Recombinant Bovine Dolichol phosphate-mannose biosynthesis regulatory protein (DPM2)

What is the structural composition of DPM2 protein?

DPM2 is an 84-amino acid membrane protein localized in the endoplasmic reticulum (ER). The protein contains two putative transmembrane domains spanning amino acid residues 11-31 and 49-69 . These transmembrane domains are critical for its function, particularly the first domain which is involved in association with DPM1 . The protein's compact structure enables it to serve as a stabilizing element within the heterotrimeric DPM synthase complex, which consists of DPM1, DPM2, and DPM3 .

The transmembrane topology of DPM2 is crucial for its regulatory role, as specific amino acid residues like tyrosine at position 23 (Tyr23) in the first transmembrane domain are highly conserved throughout evolution, indicating their essential role in maintaining proper protein function . This structural conservation suggests selective pressure to preserve DPM2's ability to properly interact with other components of the glycosylation machinery.

What are the primary functions of DPM2 in cellular metabolism?

DPM2 serves as a critical regulatory component of the heterotrimeric dolichol-phosphate-mannose (DPM) synthase complex, which catalyzes the synthesis of dolichol-P-mannose . This complex plays essential roles in multiple glycosylation pathways, including:

• N-glycosylation of proteins

• O-mannosylation

• C-mannosylation

• Formation of glycosyl-phosphatidylinositol (GPI) anchors

DPM2's primary function appears to be stabilizing the DPM synthase complex, particularly by stabilizing the DPM3 protein . This stabilization is essential for proper enzymatic function of the complex. Additionally, DPM2 is required for the correct localization of DPM1 to the ER, which is critical for the synthetic activity of the complex . Without DPM2, the complex cannot properly assemble or function, leading to defects in various glycosylation pathways.

How does DPM2 interact with other components of the DPM synthase complex?

DPM2 forms specific interactions with both DPM1 and DPM3 within the heterotrimeric DPM synthase complex. The first transmembrane domain of DPM2 (amino acids 11-31) is particularly important for its association with DPM1 . Specifically, amino acid residues Phe21 and Tyr23 in this domain are critical, as substitutions at these positions can abolish DPM2's ability to associate with DPM1 .

The interaction between DPM2 and other complex components appears to serve two primary functions:

• Stabilization: DPM2 stabilizes the entire synthase complex, particularly the DPM3 protein, ensuring that all components maintain their proper conformation and activity .

• Localization: DPM2 is essential for the correct ER localization of DPM1, which is critical because the enzymatic activity must occur at the ER membrane where dolichol phosphate is accessible .

These interactions create a functional unit capable of efficiently catalyzing the synthesis of dolichol-P-mannose, which serves as a mannosyl donor for various glycosylation pathways.

What are the recommended protocols for expressing and purifying recombinant DPM2?

For effective expression and purification of recombinant DPM2, researchers should consider the following methodological approach based on recent successful experimental designs:

Expression System Preparation:

• Amplify the DPM2 coding sequence from an appropriate cellular source (e.g., HCT116 cells) using primers targeting the full coding region: 5′-ATG GCC ACG GGG ACA GAC-3′ and 5′-TCA CTG AGC CTT CTT GGT CAC TCTC-3′ with high-fidelity DNA polymerase .

• Insert the amplified fragment into an expression vector (e.g., pcDNA3.1(+)) using appropriate restriction sites (BamHI and EcoRI) .

• Verify correct insertion and sequence by Sanger sequencing.

Cell Culture and Transfection:

• Culture host cells (HCT116 cells or similar mammalian cell lines) in DMEM supplemented with 10% fetal bovine serum .

• Transfect cells with the verified DPM2-containing plasmid using a lipofection reagent such as Lip3000 at a concentration of 1 μg plasmid per well in a 6-well plate format .

• Allow 24-48 hours for protein expression.

Protein Extraction and Purification:

• Lyse cells in 1% NP-40 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.5% sodium deoxycholate) for 30 minutes on ice .

