Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland.
Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland; Nextech Invest, Bahnhofstrasse 18, 8001, Zurich, Switzerland.
Biochimie. 2021 Apr;183:89-99. doi: 10.1016/j.biochi.2020.12.003. Epub 2020 Dec 9.
Despite biochemical and genetic testing being the golden standards for identification of proximal urea cycle disorders (UCDs), genotype-phenotype correlations are often unclear. Co-occurring partial defects affecting more than one gene have not been demonstrated so far in proximal UCDs. Here, we analyzed the mutational spectrum of 557 suspected proximal UCD individuals. We probed oligomerizing forms of NAGS, CPS1 and OTC, and evaluated the surface exposure of residues mutated in heterozygously affected individuals. BN-PAGE and gel-filtration chromatography were employed to discover protein-protein interactions within recombinant enzymes. From a total of 281 confirmed patients, only 15 were identified as "heterozygous-only" candidates (i.e. single defective allele). Within these cases, the only missense variants to potentially qualify as dominant negative triggers were CPS1 p.Gly401Arg and NAGS p.Thr181Ala and p.Tyr512Cys, as assessed by residue oligomerization capacity and surface exposure. However, all three candidates seem to participate in critical intramolecular functions, thus, unlikely to facilitate protein-protein interactions. This interpretation is further supported by BN-PAGE and gel-filtration analyses revealing no multiprotein proximal urea cycle complex formation. Collectively, genetic analysis, structural considerations and in vitro experiments point against a prominent role of dominant negative effects in human proximal UCDs.
尽管生化和基因检测是鉴定近端尿素循环障碍（UCD）的黄金标准，但基因型-表型相关性通常不明确。迄今为止，尚未在近端 UCD 中证明同时存在影响多个基因的部分缺陷。在这里，我们分析了 557 名疑似近端 UCD 个体的突变谱。我们探测了 NAGS、CPS1 和 OTC 的寡聚形式，并评估了杂合受影响个体中突变残基的表面暴露。BN-PAGE 和凝胶过滤层析用于发现重组酶内的蛋白质-蛋白质相互作用。在总共 281 名确诊患者中，只有 15 名被鉴定为“仅杂合子”候选者（即单个缺陷等位基因）。在这些病例中，唯一有资格成为显性负触发物的错义变体是 CPS1 p.Gly401Arg 和 NAGS p.Thr181Ala 和 p.Tyr512Cys，这是通过残基寡聚能力和表面暴露来评估的。然而，所有这三种候选物似乎都参与了关键的分子内功能，因此不太可能促进蛋白质-蛋白质相互作用。这一解释进一步得到 BN-PAGE 和凝胶过滤分析的支持，该分析显示没有形成多蛋白近端尿素循环复合物。总的来说，遗传分析、结构考虑和体外实验表明，显性负效应在人类近端 UCD 中没有突出作用。
