From genotype to phenotype: Early prediction of disease severity in argininosuccinic aciduria

Abstract

Argininosuccinic aciduria (ASA) is an inherited urea cycle disorder and has a highly variable phenotypic spectrum ranging from individuals with lethal hyperammonemic encephalopathy, liver dysfunction, and cognitive deterioration, to individuals with a mild disease course. As it is difficult to predict the phenotypic severity, we aimed at identifying a reliable disease prediction model. We applied a biallelic expression system to assess the functional impact of pathogenic argininosuccinate lyase (ASL) variants and to determine the enzymatic activity of ASL in 58 individuals with ASA. This cohort represented 42 ASL gene variants and 42 combinations in total. Enzymatic ASL activity was compared with biochemical and clinical endpoints from the UCDC and E-IMD databases. Enzymatic ASL activity correlated with peak plasma ammonium concentration at initial presentation and with the number of hyperammonemic events (HAEs) per year of observation. Individuals with ≤9% of enzymatic activity had more severe initial decompensations and a higher annual frequency of HAEs than individuals above this threshold. Enzymatic ASL activity also correlated with the cognitive outcome and the severity of the liver disease, enabling a reliable severity prediction for individuals with ASA. Thus, enzymatic activity measured by this novel expression system can serve as an important marker of phenotypic severity.

Keywords: argininosuccinic aciduria; clinical outcome; disease course; enzymatic ASL activity; predictive biomarker.

Conflict of interest statement

Conflict of Interest Statement

All other authors declare that they have no conflict of interest.

© 2020 Wiley Periodicals, Inc.

Figures

Figure 1.. Overview of relative mRNA expression levels per variant combination.

COS-7 cells were transfected with 2.5 μg of each FLAG-and MYC-tagged ASL expression vectors and 1 μg of β-galactosidase reporter plasmid, cultured for 48 hours and subjected to quantitative analysis of mRNA expression applying qRT-PCR. Data are expressed as fold-change (mean +/− SD) normalized to the relative expression of the respective ASL wildtype plasmids (A-E; n=3 for each experiment). Red columns illustrate FLAG-tagged expression vectors, blue columns represent MYC-tagged plasmids. Variants associated with a premature stop codon are indicated by an asterisk (*).

Figure 2.. Overview of protein expression levels per variant combination.

COS-7 cells were transfected with 2.5 μg of each FLAG-and MYC-tagged ASL expression vectors and 1 μg of β-galactosidase reporter plasmid, cultured for 48 hours and subjected to protein expression analysis using standard Western blot technique. Briefly, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on nitrocellulose membranes using the Trans-Blot® Turbo Transfer System (BioRad). Expression of FLAG-or MYC-tagged ASL variants was visualized using anti-FLAG-or anti-MYC antibodies on two identical gels, which were carried in parallel (A-E). Equal protein loading in cell lysates was confirmed by immunoblotting using an anti-β-actin antibody. Activity of β-galactosidase per variant combination was measured applying the β-galactosidase enzyme assay system (Promega). Data are expressed as mean in mU/mg protein (A-E, n=3). Of note, full-size images of anti-FLAG and anti-MYC stainings per variant combination are depicted in Supp. Figure S1. Variants associated with a premature stop codon are indicated by an asterisk (*). Red letters illustrate FLAG-tagged expression vectors, blue letters represent MYC-tagged plasmids.

Figure 3.. Overview of enzymatic ASL activities per variant combination.

COS-7 cells were transfected with 2.5 μg of each FLAG-and MYC-tagged ASL expression vectors and 1 μg of β-galactosidase reporter plasmid, cultured for 48 hours and enzymatic ASL activities determined applying a spectrophotometric assay as described under Material and methods. Data are expressed as mean +/− SD in % of ASL wildtype activity (A-E, n=3 for each experiment). Variants associated with a premature stop codon are indicated by an asterisk (*).

Figure 4.. Enzymatic ASL activity correlates with initial NH4+max as well as annual frequency and severity of HAEs.

(A) NH4+max (μmol/l) subject to enzymatic ASL activity as determined in the mammalian biallelic expression system. Each point represents a single patient (n=46). Gray line displays estimated regression curve. GAM analysis, p<0.001, R2=0.36. (B) Boxplot illustrating NH4+max (μmol/l) with a enzymatic activity below or equal to 7.9% (n=17) and above 7.9% (n=29). Data are shown as median (black thick line) and mean (triangle), length of the box corresponds to interquartile range (IQR), upper and lower whiskers correspond to max. 1.5 x IQR, each point represents an outlier. Recursive partitioning, p<0.001. (C) Number of HAEs (NH4+max ≥ 100 μmol/l) per year subject to enzymatic ASL activity (%). Each point represents a single patient (n=35). Gray line displays estimated regression curve. GAM analysis, p<0.001, R2=0.39. (D) NH4+max during most severe HAE subject to enzymatic ASL activity (%). Each point represents a single patient (n=19). Gray line displays estimated regression curve. GAM analysis, p=0.019, R2=0.32. (E) Boxplot illustrating number of HAEs (NH4+max ≥ 100 μmol/l) per year with a enzymatic ASS1 activity below or equal to 8.7% (n=10) or above 8.7% (n=25). Data are shown as median (black thick line) and mean (triangle), length of the box corresponds to IQR, upper and lower whiskers correspond to max. 1.5 x IQR, each point represents an outlier. Recursive partitioning, p=0.004. ASL, argininosuccinate lyase.

Figure 5.. Neurocognitive outcome is associated with enzymatic ASL activity.

(A) Cognitive SDS subject to enzymatic ASL activity (%). Each point represents a single patient (n=30). Gray line indicates linear regression curve. Linear regression, p=0.032, R2=0.15. (B) Cognitive SDS subject to enzymatic ASS1 activity (%). Each point represents a single patient (n=34). Gray line indicates linear regression curve. Linear regression, p=0.029, R2=0.14. Figure adapted from (Zielonka, Kolker, et al., 2019). (C) Boxplot illustrating cognitive SDS with an enzymatic ASL activity below or equal to 24.3% (n=11) and above 24.3% (n=19). Data are shown as median (black thick line) and mean (triangle), length of the box corresponds to IQR, upper and lower whiskers correspond to max. 1.5 x IQR. Recursive partitioning, p=0.034. (D) Levelplot for cognitive SDS, enzymatic ASL activity (%) and age at testing (years). Cognitive SDS values are indicated by color coding in grading from blue to red with descending cognitive SDS. ASL, argininosuccinate lyase; ASS1, argininosuccinate synthetase 1.