Sleeping Beauty mRNA-LNP enables stable rAAV transgene expression in mouse and NHP hepatocytes and improves vector potency.

Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.

Severity-adjusted evaluation of liver transplantation on health outcomes in urea cycle disorders.

PURPOSE: Liver transplantation (LTx) is performed in individuals with urea cycle disorders when medical management (MM) insufficiently prevents the occurrence of hyperammonemic events. However, there is a paucity of systematic analyses on the effects of LTx on health-related outcome parameters compared to individuals with comparable severity who are medically managed. METHODS: We investigated the effects of LTx and MM on validated health-related outcome parameters, including the metabolic disease course, linear growth, and neurocognitive outcomes. Individuals were stratified into "severe" and "attenuated" categories based on the genotype-specific and validated in vitro enzyme activity. RESULTS: LTx enabled metabolic stability by prevention of further hyperammonemic events after transplantation and was associated with a more favorable growth outcome compared with individuals remaining under MM. However, neurocognitive outcome in individuals with LTx did not differ from the medically managed counterparts as reflected by the frequency of motor abnormality and cognitive standard deviation score at last observation. CONCLUSION: Whereas LTx enabled metabolic stability without further need of protein restriction or nitrogen-scavenging therapy and was associated with a more favorable growth outcome, LTx-as currently performed-was not associated with improved neurocognitive outcomes compared with long-term MM in the investigated urea cycle disorders.

Gene therapy for urea cycle defects: An update from historical perspectives to future prospects.

Urea cycle defects (UCDs) are severe inherited metabolic diseases with high unmet needs which present a permanent risk of hyperammonaemic decompensation and subsequent acute death or neurological sequelae, when treated with conventional dietetic and medical therapies. Liver transplantation is currently the only curative option, but has the potential to be supplanted by highly effective gene therapy interventions without the attendant need for life-long immunosuppression or limitations imposed by donor liver supply. Over the last three decades, pioneering genetic technologies have been explored to circumvent the consequences of UCDs, improve quality of life and long-term outcomes: adenoviral vectors, adeno-associated viral vectors, gene editing, genome integration and non-viral technology with messenger RNA. In this review, we present a summarised view of this historical path, which includes some seminal milestones of the gene therapy's epic. We provide an update about the state of the art of gene therapy technologies for UCDs and the current advantages and pitfalls driving future directions for research and development.

Impact of citrulline substitution on clinical outcome after liver transplantation in carbamoyl phosphate synthetase 1 and ornithine transcarbamylase deficiency.

Carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamylase (OTC) deficiencies are rare urea cycle disorders, which can lead to life-threatening hyperammonemia. Liver transplantation (LT) provides a cure and offers an alternative to medical treatment and life-long dietary restrictions with permanent impending risk of hyperammonemia. Nevertheless, in most patients, metabolic aberrations persist after LT, especially low plasma citrulline levels, with questionable clinical impact. So far, little is known about these alterations and there is no consensus, whether l-citrulline substitution after LT improves patients' symptoms and outcomes. In this multicentre, retrospective, observational study of 24 patients who underwent LT for CPS1 (n = 11) or OTC (n = 13) deficiency, 25% did not receive l-citrulline or arginine substitution. Correlation analysis revealed no correlation between substitution dosage and citrulline levels (CPS1, p = 0.8 and OTC, p = 1). Arginine levels after liver transplantation were normal after LT independent of citrulline substitution. Native liver survival had no impact on mental impairment (p = 0.67). Regression analysis showed no correlation between l-citrulline substitution and failure to thrive (p = 0.611) or neurological outcome (p = 0.701). Peak ammonia had a significant effect on mental impairment (p = 0.017). Peak plasma ammonia levels correlate with mental impairment after LT in CPS1 and OTC deficiency. Growth and intellectual impairment after LT are not significantly associated with l-citrulline substitution.

Implications of Genetic Factors Underlying Mouse Hydronephrosis: Cautionary Considerations on Phenotypic Interpretation in Genetically Engineered Mice.

