Disease-causing mutations in the promoter and enhancer of the ornithine transcarbamylase gene

Abstract

The ornithine transcarbamylase (OTC) gene is on the X chromosome and its product catalyzes the formation of citrulline from ornithine and carbamylphosphate in the urea cycle. About 10%-15% of patients, clinically diagnosed with OTC deficiency (OTCD), lack identifiable mutations in the coding region or splice junctions of the OTC gene on routine molecular testing. We collected DNA from such patients via retrospective review and by prospective enrollment. In nine of 38 subjects (24%), we identified a sequence variant in the OTC regulatory regions. Eight subjects had unique sequence variants in the OTC promoter and one subject had a novel sequence variant in the OTC enhancer. All sequence variants affect positions that are highly conserved in mammalian OTC genes. Functional studies revealed reduced reporter gene expression with all sequence variants. Two sequence variants caused decreased binding of the HNF4 transcription factor to its mutated binding site. Bioinformatic analyses combined with functional assays can be used to identify and authenticate pathogenic sequence variants in regulatory regions of the OTC gene, in other urea cycle disorders or other inborn errors of metabolism.

Keywords: enhancer mutation; gene expression gene regulation; hyperammonemia; ornithine transcarbamylase; ornithine transcarbamylase deficiency; promoter mutation; urea cycle.

Figure 1.

Promoter region of the human OTC gene. A. Sequence of the human OTC promoter. Sequence variants identified in this study are shown in red and underlined typeface. Angled arrows indicate transcription start sites in human OTC gene. Sequence variants found in study subjects are: NM_000531.5(OTC_v001):c.−106C>A, NM_000531.5(OTC_v001):c.−116C>T, NM_000531.5(OTC_v001):c.−115C>T, NM_000531.5(OTC_v001):c.−139A>G, NM_000531.5(OTC_v001):c.−142G>A, NM_000531.5(OTC_v001):c.−157T>G, NM_000531.5(OTC_v001):c.−9384G>T. B. LOGO representation of the multiple sequence alignment of OTC promoters from 36 mammals. The height of each letter corresponds to its conservation. Sequences that correspond to experimentally identified transcription factor binding sites in the rat Otc promoter are highlighted in yellow. Known transcription factor binding sites in the rat Otc promoter are shown in cyan and predicted transcription factor binding sites are shown in green.

Figure 2.

Liver-specific enhancer (LSE) of the human OTC gene. A. Sequence of the human LSE. Sequence variant identified in this study is shown in red and underlined typeface. B. LOGO representation of the multiple sequence alignment of OTC enhancers from 37 mammals. The height of each letter corresponds to its conservation. Sequences that correspond to experimentally identified transcription factor binding sites in the rat Otc enhancer are highlighted in yellow. Known transcription factor binding sites in the rat Otc promoter are shown in cyan and predicted transcription factor binding sites are shown in green.

Figure 3.

Effects of promoter sequence variants found in study subjects on reporter gene expression in HepG2 (A) and HuH-7 (B) cell lines. All results are averages of either two or three independent experiments, each carried out in triplicate. Results are shown as mean ± SEM. One asterisk (*) indicates P≤0.05, three asterisks (***) indicate P≤0.001, and four asterisks (****) indicate P≤0.0001.

Figure 4.

Effect of the c.−9383G>T sequence variant on expression of the luciferase gene in HepG2 (A) and HuH-7 (B) cell lines. All results are averages of three independent experiments, each carried out in triplicate. Results are shown as mean ± SEM. One asterisk (*) indicates P≤0.05, two asterisks (**) indicate P≤0.01, and four asterisks (****) indicate P≤0.0001.

Figure 5.

Reduced binding of HNF4 to sites with c.−106C>A and c.−115C>T sequence variants. DNA pull-down assays were used to compare binding of HNF4 to its wild-type and mutated binding sites. Lanes: 1 – biotinylated DNA with wild-type binding site; 2 – unlabeled DNA with wild-type binding site; 3 – non-specific biotinylated DNA; 4 – pull-down without DNA; 5 – biotinylated binding site containing c.−106C>A variant; 6 – non-biotinylated binding site containing c.−106C>A variant; 7 – biotinylated binding site containing c.−115C>T variant; 8 – non-biotinylated binding site containing c.−115C>T variant. Results are representative of three independent experiments.