Research Models for O-GlcNAc Transferase Congenital Disorder of Glycosylation (OGT-CDG)

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Disclaimer: the following is Google Gemini Deep Research-generated text, not checked by research professionals. Use with caution. References are provided in the end of the post. If anything needs to be changed or removed – please contact Andrey Skripkin

I. Introduction to OGT-CDG and the Importance of Research Models

A. OGT-CDG/OGT-XLID Overview

O-GlcNAc Transferase Congenital Disorder of Glycosylation (OGT-CDG), also referred to as OGT-linked X-linked Intellectual Disability (OGT-XLID), is a syndromic neurodevelopmental disorder arising from genetic variants in the X-linked OGT gene.1 The OGT gene encodes the O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), a unique and essential enzyme responsible for catalyzing the addition of a single N-acetylglucosamine (O-GlcNAc) sugar moiety onto serine and threonine residues of thousands of nuclear and cytoplasmic proteins.4 This dynamic post-translational modification (PTM), known as O-GlcNAcylation, is reversed by the enzyme O-GlcNAcase (OGA), creating a rapid cycling system analogous in some respects to phosphorylation.1

The fundamental importance of OGT is underscored by its evolutionary conservation and essentiality for viability and development across diverse species. Complete knockout of Ogt in mice results in embryonic lethality at the blastocyst stage.5 Similarly, loss of OGT function is lethal or causes severe developmental defects in zebrafish and Drosophila melanogaster.5 This essentiality highlights the critical role of O-GlcNAcylation in fundamental cellular processes.

Clinically, OGT-CDG presents with considerable heterogeneity, though core features often include intellectual disability (ID), global developmental delay, and significantly restricted language skills.2 Affected individuals, predominantly males due to the X-linkage, frequently exhibit dysmorphic features such as clinodactyly (curved fifth finger), characteristic craniofacial features (e.g., high forehead, broad nasal root, long philtrum), and sometimes microcephaly, brain abnormalities (e.g., thin corpus callosum), and eye abnormalities.2 The variants causing OGT-CDG are typically missense mutations, found in both the N-terminal tetratricopeptide repeat (TPR) domain, involved in substrate recognition and protein interactions, and the C-terminal catalytic domain.1

B. Rationale for Diverse Research Models

Understanding the complex pathophysiology linking specific OGT variants to the diverse clinical manifestations of OGT-CDG necessitates the use of multiple, complementary research models.1 While early studies involving gene knockouts established the fundamental requirement for OGT in survival and development 5, these models do not fully recapitulate the situation in OGT-CDG patients, who harbor missense variants rather than null alleles.1 Therefore, developing models that carry specific patient-associated variants is a critical step toward dissecting the disease mechanisms relevant to human patients, moving beyond the study of basic OGT biology.2

The clinical heterogeneity observed in OGT-CDG 1 further emphasizes the need for a range of model systems. Different variants, located in distinct functional domains (TPR vs. catalytic), might disrupt different aspects of OGT function—such as catalytic activity, substrate specificity, protein-protein interactions mediated by the TPR domain, or the non-catalytic processing of Host Cell Factor 1 (HCF1)—to varying degrees.5 Investigating these possibilities requires models spanning different levels of biological complexity: from in vitro biochemical assays and cellular systems (including patient-derived cells and engineered stem cells) to probe molecular mechanisms and cellular homeostasis, to whole organisms (like mice and flies) to study developmental processes, systemic effects, and behavioral phenotypes relevant to ID.1 Key research groups, including those led by Daan van Aalten, Lance Wells, Stephanie Olivier-Van Stichelen, Gerald Hart, and Natasha E. Zachara, have been instrumental in generating and characterizing many of the models currently used to investigate OGT-CDG.1

II. Consolidated Overview: Table of OGT-CDG Research Models

A. Introduction to the Table

The following table provides a consolidated summary of the primary research models developed for studying OGT-CDG, as identified through a review of publicly available literature and research resources. This compilation aims to serve as a practical reference for researchers seeking information on the tools available for investigating the molecular, cellular, and organismal consequences of specific OGT variants. The table highlights the specific variants modeled, the biological system employed, key findings or applications, the originating laboratory or institution, and the known availability status. This centralized view allows for rapid identification of existing models relevant to particular variants or research questions and helps to pinpoint areas where model development may be needed.

