Automated grape leaf nutrition deficiency disease detection and classification Equilibrium Optimizer with deep transfer learning model.

Our approach includes picture preprocessing, feature extraction utilizing the SqueezeNet model, hyperparameter optimisation utilising the Equilibrium Optimizer (EO) algorithm, and classification utilising a Stacked Autoencoder (SAE) model. Each of these processes is carried out in a series of separate steps. During the image preprocessing stage, contrast limited adaptive histogram equalisations (CLAHE) is utilized to improve the contrasts, and Adaptive Bilateral Filtering (ABF) to get rid of any noise that may be present. The SqueezeNet paradigm is utilized to obtain relevant characteristics from the pictures that have been preprocessed, and the EO technique is utilized to fine-tune the hyperparameters. Finally, the SAE model categorises the diseases that affect the grape leaf. The simulation analysis of the EODTL-GLDC technique tested New Plant Diseases Datasets and the results were inspected in many prospects. The results demonstrate that this model outperforms other deep learning techniques and methods that are more often related to machine learning. Specifically, this technique was able to attain a precision of 96.31% on the testing datasets and 96.88% on the training data set that was split 80:20. These results offer more proof that the suggested strategy is successful in automating the detection and categorization of grape leaf diseases.

Clinical features and CPS1 variants in Chinese patients with carbamoyl phosphate synthetase 1 deficiency.

BACKGROUND: Carbamoyl phosphate synthetase 1 (CPS1) deficiency (OMIM 237300), an autosomal recessive rare and severe urea cycle disorder, is associated with hyperammonemia and high mortality. METHODS: Herein we present 12 genetic variants identified in seven clinically well-characterized Chinese patients with CPS1 deficiency who were admitted to the Children's Medical Center of Peking University First Hospital from September 2014 to August 2023. RESULTS: Seven patients (two male and five female patients including two sisters) experienced symptoms onset between 2 days and 13 years of age, and they were diagnosed with CPS1 deficiency between 2 months and 20 years. Peak blood ammonia levels ranged from 160 to 1,000 µmol/L. Three patients showed early-onset CPS1 deficiency, with only one surviving after treatment with sodium phenylbutyrate, N-carbamoyl-L-glutamate, and liver transplantation at 4 months, showing a favorable outcome. The remaining four patients had late-onset CPS1 deficiency, presenting with mental retardation, psychiatric symptoms, and self-selected low-protein diets. Among the 12 CPS1 variants identified in these patients, 10 were novel, with all patients exhibiting compound heterozygosity for CPS1 mutant alleles. Seven variants (c.149T > C, c.616 A > T, c.1145 C > T, c.1294G > A, c.3029 C > T, c.3503 A > T, and c.3793 C > T) resulted in single amino acid substitutions. Three frameshift variations (c.2493del, c.3067dup, and c.3241del) were identified, leading to enzyme truncation. One mutation (c.3506_3508del) caused an in-frame single amino acid deletion, while another (c.2895 + 2T > C) resulted in aberrant splicing. CONCLUSIONS: Except for two known variants, all other variants were identified as novel. No hotspot variants were observed among the patients. Our data contribute to expanding the mutation spectrum of CPS1.

Unraveling the therapeutic potential of carbamoyl phosphate synthetase 1 (CPS1) in human diseases.

CPS1, the rate-limiting enzyme that controls the first reaction of the urea cycle, is responsible for converting toxic ammonia into non-toxic urea in mammals. While disruption of the functions of CPS1 leads to elevated ammonia and nerve damage in the body, mainly manifested as urea cycle disorder. Moreover, accumulating evidence has recently revealed that CPS1 is involved in a variety of human diseases, including CPS1D, cardiovascular disease, cancers, and others. In particular, CPS1 expression varies among cancers, being overexpressed in some cancers and downregulated in others, suggesting that CPS1 may be a promising cancer therapeutic target. In addition, some small-molecule inhibitors of CPS1 have been reported, which have not been confirmed experimentally in malignancies, meaning their future role is far from certain. In this review, we describe the structure and function of CPS1, highlight its important roles in various human diseases, and further discuss the potential diagnostic and therapeutic implications of small molecule compounds targeting CPS1.

Long-term follow-up of children with carbamoyl phosphate synthase 1 deficiency detected in newborn screening. / æ–°ç”Ÿå„¿ç­›æŸ¥å‘çŽ°çš„æ°¨ç”²é °ç£·é ¸åˆæˆé ¶1ç¼ºä¹ç—‡æ‚£å„¿é•¿æœŸéšè®¿ç ”ç©¶.

