Discussion
This report describes four new non-coding sequence variants in theNAGS gene that can cause reduced NAGS expression and NAGSD. None of the four sequence variants have been previously reported and all four reduced luciferase activity in reporter gene assays. Two of the sequence variants are located in a conserved region of the firstNAGS intron and define a novel regulatory element of theNAGS gene. This novel regulatory element binds transcription factors HNF4α, RXR α and Sp1 in human liver based on the data from the ENCODE project. First introns of many human genes often harbor regulatory elements based on their conservation in mammalian genomes, presence of DNase hypersensitive sites and epigenetic histone modifications indicative of active regulatory elements (Jo & Choi, 2019; Park, Hannenhalli, & Choi, 2014). Several regulatory elements found within first introns bind Sp1 transcription factor (Beaulieu et al., 2011; Bornstein, McKay, Morishima, Devarayalu, & Gelinas, 1987; Guerin, Leclerc, Verreault, Labrie, & Luu-The, 1995; Liska, Robinson, & Bornstein, 1992), similar to the regulatory element within first intron of human NAGS gene identified in this study.
The c.427-218A>C and c.426+326G>A sequence variants both affect highly conserved base pairs in the RXRα binding site suggesting a role for this transcription factor in the regulation of NAGS expression. Decreased reporter gene activity in cells transfected with constructs containing sequence variants within RXRα binding site, which is consistent with decreased NAGS expression in patients with the two sequence variants, suggests that RXRα acts as transcriptional activator of NAGS . RXRα transcription factor is a nuclear receptor that binds vitamin A metabolites 9-cis -retinoic acid (Evans & Mangelsdorf, 2014) and 9-cis -13,14-dihydroretinoic acid, which is a better candidate for physiological RXRα ligand because it has been detected in the liver (Krezel, Ruhl, & de Lera, 2019). RXRα regulates transcription either as a homodimer or heterodimer with retinoic acid receptor, thyroid receptor or vitamin D receptor. Ongoing efforts of the ENCODE project may reveal whether RXRα binds as a homo- or heterodimer to regulatory element in the first intron of humanNAGS gene. There are no reports of regulation of urea cycle enzymes by vitamin D. Both vitamin A and thyroid hormone play a role in the protein metabolism of rats. Vitamin A deficiency results in increased protein catabolism and higher expression of urea cycle genes and enzymes in adult and growing rats (Esteban-Pretel et al., 2010; McClintick et al., 2006). This effect of vitamin A deficiency on expression of urea cycle genes is likely an indirect consequence of increased protein catabolism, does not exclude activation of NAGSexpression by RXRα and can be consistent with decreased expression ofNAGS due to sequence variants that may decrease binding of receptors for vitamin A and its metabolites. Manipulation of the thyroid hormone levels in rats affect abundance of urea cycle enzymes, but direction of the change depends on the duration of hypothyroidism and control of food intake by experimental animals. Prolonged hypothyroidism in rats, lasting 4-7 weeks, resulted in increased abundance of urea cycle enzymes and capacity to produce urea probably due to decreased food intake and weight loss in the hypothyroid animals (Marti, Portoles, Jimenez-Nacher, Cabo, & Jorda, 1988; Silvestri et al., 2006). In a different set of studies, hypothyroidism lasting two weeks led to increased production of urea and urea cycle intermediates, including NAG. However, neither hypo- nor hyperthyroidism led to changes in expression of urea cycle genes in mouse liver (Feng, Jiang, Meltzer, & Yen, 2000; Flores-Morales et al., 2002) as well as abundance and activity of urea cycle enzymes in rat liver (Hayase, Naganuma, Koie, & Yoshida, 1998; Hayase, Yonekawa, Yokogoshi, & Yoshida, 1991; Hayase, Yonekawa, & Yoshida, 1992, 1993; Hayase & Yoshida, 1995). This suggests that thyroid hormone receptors are unlikely to regulate expression of NAGS by forming heterodimers with RXRα.