Hydronephrosis, the dilation of kidneys due to abnormal urine retention, occurs spontaneously in certain inbred mouse strains. In humans, its occurrence is often attributed to acquired urinary tract obstructions in adults, whereas in children, it can be congenital. However, the genetic factors underlying hydronephrosis pathogenesis remain unclear. We investigated the cause of hydronephrosis by analyzing tetraspanin 7 (Tspan7) gene-modified mice, which had shown a high incidence of hydronephrosis-like symptoms. We found that these mice were characterized by low liver weights relative to kidney weights and elevated blood ammonia levels, suggesting liver involvement in hydronephrosis. Gene expression analysis of the liver suggested that dysfunction of ornithine transcarbamylase (OTC), encoded by the X chromosome gene Otc and involved in the urea cycle, may contribute as a congenital factor in hydronephrosis. This OTC dysfunction may be caused by genomic mutations in X chromosome genes contiguous to Otc, such as Tspan7, or via the genomic manipulations used to generate transgenic mice, including the introduction of Cre recombinase DNA cassettes and cleavage of loxP by Cre recombinase. Therefore, caution should be exercised in interpreting the hydronephrosis phenotype observed in transgenic mice as solely a physiological function of the target gene.

Hypoosmosis alters hepatocyte mitochondrial morphology and induces selective release of carbamoyl phosphate synthetase 1.

Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury.NEW & NOTEWORTHY Exposure of isolated mouse livers, primary cultured hepatocytes, or mice to hypoosmosis/hyponatremia conditions induces significant mitochondrial shape alterations accompanied by preferential release of the mitochondrial matrix protein CPS1, a urea cycle enzyme. In contrast, the intermembrane space protein, cytochrome c, and two other matrix proteins, including the urea cycle enzyme ornithine transcarbamylase, remain preferentially retained in mitochondria. Therefore, hepatocyte CPS1 manifests unique mitochondrial stress response compartmentalization and is a sensitive sensor of mitochondrial hypoosmotic/hyponatremic injury.

Anesthesia management protocol for liver transplantation as treatment for ornithine transcarbamylase deficiency.

BACKGROUND: Ornithine transcarbamylase deficiency is an X-linked genetic disorder that induces accumulation of ammonia in the liver and is the most common urea cycle disorder. The clinical manifestation of ornithine transcarbamylase deficiency is hyperammonemia that causes irreversible neurological damage. Liver transplantation is a curative therapy for ornithine transcarbamylase deficiency. The aim of this study is to suggest, from our previous experience, an anesthesia management protocol of liver transplantation for ornithine transcarbamylase deficiency, particularly focused on liver transplantation for cases with uncontrolled hyperammonemia. METHOD: We retrospectively reviewed our anesthesia-related experience in all cases of liver transplantation for ornithine transcarbamylase deficiency in our center. RESULTS: Twenty-nine liver transplantation cases for ornithine transcarbamylase deficiency were found between November 2005 and March 2021 in our center. Of these, 25 cases were stable through the perioperative period. However, 2 cases with carrier donor graft had hyperammonemia after liver transplantation. Another two cases had uncontrolled hyperammonemia before liver transplantation, even with continuous hemodialysis. They underwent life-saving liver transplantation. Their metabolic status stabilized after the anhepatic phase. CONCLUSION: Liver transplantation for cases with uncontrolled hyperammonemia can be performed with proper management. Second, liver transplantation with carrier donors should be avoided because of the risk of postoperative recurrence.

Acute onset of ornithine transcarbamylase deficiency after total anomalous pulmonary venous connection repair to a 2-day-old neonate.

Ornithine transcarbamylase deficiency is an X-linked disorder which results in the accumulation of ammonia causing irritability and vomiting. Acute hyperammonemia requires rapid and intensive intervention. However, as those clinical features are non-specific and commonly seen in peri-operative situation, ornithine transcarbamylase deficiency could be difficult to diagnose prior to and post-emergency cardiac surgery. We report a 2-day-old male neonate who was diagnosed with ornithine transcarbamylase deficiency presenting hyperammonemia and severe heart failure after total anomalous pulmonary venous connection repair.

