deficiency. Loss of NGLY1 function resulted in developmental delay and lethality. We used RNAseq to determine which processes are misregulated in the absence of NGLY1. Transcriptome analysis showed no evidence of ER stress upon NGLY1 knockdown. However, loss of NGLY1 resulted in a strong signature of NRF1 dysfunction among downregulated genes, as evidenced by an enrichment of genes encoding proteasome components and proteins involved in oxidation-reduction. A number of transcriptome changes also suggested potential therapeutic interventions, including dysregulation of GlcNAc synthesis and upregulation of the heat shock response. We show that increasing the function of both pathways rescues lethality. Together, transcriptome analysis in a Drosophila model of NGLY1 deficiency provides insight into potential therapeutic approaches.

Mitochondrial respiratory chain (RC) diseases and congenital disorders of glycosylation (CDG) share extensive clinical overlap but are considered to have distinct cellular pathophysiology. Here, we demonstrate that an essential physiologic connection exists between cellular N-linked deglycosylation capacity and mitochondrial function. Following identification of altered muscle and liver mitochondrial amount and function in two children with a CDG subtype caused by NGLY1 deficiency, we evaluated mitochondrial physiology in NGLY1 disease human fibroblasts, and in NGLY1-knockout mouse embryonic fibroblasts and C. elegans. Across these distinct evolutionary models of cytosolic NGLY1 deficiency, a consistent disruption of mitochondrial physiology was present involving modestly reduced mitochondrial content with more pronounced impairment of mitochondrial membrane potential, increased mitochondrial matrix oxidant burden, and reduced cellular respiratory capacity. Lentiviral rescue restored NGLY1 expression and mitochondrial physiology in human and mouse fibroblasts, confirming that NGLY1 directly influences mitochondrial function. Overall, cellular deglycosylation capacity is shown to be a significant factor in mitochondrial RC disease pathogenesis across divergent evolutionary species., (Copyright © 2017. Published by Elsevier B.V.)

N-Glycanase deficiency, or NGLY1 deficiency, is an extremely rare human genetic disease. N-Glycanase, encoded by the gene NGLY1, is an important enzyme involved in protein deglycosylation of misfolded proteins. Deglycosylation of misfolded proteins precedes the endoplasmic reticulum (ER)-associated degradation (ERAD) process. NGLY1 patients produce little or no N-glycanase (Ngly1), and the symptoms include global developmental delay, frequent seizures, complex hyperkinetic movement disorder, difficulty in swallowing/aspiration, liver dysfunction, and a lack of tears. Unfortunately, there has not been any therapeutic option available for this rare disease so far. Recently, a proposed molecular mechanism for NGLY1 deficiency suggested that endo-β-N-acetylglucosaminidase (ENGase) inhibitors may be promising therapeutics for NGLY1 patients. Herein, we performed structure-based virtual screening utilizing FDA-approved drug database on this ENGase target to enable repurposing of existing drugs. Several Proton Pump Inhibitors (PPIs), a series of substituted 1H-benzo [d] imidazole, and 1H-imidazo [4,5-b] pyridines, among other scaffolds, have been identified as potent ENGase inhibitors. An electrophoretic mobility shift assay was employed to assess the inhibition of ENGase activity by these PPIs. Our efforts led to the discovery of Rabeprazole Sodium as the most promising hit with an IC 50 of 4.47±0.44μM. This is the first report that describes the discovery of small molecule ENGase inhibitors, which can potentially be used for the treatment of human NGLY1 deficiency., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.

Purpose: The cytosolic enzyme N-glycanase 1, encoded by NGLY1, catalyzes cleavage of the β-aspartyl glycosylamine bond of N-linked glycoproteins, releasing intact N-glycans from proteins bound for degradation. In this study, we describe the clinical spectrum of NGLY1 deficiency (NGLY1-CDDG)., Methods: Prospective natural history protocol., Results: In 12 individuals ages 2 to 21 years with confirmed, biallelic, pathogenic NGLY1 mutations, we identified previously unreported clinical features, including optic atrophy and retinal pigmentary changes/cone dystrophy, delayed bone age, joint hypermobility, and lower than predicted resting energy expenditure. Novel laboratory findings include low cerebral spinal fluid (CSF) total protein and albumin and unusually high antibody titers toward rubella and/or rubeola following vaccination. We also confirmed and further quantified previously reported findings noting that decreased tear production, transient transaminitis, small feet, a complex hyperkinetic movement disorder, and varying degrees of global developmental delay with relatively preserved socialization are the most consistent features., Conclusion: Our prospective phenotyping expands the clinical spectrum of NGLY1-CDDG, offers prognostic information, and provides baseline data for evaluating therapeutic interventions.Genet Med 19 2, 160-168.

Pediatric palliative care providers often care for children with rare, poorly understood diseases. In addition to grappling with a life-limiting diagnosis, families face complexity in decision making stemming from the prognostic uncertainty surrounding their child's rare condition. We discuss several unique challenges, illustrated through case studies of three children who shared the rare diagnosis of congenital disorder of glycosylation.

N-Glycanase 1, encoded by NGLY1, catalyzes the deglycosylation of misfolded N-linked glycoproteins retrotranslocated into the cytosol. We identified nine cases with mutations in NGLY1. The patients show developmental delay, seizures, peripheral neuropathy, abnormal liver function and alacrima (absence of tears). The mutations in NGLY1 resulted in the absence of N-glycanase 1 protein in patient-derived fibroblasts. Applying a recently established cellular deglycosylation-dependent Venus fluorescence assay, we found that patient fibroblasts had dramatically reduced fluorescence, indicating a pronounced reduction in N-glycanase enzymatic activity. Using this assay, we could find no evidence of other related activities. Our findings reveal that NGLY1 mutations destroy both N-glycanase 1 protein and enzymatic activity., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)