Introduction
N-acetylglutamate synthase deficiency (NAGSD, MIM# 237310) is an
autosomal recessive urea cycle disorder caused by pathogenic variants in
the N-acetylglutamate synthase (NAGS ) gene. Its product, the NAGS
enzyme (EC 2.3.1.1) catalyzes formation of N-acetylglutamate (NAG),
which is essential for the activity of carbamylphosphate synthetase 1
(CPS1, EC 6.3.4.16), the rate-limiting enzyme of the urea cycle
(Waterlow, 1999). NAGSD is the only urea cycle disorder that can be
effectively treated with a drug N-carbamylglutamate (NCG), a stable NAG
analog, which binds to and activates CPS1 to completely restore
ureagenesis in NAGS deficient patients (Caldovic et al., 2004;
Grisolia & Cohen, 1952, 1953; Haberle, 2011). NAGSD can manifest either
within hours after birth if there is complete loss of NAGS function or
at any time thereafter in patients with residual NAGS activity. Symptoms
of NAGSD include lethargy progressing to coma and death with laboratory
studies showing elevated blood ammonia and glutamine, and low citrulline
concentrations (Ah Mew & Caldovic, 2011; Haberle, 2011).
The human NAGS gene, located on chromosome 17, has seven exons
that encode a 534 amino acid protein and a regulatory region that
extends at least 3 kb upstream of the first exon (Caldovic et al., 2002;
Elpeleg, Shaag, Ben-Shalom, Schmid, & Bachmann, 2002; Haberle et al.,
2003; Heibel et al., 2011). Expression of the NAGS gene is
controlled by the promoter and -3kb enhancer, which were identified
based on their conservation in mammalian NAGS genes (Heibel et
al., 2011; Heibel et al., 2012). The NAGS promoter binds the
transcription factors specificity protein 1 (Sp1), cAMP response element
binding (CREB) and farnesoid X receptor (FXR) (Heibel et al., 2012;
Renga et al., 2011). The -3 kb NAGS enhancer binds transcription
factors hepatocyte nuclear factor 1 (HNF1) and nuclear factor Y (NF-Y)
and directs liver-specific expression of the NAGS gene (Heibel et
al., 2012).
The establishment of accurate molecular diagnostic methods for NAGSD is
clinically important because the biochemical phenotype cannot be easily
differentiated from other proximal urea cycle disorders, but unlike
these other disorders, NAGSD can be effectively treated with NCG (Ah Mew
& Caldovic, 2011; Haberle, 2011). Although most of the sequence
variants causing NAGSD have been found in the NAGS coding region
(Al Kaabi & El-Hattab, 2016; Caldovic, Morizono, & Tuchman, 2007; Kim
et al., 2015; Sancho-Vaello et al., 2016; van de Logt, Kluijtmans,
Huigen, & Janssen, 2017), three pathogenic variants have been found in
the non-coding regions (Heibel et al., 2012; Sonaimuthu et al., 2021;
Williams et al., 2018). Therefore, understanding of the NAGS gene
regulation and the function of its regulatory elements is essential for
confirming the pathological nature of sequence variants found in
patients. In this study, we identify a novel regulatory element in the
first intron of the NAGS gene based on the sequence variants
found in patients with NAGSD and describe two new pathogenic sequence
variants in the -3 kb enhancer of the NAGS gene.