The clinical symptoms of OTCD are non-specific and often misdiagnosed as gastrointestinal disease or liver disease, food or drug poisoning, or central nervous system infection. This can delay diagnosis and treatment, leading to a high mortality rate [6,7,8]. Hyper-ammonemia is a critical diagnostic indicator. In this study, five male patients presented with early-onset symptoms. Clinical manifestations of affected subjects include eating difficulties, drowsiness, groaning, and seizures. The main symptoms in our late-onset patients were poor appetite, vomiting, lethargy and abnormal liver function.

Blood ammonia levels should be promptly tested in patients with unexplained gastrointestinal symptoms, abnormal liver function, or encephalopathy. Early-onset patients exhibited significantly increased ammonia levels, while late-onset ones exhibited greater variability. Among our late-onset patients, two with significantly increased blood ammonia levels died despite active treatment, while two children who underwent liver transplantation survived. One patient (P7) with moderately increased blood ammonia levels improved after receiving low protein diet and arginine treatment, but later presented with recurrent hyperammonemia and mild intellectual disability. These cases underscore the high mortality rate associated with OTCD and highlight the crucial role of blood ammonia levels in determining the disease severity and prognosis.

The correlation between OTCD and liver complications is increasingly recognized, with over 50% of symptomatic OTCD patients experiencing such condition [9,10,11,12]. In some instances, acute liver diseases(ALF) may be the first sign of OTCD [10, 12]. Furthermore, patients with acute hyperammonemia may experience recurrent episodes of ALF [11, 13]. ALF is the most common complication in male newborns with severe OTCD [10, 11]. In this study, all four girls with late-onset OTCD had ALF, whereas only one of five boys with early-onset OTCD experienced ALF. More recently, a comprehensive review has summarized that acute liver failure is a more frequent observation in OTCD, but the specific underlying mechanisms are still not well elucidated [9]. Moreover, a late-onset patient with a peak blood ammonia level of 222 mol/L developed severe ALF, indicating that the blood ammonia level is not always the direct cause of liver failure. Studies have proposed that the inhibition of liver protein synthesis rather than nonspecific cell death due to hyperammonemia is a potential mechanism in hyperammonemia-induced ALF [11]. One study showed that patients had a high rate of liver hyperechogenicity, and more precisely in the 53% of female patients and 42% of male ones [9]. In our study, five patients underwent color Doppler ultrasound examination of the liver, of which three showed a hyperechoic liver and one did not. The specific significance of liver hyperechogenicity is unclear.Further research is needed to investigate the underlying mechanisms of liver disease in OTCD to provide better treatment strategies for this complication.

The interaction between genomic and epigenetic factors leads to different phenotypes of disease [14],OTC mutations show high heterogeneity [1, 6]. In this study, nine OTC gene variants were detected in ten patients: seven were previously reported, while two variants, c.1033del and c.167T > A are novel. The heterozygous deletion of c.1033del resulted in a frameshift mutation, leading to the formation of a truncated protein (p.Y345Tfs*50) and a loss-of-function variant, identified by ACMG as pathogenic (PVS1 + PS2_Moderate + PM2). The c.167T > A missense variant is located in a highly conserved region of OTC enzymes at the junction of three crucial helices (helices 1, 5, and 11) that bridge the two domains of the protein [15]. Therefore, this variant may affect interdomain alignment, thus affecting enzyme activity, as well as a mutation in the pathogenic amino acid residue p.M56T at this locus, which has been reported to be related to late-onset OTCD [15,16,17]. According to the pathogenicity classification of ACMG, this is likely to be a pathogenic mutation (PM1 + PM2 + PM5 + PP3).

Disease severity depends on OTC activity, which is influenced by the type and site of mutations [16, 18] and environmental factors, including daily protein intake [7, 8]. The complete loss of OTC function caused by large deletions, frameshifts, or nonsense mutations often leads to a severe neonatal onset [16, 17]. This study found P8 with the deletion of exon 5 of the OTC gene exhibiting early-onset, while two patients (P1 and P2) with frameshift mutations who showed severe late-onset. The c.140del variant of P2 has also been reported in a Chinese female patient who died after drug treatment [6].

Some missense mutations can cause a complete loss of OTC function, leading to severe neonatal-onset diseases in hemizygous males and most symptomatic heterozygous females. Missense mutations which retain some OTC activity can cause late-onset in hemizygous males [16, 17]. In this study, we detected the missense mutation c.1028 C > G in P3, which has a relatively mild late-onset clinical presentation. This mutation was previously found in a male late-onset patient without neurological damage [19], suggesting that it may partially preserve OTC activity and present as late-onset in male subjects. P225L is a relatively common mutation in the Chinese OTCD population [6]. It has been identified in several male patients with early-onset, all of whom experienced severe hyperammonemia and died during the neonatal period [20,21,22,23,24]. This indicates that the P225L variant is associated with severe male early-onset. Although the correlation between genotype and phenotype is not yet clear, genetic testing can support the clinician in providing a more precise diagnostic evaluation and solid evidence for counselling to the affected families [25], and it should facilitate the functional study on the proteins in urea cycle.

In this study, among the seven patients with parental validation, five had mutations in healthy mothers, including two female patients with severe hyperammonemia whose mothers were also healthy. Members of the same family with the same mutation can have great differences in phenotype. Moreover, the clinical manifestations of OTCD in affected women within the same family may differ [20, 26] due to random X chromosome inactivation or mosaicism, epigenetics, diet, lifestyle, nutrition, and other factors [2, 27]. This highlights the diversity of clinical manifestations of late-onset OTCD and the multi-factor influence of the OTC gene, which further complicates the prenatal diagnosis of female heterozygotes.