B. Table 1: Current Research Models for OGT-CDG

Model TypeSpecific Model/Line IDGene Variant(s)Key Phenotypic Characteristics / Research ApplicationOriginating Lab/InstitutionAvailability Status & Contact InfoKey Reference(s)
Patient FibroblastsPatient 1 FibroblastsR284P (TPR)Reduced OGT/OGA levels, normal global O-GlcNAc, altered HCF1 maturation. Used for studying cellular O-GlcNAc homeostasis and HCF1 processing.Lefeber Lab (Radboudumc) / van Aalten Lab (Dundee/Aarhus)Likely restricted (patient material); Contact D. Lefeber (Dirk.Lefeber@Radboudumc.nl) or D. van Aalten (daan@mbg.au.dk).20
Patient FibroblastsPatient 2 Fibroblastsc.463-6T>G (Splicing defect, TPR region)Aberrant mRNA splicing, no stable truncated protein detected, reduced OGT/OGA levels, normal global O-GlcNAc. Used for studying cellular O-GlcNAc homeostasis.Lefeber Lab (Radboudumc) / van Aalten Lab (Dundee/Aarhus)Likely restricted (patient material); Contact D. Lefeber or D. van Aalten.20
Patient LymphoblastsK9427 (P1, P2)L254F (TPR)Decreased OGT/OGA levels, normal global O-GlcNAc, unstable OGT protein. Used for initial characterization of variant effects on protein stability and cellular homeostasis.Wells Lab (UGA CCRC) / Schwartz Lab (Greenwood Genetic Center)Possible via Greenwood Genetic Center or contact L. Wells (lwells@ccrc.uga.edu) / C. Schwartz (ceschwartz@ggc.org).16
mESCOGT-sfGFP reporterEndogenous Ogt tagged w/ sfGFPFluorescent reporter system for high-throughput screening of OGT variant effects on O-GlcNAc homeostasis (feedback response). OGT-CDG variants show reduced disruption vs WT/gnomAD.van Aalten Lab (Aarhus)Available upon request from D. van Aalten (daan@mbg.au.dk).1
mESCCRISPR-editedC921Y (Catalytic)Loss of catalytic activity, decreased O-GlcNAc, reduced Oct4/Sox2/ALP levels, impaired self-renewal capacity. Model for studying early developmental etiology.van Aalten Lab (Aarhus/Dundee)Likely available upon request from D. van Aalten (daan@mbg.au.dk).7
mESCCRISPR-editedN648Y (Catalytic)Significant reduction in global O-GlcNAcylation. Used for studying catalytic domain variant effects.Pravata et al. / van Aalten Lab (Dundee)Availability unclear; potentially via D. van Aalten.12
hESCRUES-1 CRISPR-editedL254F, A259T, R284P, E339G (TPR variants)Modest declines in stability/activity, significant dysregulation of genes involved in cell fate determination and signaling pathways. Model for interactome/transcriptome studies.Applied StemCell (service for Wells Lab, UGA CCRC)Availability unclear; Contact L. Wells (lwells@ccrc.uga.edu). Transcriptomic data in GEO (GSE110616).6
iPSCCRISPR-editedUnspecified OGT-CDG variant (likely C921Y based on lab’s other work)Decreased OGT/OGA protein levels, normal stemness/pluripotency, significant differences in O-GlcNAc homeostasis upon differentiation into ectoderm. Model for neuroectoderm development.van Aalten Lab (Aarhus)Availability unclear; Contact D. van Aalten (daan@mbg.au.dk).27
MouseC57BL/6J Allelic SeriesH568A, Y851A, T931A, Q849N (Catalytic Hypomorphs)Graded reduction in OGT catalytic activity; severity of embryonic lethality/delay scales with catalytic impairment; sexually dimorphic effects; ERV desilencing. Demonstrates catalysis is essential.Boulard Lab (EMBL Rome)Availability unclear; Contact M. Boulard. RNA-Seq data in Biostudies (E-MTAB-13298, etc.). Code on GitHub.4
MouseC57BL/6J CRISPR Knock-in (OGT^C921Y^)C921Y (Catalytic)Viable model. Decreased brain O-GlcNAc, OGT, OGA levels. Lower body weight/fat mass, short stature, microcephaly. Reduced levels of DDX3X. Recapitulates human features.van Aalten Lab (Aarhus/Dundee) / Univ. Edinburgh Transgenic CoreAvailability unclear; Contact D. van Aalten (daan@mbg.au.dk). Procedures under UK Home Office licenses.2
MouseC57BL/6J CRISPR Knock-inN648Y, N567K (Catalytic)Viable models carrying catalytically impaired OGT-CDG variants. Allows in vivo study of these specific mutations.van Aalten Lab (Aarhus/Dundee) / Univ. Edinburgh Transgenic CoreAvailability unclear; Contact D. van Aalten (daan@mbg.au.dk).52
Fly (Drosophila)CRISPR Knock-in (sxc^N595K^)N595K (Human N567K equivalent)Reduced global O-GlcNAcylation. Defects in habituation learning and synaptogenesis. Model for studying catalytic variant effects on behavior/neurons.van Aalten Lab (Aarhus/Dundee)Available upon request from D. van Aalten (daan@mbg.au.dk).17
Fly (Drosophila)CRISPR Knock-in (sxc^C941Y^)C941Y (Human C921Y equivalent)Reduced O-GlcNAc throughout development. Neuronal arborization defects, sleep defects. Phenotypes rescuable by OGA inhibition (genetic/pharmacological).van Aalten Lab (Aarhus/Dundee)Available upon request from D. van Aalten (daan@mbg.au.dk).17
Fly (Drosophila)CRISPR Knock-in (sxc^R313P^)R313P (Human R284P equivalent)Habituation learning deficit. Model for studying TPR variant effects on cognition-relevant behavior.van Aalten Lab (Dundee)Likely available upon request from D. van Aalten. Crossed into VDRC background.19
Fly (Drosophila)CRISPR Knock-in (sxc^A348T^)A348T (Human A319T equivalent)Habituation learning deficit. Model for studying TPR variant effects on cognition-relevant behavior.van Aalten Lab (Dundee)Likely available upon request from D. van Aalten. Crossed into VDRC background.19
Fly (Drosophila)CRISPR Knock-in (sxc^L283F^)L283F (Human L254F equivalent)Poor initial motor response (jump), precluding habituation assessment. Suggests potential motor system involvement for this variant.van Aalten Lab (Dundee)Likely available upon request from D. van Aalten. Crossed into VDRC background.19
Fly (Drosophila)Hypomorphic Allele (sxc^H537A^)H537A (Catalytic Hypomorph)Diminished catalytic activity. Habituation learning deficit. Used as a tool to study effects of reduced OGT activity.van Aalten Lab (Dundee)Likely available upon request from D. van Aalten.17
ZebrafishMorpholino Knockdown (ogt)General Loss-of-Function (LoF)Impaired embryonic growth, reduced brain size, developmental defects. Establishes essential role of OGT in vertebrate development.Dougan Lab (UGA) / OthersN/A (Method, not stable line). Morpholinos commercially available.8
C. elegansMutant Strains (ogt-1, oga-1)General LoF/GoF of O-GlcNAc cyclingAltered Pol II distribution, neuronal protection against toxicity, effects on axon regeneration via metabolic pathways (insulin signaling, glycolysis, mitochondria). Model for basic O-GlcNAc biology.Various labs (e.g., Gabel Lab, BU)Mutant strains often available via Caenorhabditis Genetics Center (CGC).19