OBJECTIVES: To investigate genotype-phenotype characteristics and long-term prognosis of neonatal carbamoyl phosphate synthetase 1 (CPS1) deficiency among children through newborn screening in Zhejiang province. METHODS: The clinical and follow-up data of children with CPS1 deficiency detected through neonatal screening and confirmed by tandem mass spectrometry and genetic testing in Zhejiang Province Newborn Disease Screening Center from September 2013 to August 2023 were retrospectively analyzed. RESULTS: A total of 4 056 755 newborns were screened and 6 cases of CPS1 deficiency were diagnosed through phenotypic and genetic testing. Ten different variations of CPS1 genewere identified in genetic testing, including 2 known pathogenic variations (c.2359C>T and c.1549+1G>T) and 8 unreported variations (c.3405-1G>T, c.2372C>T, c.1436C>T, c.2228T>C, c.2441G>A, c.3031G>A, c.3075T>C and c.390-403del). All patients had decreased citrulline levels (2.72-6.21 µmol/L), and varying degrees of elevated blood ammonia. The patients received restricted natural protein intake (special formula), arginine and supportive therapy after diagnosis, and were followed-up for a period ranging from 9 months to 10 years. Three patients experienced hyperammonemia, and one patient each had attention deficit hyperactivity disorder, transient facial twitching and increased muscle tone. One patient died, while the other five surviving patients had normal scores of the Ages & Stages Questionnaires (ASQ) and Griffiths Development Scales up to the present time; 4 cases had combined height or weight lag and one case was normal in height and weight. CONCLUSIONS: Low citrulline levels and hyperammonemia are common in CPS1 deficiency patients in Zhejiang. Most gene variants identified were specific to individual families, and no hotspot mutations were found. Early diagnosis through newborn screening and following standardized treatment can significantly improve the prognosis of the patients.

Split AAV-Mediated Gene Therapy Restores Ureagenesis in a Murine Model of Carbamoyl Phosphate Synthetase 1 Deficiency.

The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.

CPS1: Looking at an ancient enzyme in a modern light.

The mammalian urea cycle (UC) is responsible for siphoning catabolic waste nitrogen into urea for excretion. Disruptions of the functions of any of the enzymes or transporters lead to elevated ammonia and neurological injury. Carbamoyl phosphate synthetase 1 (CPS1) is the first and rate-limiting UC enzyme responsible for the direct incorporation of ammonia into UC intermediates. Symptoms in CPS1 deficiency are typically the most severe of all UC disorders, and current clinical management is insufficient to prevent the associated morbidities and high mortality. With recent advances in basic and translational studies of CPS1, appreciation for this enzyme's essential role in the UC has been broadened to include systemic metabolic regulation during homeostasis and disease. Here, we review recent advances in CPS1 biology and contextualize them around the role of CPS1 in health and disease.

Molecular, biochemical, and clinical analyses of five patients with carbamoyl phosphate synthetase 1 deficiency.

BACKGROUND: Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is a rare urea cycle disorder. The aim of this study was to present the clinical findings, management, biochemical data, molecular genetic analysis, and short-term prognosis of five children with CPS1D. METHODS: The information of five CPS1D patients was retrospectively studied. We used targeted next-generation sequencing to identify carbamoyl phosphate synthetase 1 (CPS1) variants in patients suspected to have CPS1D. Candidate mutations were validated by Sanger sequencing. In silico and structure analyses were processed for the pathogenicity predictions of the identified mutations. RESULTS: The patients had typically clinical manifestations and biochemical data of CPS1D. Genetic analysis revealed nine mutations in the CPS1 gene, including recurrence of c.1145C > T, five of which were firstly reported. Seven mutations were missense changes, while the remaining two were predicted to create premature stop codons. In silico and structure analyses showed that these genetic lesions were predicted to affect the function or stability of the enzyme. CONCLUSION: We reported five cases of CPS1D. Five novel mutations of CPS1 gene were found. Mutations of CPS1 have private nature, and most of them are missense compound heterozygous. The mutation affecting residue predicted to interfere the catalytic sites, the internal tunnel, or the regulatory domain results in severe phenotype.

A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia.

The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 -/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder.

Hepatic glutamine synthetase augmentation enhances ammonia detoxification.

The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-alpha-fetoprotein), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia.

A liver-humanized mouse model of carbamoyl phosphate synthetase 1-deficiency.

