Mutations and Polymorphisms in the HumanArgininosuccinate Lyase(ASL) Gene

Abstract

Argininosuccinate lyase deficiency (ASLD) is caused by a defect of the urea cycle enzyme argininosuccinate lyase (ASL) encoded by the ASL gene. Patients often present early after birth with hyperammonemia but can also manifest outside the neonatal period mainly triggered by excessive protein catabolism. Clinical courses comprise asymptomatic individuals who only excrete the biochemical marker, argininosuccinic acid, in urine, and patients who succumb to their first hyperammonemic decompensation. Some patients without any hyperammonemia develop severe neurological disease. Here, we are providing an update on the molecular basis of ASLD by collecting all published (n = 67) as well as novel mutations (n = 67) of the ASL gene. We compile data on all 160 different genotypes ever identified in 223 ASLD patients, including clinical courses whenever available. Finally, we are presenting structural considerations focusing on the relevance of mutations for ASL homotetramer formation. ASLD can be considered as a panethnic disease with only single founder mutations identified in the Finnish (c.299T>C, p.Ile100Thr) and Arab (c.1060C>T, p.Gln354*) population. Most mutations are private with only few genotypes recurring in unrelated patients. The majority of mutations are missense changes including some with more frequent occurrence such as p.Arg12Gln, p.Ile100Thr, p.Val178Met, p.Arg186Trp, p.Glu189Gly, p.Gln286Arg, and p.Arg385Cys.

Unstable argininosuccinate lyase in variant forms of the urea cycle disorder argininosuccinic aciduria

“…1). Of the total 13 mutations, 11 (p.Arg12Gln (c.35G>A), p.Asp31Asn (c.91G>A), p.Arg95Cys (c.283C > T), p.Ile100Thr (c.299 T > C), p.Val178Met (c.532G > A), p.Glu189Gly (c.566A > G), p.Arg193Trp (c.577C > T), p.Val335Leu (c.1003G > T), p.Arg379Cys (c.1135C>T), p.Arg385Cys (c.1153C>T) and p.Arg445Pro (c.1334G > C)) are, according to literature (Balmer et al 2014), always associated with a variant clinical course, defined as late onset and/or mild clinical and biochemical phenotype. These 11 mutations as well as the two severe mutations compile to a list of over 60 genotypes that are provided, together with the available clinical information, in Supplemental Table 1.…”

“…The amino acid substitutions p.Ile100Thr and p.Arg379Cys belong to the two most frequent changes in ASLD that were initially described in Finish patients . Notably, the mutations c.1153C>T (p.Arg385Cys) and c.1154G>T (p.Arg385Leu) affect the same amino acid but are reported to result in variant and severe clinical courses, respectively (Balmer et al 2014).…”

“…Cell extracts (30 μg total protein) were separated by 10 % denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to nitrocellulose transfer membranes (Whatman GmbH, Dassel, Germany). The primary polyclonal antibody anti-ASL (GeneTex, Irvine CA, USA), recognizing EV, empty vector; WT, wild-type a All known genotypes to each missense mutation are published in (Balmer et al 2014) and are listed as well in Supplementary Table 1 b ASL activities were measured under standard conditions using 13.6 mM argininosuccinate and given as experimental activity versus the total protein content in the extract in mIU/mg protein c ASL protein content was given as percentage of WT to estimate the expressed ASL mutant protein present in the extract compared to that of WT based on GAPDH by densitometry. It was determined by the following quotient: (ASL band for the mutant/GAPDH band for mutant)/(ASL band for WT/ GAPDH band for WT) using Western blot analysis d The specific activity of pure ASL given as percentage of WT was determined by the ratio of ASL mutant activity/ASL WT activity/ASL protein content indicating the relative enzyme activity of the expressed ASL mutant protein compared to that of the WT enzyme after normalization of the expressed ASL protein levels based on GAPDH by densitometry estimation.…”

“…In order to investigate the molecular characteristics of all known naturally occurring ASL missense mutations associated with a variant clinical course in ASA patients (Balmer et al 2014), we first constructed the recombinant ASL WT and mutant plasmids followed by introducing them into 293 T cells, respectively. To check whether the recombinant diverse ASL mutations can be expressed at the protein level, Western blot analysis was performed under denaturing conditions which yielded expression of all recombinant ASL mutant proteins (Fig.…”

“…Recently, an ASL pseudogene, which includes sequences from intron two to intron three, was identified upstream of the human ASL gene on chromosome 7 (Trevisson et al 2007). Mutations are spread almost all over the ASL gene and have recently been reviewed (Balmer et al 2014). Several attempts have been made to accomplish a prognostic marker and improve our understanding of the biochemical and clinical variability of ASLD.…”