Functional testing of sequence variants in the -3kb
enhancer of the NAGS gene
Subjects 1 and 3 had deleterious sequence variants in the first intron
and coding sequence of their NAGS genes; we used functional
testing of the sequence variants found in the -3 kb enhancer of theirNAGS genes to establish molecular diagnosis of NAGSD. Epigenetic
mark H3K27Ac of the NAGS -3 kb enhancer in the human liver
indicate active enhancer status while H3K4me3 epigenetic mark,
characteristic of active promoters (Kouzarides, 2007), of the -3 kb
enhancer suggests that it may regulate NAGS gene expression
through initiation of enhancer mRNA (Figure 3A) (Natoli & Andrau,
2012). Query of the ENCODE database revealed that -3 kb enhancer harbors
conserved GR binding site and binds transcription factors HNF4α, RXR α,
Sp1, and YY1 in addition to previously identified HNF1, NF-Y binding
sites (Heibel et al., 2012) and NF1C conserved site (Williams et al.,
2018) (Figure 3). The c.-3065A>T sequence variant is
located within HNF1 binding site (Figure 3B), one base pair upstream of
the previously identified pathogenic NAGS sequence variant
(Heibel et al., 2011) while the c.-3098C>T sequence variant
resides in the predicted GR binding site (Figure 3B).
Reporter gene assays were used to determine whether
c.-3065A>T and c.-3098C>T sequence variants
can affect gene expression in HuH-7 and HepG2 cells. Base pair changes
that correspond to the two -3 kb enhancer variants found in subjects 1
and 3 were introduced into minP-E plasmid (Table 1) followed by
transfection of the plasmids into either HuH-7 or HepG2 cells. The
minP-E plasmid was used as a positive control; cells transfected with
plasmid containing either only minimal promoter or promoter-less plasmid
were used as negative controls. Depending on the experiment, luciferase
activity increased approximately 8 to 10-fold in the HuH-7 cells and 19
to 29-fold in the HepG2 cells when -3 kb enhancer was placed upstream of
the minimal promoter (Figure 4). Luciferase activity was slightly above
background level in the presence of the c.-3065A>T sequence
variant in both HuH-7 and HepG2 cells (Figure 4A-B) strongly suggesting
that this sequence variant can have deleterious effect on theNAGS gene expression. In the presence of the
c.-3098C>T sequence variant luciferase activity was reduced
by approximately one half in the HuH-7 cells and by about two thirds in
the HepG2 cells compared to cells expressing plasmid with the wild-type
-3 kb enhancer (Figure 4C-D). This suggests that the
c.-3098C>T sequence variant likely has a negative effect on
the NAGS gene expression.