N-carbamylglutamate enhancement of ureagenesis leads to discovery of a novel deleterious mutation in a newly defined enhancer of the NAGS gene and to effective therapy

Abstract

NAGS catalyzes the conversion of glutamate and acetyl-CoA to N-acetylglutamate (NAG) the essential allosteric activator of carbamyl phosphate synthetase I, the first urea cycle enzyme in mammals. A 17-year-old female with recurrent hyperammonemia attacks, the cause of which remained undiagnosed for 8 years in spite of multiple molecular and biochemical investigations, showed markedly enhanced ureagenesis (measured by isotope incorporation) in response to N-carbamylglutamate (NCG). This led to sequencing of the regulatory regions of the NAG synthase (NAGS) gene and identification of a deleterious single-base substitution in the upstream enhancer. The homozygous mutation (-3063C>A), affecting a highly conserved nucleotide within the Hepatic Nuclear Factor 1 (HNF-1) binding site, was not found in SNP databases and in a screen of 1086 alleles from a diverse population. Functional assays demonstrated that this mutation decreases transcription and binding of HNF-1 to the NAGS gene, while a consensus HNF-1 binding sequence enhances binding to HNF-1 and increases transcription. Oral daily NCG therapy appears to have restored ureagenesis in this patient, normalizing her biochemical markers, and allowing discontinuation of alternate pathway therapy and normalization of her diet with no recurrence of hyperammonemia.

Carglumic acid for the treatment ofN-acetylglutamate synthase deficiency and acute hyperammonemia

“…In 1981, the first patient suffering from NAGSD was described [5] and since then a small number of clinical reports have described further cases [6,[9][10][11][12][13][14][15][16][17][18][19][20]. NAGSD most often leads to neonatal onset and life-threatening hyperammonemia [5,21] but late-onset (defined as outside the neonatal period) patients are also described [17,19,20,22].…”

“…NAGSD most often leads to neonatal onset and life-threatening hyperammonemia [5,21] but late-onset (defined as outside the neonatal period) patients are also described [17,19,20,22]. The incidence of NAGSD is not known precisely but the disorder has to be considered as extremely rare, occurring in <1 per 100,000 newborns [8].…”

Stable isotopes in the diagnosis and treatment of inherited hyperammonemia

“…In addition, hepatic enzyme assay showed normal activity of CPS1 and OTC. The [1-13 C] acetate procedure revealed a response to NCG (Figure 2) [38] which was similar to that of the NAGS deficient subject described above [28]. This made a compelling case for a diagnosis of NAGS deficiency, despite the initial negative molecular genetic testing.…”

“…Molecular and enzymatic testing had failed to reveal a diagnosis. However, the marked augmentation in urea production following an NCG trial shown here prompted a search in the non-coding regions of the NAGS gene, and culminated in the identification of a mutation in the NAGS enhancer [38].…”

“…Interestingly, a marked increase in ureagenesis in response to NCG therapy was crucial in the diagnosis of a young woman with NAGS deficiency [38]. We evaluated a young patient in whom biochemical markers suggested a proximal urea cycle disorder, but in whom an allopurinol challenge was negative, and molecular diagnostic testing of NAGS, CPS1 and OTC failed to disclose a mutation.…”