• Centrifuge at 12,000 rpm for 10 minutes to remove cellular debris.

• For tagged protein variants, employ affinity chromatography (e.g., anti-Flag for Flag-tagged constructs).

• Verify protein expression by SDS-PAGE (12% gels) and western blotting using appropriate antibodies.

This protocol can be adapted for generating both wild-type DPM2 and variant forms for comparative studies of protein function and stability.

What methods are most effective for studying DPM2 localization and protein-protein interactions?

For investigating DPM2 localization and protein-protein interactions, researchers should implement the following complementary approaches:

Subcellular Localization Analysis:

• Immunofluorescence Microscopy: Fix cells with 3% paraformaldehyde for 15 minutes, permeabilize with 0.1% Triton X-100/PBS for 15 minutes, and block with 3% BSA/PBS for 60 minutes. Incubate with anti-DPM2 antibody and organelle markers (e.g., anti-PDI for ER localization) for 60 minutes, followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488 and 555). Counterstain with DAPI and visualize using confocal microscopy .

• Subcellular Fractionation: Separate cellular compartments through differential centrifugation and analyze DPM2 distribution by immunoblotting of fraction samples.

Protein-Protein Interaction Studies:

• Co-immunoprecipitation: Use anti-DPM2 antibodies to pull down protein complexes, followed by immunoblotting for potential interaction partners (DPM1, DPM3).

• Proximity Ligation Assay: Apply this technique to visualize and quantify interactions between DPM2 and other components of the DPM synthase complex in situ.

• Site-Directed Mutagenesis: Generate variants of DPM2 targeting specific residues in the transmembrane domains (particularly focusing on Tyr23 and other conserved residues) to assess their impact on protein interactions . This approach can effectively map the interaction interface between DPM2 and other proteins.

These methodologies allow for comprehensive characterization of both the subcellular distribution of DPM2 and its interactions with other proteins, providing insights into the functional significance of these spatial arrangements and molecular associations.

How can researchers assess the functional impact of DPM2 variants?

To assess the functional impact of DPM2 variants, researchers should implement a multi-faceted experimental approach:

Generation of DPM2 Variants:

• Employ site-directed mutagenesis using overlap PCR to introduce specific mutations. For example, to generate G66E-DPM2, use primers 5′-GAC GCT CCT GTT TGT GGA ACT GTT CAT CTC-3′ and 5′-TCC ACA AAC AGG AGC AGC AGG AGG CCTG-3′ .

• Verify all constructs through Sanger sequencing to confirm the presence of the desired mutation.

Expression Analysis:

• Measure mRNA expression levels via quantitative RT-PCR using primers specific to DPM2 (e.g., DPM2-F: CTT GCC ATT CAT CGA CAG TCAG; DPM2-R: CTT GGT CAC TCT CTT GGT CTTC) .

• Assess protein expression through western blotting using anti-DPM2 or anti-tag antibodies if the construct contains an epitope tag (e.g., Flag) .

Functional Assessments:

• Glycosylation Marker Analysis: Measure levels of glycosylation markers such as ICAM1, a universal biomarker for hypoglycosylation in cells expressing DPM2 variants .

• Protein Stability Assessment: Perform cycloheximide chase assays to determine if variants affect protein half-life.

• Complex Formation Analysis: Assess the ability of DPM2 variants to form complexes with DPM1 and DPM3 through co-immunoprecipitation experiments.

• Enzymatic Activity: Measure DPM synthase activity in cells expressing wild-type versus variant DPM2 by assessing the incorporation of radioactively labeled mannose into dolichol phosphate.

The table below summarizes key findings from previous studies comparing wild-type DPM2 with variant forms:

This systematic approach allows researchers to comprehensively characterize the molecular, cellular, and functional consequences of DPM2 variants, providing insights into structure-function relationships and potential pathogenic mechanisms.