Two of the sequence variants were found in the -3 kb enhancer of theNAGS gene. We queried ENCODE project database for epigenetic marks found in this region in the human liver. Acetylation of the lysine 27 of the histone H3 in this region indicates that it is an active enhancer of the human NAGS gene. The lysine 4 of the histone H3 is tri-methylated in the -3 kb enhancer. Although this epigenetic mark indicates active promoters, many enhancers can bind RNA polymerase II and initiate transcription of enhancer RNA (eRNA) (Natoli & Andrau, 2012). Closer inspection of the Transcription Factor ChIP-Seq track of the UCSC Genome Browser revealed that RNA polymerase II binds to the -3 kb NAGS enhancer in HepG2 cells and likely initiates transcription of an eRNA from this region. This may explain the inability of the -3 kb enhancer to act in the orientation independent manner (Heibel et al., 2012).
The c.-3065A>T sequence variant affects a base pair that is highly conserved in mammals and located in the HNF1 transcription factor binding site. Negative effect of the c.-3065A>T sequence variant on HNF1 binding to the -3kb enhancer is a likely explanation for the deleterious effect of this variant. A sequence variant that reduces binding of HNF1 to -3 kb enhancer and located immediately downstream of the c.-3065A>T was found in a patient with NAGSD (Heibel et al., 2011). Two pathogenic sequence variants found in the HNF1 binding site of the -3 kb enhancer stress the importance of this transcription factor for expression of the NAGS gene and normal ureagenesis.
The second variant found in the -3 kb enhancer is located in the predicted GR binding site. This variant reduced luciferase activity presumably through reduced binding of GR to its binding site in the -3 kb enhancer. Circadian fluctuations of glucocorticoid secretion regulate expression of urea cycle genes and enzymes during feeding and fasting periods to accommodate removal of excess ammonia that is released as amino acids enter gluconeogenesis (Luna-Moreno, Garcia-Ayala, & Diaz-Munoz, 2012). The role of glucocorticoids in regulation of ureagenesis was revealed through decreased abundance and activity of urea cycle enzymes in adrenalectomized rats (Hazra, DuBois, Almon, Snyder, & Jusko, 2008; McLean & Gurney, 1963). GR binds to regulatory elements and activates expression of the rat Cps1 gene (Christoffels et al., 1998; Christoffels et al., 2000; Christoffels, van den Hoff, Moorman, & Lamers, 1995; Schoneveld, Gaemers, Hoogenkamp, & Lamers, 2005). Regulation of other urea cycle genes by GR is indirect and requires ongoing protein synthesis of transcription factors that directly regulate rat ornithine transcarbamylase, argininosuccinate synthetase 1, argininosuccinate lyase and arginase 1 (Gebhardt & Mecke, 1979; Lin, Snodgrass, & Rabier, 1982; Morris & Kepka-Lenhart, 2002; Nebes & Morris, 1988; Ulbright & Snodgrass, 1993). A role for GR in regulation of ureagenesis in humans is supported by the observation of abnormal concentrations of urea cycle intermediates and low urea concentration in the blood of patients with Addison’s disease (Okun et al., 2015) and in patients receiving prednisolone treatment (Wolthers, Hamberg, Grofte, & Vilstrup, 2000). NAGSD in one of our patients and functional tests of the c.-3098C>T variant, located in the predicted GR binding site, suggest that GR might directly regulateNAGS expression. Unfortunately, the data about GR binding to DNA in human liver are not yet available in the ENCODE database.
Molecular diagnosis of NAGSD is important because it is the only urea cycle disorder that can be effectively treated with a drug (Caldovic et al., 2004; Haberle, 2011). Two sequence variants found in the first intron define a new NAGS regulatory element that binds and implicates transcription factors HNF4α and RXRα in the regulation ofNAGS expression and ureagenesis. The four non-coding sequence variants that cause NAGS deficiency reported here bring the total number of non-coding, disease-causing NAGS variants to seven, which is almost 14% of deleterious NAGS sequence variants (Al Kaabi & El-Hattab, 2016; Bijarnia-Mahay et al., 2018; Cartagena et al., 2013; Cavicchi et al., 2018; Heibel et al., 2011; van de Logt et al., 2017; Williams et al., 2018). This underscores the importance of analyzing both coding and non-coding regions of the NAGS gene, which is amenable to both Sanger and next generation sequencing, for the presence of disease-causing sequence variants.