A liver-humanized mouse model for CPS1-deficiency was generated by the high-level repopulation of the mouse liver with CPS1-deficient human hepatocytes. When compared with mice that are highly repopulated with CPS1-proficient human hepatocytes, mice that are repopulated with CPS1-deficient human hepatocytes exhibited characteristic symptoms of human CPS1 deficiency including an 80% reduction in CPS1 metabolic activity, delayed clearance of an ammonium chloride infusion, elevated glutamine and glutamate levels, and impaired metabolism of [15 N]ammonium chloride into urea, with no other obvious phenotypic differences. Because most metabolic liver diseases result from mutations that alter critical pathways in hepatocytes, a model that incorporates actual disease-affected, mutant human hepatocytes is useful for the investigation of the molecular, biochemical, and phenotypic differences induced by that mutation. The model is also expected to be useful for investigations of modified RNA, gene, and cellular and small molecule therapies for CPS1-deficiency. Liver-humanized models for this and other monogenic liver diseases afford the ability to assess the therapy on actual disease-affected human hepatocytes, in vivo, for long periods of time and will provide data that are highly relevant for investigations of the safety and efficacy of gene-editing technologies directed to human hepatocytes and the translation of gene-editing technology to the clinic.

Conditional disruption of hepatic carbamoyl phosphate synthetase 1 in mice results in hyperammonemia without orotic aciduria and can be corrected by liver-directed gene therapy.

Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.

Two novel CPS1 mutations in a case of carbamoyl phosphate synthetase 1 deficiency causing hyperammonemia and leukodystrophy.

BACKGROUND: Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is a rare autosomal recessive disorder of the urea cycle, mostly characterized by hyperammonemia and the concomitant leukodystrophy. The onset of CPS1D can be at any age, and the clinical manifestations are variable and atypical. Genetic tests are indispensable for accurate diagnosis of CPS1D on the basis of biochemical tests. METHODS: Blood tandem mass spectrometric analysis and urea organic acidemia screening were performed on a Chinese neonatal patient with low activity, recurrent seizures, and hyperammonemia. Next-generation sequencing and Sanger sequencing were followed up for making a definite diagnosis. Bioinformatics tools were used for the conservation analysis and pathogenicity predictions of the identified mutations. RESULTS: Increased lactate in urea and decreased citrulline in blood were detected in the patient. Two novel mutations (c.173G>T, p.G58V in exon 2 and c.796G>A, p.G266R in exon 8) in CPS1 identified in the neonatal patient were found through coseparation verification. Both of the two mutations were predicted to be deleterious, and the two relevant amino acids exerted highly evolutionarily conserved. The final diagnosis of the patient was compound heterozygous CPS1D. CONCLUSION: This study described the specific clinical characteristics and the variations of physiological and biochemical indices in a Chinese neonatal patient with CPS1D, which facilitated the diagnosis and mechanism research of the disease. Two novel causative missense mutations were identified, which enriched the mutation spectrum of CPS1D in China and worldwide. Advice of prenatal diagnosis was given to the family for a new pregnancy.

Carbamoyl phosphate synthetase 1 deficiency diagnosed by whole exome sequencing.

BACKGROUND: Carbamoyl Phosphate Synthetase 1 deficiency (CPS1D) is a rare autosomal recessive inborn metabolic disease characterized mainly by hyperammonemia. The fatal nature of CPS1D and its similar symptoms with other urea cycle disorders (UCDs) make its diagnosis difficult, and the molecular diagnosis is hindered due to the large size of the causative gene CPS1. Therefore, the objective of the present study was to investigate the clinical applicability of exome sequencing in molecular diagnosis of CPS1D in Chinese population. METHODS: We described two Chinese neonates presented with unconsciousness and drowsiness due to deepening encephalopathy with hyperammonemia. Whole exome sequencing was performed. Candidate mutations were validated by Sanger sequencing. In-silicon analysis was processed for the pathogenicity predictions of the identified mutations. RESULTS: Two compound heterozygous mutations in the gene carbamoyl phosphate synthetase 1(CPS1) were identified. One is in Case 1 with two novel missense mutations (c.2537C>T, p. Pro846Leu and c.3443T>A, p.Met1148Lys), and the other one is in Case 2 with a novel missense mutation (c.1799G>A, p.Cys600Tyr) and a previously reported 12-bp deletion (c.4088_4099del, p.Leu 1363_Ile1366del). Bioinformatics deleterious predictions indicated pathogenicity of the missense mutations. Conversation analysis and homology modeling showed that the substituted amino acids were highly evolutionary conserved and necessary for enzyme stability or function. CONCLUSION: The present study initially and successfully applied whole exome sequencing to the molecular diagnosis of CPS1D in Chinese neonates, indicating its applicability in cost-effective molecular diagnosis of CPS1D. Three novel pathogenic missense mutations were identified, expanded the mutational spectrum of the CPS1 gene.