What are the known pathogenic variants of DPM2 and their clinical manifestations?

DPM2 mutations are associated with a rare form of congenital disorders of glycosylation (CDG), specifically classified as DPM2-CDG. Based on current literature, six cases from four families with five distinct variants have been identified :

Patients with mutations in DPM2 typically present with a spectrum of clinical manifestations including:

• Developmental delay and intellectual disability ranging from mild to severe

• Hypotonia (decreased muscle tone)

• Seizures, which can be intractable in severe cases

• Dysmorphic features

• Multiple system involvement (nervous system, liver, heart, muscular, eyes, and immune system)

Notably, patients with the Tyr23Cys variant (homozygous or compound heterozygous) exhibit more severe phenotypes compared to patients with variants in the second transmembrane domain, suggesting a potential genotype-phenotype correlation . This variant is located in the first transmembrane domain, which is critical for DPM2's interaction with DPM1, explaining the severity of this mutation's impact.

How do researchers establish genotype-phenotype correlations for DPM2 variants?

Establishing genotype-phenotype correlations for DPM2 variants requires a systematic and multidisciplinary approach:

Clinical Characterization:

• Comprehensive clinical evaluation of patients with DPM2 variants, including detailed neurological assessment, developmental milestones, imaging studies, and evaluation of multiple organ systems.

• Standardized assessment tools to quantify the severity of symptoms.

• Longitudinal follow-up to document disease progression.

Genetic Analysis:

• Whole-Exome Sequencing (WES): Using platforms like Illumina NovaSeq 6000 with subsequent data analysis through tools such as GATK for variant identification .

• Variant Confirmation: Validating identified variants through Sanger sequencing in patients and family members to establish inheritance patterns.

• Conservation Analysis: Evaluating evolutionary conservation of affected amino acid residues using software like MEGA to assess potential functional significance .

Functional Studies:

• In vitro expression of wild-type and mutant DPM2 proteins to compare expression levels, subcellular localization, and protein-protein interactions .

• Assessment of glycosylation markers like ICAM1 to evaluate functional consequences of variants .

• Structural analysis of variants to determine their position relative to functional domains (e.g., transmembrane domains).

Correlation Analysis:

• Systematic comparison of clinical severity with the molecular impact of variants.

• Consideration of the variant's location within the protein structure (e.g., first vs. second transmembrane domain).

• Analysis of the biochemical properties of the substituted amino acids.

Current evidence suggests a potential correlation between variant location and disease severity, with mutations in the first transmembrane domain (e.g., Tyr23Cys) associated with more severe phenotypes compared to mutations in the second transmembrane domain . This correlation likely reflects the critical role of the first transmembrane domain in mediating DPM2's interaction with DPM1, highlighting the importance of protein-protein interactions for DPM2 function.

What glycosylation biomarkers are most informative for monitoring DPM2 deficiency?

For monitoring DPM2 deficiency, several glycosylation biomarkers provide valuable insights into the functional consequences of impaired DPM synthase activity:

Primary Biomarkers:

• ICAM1 (Intercellular Adhesion Molecule 1): This serves as a universal biomarker for hypoglycosylation in patients with congenital disorders of glycosylation (CDG). Decreased ICAM1 levels have been observed in cells expressing pathogenic DPM2 variants (both Tyr23Cys and Gly66Glu), making it a sensitive indicator of DPM2 dysfunction . ICAM1 levels can be assessed through western blotting using specific antibodies (e.g., Proteintech, 60299-1-Ig at 1:3000 dilution) .

• Transferrin Glycoforms: Analysis of transferrin glycosylation patterns through isoelectric focusing or mass spectrometry can reveal characteristic abnormalities in N-glycosylation.

• Alpha-Dystroglycan Glycosylation: Since DPM2 is involved in O-mannosylation, assessment of alpha-dystroglycan glycosylation through immunofluorescence or western blotting can provide insights into disruption of this pathway.

