Gatticchi Leonardo, Bellezza Ilaria, Rumsby Gill, Glover Michelle, Cellini Barbara
Department of Medicine and Surgery, University of Perugia, Perugia, Italy.
Department of Medicine and Surgery, University of Perugia, Perugia, Italy.
J Biol Chem. 2025 Jul 17;301(8):110494. doi: 10.1016/j.jbc.2025.110494.
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 the 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 the 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 the 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 its 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 the diagnosis of PH1.
1型原发性高草酸尿症（PH1）是由丙氨酸：乙醛酸转氨酶（AGT1）功能缺陷引起的，导致草酸盐积聚。PH1通过检测AGXT基因中的双等位基因致病性/可能致病性变异来诊断。然而，基因筛查的广泛应用导致患者中新型变异的识别增加，但准确确定其致病性仍然是一项挑战。由于计算机分析工具可能会给出误导性结果，因此需要在疾病模型中进行功能分析以确诊。在此，为了帮助对意义未明的AGXT变异（VUS）进行临床评估，我们基于PH1细胞模型构建了一个平台，该模型由内源性AGXT基因敲除的HepG2细胞组成，并感染编码每个变异的慢病毒载体。我们生成了表达AGT1两种多态形式（主要：AGT1-Ma和次要：AGT1-Mi）的稳定克隆作为阴性对照；五个经过验证的致病性形式作为阳性对照；13个根据美国医学遗传学与基因组学学会指南分类为VUS、良性/可能良性或相互矛盾的变异。我们分析了每个克隆的AGT1特异性活性、蛋白质水平和细胞内定位，以及其乙醛酸解毒能力，作为功能测定。对数据进行无偏全局分析允许对非致病性和经过验证的致病性变异进行明确聚类，从而提供每个变异可能致病性的信息。因此，我们的细胞平台是一种重要工具，可应用于大量AGXT变异以支持PH1的诊断。