Abbreviations: mESC (mouse Embryonic Stem Cell), hESC (human Embryonic Stem Cell), iPSC (induced Pluripotent Stem Cell), OGT (O-GlcNAc Transferase), OGA (O-GlcNAcase), O-GlcNAc (O-linked β-N-acetylglucosamine), TPR (Tetratricopeptide Repeat), HCF1 (Host Cell Factor 1), sfGFP (superfolder Green Fluorescent Protein), WT (Wild-Type), gnomAD (Genome Aggregation Database), GEO (Gene Expression Omnibus), ERV (Endogenous Retrovirus), UGA CCRC (University of Georgia Complex Carbohydrate Research Center), BU (Boston University), VDRC (Vienna Drosophila Resource Center), N/A (Not Applicable).*

C. Utility of the Consolidated Model Data

The compilation presented in Table 1 offers significant value to the OGT-CDG research community. By juxtaposing the phenotypic data derived from different models alongside the specific OGT variant studied, researchers can identify potential convergences and divergences in disease mechanisms. For instance, the observation of compensatory OGA downregulation in patient-derived cells 16 and also in the brains of C921Y knock-in mice 2 suggests a conserved homeostatic response across systems. Conversely, comparing the effects of TPR versus catalytic domain mutants in flies reveals shared habituation deficits but potentially different impacts on motor function 19, hinting at domain-specific consequences. Such comparisons are crucial for building a comprehensive understanding of OGT-CDG pathophysiology, distinguishing core disease mechanisms from model-specific artifacts, and guiding the selection of appropriate models for specific research questions.

