HepG2 PMM2-CDG knockout model: A versatile platform for variant and therapeutic evaluation

Area of Science:

• Biochemistry
• Genetics
• Cell Biology

Background:

• Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common congenital disorder of glycosylation, an autosomal recessive condition resulting from PMM2 gene variants.
• This multisystemic syndrome currently lacks a cure, necessitating the development of effective therapeutic strategies.
• Existing preclinical models face limitations, particularly regarding high lethality, hindering treatment efficacy evaluation.

Purpose of the Study:

• To generate a robust cellular disease model for PMM2-CDG.
• To validate the model's capacity for studying PMM2 variants of unknown significance.
• To facilitate the assessment of novel therapeutic strategies for PMM2-CDG.

Main Methods:

• CRISPR-Cas9 gene editing was employed to knockout the PMM2 gene in the HepG2 hepatoma cell line.
• The generated PMM2-deficient HepG2 cells were characterized for PMM2 protein and mRNA levels, enzymatic activity, and glycoprotein alterations.
• The model's utility was assessed by evaluating known PMM2 disease-causing variants.

Main Results:

• A PMM2-knockout HepG2 cellular model was successfully generated, exhibiting a complete absence of PMM2 protein and mRNA.
• The model demonstrated a significant 90% decrease in PMM enzymatic activity, characteristic of PMM2-CDG.
• Altered patterns of ICAM-1, LAMP1, and A1AT glycoproteins were observed, mirroring the PMM2-CDG phenotype.
• The model effectively recapitulated disease features, validating its utility for variant assessment.

Conclusions:

• The CRISPR-Cas9-generated HepG2 PMM2 knockout model accurately replicates the PMM2-CDG phenotype.
• This cellular model serves as a valuable tool for studying PMM2 clinical variants and understanding disease mechanisms.
• The model holds promise for advancing diagnostic insights and evaluating therapeutic strategies for PMM2-CDG.