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.