III. Detailed Summary by Model Type

A. Cell-Based Models

Cell-based systems represent foundational tools for dissecting the molecular and cellular consequences of OGT-CDG variants. These range from primary cells derived directly from patients to genetically engineered stem cell lines.

1. Patient-Derived Lines (Fibroblasts, Lymphoblasts)

Primary cells, such as skin fibroblasts and Epstein-Barr virus-transformed lymphoblastoid cell lines, obtained from individuals with OGT-CDG offer the most direct human context for investigation.1 Studies utilizing these cells have provided initial insights into the effects of specific patient mutations.

Work by the Wells laboratory (University of Georgia) and collaborators characterized lymphoblastoid cell lines (K9427) from a family with the OGT L254F variant located in the TPR domain.16 These cells exhibited decreased levels of both OGT and OGA protein, yet maintained apparently normal global levels of O-GlcNAcylation. Biochemical studies indicated that the L254F mutation rendered the OGT protein unstable.16

Similarly, research from the Lefeber (Radboud University Medical Center) and van Aalten (then University of Dundee) labs examined fibroblasts from patients with the TPR variant R284P and a splicing defect (c.463-6T>G) affecting the TPR region.20 Both patient cell lines showed reduced levels of OGT protein and a corresponding decrease in OGA protein levels, while global O-GlcNAc levels appeared largely unaffected. Notably, the R284P variant demonstrated reduced ability to process HCF1 in vitro, suggesting that disruption of this OGT function, in addition to potential subtle effects on glycosylation, might contribute to the XLID phenotype.20 The splicing variant resulted in aberrant mRNA but no detectable stable truncated protein.25

A consistent finding across these patient-derived cell studies is the coordinate downregulation of OGA protein levels in response to reduced functional OGT.16 This observation points towards a robust cellular homeostatic mechanism that attempts to buffer global O-GlcNAcylation levels. The fact that global O-GlcNAc appears relatively normal in these specific patient cell contexts, despite the presence of pathogenic variants and reduced OGT/OGA, implies that the disease pathology may not solely stem from a gross reduction in overall O-GlcNAcylation. Instead, it might arise from more subtle defects, such as altered glycosylation of specific critical substrates, disruption of protein-protein interactions mediated by the OGT TPR domain 5, or impaired HCF1 processing.20 While invaluable for their human relevance, patient-derived lines can be limited by availability, finite lifespan (fibroblasts), potential variability introduced during transformation (lymphoblasts), and inherent genetic background differences between individuals, which can complicate comparisons.1

2. Engineered Stem Cell Lines (mESC, hESC, iPSC)

Engineered stem cell lines, including mouse embryonic stem cells (mESC), human ESC (hESC), and induced pluripotent stem cells (iPSC), offer significant advantages for studying OGT-CDG. Their pluripotency allows investigation of early developmental processes and differentiation into relevant cell types (e.g., neurons), which is crucial for understanding a neurodevelopmental disorder.10 Furthermore, the use of genome editing technologies like CRISPR/Cas9 enables the creation of cell lines carrying specific OGT variants alongside perfectly matched isogenic controls, eliminating confounding genetic background variations.1