Treatment and management for children with urea cycle disorder in chronic stage. / å°¿ç´ å¾ªçŽ¯éšœç¢æ‚£å„¿æ ¢æ€§æœŸæ²»ç–—å’Œç®¡ç†.

Urea cycle disorder (UCD) is a group of inherited metabolic diseases with high disability or fatality rate, which need long-term drug treatment and diet management. Except those with Citrin deficiency or liver transplantation, all pediatric patients require lifelong low protein diet with safe levels of protein intake and adequate energy and lipids supply for their corresponding age; supplementing essential amino acids and protein-free milk are also needed if necessary. The drugs for long-term use include nitrogen scavengers (sodium benzoate, sodium phenylbutyrate, glycerol phenylbutyrate), urea cycle activation/substrate supplementation agents (N-carbamylglutamate, arginine, citrulline), etc. Liver transplantation is recommended for pediatric patients not responding to standard diet and drug treatment, and those with severe progressive liver disease and/or recurrent metabolic decompensations. Gene therapy, stem cell therapy, enzyme therapy and other novel technologies may offer options for treatment in UCD patients. The regular biochemical assessments like blood ammonia, liver function and plasma amino acid profile are needed, and physical growth, intellectual development, nutritional intake should be also evaluated for adjusting treatment in time.

[Neonate-onset ornithine transcarbamylase deficiency]. / æ–°ç”Ÿå„¿é¸Ÿæ°¨é ¸æ°¨ç”²é °åŸºè½¬ç§»é ¶ç¼ºä¹ç—‡.

The male neonate in this case study was admitted to the hospital at 15 hours of age due to respiratory distress for 15 hours and poor response for 3 hours after resuscitation from asphyxia. The neonate was highly unresponsive, with central respiratory failure and seizures. Serum ammonia was elevated (>1 000 µmol/L). Blood tandem mass spectrometry revealed a significant decrease in citrulline. Rapid familial whole genome sequencing revealed OTC gene mutations inherited from the mother. Continuous hemodialysis filtration and other treatments were given. Neurological assessment was performed by cranial magnetic resonance imaging and electroencephalogram. The neonate was diagnosed with ornithine transcarbamylase deficiency combined with brain injury. He died at 6 days of age after withdrawing care. This article focuses on the differential diagnosis of neonatal hyperammonemia and introduces the multidisciplinary management of inborn error of metabolism.

A simple screening method for heterozygous female patients with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD), caused by X-linked OTC mutations, is characterized by life-threatening hyperammonemia. Heterozygous female patients are often asymptomatic and usually have milder disease than affected male patients, but can have higher morbidity and mortality rates if the disease progresses prior to diagnosis. Our purpose was to establish a screening method for female heterozygotes with OTCD. We retrospectively identified female patients who underwent plasma amino acid analysis at the National Center for Child Health and Development, using data from electronic medical records from March 2002 to September 2021. We extracted patient age, medical history, and biochemical data, including plasma amino acid levels. Patients were categorized into several groups according to their underlying diseases; those with underlying diseases that could potentially affect plasma amino acid levels, such as mitochondrial disease or short bowel syndrome, were excluded, except for untreated OTCD. Biochemical values were compared between OTCD patients and others using the Mann-Whitney U test. The receiver operator characteristic analysis was performed to assess the diagnostic capability for detecting OTCD in each subject. For patients with multiple test data, the most recent of the measurement dates was used in the analysis. The data sets of 976 patients were included. There were significant differences in values of glutamine, citrulline, arginine, and ammonia, but the diagnostic capacity of each alone was inadequate. By contrast, the (glutamine + glycine)/(citrulline + arginine) ratio was appropriate for discriminating heterozygous female patients with OTCD, with a sensitivity of 100% and specificity of 98.6% when the cutoff level was 15.8; the AUC for this discrimination was 0.996 (95% confidence interval, 0.992 to 1.000). These findings could help identify heterozygous female patients with OTCD before the onset of clinical disease.

Moving towards a novel therapeutic strategy for hyperammonemia that targets glutamine metabolism.