Analytical Methods:

• Western Blotting: For quantitative assessment of glycoprotein levels like ICAM1 .

• Flow Cytometry: To evaluate surface expression of glycoproteins in patient cells.

• Mass Spectrometry: For comprehensive analysis of glycan structures on multiple glycoproteins.

• Lectin Binding Assays: To detect specific glycan structures affected by DPM2 deficiency.

A comprehensive glycosylation assessment should include multiple biomarkers to capture the diverse glycosylation pathways affected by DPM2 deficiency, including N-glycosylation, O-mannosylation, C-mannosylation, and GPI-anchor biosynthesis. This multi-marker approach provides a more complete picture of the functional consequences of DPM2 variants and can help monitor disease progression or therapeutic response.

How do researchers address data contradictions when studying DPM2 expression and function?

When confronting contradictory data in DPM2 research, scientists should implement a structured approach to resolve inconsistencies and enhance data quality:

Contradiction Pattern Analysis:
Researchers can apply the (α, β, θ) notation system to classify contradiction patterns in DPM2 experimental data :

• α: Number of interdependent items (e.g., mRNA level, protein expression, ICAM1 levels)

• β: Number of contradictory dependencies defined by domain experts

• θ: Minimal number of required Boolean rules to assess these contradictions

This framework helps identify the complexity of contradictions present in experimental datasets and guides the development of appropriate resolution strategies .

Specific Contradiction Resolution Strategies:

• Expression-Function Discrepancies: For variants like Tyr23Cys where protein expression levels may appear normal despite functional impairment, investigate protein-protein interactions and complex formation rather than focusing solely on expression levels . For example, despite normal expression, Tyr23Cys DPM2 shows reduced ICAM1 levels, indicating functional impairment through mechanisms other than protein expression .

• Transfection Efficiency Variability: When comparing expression levels between wild-type and variant DPM2 constructs, normalize data using appropriate housekeeping genes and include multiple internal controls . Consider using stable cell lines rather than transient transfection to minimize expression variability.

• Individual Variation in Patient Samples: When analyzing patient-derived samples showing unexpected values (e.g., higher DPM2 mRNA levels in affected individuals), incorporate larger sample sizes and implement statistical methods to account for individual variations .

• In Vitro vs. In Vivo Discrepancies: Validate findings using complementary methodologies and different cell types or model systems to ensure reproducibility across experimental contexts.

What are the optimal cell models for studying DPM2 functionality in glycosylation pathways?

Selecting appropriate cell models is critical for investigating DPM2 functionality in glycosylation pathways, with each model offering distinct advantages:

Primary Cell Models:

• Patient-Derived Primary Cells: Peripheral blood mononuclear cells (PBMCs) from patients with DPM2 mutations provide directly relevant models for studying disease pathophysiology . These cells maintain the genetic background of affected individuals, enabling analysis of DPM2 variant effects in their native context.

• Primary Fibroblasts: These cells are readily obtainable from patient skin biopsies and maintain stable glycosylation patterns, making them suitable for long-term studies of DPM2 function.

Established Cell Lines:

• HCT116 Cells: These human colorectal carcinoma cells have been successfully used for transfection and expression of DPM2 variants . They provide a consistent background for comparative studies of wild-type and mutant DPM2 proteins.

• CHO Mutant Cell Lines: The Lec15 mutant Chinese hamster ovary (CHO) cells, which are defective in DPM2, serve as an excellent model system for complementation studies . These cells offer a clean background for investigating DPM2 function without interference from endogenous protein.

• Thy-1negative Class E Mutant Mouse Lymphoma Cells: These cells, defective in DPM1, provide a complementary model for studying the DPM synthase complex and interactions between DPM1 and DPM2 .

Emerging Models:

• iPSC-Derived Cell Types: Patient-specific induced pluripotent stem cells can be differentiated into various cell types relevant to DPM2-associated disorders, including neurons, cardiomyocytes, and hepatocytes, enabling tissue-specific studies of glycosylation defects.

