Figure Legends
Figure 1. Sequence variants in the novel regulatory
element in the NAGS intron 1. A. The phastCons track of the UCSC
Genome Browser showing conserved regions in the NAGS gene (top)
and map of the NAGS gene (bottom) showing genomic region
chr17:42,081,993 – 42,086,412 of the GRCh37/hg19 human genome assembly
with exons as gray boxes, introns as gray lines and predictedcis -acting conserved regulatory element in the NAGS intron
1 as purple box. B. Genomic region chr17:42,082,368-42,083,068
of the GRCh37/hg19 human genome assembly. Results of the ENCODE ChIP-Seq
experiments showing binding of transcription factors HNF4α (blue), RXRα
(orange) and Sp1 (green) to NAGS intron 1 in the human liver
tissue (top) and phastCons track of the UCSC Genome Browser
showing conserved region within NAGS intron 1 (bottom) .C. LOGO alignment of the conserved intronic sequences from 20
mammals. Magenta – pathogenic sequence variants. Blue – HNF4α binding
site. Orange – RXRα binding site. Green – Sp1 binding site.
Figure 2. Functional testing of the sequence variants
found in NAGS intron 1 from patients with NAGSD. Relative luciferase
activity in HuH-7 (A) and HepG2 (B) cells transfected
with the promoter-less plasmid pGL4.10[luc2 ] (Bkgd), plasmid
with NAGS promoter controlling luciferase expression (Prom-Luc),
plasmid with NAGS promoter controlling luciferase fused to the
coding sequence of NAGS exon 1 (Prom-Ex1-Luc), plasmid withNAGS promoter controlling luciferase fused to the NAGSexon and intron 1 (Prom-Ex1-Int1-Luc) and plasmids with intronic
mutations found in patients with NAGS deficiency
(c.427-218A>C and c.426+326G>A). Data
represent mean ± SEM of n=9 measurements. ** indicate p<0.01,
**** indicates p<0.0001.
Figure 3. Sequence variants in the -3kb enhancer of theNAGS gene. A. Genomic region chr17:42,078,635-42,079,129 of the
GRCh37/hg19 human genome assembly. Results of the ENCODE ChIP-Seq
experiments showing histone modifications (top) and binding of
transcription factors HNF4α (blue), RXRα (orange), Sp1 (green) and YY1
(purple) to -3kb enhancer in the human liver tissue (bottom) .B. LOGO alignment of the -3kb enhancer sequences from 25
mammals. Confirmed HNF1 and NF-Y binding sites are highlighted in
yellow. Conserved GR and NF1C binding sites are highlighted in gray.
Magenta – pathogenic sequence variants reported here. Dark blue –
location of the previously reported c.-3026C>T and
c.-3064C>A sequence variants. Blue – HNF4α binding site.
Orange – RXRα binding site. Green – Sp1 binding site. Purple – YY1
binding site.
Figure 4. Functional testing of the sequence variants
found in the NAGS -3kb enhancer from patients with NAGSD. Effect
of the c.-3065A>T sequence variant on luciferase gene
expression was tested in HuH-7 (A) and HepG2 (B)cells. Effect of the c.-3098C>T sequence variant on
luciferase gene expression was tested in HuH-7 (C) and HepG2(D) cells. Cells were transfected with the promoter-less
plasmid pGL4.10[luc2 ] (Bkgd), plasmid with minimal eukaryotic
promoter controlling luciferase expression (minP), plasmid harboring
-3kb NAGS enhancer and minimal promoter controlling luciferase
expression (minP_E), and plasmids with enhancer mutations found in
patients with NAGS deficiency (c.-3065A>T and
c.-3098C>T). Data represent mean ± SEM of n=9 measurements.
* indicate p<0.05, *** indicate p<0.001, ****
indicate p<0.0001.