Primary hyperoxaluria type 1 (PH1) is caused by the functional deficit of alanine:glyoxylate aminotransferase (AGT1), resulting in a build-up of oxalate. PH1 is diagnosed through detection of biallelic pathogenic/likely pathogenic variations in the AGXT gene. However, the widespread availability of genetic screening has led to an increased identification of novel variants in patients, yet precisely determining their pathogenicity remains a challenge. Since in silico tools can give misleading results, functional analyses in disease models are needed to confirm diagnosis.

Here, to help the clinical assessment of AGXT variants of uncertain significance (VUS), we implemented a platform based on a cellular model of PH1, made up of HepG2 cells with endogenous AGXT gene knocked out and infected with lentiviral vectors encoding each variant. We generated stable clones expressing the two polymorphic forms of AGT1 (major: AGT1-Ma and minor: AGT1-Mi) as negative controls; five validated pathogenic forms as positive controls; 13 variants classified according to the American College of Medical Genetics and Genomics guidelines as VUS, benign/likely benign, or conflicting. We analyzed each clone for AGT1 specific activity, protein levels, and intracellular localization, as well as for their glyoxylate detoxification ability, as a functional assay. An unbiased global analysis of the data allowed unambiguous clustering of both non-pathogenic and validated pathogenic variants, thus providing information on the possible pathogenicity of each variant. As such, our cell platform represents an important tool that could be applied to a large number of AGXT variants to support diagnosis of PH1.