Several mESC models have been developed. The van Aalten laboratory (Aarhus University) created an innovative OGT-sfGFP knock-in reporter mESC line.1 In this system, endogenous OGT is tagged with superfolder GFP (sfGFP). The fluorescence intensity reflects OGT protein levels, which are subject to feedback regulation based on cellular O-GlcNAc status. This line provides a high-throughput platform to screen the functional impact of OGT variants by measuring their ability to disrupt O-GlcNAc homeostasis; OGT-CDG variants generally induced a lower feedback response (less reduction in sfGFP signal upon overexpression) compared to wild-type or benign variants from the gnomAD database.1 This reporter system represents a significant technological advance for the functional classification of the rapidly increasing number of OGT variants identified in patients, addressing the bottleneck created by slower, more costly traditional characterization methods.1 The same group also generated an mESC line carrying the catalytic domain variant C921Y.43 These cells exhibited loss of catalytic activity, decreased global O-GlcNAcylation, and importantly, showed reduced expression of pluripotency markers (Oct4, Sox2) and alkaline phosphatase (ALP), indicating impaired self-renewal capacity.18 This finding directly links an OGT-CDG variant to a potential defect in early stem cell function. Additionally, an mESC model for the N648Y catalytic variant was reported to show a significant reduction in modified proteins.12

In human ESCs, the Wells laboratory utilized CRISPR/Cas9 to engineer the male RUES-1 line to express several TPR domain variants (L254F, A259T, R284P, E339G) associated with XLID.13 While biochemical characterization revealed only modest effects on the stability or in vitro activity of these variants, transcriptomic analysis uncovered significant shared dysregulation of genes and pathways involved in early development and cell fate determination.13 This highlights the power of stem cell models, particularly hESCs and iPSCs, to uncover pathogenic mechanisms related to gene expression control that might be missed by purely biochemical assays or in non-developmental cell types. The observed transcriptomic changes, even with subtle biochemical defects, suggest that OGT’s role in regulating gene expression—potentially through interactions with chromatin modifiers 6 or via HCF1 processing 2—is particularly critical during early development and differentiation.

More recently, an iPSC model of OGT-CDG (variant unspecified in the abstract, potentially C921Y) was developed by the van Aalten group.48 Undifferentiated OGT-CDG iPSCs showed decreased OGT and OGA protein levels but maintained normal stemness markers. However, upon differentiation towards the ectodermal lineage, significant differences in O-GlcNAc homeostasis emerged compared to isogenic controls.48 This suggests that the neuroectoderm, the precursor to the nervous system, may be particularly vulnerable to perturbations caused by OGT-CDG variants, providing a potential explanation for the predominantly neurological phenotype. General protocols for differentiating iPSCs into relevant lineages like neural crest cells are established.61

B. Mouse Models

Mouse models provide an essential platform for studying the systemic effects, developmental trajectories, and behavioral consequences of OGT-CDG variants in vivo.

1. OGT Knockout and Hypomorphs

As mentioned, complete deletion of Ogt in mice is embryonic lethal, precluding its use for studying postnatal phenotypes associated with OGT-CDG.2 Conditional knockouts targeted to specific brain cell populations have highlighted OGT’s roles in neurodevelopment and neuronal survival but often also result in severe phenotypes or lethality.10

A significant advance came from the Boulard laboratory (EMBL Rome), which generated a murine allelic series carrying specific point mutations in the OGT catalytic domain (H568A, Y851A, T931A, Q849N) designed to cause graded reductions in enzymatic activity in vivo.4 Analysis of these hypomorphic models demonstrated unequivocally that the severity of embryonic lethality and developmental delay scaled directly with the degree of catalytic impairment.4 This provides strong in vivo evidence that the O-GlcNAc modification itself, and its level, is critically required for normal mouse embryonic development, supporting the hypothesis that reduced O-GlcNAcylation is a key pathogenic factor, at least for catalytic domain variants. These models also revealed unexpected roles for OGT/O-GlcNAc levels in silencing endogenous retroviruses and uncovered sexually dimorphic sensitivity to OGT impairment, with male embryos being more affected.4

2. Specific OGT-CDG Variant Knock-in Mice

The generation of viable knock-in (KI) mouse lines carrying specific OGT-CDG patient variants represents a major breakthrough for the field.2 These models allow for the investigation of OGT-CDG pathophysiology in a whole-organism context throughout postnatal life.

