N-glycanase 1 Deficiency Research Articles

NGLY1 mutations cause protein aggregation in human neurons

Biallelic mutations in the gene that encodes the enzyme N-glycanase 1 (NGLY1) cause a rare disease with multi-symptomatic features including developmental delay, intellectual disability, neuropathy, and seizures. NGLY1's activity in human neural cells is currently not well understood. To understand how NGLY1 gene loss leads to the specific phenotypes of NGLY1 deficiency, we employed direct conversion of NGLY1 patient-derived induced pluripotent stem cells (iPSCs) to functional cortical neurons. Transcriptomic, proteomic, and functional studies of iPSC-derived neurons lacking NGLY1 function revealed several major cellular processes that were altered, including protein aggregate-clearing functionality, mitochondrial homeostasis, and synaptic dysfunctions. These phenotypes were rescued by introduction of a functional NGLY1 gene and were observed in iPSC-derived mature neurons but not astrocytes. Finally, laser capture microscopy followed by mass spectrometry provided detailed characterization of the composition of protein aggregates specific to NGLY1-deficient neurons. Future studies will harness this knowledge for therapeutic development.

NGLY1 deficiency: a prospective natural history study.

N-glycanase 1 (NGLY1) deficiency is a debilitating, ultra-rare autosomal recessive disorder caused by loss of function of NGLY1, a cytosolic enzyme that deglycosylates other proteins. It is characterized by severe global developmental delay and/or intellectual disability, hyperkinetic movement disorder, transient elevation of transaminases, (hypo)alacrima and progressive, diffuse, length-dependent sensorimotor polyneuropathy. A prospective natural history study (NHS) was conducted to elucidate clinical features and disease course. Twenty-nine participants were enrolled (15 onsite, 14 remotely) and followed for up to 32months, representing ~29% of the ~100 patients identified worldwide. Participants exhibited profound developmental delays, with almost all developmental quotients below 20 on the Mullen Scales of Early Learning, well below the normative score of 100. Increased difficulties with sitting and standing suggested decline in motor function over time. Most patients presented with (hypo)alacrima and reduced sweat response. Pediatric quality of life was poor except for emotional function. Language/communication and motor skill problems including hand use were reported by caregivers as the most bothersome symptoms. Levels of the substrate biomarker, GlcNAc-Asn (aspartylglucosamine; GNA), were consistently elevated in all participants over time, independent of age. Liver enzymes were elevated for some participants but improved especially in younger patients and did not reach levels indicating severe liver disease. Three participants died during the study period. Data from this NHS informs selection of endpoints and assessments for future clinical trials for NGLY1 deficiency interventions. Potential endpoints include GNA biomarker levels, neurocognitive assessments, autonomic and motor function (particularly hand use), (hypo)alacrima and quality of life.

AAV9-NGLY1 gene replacement therapy improves phenotypic and biomarker endpoints in a rat model of NGLY1 Deficiency

N-glycanase 1 (NGLY1) Deficiency is a progressive, ultra-rare, autosomal recessive disorder with no approved therapy and five core clinical features: severe global developmental delay, hyperkinetic movement disorder, elevated liver transaminases, alacrima, and peripheral neuropathy. Here, we confirmed and characterized the Ngly1 -/- / rat as a relevant disease model. GS-100, a gene therapy candidate, is a recombinant, single-stranded adeno-associated virus (AAV) 9 vector designed to deliver a functional copy of the human NGLY1 gene. Using the Ngly1 -/- rat, we tested different administration routes for GS-100: intracerebroventricular (ICV), intravenous (IV), or the dual route (IV+ ICV). ICV and IV+ ICV administration resulted in widespread biodistribution of human NGLY1 DNA and corresponding mRNA and protein expression in CNS tissues. GS-100 delivered by ICV or IV+ ICV significantly reduced levels of the substrate biomarker N-acetylglucosamine-asparagine (GlcNAc-Asn or GNA) in CSF and brain tissue compared with untreated Ngly1-/- rats. ICV and IV+ ICV administration of GS-100 resulted in behavioral improvements in rotarod and rearing tests, whereas IV-only administration did not. IV+ ICV did not provide additional benefit compared with ICV administration alone.These data provide evidence that GS-100 could be an effective therapy for NGLY1 Deficiency using the ICV route of administration.

