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Abstract: FR-PO593

Functional Analysis of AGXT1 Missense Variants of Uncertain Significance in HepG2 Cells: Association with Primary Hyperoxaluria Type I

Session Information

Category: Genetic Diseases of the Kidneys

  • 1202 Genetic Diseases of the Kidneys: Non-Cystic


  • Cellini, Barbara, Universita degli Studi di Perugia Dipartimento di Medicina e Chirurgia, Perugia, Umbria, Italy
  • Gatticchi, Leonardo, Universita degli Studi di Perugia Dipartimento di Medicina e Chirurgia, Perugia, Umbria, Italy
  • Bellezza, Ilaria, Universita degli Studi di Perugia Dipartimento di Medicina e Chirurgia, Perugia, Umbria, Italy

Primary Hyperoxaluria Type I (PH1) is caused by functional deficit of alanine:glyoxylate aminotransferase (AGT1), a liver enzyme that detoxifies glyoxylate, thus preventing its conversion into oxalate. The hallmark of PH1 is the progressive deposition of calcium oxalate resulting in kidney stones leading to ESRD and systemic oxalosis. The diagnosis is established on a proband with hyperoxaluria or hyperoxalemia by the presence of biallelic pathogenic or likely pathogenic variations in the AGXT1 gene encoding AGT1, 67% of which are missense. Although the pathogenicity of some genetic variants has been clearly defined, the clinical assessment of variants of uncertain significance (VUS) represents a big challenge. Our work is aimed at determining pathogenicity of AGXT1 missense variants in a cellular model of disease.


We used hepatocarcinoma-derived human HepG2 cell line knocked-out for the AGXT1 gene utilizing CRISPR/Cas9 technology, since they exhibit conserved glyoxylate/oxalate metabolism. By lentiviral infection we created and analyzed stable clones of AGXT1-KO HepG2 cells expressing the two polymorphic forms of AGT1 (AGT1-Ma and AGT1-Mi). Eight VUS were expressed on the AGT1-Ma background and three on the AGT1-Mi background. We validated our experimental setting to mimic the liver expression of AGT1 and investigated the effects of amino acid changes at the protein level.


The p.Pro28Ser, p.Arg118Cys, p.Asp129His, p.Ala248Val, p.Arg317Trp mutations on AGT1-Ma, and the p.Glu274Asp, p.Ile279Thr, p.Arg289Cys mutations on AGT-Mi led to a biologically meaningful reduction in protein levels and activity, denoting the variants are likely pathogenic. On the other hand, the p.Pro314= mutation did not significantly affect AGT1 protein levels and activity, while p.Ala186Val and p.Arg197Gln cause only a partial reduction, thus suggesting that they could be benign or likely-benign.


We developed a platform to predict the pathogenicity of newly identified AGXT1 variants that could have a significant clinical implication as potential tool to support the diagnosis of PH1 and establish genotype/phenotype correlations.


  • Commercial Support – Novo Nordisk Inc.