Secondary NAD⁺ deficiency in the inherited defect of glutamine synthetase

Abstract

Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis

“…The increase of NAD + in leucocytes (described above) also supported the beneficial effects of the treatment at the cellular level [5,31]. …”

“…Furthermore, this observation could open the possibility of a novel therapeutic intervention to alleviate the devastating phenotype in GS deficiency. Exogenous supplementation with either glutamine or NAD + precursors (nicotinamide or nicotinate) in fibroblasts in vitro normalized the intracellular NAD + concentration [31]. The same was observed in vivo when the GS-deficient patient received glutamine over four weeks, resulting in a marked increase in NAD + levels in leukocytes [31].…”

“…In two patients (patients 1 and 2), GS activity was strongly reduced, as measured in immortalized lymphocytes from patient 1 and in COS7 cells transfected with the mutated GS from patient 2 [2,31]. Investigations on fibroblasts from patients 1 and 2 were not possible, since they failed to grow (other GS-deficient fibroblasts required a high-glutamine-containing medium to grow).…”

“…Thus, removal of this arginine will presumably hamper ATP binding, explaining the decreased enzyme activity observed in both mutant forms [2,5]. Besides, since p.Arg324Cys and p.Arg324Ser had increased expression levels in cell culture studies [2,31], these mutants probably lead to lower catalytic efficiency. It is also to be highlighted that, in homozygosis, p.Arg324Cys was fatal in the neonatal period, while p.Arg324Ser was found in a patient still alive at the age of 6 years.…”

“…We had the opportunity to investigate the availability of NAD + in one patient with inherited GS deficiency (patient 3) to follow-up on the hypothesis of a NAD + deficiency in patients with systemic glutamine deficiency. Unsurprisingly, NAD + was found to be severely lacking in cultured fibroblasts, immortalized peripheral blood stem cells, and in leukocytes both in vitro and in vivo [31]. Consequently, it was speculated that the NAD + deficiency contributed to the global severity of the disease.…”