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.