Comparative proteomics reveals elevated CCN2 in NGLY1-deficient cells

N-glycanase 1(NGLY1) catalyzes the removal of N-linked glycans from newly synthesized or misfolded protein. NGLY1 deficiency is a recently diagnosed rare genetic disorder. The affected individuals present a broad spectrum of clinical features. Recent studies explored several possible molecular mechanisms of NGLY1 deficiency including defects in proteostasis, mitochondrial homeostasis, innate immunity, and water/ion transport. We demonstrate abnormal accumulation of endoplasmic reticulum-associated degradation (ERAD) substrates in NGLY1-deficient cells. Global quantitative proteomics discovered elevated levels of endogenous proteins in NGLY1-defective human and mouse cells. Further biological validation assays confirmed the altered abundance of several key candidates that were subjected to isobarically labeled proteomic analysis. CCN2 was selected for further analysis due to its significant increase in different cell models of NGLY1 deficiency. Functional assays show elevated CCN2 and over-stimulated TGF-β signaling in NGLY1-deficient cells. Given the important role of CCN2 and TGF-β pathway in mediating systemic fibrosis, we propose a potential link of increased CCN2 and TGF-β signaling to microscopic liver fibrosis in NGLY1 patients.

An in vivo drug repurposing screen and transcriptional analyses reveals the serotonin pathway and GSK3 as major therapeutic targets for NGLY1 deficiency.

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.

NGLY1 Deficiency, a Congenital Disorder of Deglycosylation: From Disease Gene Function to Pathophysiology.

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.

Deficiency of N-glycanase 1 perturbs neurogenesis and cerebral development modeled by human organoids

Mutations in N-glycanase 1 (NGLY1), which deglycosylates misfolded glycoproteins for degradation, can cause NGLY1 deficiency in patients and their abnormal fetal development in multiple organs, including microcephaly and other neurological disorders. Using cerebral organoids (COs) developed from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), we investigate how NGLY1 dysfunction disturbs early brain development. While NGLY1 loss had limited impact on the undifferentiated cells, COs developed from NGLY1-deficient hESCs showed defective formation of SATB2-positive upper-layer neurons, and attenuation of STAT3 and HES1 signaling critical for sustaining radial glia. Bulk and single-cell transcriptomic analysis revealed premature neuronal differentiation accompanied by downregulation of secreted and transcription factors, including TTR, IGFBP2, and ID4 in NGLY1-deficient COs. NGLY1 malfunction also dysregulated ID4 and enhanced neuronal differentiation in CO transplants developed in vivo. NGLY1-deficient CO cells were more vulnerable to multiple stressors; treating the deficient cells with recombinant TTR reduced their susceptibility to stress from proteasome inactivation, likely through LRP2-mediated activation of MAPK signaling. Expressing NGLY1 led to IGFBP2 and ID4 upregulation in CO cells developed from NGLY1-deficiency patient’s hiPSCs. In addition, treatment with recombinant IGFBP2 enhanced ID4 expression, STAT3 signaling, and proliferation of NGLY1-deficient CO cells. Overall, our discoveries suggest that dysregulation of stress responses and neural precursor differentiation underlies the brain abnormalities observed in NGLY1-deficient individuals.

Patient-derived gene and protein expression signatures of NGLY1 deficiency.

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.

GlcNAc-Asn is a biomarker for NGLY1 deficiency.

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.

