Biochemical diagnosis of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism, comprising over 160 described diseases to this day. CDG are characterized by a dysfunctional glycosylation process, with molecular defects localized in the cytosol, the endoplasmic reticulum, or the Golgi apparatus. Depending on the CDG, N-glycosylation, O-glycosylation and/or glycosaminoglycan synthesis can be affected. Various proteins, lipids, and glycosylphosphatidylinositol anchors bear glycan chains, with potential impacts on their folding, targeting, secretion, stability, and thus, functionality. Therefore, glycosylation defects can have diverse and serious clinical consequences. CDG patients often present with a non-specific, multisystemic syndrome including neurological involvement, growth delay, hepatopathy and coagulopathy. As CDG are rare diseases, and typically lack distinctive clinical signs, biochemical and genetic testing bear particularly important and complementary diagnostic roles. Here, after a brief introduction on glycosylation and CDG, we review historical and recent findings on CDG biomarkers and associated analytical techniques, with a particular emphasis on those with relevant use in the specialized clinical chemistry laboratory. We provide the reader with insights and methods which may help them properly assist the clinician in navigating the maze of glycosylation disorders.

Novel insight into FCSK-congenital disorder of glycosylation through a CRISPR-generated cell model.

BACKGROUND: FCSK-congenital disorder of glycosylation (FCSK-CDG) is a recently discovered rare autosomal recessive genetic disorder with defective fucosylation due to mutations in the fucokinase encoding gene, FCSK. Despite the essential role of fucokinase in the fucose salvage pathway and severe multisystem manifestations of FCSK-CDG patients, it is not elucidated which cells or which types of fucosylation are affected by its deficiency. METHODS: In this study, CRISPR/Cas9 was employed to construct an FCSK-CDG cell model and explore the molecular mechanisms of the disease by lectin flow cytometry and real-time PCR analyses. RESULTS: Comparison of cellular fucosylation by lectin flow cytometry in the created CRISPR/Cas9 FCSK knockout and the same unedited cell lines showed no significant change in the amount of cell surface fucosylated glycans, which is consistent with the only documented previous study on different cell types. It suggests a probable effect of this disease on secretory glycoproteins. Investigating O-fucosylation by analysis of the NOTCH3 gene expression as a potential target revealed a significant decrease in the FCSK knockout cells compared with the same unedited ones, proving the effect of fucokinase deficiency on EGF-like repeats O-fucosylation. CONCLUSION: This study expands insight into the FCSK-CDG molecular mechanism; to the best of our knowledge, it is the first research conducted to reveal a gene whose expression level alters due to this disease.

Congenital disorders of glycosylation with multiorgan disruption and immune dysregulation caused by compound heterozygous variants in MAN2B2.

BACKGROUND: Congenital disorders of glycosylation (CDG) are a type of inborn error of metabolism (IEM) resulting from defects in glycan synthesis or failed attachment of glycans to proteins or lipids. One rare type of CDG is caused by homozygous or compound heterozygous loss-of-function variants in mannosidase alpha class 2B member 2 (MAN2B2). To date, only two cases of MAN2B2-CDG have been reported worldwide. METHODS: Trio whole-exome sequencing (Trio-WES) was conducted to screen for candidate variants. N-glycan profiles were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MAN2B2 expression was evaluated by western blotting. MX dynamin like GTPase 1 (MX1) function was estimated via Thogoto virus (THOV) minireplicon assay. RESULTS: Trio-WES identified compound heterozygous MAN2B2 (hg19, NM_015274.1) variants (c.384G>T; c.926T>A) in a CDG patient. This patient exhibited metabolic abnormalities, symptoms of digestive tract dysfunction, infection, dehydration, and seizures. Novel immune dysregulation characterized by abnormal lymphocytes and immunoglobulin was observed. The MAN2B2 protein level was not affected, while LC-MS/MS showed obvious disruption of N-glycans and N-linked glycoproteins. CONCLUSION: We described a CDG patient with novel phenotypes and disruptive N-glycan profiling caused by compound heterozygous MAN2B2 variants (c.384G>T; c.926T>A). Our findings broadened both the genetic and clinical spectra of CDG.