• CRISPR-Engineered Cell Lines: Genome-edited cell lines with specific DPM2 variants offer isogenic comparisons, minimizing confounding factors from different genetic backgrounds.

The selection of cell models should be guided by the specific research question, with consideration given to the glycosylation pathways of interest, the availability of appropriate controls, and the technical feasibility of required experimental manipulations. A comprehensive approach often involves using multiple complementary models to validate findings across different cellular contexts.

What are the methodological challenges in purifying functional DPM synthase complex containing DPM2?

Purification of the functional heterotrimeric DPM synthase complex presents several methodological challenges due to its membrane association and the intricate interactions between its components:

Membrane Protein Solubilization Challenges:

• DPM2 contains two transmembrane domains that anchor it to the ER membrane , necessitating careful selection of detergents that effectively solubilize the protein while preserving its native conformation and interactions.

• Commonly used detergents include:

  • Mild non-ionic detergents (e.g., Digitonin, DDM)

  • Zwitterionic detergents (e.g., CHAPS)

  • Newer amphipathic polymers (e.g., SMA copolymers)

Each solubilization method must be optimized to maintain the integrity of the heterotrimeric complex.

Complex Stability Considerations:

• The interactions between DPM1, DPM2, and DPM3 are sensitive to purification conditions, with DPM2 playing a critical role in stabilizing the complex .

• The complex may dissociate during purification steps, particularly during chromatographic separations, requiring gentle handling and optimization of buffer conditions (pH, ionic strength, presence of stabilizing agents).

Functional Assessment Challenges:

• Enzymatic activity assays for purified DPM synthase complex require:

  • Appropriate substrates (dolichol phosphate, GDP-mannose)

  • Suitable membrane environment or reconstitution system

  • Sensitive detection methods for product formation

• Activity may be lost during purification, necessitating rapid processing and inclusion of protease inhibitors and stability-enhancing additives.

Advanced Purification Strategies:

• Tandem Affinity Purification: Using dual tags on different complex components to ensure isolation of intact complexes.

• Mild Solubilization Followed by Native PAGE: To preserve native interactions within the complex.

• Reconstitution into Nanodiscs or Liposomes: To provide a membrane-like environment for the purified complex, potentially enhancing stability and activity.

• Co-expression Systems: Simultaneous expression of all three components (DPM1, DPM2, DPM3) in appropriate stoichiometry to facilitate complex formation prior to purification.

These methodological challenges underscore the need for careful optimization of purification protocols specific to the DPM synthase complex, with particular attention to maintaining the critical stabilizing function of DPM2 throughout the purification process.

What are the emerging techniques for investigating DPM2's role in tissue-specific glycosylation patterns?

Recent advances in glycobiology and protein analysis have yielded innovative approaches for elucidating DPM2's tissue-specific functions:

Advanced Imaging Techniques:

• Super-Resolution Microscopy: Techniques such as STORM, PALM, or STED microscopy can visualize DPM2 localization within the ER at nanometer resolution, revealing potential tissue-specific distribution patterns and co-localization with other glycosylation machinery components.

• Live-Cell Imaging with Fluorescent DPM2 Fusions: Enables real-time monitoring of DPM2 dynamics in different tissue contexts, particularly when combined with organelle-specific markers.

Glycoproteomics Approaches:

• Tissue-Specific Glycoproteomic Profiling: Mass spectrometry-based methods combining glycan and protein analysis to identify tissue-specific glycosylation patterns dependent on DPM2 function.

• Glycan Remodeling Assays: Pulse-chase experiments with isotopically labeled mannose to track tissue-specific rates of mannose incorporation into glycans dependent on dolichol phosphate-mannose.

Genetic Manipulation in Model Systems:

• Tissue-Specific DPM2 Knockout/Knockdown: Using Cre-lox systems or tissue-specific promoters to modulate DPM2 expression in specific tissues of model organisms.