The van Aalten laboratory (Aarhus/Dundee) successfully generated a KI mouse model carrying the C921Y mutation, a catalytically impaired variant found in OGT-CDG patients.2 Unlike Ogt knockout mice, OGT^C921Y^ mice are viable and fertile.11 Phenotypic characterization revealed several features mirroring the human condition: the mice exhibit lower body weight associated with reduced fat mass, shorter stature (naso-anal length), and microcephaly.2 At the molecular level, brains of OGT^C921Y^ mice show altered O-GlcNAc homeostasis with decreased global O-GlcNAcylation and reduced levels of both OGT and OGA proteins 2, consistent with findings in patient cells. Furthermore, this model showed reduced levels of DDX3X 10, another X-linked intellectual disability gene product, suggesting a potential downstream pathway affected in OGT-CDG. The viability and relevant phenotypes of the OGT^C921Y^ mouse make it an invaluable tool for studying genotype-phenotype correlations, investigating neurodevelopmental mechanisms, exploring behavior, and potentially testing therapeutic interventions in vivo.2

The same group also reported the generation of viable KI mouse lines for two other catalytic domain variants, N648Y and N567K.52 The availability of multiple distinct OGT-CDG KI mouse models will allow for comparative studies to understand potential variant-specific effects in vivo.

C. Drosophila Models

Drosophila melanogaster (fruit fly) serves as a powerful genetic model organism for studying OGT function and OGT-CDG, owing to its sophisticated genetic toolkit, short generation time, and the presence of a highly conserved OGT ortholog encoded by the Polycomb group gene super sex combs (sxc).5 Similar to mammals, complete loss of sxc function is lethal and causes developmental patterning defects.5

The van Aalten lab has leveraged CRISPR/Cas9 to generate several Drosophila lines carrying mutations equivalent to human OGT-CDG variants.17 Models for catalytic domain variants, including sxc^N595K^ (human N567K) and sxc^C941Y^ (human C921Y), exhibit reduced global O-GlcNAcylation levels.17 These flies display significant phenotypes relevant to neurodevelopmental disorders, including defects in neuronal arborization (branching), abnormal sleep patterns, and impaired habituation, a simple form of non-associative learning often disrupted in ID models.17

Fly models carrying mutations equivalent to TPR domain variants, such as sxc^R313P^ (human R284P) and sxc^A348T^ (human A319T), also show deficits in habituation learning.19 Interestingly, the sxc^L283F^ model (human L254F) displayed a poor initial jump response, preventing assessment of habituation but suggesting a potential impact on motor systems or sensory processing for this specific TPR variant.19

Crucially, studies in the catalytic mutant fly models (sxc^N595K^, sxc^C941Y^) demonstrated that the observed defects in habituation, sleep, and synaptogenesis could be rescued or ameliorated by either genetically removing OGA activity or pharmacologically inhibiting OGA using compounds like Thiamet-G.17 This provides compelling evidence that these behavioral and neuronal phenotypes are directly linked to the reduction in O-GlcNAcylation levels caused by the hypomorphic OGT variants. The ability to rescue these phenotypes by increasing O-GlcNAc levels strongly implicates hypo-O-GlcNAcylation as a key driver of the cognitive deficits associated with these variants and importantly, suggests that targeting OGA to boost O-GlcNAcylation could be a viable therapeutic strategy for OGT-CDG.17 The fly models, therefore, not only replicate relevant behavioral deficits but also provide a platform for exploring therapeutic approaches.

D. Zebrafish Models

Zebrafish (Danio rerio) are another valuable vertebrate model system frequently used in developmental biology and disease modeling. Studies using morpholino-mediated knockdown have established that OGT is essential for normal zebrafish development.8 Depletion of ogt results in impaired embryonic growth, a shortened body axis, and notably, reduced brain size.14 These findings confirm the conserved role of OGT in vertebrate development, particularly in the formation of the central nervous system.

However, the zebrafish system presents some complexity regarding OGT, as the zebrafish genome contains two distinct ogt genes, ogta and ogtb, arising from teleost-specific genome duplication events. These genes produce multiple transcripts, and not all encoded protein isoforms possess conventional OGT catalytic activity.59

Currently, based on the reviewed literature, specific OGT-CDG patient variant models (e.g., knock-ins) have not been reported in zebrafish.5 Research in this model has primarily focused on understanding the consequences of general OGT loss-of-function through knockdown approaches or investigating broader O-GlcNAc biology.59 While zebrafish offer advantages for studying vertebrate development, the absence of specific OGT-CDG variant models represents a potential gap in the available toolkit. This may be partly due to the challenges associated with modeling heterozygous or hemizygous human mutations in the context of duplicated ogt genes with potentially overlapping or distinct functions.

