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.