• CRISPR Base Editing: Introduction of specific DPM2 variants to study their tissue-specific consequences without disrupting the entire gene.

Systems Biology Approaches:

• Integrated Multi-Omics Analysis: Combining transcriptomics, proteomics, and glycomics data from different tissues to identify tissue-specific pathways and processes regulated by DPM2.

• Network Analysis: Computational methods to identify tissue-specific interaction partners and regulatory networks involving DPM2.

Organoid and 3D Culture Systems:

• Tissue-Specific Organoids: Development of organ-specific 3D cultures from cells with DPM2 variants to study tissue-specific glycosylation defects in more physiologically relevant models.

• Multi-Tissue Organoid Systems: Co-culture systems to investigate tissue interactions influenced by DPM2-dependent glycosylation.

These emerging techniques offer unprecedented opportunities to elucidate the tissue-specific roles of DPM2 in glycosylation pathways, potentially explaining the variable organ involvement observed in patients with DPM2 mutations and informing the development of targeted therapeutic approaches.

What therapeutic approaches are being explored for addressing DPM2-related glycosylation disorders?

While therapeutic development for DPM2-related glycosylation disorders remains in early stages, several promising approaches warrant further investigation:

Gene Therapy Approaches:

• AAV-Mediated Gene Delivery: Adeno-associated viral vectors carrying functional DPM2 genes could potentially restore glycosylation function in affected tissues, particularly in the central nervous system where many DPM2-related symptoms manifest.

• mRNA Therapeutics: Delivery of synthetic DPM2 mRNA encapsulated in lipid nanoparticles represents a potentially renewable source of functional protein without genomic integration.

Protein Stabilization Strategies:

• Pharmacological Chaperones: Small molecules that could stabilize mutant DPM2 proteins, particularly those with missense mutations that affect protein folding rather than catalytic function.

• Proteasome Inhibitors: For variants where accelerated degradation is the primary pathogenic mechanism, selective proteasome inhibition might increase effective protein levels.

Metabolic Bypass Strategies:

• Alternative Mannose Donors: Development of compounds that could serve as alternative mannose donors in glycosylation pathways, bypassing the need for dolichol phosphate-mannose.

• Downstream Pathway Modulation: Identification of interventions targeting downstream effects of impaired glycosylation, such as compounds that stabilize underglycosylated proteins or enhance their trafficking.

Glycan Supplementation Approaches:

• Mannose Supplementation: Similar to approaches used in phosphomannose isomerase deficiency, dietary mannose supplementation might increase substrate availability for residual DPM synthase activity.

• Glycan Precursor Administration: Development of cell-permeable glycan precursors that could enter glycosylation pathways downstream of the DPM2-dependent steps.

Combination Therapies:

• Coordinated Multi-target Approach: Addressing both the primary defect in DPM2 function and secondary consequences through complementary therapeutic modalities.

• Personalized Medicine Strategies: Tailoring therapeutic approaches based on specific DPM2 variants and their functional consequences.

These emerging therapeutic strategies require further validation in appropriate model systems before clinical translation, with particular attention to tissue-specific effects and potential differential responses based on the nature of the underlying DPM2 variants.

How can high-throughput screening methods be optimized for identifying DPM2 modulators?

Optimizing high-throughput screening (HTS) approaches for identifying DPM2 modulators requires systematic development of appropriate assays and screening cascades:

Primary Screening Assay Development:

• Reporter-Based Glycosylation Assays: Development of cell lines expressing reporter proteins whose activity or localization depends on proper glycosylation (e.g., secreted luciferase with N-glycosylation sites, fluorescent proteins with glycosylation-dependent folding).

• ICAM1 Expression Screens: Since ICAM1 serves as a universal biomarker for hypoglycosylation in CDG , high-content imaging or flow cytometry-based assays measuring ICAM1 surface expression could provide a functional readout for DPM2 activity.