[Detection of CPS1 gene mutation in a neonate with carbamoyl phosphate synthetase I deficiency].

OBJECTIVE: To explore the genetic basis for a neonate featuring hyperammonemia. METHODS: The patient was examined and tested by tandem mass spectrometry and next generation sequencing (NGS). Suspected mutations were confirmed by Sanger sequencing of the proband and her parents. Potential impact of the mutation was predicted with SIFT, PolyPhen-2 and MutationTaste software. RESULTS: Plasma ammonia and alanine were significantly increased in the proband, while serum citrulline was decreased. The neonate was found to harbor compound heterozygous mutations of the CPS1 gene [c.1631C>T(p.T544M) and c.1981G>T(p.G661C)], which were respectively inherited from her father and mother. CONCLUSION: The carbamoyl phosphate synthetase I deficiency of the proband can probably be attributed to the mutations of the CPS1 gene. Above finding has expanded the spectrum of CPS1 mutations in association with carbamoyl phosphate synthetase I deficiency.

Precision medicine in rare disease: Mechanisms of disparate effects of N-carbamyl-l-glutamate on mutant CPS1 enzymes.

This study documents the disparate therapeutic effect of N-carbamyl-l-glutamate (NCG) in the activation of two different disease-causing mutants of carbamyl phosphate synthetase 1 (CPS1). We investigated the effects of NCG on purified recombinant wild-type (WT) mouse CPS1 and its human corresponding E1034G (increased ureagenesis on NCG) and M792I (decreased ureagenesis on NCG) mutants. NCG activates WT CPS1 sub-optimally compared to NAG. Similar to NAG, NCG, in combination with MgATP, stabilizes the enzyme, but competes with NAG binding to the enzyme. NCG supplementation activates available E1034G mutant CPS1 molecules not bound to NAG enhancing ureagenesis. Conversely, NCG competes with NAG binding to the scarce M792I mutant enzyme further decreasing residual ureagenesis. These results correlate with the respective patient's response to NCG. Particular caution should be taken in the administration of NCG to patients with hyperammonemia before their molecular bases of their urea cycle disorders is known.

Carbamoylphosphate synthetase 1 (CPS1) deficiency: clinical, biochemical, and molecular characterization in Malaysian patients.

UNLABELLED: Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a rare autosomal recessive disorder of ureagenesis presenting as life-threatening hyperammonemia. In this study, we present the main clinical features and biochemical and molecular data of six Malaysian patients with CPS1 deficiency. All the patients have neonatal-onset symptoms, initially diagnosed as infections before hyperammonemia was recognized. They have typical biochemical findings of hyperglutaminemia, hypocitrullinemia, and low to normal urinary excretion of orotate. One neonate succumbed to the first hyperammonemic decompensation. Five neonatal survivors received long-term treatment consisting of dietary protein restriction and ammonia-scavenging drugs. They have delayed neurocognitive development of varying severity. Genetic analysis revealed eight mutations in CPS1 gene, five of which were not previously reported. Five mutations were missense changes while another three were predicted to create premature stop codons. In silico analyses showed that these new mutations affected different CPS1 enzyme domains and were predicted to interrupt interactions at enzyme active sites, disturb local enzyme conformation, and destabilize assembly of intact enzyme complex. CONCLUSION: All mutations are private except one mutation; p.Ile1254Phe was found in three unrelated families. Identification of a recurrent p.Ile1254Phe mutation suggests the presence of a common and unique mutation in our population. Our study also expands the mutational spectrum of the CPS1 gene.

Loss of Hep Par 1 immunoreactivity in the livers of patients with carbamoyl phosphate synthetase 1 deficiency.

