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.