E. Other Models (e.g., C. elegans)

The nematode Caenorhabditis elegans is a powerful model for investigating fundamental biological processes due to its genetic simplicity, well-defined cell lineage, and rapid lifecycle.64 Research in C. elegans has provided valuable insights into the conserved roles of O-GlcNAc signaling (ogt-1 and oga-1 being the orthologs) in the nervous system. For instance, studies have shown that O-GlcNAcylation can protect against proteotoxicity in worm models of neurodegenerative diseases 19 and plays a complex role in orchestrating the metabolic response during axon regeneration after injury.60 Mutants in ogt-1 or oga-1 affect various processes, including the genomic distribution of RNA Polymerase II.46

However, no studies reporting the generation or characterization of C. elegans models carrying specific human OGT-CDG variants were identified in the reviewed literature.19 While C. elegans is invaluable for dissecting conserved cellular pathways involving O-GlcNAc, its significant evolutionary distance from humans, coupled with differences in glycosylation pathways (e.g., its complex and unusual N-glycome 64), may limit its utility for directly modeling the specific nuances of the human OGT-CDG syndrome. Its primary strength likely lies in uncovering basic mechanisms of O-GlcNAc function rather than precise disease replication.

IV. Model Availability and Access for Researchers

A. General Accessibility Landscape

The accessibility of the various OGT-CDG research models described above varies considerably. A significant portion of the specific engineered cell lines (mESC, hESC, iPSC) and animal models (mice, flies) appear to be primarily maintained within the laboratories that generated them, notably the groups of Daan van Aalten (Aarhus University, formerly University of Dundee) 2, Lance Wells (University of Georgia CCRC) 34, Michel Boulard (EMBL Rome) 4, and Dirk Lefeber (Radboudumc).20

Consequently, accessing these valuable resources often necessitates direct contact with the principal investigators. Contact information, such as email addresses for Daan van Aalten (daan@mbg.au.dk) 1 and Lance Wells (lwells@ccrc.uga.edu) 16, is typically available in corresponding publications or via institutional websites. Acquisition of materials usually involves the execution of a Material Transfer Agreement (MTA) between institutions.

In some instances, availability is explicitly stated within publications. For example, the van Aalten lab has indicated that the OGT-sfGFP reporter mESC line 1 and their generated Drosophila genotypes 17 are available upon request. Such clear statements greatly facilitate resource sharing.

Patient-derived materials, such as lymphoblastoid cell lines or fibroblasts, may have more complex access routes. For example, lymphoblastoid lines related to the L254F variant originated from samples collected via the Greenwood Genetic Center 16, which might be a point of contact, although ethical approvals and patient consent govern the sharing of such materials.20

Regarding public repositories, while some standard fly stocks (e.g., RNAi lines, background controls) used in OGT-CDG research can be obtained from resources like the Vienna Drosophila Resource Center (VDRC) 19, the specific CRISPR-generated OGT-CDG mutant fly lines generally require direct contact with the generating lab.19 There was no mention in the reviewed materials of specific OGT-CDG patient variant cell lines being deposited in widely used repositories like the American Type Culture Collection (ATCC) or Addgene. Similarly, while data (e.g., RNA-Seq) from some model studies are deposited in public databases like GEO or Biostudies 4, the physical mouse models themselves are typically requested from the generating lab or facility.2

The current reliance on direct lab-to-lab sharing for many specific OGT-CDG models, while standard practice in biomedical research, can potentially create bottlenecks. The process of identifying the correct contact, negotiating MTAs, and arranging shipments can slow down the initiation of research for groups wishing to utilize these specialized tools. This may limit the speed and breadth of investigation across the wider research community compared to models readily available from centralized repositories. Nonetheless, the explicit statements of availability upon request for key resources like the reporter mESC line 1 and specific fly lines 17 are highly valuable. Such transparency removes ambiguity for potential users and sets a positive precedent, encouraging the utilization of validated tools and fostering collaboration, which ultimately benefits the entire OGT-CDG research field.

V. Key Insights and Concluding Remarks

A. Synthesis of Current OGT-CDG Modeling

Significant progress has been achieved in developing a diverse portfolio of research models to investigate OGT-CDG. The field has effectively transitioned from initial studies focused on the essentiality of OGT using knockout approaches to creating sophisticated systems that model specific patient-associated variants. These include patient-derived primary cells, engineered isogenic stem cell lines (mESC, hESC, iPSC), and whole-organism models in mice and Drosophila.