Patients with urea cycle disorders intermittently develop episodes of decompensation with hyperammonemia. Although such an episode is often associated with starvation and catabolism, its molecular basis is not fully understood. First, we attempted to elucidate the mechanism of such starvation-associated hyperammonemia. Using a mouse embryonic fibroblast (MEF) culture system, we found that glucose starvation increases ammonia production, and that this increase is associated with enhanced glutaminolysis. These results led us to focus on α-ketoglutarate (AKG), a glutamate dehydrogenase inhibitor, and a major anaplerotic metabolite. Hence, we sought to determine the effect of dimethyl α-ketoglutarate (DKG), a cell-permeable AKG analog, on MEFs and found that DKG mitigates ammonia production primarily by reducing flux through glutamate dehydrogenase. We also verified that DKG reduces ammonia in an NH4 Cl-challenged hyperammonemia mouse model and observed that DKG administration reduces plasma ammonia concentration to 22.8% of the mean value for control mice that received only NH4 Cl. In addition, we detected increases in ornithine concentration and in the ratio of ornithine to arginine following DKG treatment. We subsequently administered DKG intravenously to a newborn pig with hyperammonemia due to ornithine transcarbamylase deficiency and found that blood ammonia concentration declined significantly over time. We determined that this effect is associated with facilitated reductive amination and glutamine synthesis. Our present data indicate that energy starvation triggers hyperammonemia through enhanced glutaminolysis and that DKG reduces ammonia accumulation via pleiotropic mechanisms both in vitro and in vivo. Thus, cell-permeable forms of AKG are feasible candidates for a novel hyperammonemia treatment.

Corticosteroid suppresses urea-cycle-related gene expressions in ornithine transcarbamylase deficiency.

BACKGROUND: Ornithine transcarbamylase deficiency (OTCD) is most common among urea cycle disorders (UCDs), defined by defects in enzymes associated with ureagenesis. Corticosteroid administration to UCD patients, including OTCD patients, is suggested to be avoided, as it may induce life-threatening hyperammonemia. The mechanism has been considered nitrogen overload due to the catabolic effect of corticosteroids; however, the pathophysiological process is unclear. METHODS: To elucidate the mechanism of hyperammonemia induced by corticosteroid administration in OTCD patients, we analyzed a mouse model by administering corticosteroids to OTCspf-ash mice deficient in the OTC gene. Dexamethasone (DEX; 20 mg/kg) was administered to the OTCspf-ash and wild-type (WT) mice at 0 and 24 h, and the serum ammonia concentrations, the levels of the hepatic metabolites, and the gene expressions related with ammonia metabolism in the livers and muscles were analyzed. RESULTS: The ammonia levels in Otcspf-ash mice that were administered DEX tended to increase at 24 h and increased significantly at 48 h. The metabolomic analysis showed that the levels of citrulline, arginine, and ornithine did not differ significantly between Otcspf-ash mice that were administered DEX and normal saline; however, the level of aspartate was increased drastically in Otcspf-ash mice owing to DEX administration (P < 0.01). Among the enzymes associated with the urea cycle, mRNA expressions of carbamoyl-phosphate synthase 1, ornithine transcarbamylase, arginosuccinate synthase 1, and arginosuccinate lyase in the livers were significantly downregulated by DEX administration in both the Otcspf-ash and WT mice (P < 0.01). Among the enzymes associated with catabolism, mRNA expression of Muscle RING-finger protein-1 in the muscles was significantly upregulated in the muscles of WT mice by DEX administration (P < 0.05). CONCLUSIONS: We elucidated that corticosteroid administration induced hyperammonemia in Otcspf-ash mice by not only muscle catabolism but also suppressing urea-cycle-related gene expressions. Since the urea cycle intermediate amino acids, such as arginine, might not be effective because of the suppressed expression of urea-cycle-related genes by corticosteroid administration, we should consider an early intervention by renal replacement therapy in cases of UCD patients induced by corticosteroids to avoid brain injuries or fatal outcomes.

Placental Pathology in Maternal Ornithine Transcarbamylase Deficiency.

