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.