ELISA-based highly sensitive assay system for the detection of endogenous NGLY1 activity.

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 × 103 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.

Transcriptomic analysis identifies dysregulated pathways and therapeutic targets in PMM2-CDG.

PMM2-CDG (MIM # 212065), the most common congenital disorder of glycosylation, is caused by the deficiency of phosphomannomutase 2 (PMM2). It is a multisystemic disease of variable severity that particularly affects the nervous system; however, its molecular pathophysiology remains poorly understood. Currently, there is no effective treatment. We performed an RNA-seq based transcriptomic study using patient-derived fibroblasts to gain insight into the mechanisms underlying the clinical symptomatology and to identify druggable targets. Systems biology methods were used to identify cellular pathways potentially affected by PMM2 deficiency, including Senescence, Bone regulation, Cell adhesion and Extracellular Matrix (ECM) and Response to cytokines. Functional validation assays using patients' fibroblasts revealed defects related to cell proliferation, cell cycle, the composition of the ECM and cell migration, and showed a potential role of the inflammatory response in the pathophysiology of the disease. Furthermore, treatment with a previously described pharmacological chaperone reverted the differential expression of some of the dysregulated genes. The results presented from transcriptomic data might serve as a platform for identifying therapeutic targets for PMM2-CDG, as well as for monitoring the effectiveness of therapeutic strategies, including pharmacological candidates and mannose-1-P, drug repurposing.

Clinical and biochemical footprints of congenital disorders of glycosylation: Proposed nosology.

We have identified 200 congenital disorders of glycosylation (CDG) caused by 189 different gene defects and have proposed a classification system for CDG based on the mode of action. This classification includes 8 categories: 1. Disorders of monosaccharide synthesis and interconversion, 2. Disorders of nucleotide sugar synthesis and transport, 3. Disorders of N-linked protein glycosylation, 4. Disorders of O-linked protein glycosylation, 5. Disorders of lipid glycosylation, 6. Disorders of vesicular trafficking, 7. Disorders of multiple glycosylation pathways and 8. Disorders of glycoprotein/glycan degradation. Additionally, using information from IEMbase, we have described the clinical involvement of 19 organs and systems, as well as essential laboratory investigations for each type of CDG. Neurological, dysmorphic, skeletal, and ocular manifestations were the most prevalent, occurring in 81%, 56%, 53%, and 46% of CDG, respectively. This was followed by digestive, cardiovascular, dermatological, endocrine, and hematological symptoms (17-34%). Immunological, genitourinary, respiratory, psychiatric, and renal symptoms were less frequently reported (8-12%), with hair and dental abnormalities present in only 4-7% of CDG. The information provided in this study, including our proposed classification system for CDG, may be beneficial for healthcare providers caring for individuals with metabolic conditions associated with CDG.

Intranasal oxytocin suppresses seizure-like behaviors in a mouse model of NGLY1 deficiency.

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.

A complement C4-derived glycopeptide is a biomarker for PMM2-CDG.

BACKGROUNDDiagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG.METHODSUsing multistep mass spectrometry-based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag-based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion.RESULTSOf the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2.CONCLUSIONComplement C4-derived Man5GlcNAc2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs.FUNDINGU54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance).

Revisiting the immunopathology of congenital disorders of glycosylation: an updated review.