INTRODUCTION: Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder, inherited in an X-linked manner. Males are severely affected. Female phenotypes vary from asymptomatic to severe, and symptoms may be triggered by high metabolic states like childbirth. Literature on OTC deficiency in pregnancy and placental pathology is limited. METHODS: Pathology records were searched at a single referral center from 2000-2020 and identified three placental cases from two mothers heterozygous for OTC deficiency. Placental pathology and maternal and neonatal history were reviewed in detail. RESULTS: The placenta from one symptomatic mother carrying an affected male fetus showed widespread high-grade fetal vascular malperfusion (FVM) lesions of varying age. These lesions were not seen in the two placentas from the asymptomatic mother. DISCUSSION: In cases of symptomatic maternal OTC deficiency, our findings highlight the need for placental examination. Since thrombotic events in the placenta have the potential to associate with fetal and neonatal endothelial damage, a high index of suspicion for neonatal thrombosis may be warranted.

Identification of rare variants causing urea cycle disorders: A clinical, genetic, and biophysical study.

Urea cycle disorders (UCDs) are a group of rare metabolic conditions characterized by hyperammonemia and a broad spectrum of phenotypic severity. They are caused by the congenital deficiency in the eight biomolecules involved in urea cycle. In the present study, five cases of UCD were recruited and submitted to a series of clinical, biochemical, and genetic analysis with a combination of high throughput techniques. Moreover, in silico analysis was conducted on the identified missense genetic variants. Various clinical and biochemical indications (including profiles of amino acids and urinary orotic acids) of UCD were manifested by the five probands. Sequence analysis revealed nine diagnostic variants, including three novel ones, which caused Argininosuccinic aciduria (ASA) in one case, Carbamoyl phosphate synthetase 1deficiency (CPS1D) in two cases, Ornithine transcarbamylase deficiency (OTCD) in one case, and Citrin deficiency in 1case. Results of in silico biophysical analysis strongly suggested the pathogenicity of each the five missense variants and provided insight into their intramolecular impacts. In conclusion, this study expanded the genetic variation spectrum of UCD, gave solid evidence for counselling to the affected families, and should facilitate the functional study on the proteins in urea cycle.

Variants associated with urea cycle disorders in Japanese patients: Nationwide study and literature review.

Urea cycle disorders (UCDs) are inherited metabolic diseases that lead to hyperammonemia with variable clinical manifestations. Using data from a nationwide study, we investigated the onset time, gene variants, clinical manifestations, and treatment of patients with UCDs in Japan. Of the 229 patients with UCDs diagnosed and/or treated between January 2000 and March 2018, identified gene variants and clinical information were available for 102 patients, including 62 patients with ornithine transcarbamylase (OTC) deficiency, 18 patients with carbamoyl phosphate synthetase 1 (CPS1) deficiency, 16 patients with argininosuccinate synthetase (ASS) deficiency, and 6 patients with argininosuccinate lyase (ASL) deficiency. A total of 13, 10, 4, and 5 variants in the OTC, CPS1, ASS, and ASL genes were respectively identified as novel variants, which were neither registered in ClinVar databases nor previously reported. The onset time and severity in patients with UCD could be predicted based on the identified gene variants in each patient from this nationwide study and previous studies. This genetic information may help in predicting the long-term outcome and determining specific treatment strategies such as liver transplantation in patients with UCDs.

Acute liver dysfunction with delayed peak of serum aminotransferase levels as a presentation of ornithine transcarbamylase deficiency in females.

We describe 10 females with ornithine transcarbamylase (OTC) deficiency and liver dysfunction, revealing a unique pattern of hepatocyte injury in which initial hyperammonemia and coagulopathy is followed by a delayed peak in aminotransferase levels. None of the patients required urgent liver transplantation, though five eventually underwent transplant for recurrent metabolic crises. We intend that this novel observation will initiate further investigations into the pathophysiology of liver dysfunction in OTC-deficient patients, and ultimately lead to the development of therapies and prevent the need for liver transplant.

OTC deficiency in females: Phenotype-genotype correlation based on a 130-family cohort.