These models collectively cover a range of OGT-CDG variants spanning both the TPR and catalytic domains. They have revealed a spectrum of phenotypes, including alterations in OGT/OGA protein levels, changes in global or substrate-specific O-GlcNAcylation, impaired stem cell self-renewal, altered HCF1 processing, dysregulated gene expression, developmental abnormalities (e.g., microcephaly, growth restriction in mice), and behavioral deficits (e.g., learning and sleep defects in flies). Encouragingly, many phenotypes observed in these models, particularly in the viable knock-in mice, align well with clinical features reported in OGT-CDG patients.2

The various model systems offer complementary strengths: patient cells provide direct human relevance; stem cells allow interrogation of developmental processes and differentiation; mice enable the study of in vivo physiology, neurodevelopment, and behavior in a mammalian context; and flies offer rapid genetic manipulation and screening capabilities for behavioral and neuronal phenotypes.

B. Emerging Themes and Potential Gaps

Several key themes emerge from the analysis of existing OGT-CDG models. One prominent theme is the complexity of O-GlcNAc homeostasis. Multiple models, including patient cells and knock-in mice, demonstrate a compensatory downregulation of OGA in response to OGT defects, often resulting in near-normal global O-GlcNAc levels.11 This suggests that OGT-CDG pathology might frequently stem from disruption of OGT’s non-catalytic functions (e.g., protein interactions, HCF1 processing) or altered glycosylation of specific crucial substrates, rather than solely from global hypo-O-GlcNAcylation.

Despite the different primary molecular defects caused by variants in the TPR versus catalytic domains, there appears to be a convergence on certain downstream phenotypes, such as developmental delay, ID-related behavioral deficits (in flies), and potentially shared dysregulation of developmental gene expression pathways.13 This suggests that OGT function, whether catalytic or interaction-based, is critical for common pathways essential for neurodevelopment.

Furthermore, the successful rescue of behavioral and neuronal phenotypes in Drosophila catalytic mutants by inhibiting OGA provides proof-of-concept for a potential therapeutic strategy aimed at boosting O-GlcNAc levels.17

Potential gaps in the current modeling landscape include the relative scarcity of models for certain specific variants, particularly some TPR domain mutations compared to extensively studied catalytic variants like C921Y. The lack of reported specific OGT-CDG variant models in zebrafish and C. elegans also represents an area for potential future development, although technical challenges (e.g., gene duplication in zebrafish) may exist. Finally, improving the accessibility of key models through deposition in public repositories could significantly accelerate research progress [Insight 13]. A continued need exists for more sensitive methods, such as quantitative glycoproteomics, to define substrate-specific changes in O-GlcNAcylation in various models.15

C. Future Directions and Conclusion

The existing OGT-CDG research models provide a robust foundation for future investigations. Immediate next steps could involve leveraging these models for deeper mechanistic studies. This includes comprehensive interactome analyses (building on work like 5) and substrate-specific glycoproteomic profiling across different variants and model systems to pinpoint critical downstream targets and pathways disrupted in OGT-CDG. Expanding the repertoire of patient variants modeled, particularly those in the TPR domain or those with less clear functional consequences, will also be important for understanding the full spectrum of the disorder.

Further characterization of the existing mouse models, including detailed behavioral analyses and investigation of neurodevelopmental trajectories, will be crucial for understanding the in vivo consequences of OGT dysfunction relevant to ID. These models also provide essential platforms for preclinical testing of potential therapeutic strategies, such as OGA inhibitors, informed by the promising rescue data from Drosophila studies. Efforts to enhance the accessibility of validated models, perhaps through coordinated deposition in public repositories, would significantly benefit the research community.

In conclusion, the development and characterization of diverse research models have been instrumental in advancing our understanding of OGT-CDG. From patient cells providing human context to engineered stem cells illuminating developmental defects, and animal models revealing in vivo consequences and enabling behavioral studies, these tools are paving the way for uncovering the complex pathophysiology of this disorder and exploring potential therapeutic interventions. Continued refinement and application of these models hold great promise for improving the lives of individuals affected by OGT-CDG.

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