The hepatocyte paraffin 1 (Hep Par 1) antibody is widely used as a hepatocyte marker, recognizing carbamoyl phosphate synthetase 1 (CPS1), an essential component of the urea cycle. Various missense, nonsense, and frameshift mutations occur in the CPS1 gene. In neonatal patients with homozygous CPS1 deficiency (CPS1D), urea cycle defects with resulting severe hyperammonemia can be fatal, though liver transplantation provides a complete cure for CPS1D. We performed Hep Par 1 immunostaining in the explanted livers of 10 liver transplant patients with CPS1D. Seven were negative for Hep Par 1 in the hepatocytes and the other three showed normal diffuse granular cytoplasmic staining. As expected, all three Hep Par 1-positive patients had at least one missense mutation, and all four patients who had only nonsense or frameshift mutations were Hep Par 1-negative. The other three patients were unexpectedly negative for Hep Par 1, even though each had one missense mutation. These results suggest that CPS1D can be related to the loss of Hep Par 1 reactivity due to the loss of protein production, a one amino acid substitution resulting in an abortive protein product, or both. Hep Par 1 immunohistochemistry can be used as a simple method to confirm CPS1D.

Recurrence of carbamoyl phosphate synthetase 1 (CPS1) deficiency in Turkish patients: characterization of a founder mutation by use of recombinant CPS1 from insect cells expression.

Carbamoyl phosphate synthetase 1 (CPS1) deficiency due to CPS1 mutations is a rare autosomal-recessive urea cycle disorder causing hyperammonemia that can lead to death or severe neurological impairment. CPS1 catalyzes carbamoyl phosphate formation from ammonia, bicarbonate and two molecules of ATP, and requires the allosteric activator N-acetyl-L-glutamate. Clinical mutations occur in the entire CPS1 coding region, but mainly in single families, with little recurrence. We characterized here the only currently known recurrent CPS1 mutation, p.Val1013del, found in eleven unrelated patients of Turkish descent using recombinant His-tagged wild type or mutant CPS1 expressed in baculovirus/insect cell system. The global CPS1 reaction and the ATPase and ATP synthesis partial reactions that reflect, respectively, the bicarbonate and the carbamate phosphorylation steps, were assayed. We found that CPS1 wild type and V1013del mutant showed comparable expression levels and purity but the mutant CPS1 exhibited no significant residual activities. In the CPS1 structural model, V1013 belongs to a highly hydrophobic ß-strand at the middle of the central ß-sheet of the A subdomain of the carbamate phosphorylation domain and is close to the predicted carbamate tunnel that links both phosphorylation sites. Haplotype studies suggested that p.Val1013del is a founder mutation. In conclusion, the mutation p.V1013del inactivates CPS1 but does not render the enzyme grossly unstable or insoluble. Recurrence of this particular mutation in Turkish patients is likely due to a founder effect, which is consistent with the frequent consanguinity observed in the affected population.

Understanding carbamoyl phosphate synthetase (CPS1) deficiency by using the recombinantly purified human enzyme: effects of CPS1 mutations that concentrate in a central domain of unknown function.

Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is an inborn error of the urea cycle that is due to mutations in the CPS1 gene. In the first large repertory of mutations found in CPS1D, a small CPS1 domain of unknown function (called the UFSD) was found to host missense changes with high frequency, despite the fact that this domain does not host substrate-binding or catalytic machinery. We investigate here by in vitro expression studies using baculovirus/insect cells the reasons for the prominence of the UFSD in CPS1D, as well as the disease-causing roles and pathogenic mechanisms of the mutations affecting this domain. All but three of the 18 missense changes found thus far mapping in this domain in CPS1D patients drastically decreased the yield of pure CPS1, mainly because of decreased enzyme solubility, strongly suggesting misfolding as a major determinant of the mutations negative effects. In addition, the majority of the mutations also decreased from modestly to very drastically the specific activity of the fraction of the enzyme that remained soluble and that could be purified, apparently because they decreased V(max). Substantial although not dramatic increases in K(m) values for the substrates or for N-acetyl-L-glutamate were observed for only five mutations. Similarly, important thermal stability decreases were observed for three mutations. The results indicate a disease-causing role for all the mutations, due in most cases to the combined effects of the low enzyme level and the decreased activity. Our data strongly support the value of the present expression system for ascertaining the disease-causing potential of CPS1 mutations, provided that the CPS1 yield is monitored. The observed effects of the mutations have been rationalized on the basis of an existing structural model of CPS1. This model shows that the UFSD, which is in the middle of the 1462-residue multidomain CPS1 protein, plays a key integrating role for creating the CPS1 multidomain architecture leading us to propose here a denomination of "Integrating Domain" for this CPS1 region. The majority of these 18 mutations distort the interaction of this domain with other CPS1 domains, in many cases by causing improper folding of structural elements of the Integrating Domain that play key roles in these interactions.