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Kinetic mutations in argininosuccinate synthetase deficiency: characterisation and in vitro correction by substrate supplementation


Diez-Fernandez, Carmen; Wellauer, Olivia; Gemperle, Corinne; Rüfenacht, Véronique; Fingerhut, Ralph; Häberle, Johannes (2016). Kinetic mutations in argininosuccinate synthetase deficiency: characterisation and in vitro correction by substrate supplementation. Journal of Medical Genetics, 53(10):710-719.

Abstract

BACKGROUND: Citrullinemia type 1 is an autosomal-recessive urea cycle disorder caused by mutations in the ASS1 gene and characterised by increased plasma citrulline concentrations. Of the ∼90 argininosuccinate synthetase (ASS) missense mutations reported, 21 map near the substrate (aspartate or citrulline) binding site, and thus are potential kinetic mutations whose decreased activities could be amenable to substrate supplementation. This article aims at characterising these 21 ASS mutations to prove their disease-causing role and to test substrate supplementation as a novel therapeutic approach.
METHODS: We used an Escherichia coli expression system to study all potentially kinetic ASS mutations. All mutant enzymes were nickel-affinity purified, their activity and kinetic parameters were measured using tandem mass spectrometry and their thermal stability using differential scanning fluorimetry. Structural rationalisation of the effects of these mutations was performed.
RESULTS: Of the characterised mutants, 13 were totally inactive while 8 exhibited decreased affinity for aspartate and citrulline. The activity of these eight kinetic mutations could be rescued to ∼10-99% of the wild-type using high l-aspartate concentrations.
CONCLUSIONS: Substrate supplementation raised in vitro the activity of eight citrullinemia type 1 mutations with reduced affinity for aspartate. As a direct translation of these results to the clinics, we propose to further evaluate the use of oxaloacetate, a nitrogen-free aspartate precursor and already available medical food (anti-ageing and brain stimulating, not considered as a drug by the US Food and Drug Administration), in patients with citrullinemia type 1 with decreased aspartate affinity. Although only patients with kinetic mutations would benefit, oxaloacetate could offer a safe novel treatment.

Abstract

BACKGROUND: Citrullinemia type 1 is an autosomal-recessive urea cycle disorder caused by mutations in the ASS1 gene and characterised by increased plasma citrulline concentrations. Of the ∼90 argininosuccinate synthetase (ASS) missense mutations reported, 21 map near the substrate (aspartate or citrulline) binding site, and thus are potential kinetic mutations whose decreased activities could be amenable to substrate supplementation. This article aims at characterising these 21 ASS mutations to prove their disease-causing role and to test substrate supplementation as a novel therapeutic approach.
METHODS: We used an Escherichia coli expression system to study all potentially kinetic ASS mutations. All mutant enzymes were nickel-affinity purified, their activity and kinetic parameters were measured using tandem mass spectrometry and their thermal stability using differential scanning fluorimetry. Structural rationalisation of the effects of these mutations was performed.
RESULTS: Of the characterised mutants, 13 were totally inactive while 8 exhibited decreased affinity for aspartate and citrulline. The activity of these eight kinetic mutations could be rescued to ∼10-99% of the wild-type using high l-aspartate concentrations.
CONCLUSIONS: Substrate supplementation raised in vitro the activity of eight citrullinemia type 1 mutations with reduced affinity for aspartate. As a direct translation of these results to the clinics, we propose to further evaluate the use of oxaloacetate, a nitrogen-free aspartate precursor and already available medical food (anti-ageing and brain stimulating, not considered as a drug by the US Food and Drug Administration), in patients with citrullinemia type 1 with decreased aspartate affinity. Although only patients with kinetic mutations would benefit, oxaloacetate could offer a safe novel treatment.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:October 2016
Deposited On:27 Jan 2017 13:41
Last Modified:27 Jan 2017 13:41
Publisher:BMJ Publishing Group
ISSN:0022-2593
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1136/jmedgenet-2016-103937
PubMed ID:27287393

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