Abstract：
Gyrate atrophy of the choroid and retina (GACR) is caused by pathogenic biallelic variants in the gene encoding ornithine-δ-aminotransferase (OAT), and is characterized by progressive vision loss leading to blindness. OAT is a pyridoxal-5\'-phosphate (PLP) dependent enzyme that is mainly involved in ornithine catabolism, and patients with a deficiency develop profound hyperornithinemia. Therapy is aimed at lowering ornithine levels through dietary arginine restriction and, in some cases, through enhancement of OAT activity via supraphysiological dosages of pyridoxine. In this study, we aimed to extend diagnostic practices in GACR by extensively characterizing the consequences of pathogenic variants on the enzymatic function of OAT, both at the level of the enzyme itself as well as the flux through the ornithine degradative pathway. In addition, we developed an in vitro pyridoxine responsiveness assay. We identified 14 different pathogenic variants, of which one variant was present in all patients of Dutch ancestry (p.(Gly353Asp)). In most patients the enzymatic activity of OAT as well as the rate of [14C]-ornithine flux was below the limit of quantification (LOQ). Apart from our positive control, only one patient cell line showed responsiveness to pyridoxine in vitro, which is in line with the reported in vivo pyridoxine responsiveness in this patient. None of the patients harboring the p.(Gly353Asp) substitution were responsive to pyridoxine in vivo or in vitro. In silico analysis and small-scale expression experiments showed that this variant causes a folding defect, leading to increased aggregation properties that could not be rescued by PLP. Using these results, we developed a diagnostic pipeline for new patients suspected of having GACR. Adding OAT enzymatic analyses and in vitro pyridoxine responsiveness to diagnostic practices will not only increase knowledge on the consequences of pathogenic variants in OAT, but will also enable expectation management for therapeutic modalities, thus eventually improving clinical care.
摘要：
脉络膜和视网膜的回旋萎缩（GACR）是由编码鸟氨酸-δ-转氨酶（OAT）的基因中的致病性双等位基因变体引起的，其特征是进行性视力丧失导致失明。OAT是一种吡哆醛-5'-磷酸（PLP）依赖性酶，主要参与鸟氨酸分解代谢，缺乏的患者会出现严重的高鸟氨酸血症。治疗旨在通过饮食精氨酸限制降低鸟氨酸水平，在某些情况下,通过吡哆醇的超生理剂量增强OAT活性。在这项研究中,我们旨在通过广泛表征致病变体对OAT酶功能的影响来扩展GACR的诊断实践，在酶本身的水平以及通过鸟氨酸降解途径的通量。此外,我们开发了一种体外吡哆醇反应性试验。我们鉴定了14种不同的致病变异，其中一个变异存在于所有荷兰血统的患者中(p。（Gly353Asp）。在大多数患者中，OAT的酶活性以及[14C]-鸟氨酸通量的速率低于定量极限（LOQ）。除了我们的积极控制，只有一个患者细胞系在体外显示出对吡哆醇的反应，这与该患者体内报道的吡哆醇反应性一致。没有任何具有p。（Gly353Asp）替代的患者在体内或体外对吡哆醇有反应。计算机模拟分析和小规模表达实验表明，该变体会导致折叠缺陷，导致聚集特性增加，而PLP无法拯救。利用这些结果,我们为怀疑患有GACR的新患者开发了一条诊断管道.将OAT酶促分析和体外吡哆醇反应性添加到诊断实践中，不仅会增加对OAT致病变异后果的认识，但也将实现对治疗方式的期望管理，从而最终改善临床护理。