Glycosylation is a critical post-translational modification that plays a pivotal role in several biological processes, such as the immune response. Alterations in glycosylation can modulate the course of various pathologies, such as the case of congenital disorders of glycosylation (CDG), a group of more than 160 rare and complex genetic diseases. Although the link between glycosylation and immune dysfunction has already been recognized, the immune involvement in most CDG remains largely unexplored and poorly understood. In this study, we provide an update on the immune dysfunction and clinical manifestations of the 12 CDG with major immune involvement, organized into 6 categories of inborn errors of immunity according to the International Union of Immunological Societies (IUIS). The immune involvement in phosphomannomutase 2 (PMM2)-CDG - the most frequent CDG - was comprehensively reviewed, highlighting a higher prevalence of immune issues during infancy and childhood and in R141H-bearing genotypes. Finally, using PMM2-CDG as a model, we point to links between abnormal glycosylation patterns in host cells and possibly favored interactions with microorganisms that may explain the higher susceptibility to infection. Further characterizing immunopathology and unusual host-pathogen adhesion in CDG can not only improve immunological standards of care but also pave the way for innovative preventive measures and targeted glycan-based therapies that may improve quality of life for people living with CDG.

Glycosphingolipids in congenital disorders of glycosylation (CDG).

Congenital disorders of glycosylation (CDG) are a large family of rare disorders affecting the different glycosylation pathways. Defective glycosylation can affect any organ, with varying symptoms among the different CDG. Even between individuals with the same CDG there is quite variable severity. Associating specific symptoms to deficiencies of certain glycoproteins or glycolipids is thus a challenging task. In this review, we focus on the glycosphingolipid (GSL) synthesis pathway, which is still rather unexplored in the context of CDG, and outline the functions of the main GSLs, including gangliosides, and their role in the central nervous system. We provide an overview of GSL studies that have been performed in CDG and show that abnormal GSL levels are not only observed in CDG directly affecting GSL synthesis, but also in better known CDG, such as PMM2-CDG. We highlight the importance of studying GSLs in CDG in order to better understand the pathophysiology of these disorders.

Neural and metabolic dysregulation in PMM2-deficient human in vitro neural models.

Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is a rare inborn error of metabolism caused by deficiency of the PMM2 enzyme, which leads to impaired protein glycosylation. While the disorder presents with primarily neurological symptoms, there is limited knowledge about the specific brain-related changes caused by PMM2 deficiency. Here, we demonstrate aberrant neural activity in 2D neuronal networks from PMM2-CDG individuals. Utilizing multi-omics datasets from 3D human cortical organoids (hCOs) derived from PMM2-CDG individuals, we identify widespread decreases in protein glycosylation, highlighting impaired glycosylation as a key pathological feature of PMM2-CDG, as well as impaired mitochondrial structure and abnormal glucose metabolism in PMM2-deficient hCOs, indicating disturbances in energy metabolism. Correlation between PMM2 enzymatic activity in hCOs and symptom severity suggests that the level of PMM2 enzyme function directly influences neurological manifestations. These findings enhance our understanding of specific brain-related perturbations associated with PMM2-CDG, offering insights into the underlying mechanisms and potential directions for therapeutic interventions.

Resilience in patients and family caregivers living with congenital disorders of glycosylation (CDG): a quantitative study using the brief resilience coping scale (BRCS).

BACKGROUND: Patients and family caregivers living with Congenital Disorders of Glycosylation (CDG) experience a heavy burden, which can impact their resiliency and quality of life. The study's purpose was to measure the resilience levels of patients and family caregivers living with CDG using the brief resilience coping scale. METHODS: We conducted an observational, cross-sectional study with 23 patients and 151 family caregivers living with CDG. Descriptive analyses were performed to characterize patients with CDG and family caregivers' samples. Additionally, we assessed correlations between resilience and specific variables (e.g., age, academic degree, time until diagnosis) and examined resilience differences between groups (e.g., sex, marital status, occupation, professional and social support). RESULTS: GNE myopathy was the most prevalent CDG among patients, while in family caregivers was PMM2-CDG. Both samples showed medium levels of resilience coping scores. Individuals with GNE myopathy had significantly higher scores of resilience compared to patients with other CDG. Resilience was positively correlated with educational degree in patients with CDG. Family caregivers had marginally significant higher scores of resilience coping if they received any kind of professional support or had contact with other families or people with the same or similar disease, compared with unsupported individuals. CONCLUSIONS: Despite the inherited difficulties of living with a life-threatening disease like CDG, patients and family caregivers showed medium resilient coping levels. Resilience scores changed significantly considering the CDG genotype, individual's academic degree and professional and social support. These exploratory findings can empower the healthcare system and private institutions by promoting the development of targeted interventions to enhance individuals` coping skills and improve the overall well-being and mental health of the CDG community.

