Functional testing of sequence variants in the first intron of the NAGS gene
The pathogenic nature of the two sequence variants found in the first intron of the NAGS gene was confirmed using reporter gene assays in the HuH-7 and HepG2 cell lines. The c.427-218A>C and c.426+326G>A sequence variants were introduced into Prom-Ex1-Int1-Luc construct, which harbors luciferase gene fused to the promoter, exon 1 and intron 1 of the human NAGS gene (Table 1). Construct with the wild-type sequence of NAGS intron 1 was used as a control; constructs that contain NAGS promoter controlling expression of either luciferase gene (Prom-Luc; Table 1) or luciferase gene fused to the NAGS exon 1 (Prom-Ex1-Luc; Table 1) were additional controls while construct harboring only luciferase reporter gene was used as a negative control. Luciferase activity was lower in the HuH-7 cells transfected with the Prom-Ex1-Int1 construct harboring the c.427-218A>C variant while there was a trend (p=0.06, n=9) towards lower luciferase activity in in the HuH-7 cells transfected with the construct containing the c.426+326G>A sequence variant (Figure 2A). Luciferase activity was lower in the HuH-7 cells transfected with the Prom-Luc and Prom-Luc-Ex1 constructs compared to the HuH-7 cells transfected with the Prom-Ex1-Int1-Luc construct suggesting that presence of the NAGS intron 1 enhances gene expression in this cell line (Figure 2A). Reporter gene assays in the HepG2 cells revealed that presence of either c.427-218A>C or c.426+326G>A sequence variants caused reduced luciferase activity compared to the HepG2 cells transfected with the Prom-Ex1-Int1-Luc plasmid (Figure 2B). As in the HuH-7 cells, luciferase activity was higher in the HepG2 cells transfected with the Prom-Ex1-Int1-Luc plasmid than in the cells transfected with the Prom-Ex1-Luc construct (Figure 2B). However, luciferase activity was lower in the HepG2 cells transfected with plasmids that contain theNAGS exon 1 coding sequence suggesting that its translation led to decreased expression of the reporter gene in this cell line (Figure 2B). Taken together these data strongly suggest that the conserved element in the first intron of the NAGS gene can enhance its expression and that the c.427-218A>C and c.426+326G>A sequence variants cause NAGSD by reducing expression of the NAGS gene.