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N-acetyl-L-glutamate synthase (NAGS) deficiency (NAGSD), the rarest urea cycle defect, is clinically indistinguishable from carbamoyl phosphate synthetase 1 deficiency, rendering the identification ofNAGSgene mutations key for differentiation, which is crucial, as only NAGSD has substitutive therapy. Over the last 13 years, we have identified 43 patients from 33 families withNAGSmutations, of which 14 were novel. Overall, 36NAGSmutations have been found so far in 56 patients from 42 families, of which 76% are homozygous for the mutant allele. 61% of mutations are missense changes. Lack or decrease of NAGS protein is predicted for ˜1/3 of mutations. Missense mutations frequency is inhomogeneous alongNAGS: null for exon 1, but six in exon 6, which reflects the paramount substrate binding/catalytic role of the C-terminal domain (GNAT domain). Correspondingly, phenotypes associated with missense mutations mapping in the GNAT domain are more severe than phenotypes of amino acid kinase domain-mapping missense mutations. Enzyme activity and stability assays with 12 mutations introduced into pure recombinantPseudomonas aeruginosaNAGS, together with in silico structural analysis, support the pathogenic role of most NAGSD-associated mutations found. The disease-causing mechanisms appear to be, from higher to lower frequency, decreased solubility/stability, aberrant kinetics/catalysis, and altered arginine modulation.N-Acetyl-L-glutamate synthase (NAGS) deficiency (NAGSD), the rarest urea cycle defect, is clinically indistinguishable from carbamoyl phosphate synthetase 1 deficiency. Their differentiation is crucial, as only NAGSD has substitutive therapy, and relies on the identification ofNAGSgene mutations. We provide the first extensive repertoire of mutations found in human NAGSD, inferring the consequences of these mutations both experimentally using a recombinant bacterial NAGS, and by structural modeling. Our structure-function correlations can help guide prognosis and counseling in NAGSD.