Motor improvement in children with PMM2-CDG syndrome following a six-month rehabilitation treatment utilising whole-body vibration; a retrospective study.

OBJECTIVE: The aim of this study was to assess the effect of a six-month interval rehabilitation treatment on motor function of children with PMM2-CDG syndrome (#212065 Congenital disorder of glycosylation, Type Ia; CDG1A, OMIM catalogue number). METHODS: The concept 'Auf die Beine' (Center for Prevention and Rehabilitation of the University of Cologne, Germany) combines two short inpatient stays (1 to 2 weeks) with a six-month whole-body vibration (WBV) home-training program. 13 patients with PMM2-CDG syndrome participated in this concept from 2006 until 2015. Assessments at start, six months and 12 months (follow-up): Gross Motor Function Measure (GMFM-66), One-Minute Walk Test (1MWT) and instrumented gait analyses. RESULTS: The GMFM-66 (9 of 13 children) improved by 5.3 (mean) points (SD 3.2) at 12 months (p=0.0039). The 1MWT (6 of 13 children) improved by 19.17 meter (SD 16.51) after 12 months (p=0.0313). Gait analysis (9 of 13 children) measured by pathlength/distance ratio improved by -0.8 (SD 1.9) at 12 months (p=0.0195). CONCLUSION: Patients with PMM2-CDG syndrome benefit from the interval rehabilitation program 'Auf die Beine' including WBV.

Development of a fluorescence and quencher-based FRET assay for detection of endogenous peptide:N-glycanase/NGLY1 activity.

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.

Instrumented assessment of gait disturbance in PMM2-CDG adults: a feasibility analysis.

BACKGROUND: Congenital disorders of glycosylation (CDG) are genetic diseases caused by impaired synthesis of glycan moieties linked to glycoconjugates. Phosphomannomutase 2 deficiency (PMM2-CDG), the most frequent CDG, is characterized by prominent neurological involvement. Gait disturbance is a major cause of functional disability in patients with PMM2-CDG. However, no specific gait assessment for PMM2-CDG is available. This study analyses gait-related parameters in PMM2-CDG patients using a standardized clinical assessment and instrumented gait analysis (IGA). RESULTS: Seven adult patients with a molecular diagnosis of PMM2-CDG were followed-up from February 2021 to December 2022 and compared to a group of healthy control (HC) subjects, matched for age and sex. Standardized assessment of disease severity including ataxia and peripheral neuropathy along with isometric muscle strength and echo-biometry measurements at lower limbs were performed. IGA spatiotemporal parameters were obtained by means of a wearable sensor in basal conditions. PMM2-CDG patients displayed lower gait speed, stride length, cadence and symmetry index, compared to HC. Significant correlations were found among the used clinical scales and between disease severity (NCRS) scores and the gait speed measured by IGA. Variable reduction of knee extension strength and a significant decrease of lower limb muscle thickness with conserved echo intensity were found in PMM2-CDG compared to HC. CONCLUSIONS: The study elucidates different components of gait disturbance in PMM2-CDG patients and shows advantages of using wearable sensor-based IGA in this frame. IGA parameters may potentially serve as quantitative measures for follow-up or outcome quantification in PMM2-CDG.

Single-center experience of congenital disorders of glycosylation syndrome screening in Tunisia: A retrospective study over a 15-year period (2007-2021).

