Citrullinemia type I is an autosomal recessive disorder caused by mutations in the ASS1 gene, whereas ASS1 mutation carriers typically have a mutation in only one allele. While carriers are usually asymptomatic, they often show mildly elevated plasma citrulline levels. This study aims to investigate the relationship between the genetic background of ASS1 carriers and plasma citrulline levels, as well as the potential mechanisms behind the elevation. The study analyzed 99 ASS1 mutation carriers identified through neonatal screening. Clinical and genetic data were collected, including plasma citrulline levels at initial and follow-up screenings. This is the first large-scale analysis of the relationship between ASS1 mutations and plasma citrulline levels in carriers. Twenty-eight distinct variants were identified, with c.1087C > T (p.R363W) being the most common. Plasma citrulline levels in carriers were elevated, showing a rising then falling pattern over first six months, with a peak of 65.84 µmol/L during re-screening. Levels gradually declined thereafter and stabilized around 49.92 μmol/L, but consistently remained above the normal reference range. A citrulline threshold of 62.04 µmol/L effectively differentiated ASS1 carriers from Citrullinemia type I patients (AUC = 0.984). Elevated plasma citrulline levels are often seen in mutations affecting the monomer-monomer interface, though other mechanisms may be involved. This study identified 62.04 µmol/L as a reference threshold at the time of newborn screening to distinguish ASS1 carriers from Citrullinemia type I patients, observed a dynamic pattern of citrulline levels over the first six months in carriers, and suggested a potential role for dominant-negative effects in the underlying mechanism. The purpose of this overview is to: 1.. Briefly describe the clinical characteristics of urea cycle disorders; 2.. Review the genetic causes of urea cycle disorders; 3.. Review the differential diagnosis of urea cycle disorders with a focus on genetic conditions; 4.. Provide an evaluation strategy to identify the genetic cause of a urea cycle disorder in a proband (when possible); 5.. Review management of hyperammonemia and urea cycle disorders; 6.. Inform genetic counseling of family members of an individual with a urea cycle disorder and evaluation of a newborn at risk for a urea cycle disorder.

Citrullinemia type I (CTLN1) is an autosomal recessive disorder caused by variants in the arginine succinate synthase gene (ASS1). These variants result in arginine succinate synthase deficiency, leading to a disruption of the urea cycle and hyperammonemia. To date, only a handful of CTLN1 cases have been reported in China. One neonate responded poorly 30 h after birth and progressed to coma several hours later. Family history revealed that the neonate's older brother had also died a few days after birth. Biochemical tests on admission confirmed hyperammonemia and elevated levels of citrulline and urinary orotic acid-3. Genetic analysis revealed that the parents were carriers of two heterozygous variants in ASS1, c.910C>T(p.Arg304Trp) and c.839-1G>A, respectively. However, the splice site variant c.839-1G>A was not present in the control databases. Minigene analysis of the c.839-1G>A resulted in the product of r.839del [p.(Gly280Valfs*15)]. In conclusion, we have identified a case of CTLN1 and diagnosed a novel pathogenic variant in the ASS1 gene, c.839-1G>A, expanding the variant spectrum of ASS1. Currently, there are few reports of CTLN1 cases featuring such severe clinical manifestations and an onset at such a young age.

Expanded Newborn Screening (ENS) allows the early identification of many inherited metabolic diseases (IMDs) for which timely treatment can modify the natural history. For most IMDs, diagnosis by ENS is pre-clinical. However, clinical symptoms may emerge for certain conditions before screening results become available. We describe six cases of patients with early-onset IMDs born between 2013 and 2023, who were admitted or transferred to Sant'Orsola University Hospital in Bologna (Italy). Over the study period, 379,013 newborns underwent ENS in the Italian region of Emilia-Romagna. Excluding cases of congenital hypothyroidism, pre-clinical diagnoses from ENS were 410. In addition, six cases of IMD presented with early-onset clinical symptomatology, an antecedent to the outcome of newborn screening (incidence over 11 years of 1.58 cases per 100,000 infants). Among these patients, three were diagnosed with Urea Cycle Disorders (UCDs)-two with Citrullinemia type I (CIT1) and one with Argininosuccinic Acidemia (ASA); two were diagnosed with Methylmalonic Acidemia (MMA); and one was found to have Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD). Our 11-year experience with ENS has shown that clinical onset can occur between the second and fourth day of life, though rare. Even if dried blood spot (DBS) collection was performed 24-48 h after birth, the time required for sample transportation and processing would still delay result availability, making early intervention unlikely. Therefore, our experience supports performing ENS at 48-72 h, as currently implemented in Italy, while also highlighting the advantages and limitations of earlier screening.

European Research Network for evaluation and improvement of screening, Diagnosis, and treatment of Inherited disorders of Metabolism (ERNDIM) provides proficiency testing to clinical laboratories that offer biochemical testing, including urine organic acids analysis. Following an educational challenge, ERNDIM provided a mass spectrum of the 'trimethylsilyl derivative of cyclic derivative of citrulline' to participating laboratories. Though there is knowledge among some in the biochemical genetics' community, that the cyclic derivative of citrulline can be identified in urine of patients with citrullinemia type I, we could not find any published literature regarding this analyte in patient samples, nor were able to obtain any standard material. We did note two publications that proposed chemical structures for the cyclic derivative of citrulline, but neither suggested a clinical utility for this compound and the two proposed structures differed. Here, we used the mass spectrum provided by ERNDIM to retrospectively evaluate existing urine organic acids data from patients affected with conditions associated with elevated levels of plasma citrulline to learn more about this marker in urine and to correlate it with plasma citrulline data in diagnostic and follow-up samples. We document a positive correlation between the concentration of this compound in patient urine and the concentration of citrulline in concurrently collected plasma. We establish that this analyte is a good marker for citrullinemia type I but is not unique to this condition. Finally, we correlate the mass spectrum with the chemical structure originally proposed by Wilson et al. in 1978.

Citrullinemia type I disorders (CTLN1) is a genetic metabolic disease caused by argininosuccinate synthetase (ASS1) gene mutation. To date, the human genome mutation database has documented over 100 variants of the ASS1 gene. This study reported a novel deletion-insertion variant of ASS1 gene and employed various prediction tools to determine its pathogenicity. We reported a case of early-onset CTLN1. Whole exome sequencing was conducted to identify genetic mutations. We employed various structure prediction tools to generate accurate 3D models and utilized computational biology tools to elucidate the disparities between the wild-type and mutant proteins. The patient was characterized by severe clinical manifestations, including poor responsiveness, lethargy, convulsions, and cardiac arrest. Notably, the patient exhibited significantly elevated blood ammonia levels (655 μmol/L; normal reference: 10-30 μmol/L) and increased citrulline concentrations (936 μmol/L; normal reference: 5-25 μmol/L). Whole exome sequencing revealed a in-frame deletion-insertion mutation c.1128_1134delinsG in the ASS1 gene of unknown significance, which has not been previously reported. Our finding indicated that the C- terminal helix domain of the mutant protein structure, which was an important structure for ASS1 protein to form protein tetramers, was indeed more unstable than that of the wild-type protein structure. Through conducting an in silico analysis on this unique in-frame deletion-insertion variant of ASS1, our aim was to enhance understanding regarding its structure-function relationship as well as unraveling the molecular mechanism underlying CTLN1.