Biochemical Assay Development:

• DPM Synthase Activity Assays: Optimization of in vitro enzymatic assays measuring the formation of dolichol phosphate-mannose from GDP-mannose and dolichol phosphate.

• DPM2-DPM1 Interaction Assays: Development of proximity-based assays (FRET, BRET, AlphaScreen) to monitor the critical interaction between DPM2 and DPM1, particularly focusing on the first transmembrane domain of DPM2 .

Screening Cascade Design:

• Primary Screen: High-throughput cellular assay with glycosylation-dependent reporter.

• Secondary Validation: Orthogonal assays measuring specific glycosylation markers (e.g., ICAM1 levels by western blot or flow cytometry) .

• Mechanism Confirmation: Assays specifically measuring DPM2 stability, DPM complex formation, and enzymatic activity.

• Selectivity Assessment: Counterscreening against other glycosylation pathways to identify DPM2-specific modulators.

Specialized Screening Approaches:

• Variant-Specific Screens: Development of cell lines expressing specific DPM2 variants (e.g., Tyr23Cys, Gly66Glu) to identify compounds that rescue function of particular mutations .

• Fragment-Based Screening: For identifying small molecular scaffolds that interact with specific domains of DPM2, particularly the transmembrane regions involved in protein-protein interactions.

• In Silico Screening: Computational approaches targeting specific binding pockets or protein-protein interfaces, particularly if structural information for DPM2 becomes available.

Data Analysis and Compound Prioritization:

• Machine Learning Approaches: Development of predictive models based on initial screening results to prioritize compounds for follow-up.

• Structure-Activity Relationship Analysis: Systematic analysis of chemical features associated with DPM2 modulation to guide medicinal chemistry optimization.

Implementation of these optimized HTS methods would facilitate the identification of small molecules capable of enhancing DPM2 function or stabilizing DPM2 variants, potentially leading to therapeutic candidates for DPM2-related disorders.

What are the most promising model systems for studying tissue-specific impacts of DPM2 deficiency?

Advanced model systems offer sophisticated platforms for investigating the complex tissue-specific manifestations of DPM2 deficiency:

Cell-Based Model Systems:

• Patient-Derived iPSCs and Differentiated Derivatives: Generation of induced pluripotent stem cells from patients with DPM2 mutations allows differentiation into multiple tissue types affected in DPM2-CDG, including:

  • Neural cells (neurons, astrocytes, oligodendrocytes) for studying neurological manifestations

  • Cardiomyocytes for investigating cardiac involvement

  • Hepatocytes for examining liver dysfunction

  • Skeletal muscle cells for assessing hypotonia mechanisms

• Isogenic CRISPR-Engineered Cell Lines: Creation of cell lines with specific DPM2 variants introduced into the same genetic background, enabling precise comparison of variant effects across multiple differentiated tissue types.

Organoid Model Systems:

• Brain Organoids: Three-dimensional neural cultures recapitulating aspects of human brain development for studying neurodevelopmental impacts of DPM2 deficiency.

• Multi-Organ Organoid Systems: Co-culture of multiple organoid types to investigate systemic effects and organ interactions in the context of glycosylation defects.

Animal Model Systems:

• Conditional Knockout Mouse Models: Tissue-specific or inducible DPM2 knockout mice using Cre-lox technology, allowing temporal and spatial control of DPM2 expression.

• Knock-in Models of Human Variants: Introduction of specific human DPM2 variants (e.g., Tyr23Cys, Gly66Glu) into mouse models to recapitulate human disease manifestations.

• Zebrafish Models: Rapid development and optical transparency make zebrafish suitable for studying developmental aspects of DPM2 deficiency and conducting high-throughput phenotypic screens.

Comparative Model Analysis:
The table below summarizes the strengths and limitations of different model systems for studying DPM2 deficiency:

The integration of findings across these complementary model systems will provide a comprehensive understanding of the tissue-specific impacts of DPM2 deficiency, facilitating the development of targeted therapeutic approaches for affected tissues.