BACKGROUND: We report the results gathered over 15 years of screening for congenital disorders of glycosylation syndrome (CDGS) in Tunisia according to clinical and biochemical characteristics. METHODS: Our laboratory received 1055 analysis requests from various departments and hospitals, for children with a clinical suspicion of CDGS. The screening was carried out through separation of transferrin isoforms by capillary zone electrophoresis. RESULTS: During the 15-year period, 23 patients were diagnosed with CDGS (19 patients with CDG-Ia, three patients with CDG-IIx, and one patient with CDG-X). These patients included 13 boys and 10 girls aged between 3 months and 13 years, comprising 2.18 % of the total 1055 patients screened. The incidence for CDGS was estimated to be 1:23,720 live births (4.21 per 100,000) in Tunisia. The main clinical symptoms related to clinical disease state in newborn and younger patients were psychomotor retardation (91 %), cerebellar atrophy (91 %), ataxia (61 %), strabismus (48 %), dysmorphic symptoms (52 %), retinitis pigmentosa, cataract (35 %), hypotonia (30 %), and other symptoms. CONCLUSION: In Tunisia, CDGS still remains underdiagnosed or misdiagnosed. The resemblance to other diseases, especially neurological disorders, and physicians' unawareness of the existence of these diseases are the main reasons for the underdiagnosis. In routine diagnostics, the screening for CDGS by biochemical tests is mandatory to complete the clinical diagnosis.

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Progressive myoclonic epilepsy as an expanding phenotype of NGLY1-associated congenital deglycosylation disorder: A case report and review of the literature.

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.

Efficacy of oral manganese and D-galactose therapy in a patient bearing a novel TMEM165 variant.

TMEM165-CDG has first been reported in 2012 and manganese supplementation was shown highly efficient in rescuing glycosylation in isogenic KO cells. The unreported homozygous missense c.928G>C; p.Ala310Pro variant leading to a functional but unstable protein was identified. This patient was diagnosed at 2 months and displays a predominant bone phenotype and combined defects in N-, O- and GAG glycosylation. We administered for the first time a combined D-Gal and Mn2+ therapy to the patient. This fully suppressed the N-; O- and GAG hypoglycosylation. There was also striking improvement in biochemical parameters and in gastrointestinal symptoms. This study offers exciting therapeutic perspectives for TMEM165-CDG.

Neurological manifestations in PMM2-congenital disorders of glycosylation (PMM2-CDG): Insights into clinico-radiological characteristics, recommendations for follow-up, and future directions.

PURPOSE: In the absence of prospective data on neurological symptoms, disease outcome, or guidelines for system specific management in phosphomannomutase 2-congenital disorders of glycosylation (PMM2-CDG), we aimed to collect and review natural history data. METHODS: Fifty-one molecularly confirmed individuals with PMM2-CDG enrolled in the Frontiers of Congenital Disorders of Glycosylation natural history study were reviewed. In addition, we prospectively reviewed a smaller cohort of these individuals with PMM2-CDG on off-label acetazolamide treatment. RESULTS: Mean age at diagnosis was 28.04 months. Developmental delay is a constant phenotype. Neurological manifestation included ataxia (90.2%), myopathy (82.4%), seizures (56.9%), neuropathy (52.9%), microcephaly (19.1%), extrapyramidal symptoms (27.5%), stroke-like episodes (SLE) (15.7%), and spasticity (13.7%). Progressive cerebellar atrophy is the characteristic neuroimaging finding. Additionally, supratentorial white matter changes were noted in adult age. No correlation was observed between the seizure severity and SLE risk, although all patients with SLE have had seizures in the past. "Off-label" acetazolamide therapy in a smaller sub-cohort resulted in improvement in speech fluency but did not show statistically significant improvement in objective ataxia scores. CONCLUSION: Clinical and radiological findings suggest both neurodevelopmental and neurodegenerative pathophysiology. Seizures may manifest at any age and are responsive to levetiracetam monotherapy in most cases. Febrile seizure is the most common trigger for SLEs. Acetazolamide is well tolerated.