OTC deficiency, an inherited urea cycle disorder, is caused by mutations in the X-linked OTC gene. Phenotype-genotype correlations are well understood in males but still poorly known in females. Taking advantage of a cohort of 130 families (289 females), we assessed the relative contribution of OTC enzyme activity, X chromosome inactivation, and OTC gene sequencing to genetic counseling in heterozygous females. Twenty two percent of the heterozygous females were clinically affected, with episodic (11%), chronic (7.5%), or neonatal forms of the disease (3.5%). Overall mortality rate was 4%. OTC activity, ranging from 0% to 60%, did not correlate with phenotype at the individual level. Analysis of multiple samples from 4 mutant livers showed intra-hepatic variability of OTC activity and X inactivation profile (range of variability: 30% and 20%, respectively) without correlation between both parameters for 3 of the 4 livers. Ninety disease-causing variants were found, 27 of which were novel. Mutations were classified as "mild" or "severe," based on male phenotypes and/or in silico prediction. In our cohort, a serious disease occurred in 32% of females with a severe mutation, compared to 4% in females with a mild mutation (odds ratio = 1.365; P = 1.6e-06). These data should help prenatal diagnosis for heterozygous females and genetic counseling after fortuitous findings of OTC variants in pangenomic sequencing.

The role of orotic acid measurement in routine newborn screening for urea cycle disorders.

Urea cycle disorders (UCDs), including OTC deficiency (OTCD), are life-threatening diseases with a broad clinical spectrum. Early diagnosis and initiation of treatment based on a newborn screening (NBS) test for OTCD with high specificity and sensitivity may contribute to reduction of the significant complications and high mortality. The efficacy of incorporating orotic acid determination into routine NBS was evaluated. Combined measurement of orotic acid and citrulline in archived dried blood spots from newborns with urea cycle disorders and normal controls was used to develop an algorithm for routine NBS for OTCD in Israel. Clinical information and genetic confirmation results were obtained from the follow-up care providers. About 1147986 newborns underwent routine NBS including orotic acid determination, 25 of whom were ultimately diagnosed with a UCD. Of 11 newborns with OTCD, orotate was elevated in seven but normal in two males with early-onset and two males with late-onset disease. Orotate was also elevated in archived dried blood spots of all seven retrospectively tested historical OTCD patients, only three of whom had originally been identified by NBS with low citrulline and elevated glutamine. Among the other UCDs emerge, three CPS1D cases and additional three retrospective CPS1D cases otherwise reported as a very rare condition. Combined levels of orotic acid and citrulline in routine NBS can enhance the detection of UCD, especially increasing the screening sensitivity for OTCD and differentiate it from CPS1D. Our data and the negligible extra cost for orotic acid determination might contribute to the discussion on screening for proximal UCDs in routine NBS.

Humanized liver mouse model with transplanted human hepatocytes from patients with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD) is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. To develop new drugs or therapies for OTCD, it is ideal to use models that are more closely related to human metabolism and pathology. Primary human hepatocytes (HHs) isolated from two patients (a 6-month-old boy and a 5-year-old girl) and a healthy donor were transplanted into host mice (hemi-, hetero-OTCD mice, and control mice, respectively). HHs were isolated from these mice and used for serial transplantation into the next host mouse or for in vitro experiments. Histological, biochemical, and enzyme activity analyses were performed. Cultured HHs were treated with ammonium chloride or therapeutic drugs. Replacement rates exceeded 80% after serial transplantation in both OTCD mice. These highly humanized OTCD mice showed characteristics similar to OTCD patients that included increased blood ammonia levels and urine orotic acid levels enhanced by allopurinol. Hemi-OTCD mice showed defects in OTC expression and significantly low enzymatic activities, while hetero-OTCD mice showed residual OTC expression and activities. A reduction in ammonium metabolism was observed in cultured HHs from OTCD mice, and treatment with the therapeutic drug reduced the ammonia levels in the culture medium. In conclusion, we established in vivo OTC mouse models with hemi- and hetero-patient HHs. HHs isolated from the mice were useful as an in vitro model of OTCD. These OTC models could be a source of valuable patient-derived hepatocytes that would enable large scale and reproducible experiments using the same donor.