Generation of two gene corrected human isogenic iPSC lines (NCATS-CL6104 and NCATS-CL6105) from a patient line (NCATS-CL6103) carrying a homozygous p.R401X mutation in the NGLY1 gene using CRISPR/Cas9

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.

Tracing the NGLY1 footprints: insights from Drosophila

Abstract Recessive mutations in human N-glycanase 1 (NGLY1) cause a multisystem disorder with various phenotypes including global developmental delay. One of the models utilized to understand the biology of NGLY1 and the pathophysiology of NGLY1 deficiency is Drosophila melanogaster, a well-established, genetically tractable organism broadly used to study various biological processes and human diseases. Loss of the Drosophila NGLY1 homolog (Pngl) causes a host of phenotypes including developmental delay and lethality. Phenotypic, transcriptomic and genome-wide association analyses on Drosophila have revealed links between NGLY1 and several critical developmental and cellular pathways/processes. Further, repurposing screens of Food and Drug Administration (FDA)-approved drugs have identified potential candidates to ameliorate some of the Pngl-mutant phenotypes. Here, we will summarize the insights gained into the functions of NGLY1 from Drosophila studies. We hope that the current review article will encourage additional studies in Drosophila and other model systems towards establishing a therapeutic strategy for NGLY1 deficiency patients.

Reversibility of motor dysfunction in the rat model of NGLY1 deficiency

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.

An induced pluripotent stem cell line (NCATS-CL9075) from a patient carrying compound heterozygote mutations, p.R390P and p.L318P, in the NGLY1 gene

NGLY1 deficiency is a rare disorder caused by mutations in the NGLY1 gene which codes for the highly conserved N-glycanase1 (NGLY1). This enzyme functions in cytosolic deglycosylation of N- linked glycoproteins. An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 2-year-old patient carrying compound heterozygous mutations, p.R390P and p.L318P in the NGLY1 gene. This cell-based iPSC disease model provides a resource to study disease pathophysiology and to develop a cell-based disease model for drug development for NGLY1 patients.

JF1/B6F1 Ngly1-/- mouse as an isogenic animal model of NGLY1 deficiency.

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 F2 Ngly1−/− mice from (JF1×B6)F1 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.

A Drosophila screen identifies NKCC1 as a modifier of 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.

Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation.

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.

Ngly1 -/- rats develop neurodegenerative phenotypes and pathological abnormalities in their peripheral and central nervous systems.

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.

Defects in the Neuroendocrine Axis Contribute to Global Development Delay in a Drosophila Model of NGLY1 Deficiency.

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.

Transcriptome and functional analysis in a Drosophila model of NGLY1 deficiency provides insight into therapeutic approaches.

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.

New perspectives on the mutated NGLY1 enigma

The enzyme N-glycanase 1 (NGLY1) is considered a component of the endoplasmic reticulum-associated degradation (ERAD) machinery and clinical manifestations of its dysfunction include global developmental delay, a movement disorder, peripheral neuropathy, liver disorders, microcephaly, diminished reflexes and seizures. Although several mutations in NGLY1 have been identified, the relation between the defected protein and the above described pathologies is yet unknown. We hypothesised that NGLY1 failure to degrade certain proteins may result in their accumulation and overexpression and used a systems biology approach to identify proteins that may be affected by NGLY1 deficiency. Genes that interact with the NGLY1 gene according to BioGRID database of physical and genetic interactions were analysed with STRING Protein–Protein interaction database. Network analysis identified FAF1 (Fas-Associated Factor 1), an apoptosis-potentiating protein, as a possible degradation substrate of NGLY1. Examination of normal tissue microarrays demonstrated that FAF1-to-NGLY1 ratio is maximal (more than 3:1) in skeletal muscle and brain tissues microarrays. This evidence may explain the pathologies in brain and muscle tissues of patients with mutated NGLY1. To test this hypothesis, laboratory studies that will assess if FAF1 protein is overexpressed in tissues of patients with mutated NGLY1 are required.