Clinical and structural insights into potential dominant negative triggers of proximal urea cycle disorders.

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.