Investigation of citrullinemia type I variants by in vitro expression studies

Abstract

Mild citrullinemia is an allelic variant of classical citrullinemia type I also caused by deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS). Affected patients comprise a biochemical but no clinical phenotype. However, there is no reliable parameter allowing conclusions regarding the course of the disorder or its type of manifestation. The aim of this study was to test the importance of varying levels of ASS residual activities for the severity at diagnosis. Bacterial in vitro expression studies allowed the enzymatic analysis of purified wild-type and the mutant ASS proteins p.Ala118Thr (c.352G>A), p.Trp179Arg (c.535T>C), p.Val263Met (c.787G>A), p.Arg265Cys (c.793C>T), p.Met302Val (c.904A>G), p.Gly324Ser (c.970G>A), p.Gly362Val (c.1085G>T), and p.Gly390Arg (c.1168G>A). In the chosen system, classical mutations do not show any significant enzymatic activity, whereas mutations associated with a mild course yield significant ASS activity levels. The mutation p.Ala118Thr (c.352G>A) impresses by a high residual activity (62%) but a severe reduction of affinity toward the substrates citrulline and aspartate. This mutation was identified in a hitherto healthy female adult with no history of known citrullinemia who had died during the postpartum period from hyperammonemic coma. The results of this study suggest that even a high level of residual ASS activity is not a reliable prognostic marker for an uneventful clinical course. Determination of ASS residual activities, therefore, cannot help in anticipating the risk of metabolic derangement. This study should guide clinicians as well as patients with mild citrullinemia toward a lifelong awareness of the disorder.

Favorable Long-Term Outcome Following Severe Neonatal Hyperammonemic Coma in a Patient with Argininosuccinate Synthetase Deficiency

“…The c.1168G>A (p.G390R) missense ASS1 variant identified in our patient is so far the single most common variant identified in early-onset citrullinemia type I (Kobayashi et al 1990;Häberle et al 2002;Gao et al 2003;Engel et al 2009;Laróvere et al 2009). Homozygosity for this variant is usually associated with undetectable or severely reduced (less than 2%) residual enzymatic activity (Kobayashi et al 1990;Vilaseca et al 2001;Gao et al 2003;Berning et al 2008). Although associated with severe enzymatic deficiency and neonatal-onset hyperammonemia, this genotype does not systematically equate with poor long-term outcome, as demonstrated in our patient.…”

Two Hypomorphic Alleles of Mouse Ass1 as a New Animal Model of Citrullinemia Type I and Other Hyperammonemic Syndromes

“…In vitro expression studies have shown that milder forms of CTLN1 are due to kinetic variants of well-folded ASS1 enzyme. 12,27 The lack of appropriate animal models has precluded a more complete understanding of the pathogenesis and medical management of CTLN1. Although Ass1 knockout mice have been generated, homozygous knockout animals die shortly after birth, and no histopathological data are available for this model.…”

Expanded newborn screening: reducing harm, assessing benefit

“…At least 11 cases have been recorded, in which the diagnosis of citrullinaemia type I has come to light during a pregnancy or in the immediate post-partum period (Kurasawa et al 1998;Gao et al 2003;Ito et al 2004;Dimmock et al 2008;Berning et al 2008;Häberle et al 2010). One of these cases is of especial interest, as the patient, who died during the episode of decompensation, was homozygous for a mutation c.325G>A (p. Ala118Thr) which has been recorded in two other cases of pregnancy-related decom- SCADD Short-chain acylCoA dehydrogenase deficiency, PKU phenylketonuria, MGC methylglutaconyl CoA hydratase deficiency, VLCADD very-long-chain acylCoA dehydrogenase deficiency pensation, but is associated with only very slight reduction of enzyme activity, though altered kinetics (Berning et al 2008). Mutations identified in citrullinaemia type I have recently been reviewed (Engel et al 2009).…”