Ornithine Transcarbamylase Research Articles

Sericin enhances ammonia detoxification by promotes urea cycle enzyme genes and activates hepatic autophagy in relation to CARD-9/MAPK pathway

Urea cycle is an important metabolic process that initiates in liver mitochondria and converts ammonia to urea. The impairment of ammonia detoxification, both primary and secondary causes, lead to hyperammonemia, a life-threatening condition affecting to the brain. Current treatments are not enough effective. In addition, our recent proteomics study in hypercholesterolemic rat model demonstrated that sericin enhances hepatic nitrogenous waste removal through carbamoyl-phosphate synthase 1 (CPS-1), aldehyde dehydrogenase-2 (ALDH-2), and uricase proteins. However, the underlining mechanisms regard to this property is not clarified yet. Therefore, the present study aims to examine the effect of sericin on urea cycle enzyme genes (CPS-1 and ornithine transcarbamylase; OTC) and proteins (mitogen-activated protein kinase; MAPK, caspase recruitment domain-containing protein 9; CARD-9, Microtubule-associated protein light chain 3; LC-3), which relate to urea production and liver homeostasis in hepatic cell line (HepG2) and hypercholesterolemic rat treated with or without sericin. qRT-PCR, immunohistochemistry, and electron microscopy techniques were performed. In vitro study determined that high dose of sericin at 1 mg/ml increased liver detoxification enzyme (Cytochrome P450 1A2; CYP1A2 and ALDH-2) and urea cycle enzyme (CPS-1 and OTC) genes. Both in HepG2 cell and rat liver mitochondria, sericin significantly downregulated CARD-9 (apoptotic protein) expression while upregulated MAPK (hepatic homeostasis protein) and LC-3 (autophagic protein) expressions. Hence, it might be concluded that sericin promotes ammonia detoxification by both increases urea cycle enzyme genes and enhances hepatic autophagy in associated with CARD-9/MAPK pathway (as shown by their own negative relationship). This study presents another beneficial property of sericin to develop an upcoming candidate for ammonia toxicity alleviation and liver function improvement.

IPSC-derived hepatocytes as a novel tool for Ornithine Transcarbamylase Deficiency (OTCD) modelling and drug screening

iPSC-derived hepatocytes as a novel tool for Ornithine Transcarbamylase Deficiency (OTCD) modelling and drug screening

Hydroquinine Inhibits the Growth of Multidrug-Resistant Pseudomonas aeruginosa via the Suppression of the Arginine Deiminase Pathway Genes.

Hydroquinine has antimicrobial potential with demonstrated activity against several bacteria, including multidrug-resistant (MDR) P. aeruginosa reference strains. Despite this, there is limited evidence confirming the antibacterial activity of hydroquinine against clinical isolates and the underlying mechanism of action. Here, we aimed to investigate the antibacterial effect of hydroquinine in clinical P. aeruginosa strains using phenotypic antimicrobial susceptibility testing and synergistic testing. In addition, we examined the potential inhibitory mechanisms against MDR P. aeruginosa isolates using informatic-driven molecular docking analysis in combination with RT-qPCR. We uncovered that hydroquinine inhibits and kills clinical P. aeruginosa at 2.50 mg/mL (MIC) and 5.00 mg/mL (MBC), respectively. Hydroquinine also showed partial synergistic effects with ceftazidime against clinical MDR P. aeruginosa strains. Using SwissDock, we identified potential interactions between arginine deiminase (ADI)-pathway-related proteins and hydroquinine. Furthermore, using RT-qPCR, we found that hydroquinine directly affects the mRNA expression of arc operon. We demonstrated that the ADI-related genes, including the arginine/ornithine antiporter (arcD) and the three enzymes (arginine deiminase (arcA), ornithine transcarbamylase (arcB), and carbamate kinase (arcC)), were significantly downregulated at a half MIC of hydroquinine. This study is the first report that the ADI-related proteins are potential molecular targets for the inhibitory effect of hydroquinine against clinically isolated MDR P. aeruginosa strains.

Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies.

Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.

Exploring rat corpus cavernosum alterations induced by finasteride treatment and withdrawal.

Despite its efficacy for treating androgenetic alopecia, finasteride, an inhibitor of 5α-reductase (i.e., the enzyme converting testosterone, T, into dihydrotestosterone, DHT), is associated with several side effects including sexual dysfunction (e.g., erectile dysfunction). These side effects may persist after drug suspension, inducing the so-called post-finasteride syndrome (PFS). The effects of subchronic treatment with finasteride (i.e., 20 days) and its withdrawal (i.e., 1 month) in rat corpus cavernosum have been explored here. Data obtained show that the treatment was able to decrease the levels of the enzyme 5α-reductase type II in the rat corpus cavernosum with increased T and decreased DHT levels. This local change in T metabolism was linked to mechanisms associated with erectile dysfunction. Indeed, by targeted metabolomics, we reported a decrease in the nitric oxide synthase (NOS) activity, measured by the citrulline/arginine ratio and confirmed by the decrease in NO2 levels, and a decrease in ornithine transcarbamylase (OTC) activity, measured by citrulline/ornithine ratio. Interestingly, the T levels are negatively correlated with NOS activity, while those of DHT are positively correlated with OTC activity. Finasteride treatment also induced alterations in the levels of other molecules involved in the control of penile erection, such as norepinephrine and its metabolite, epinephrine. Indeed, plasma levels of norepinephrine and epinephrine were significantly increased and decreased, respectively, suggesting an impairment of these mediators. Interestingly, these modifications were restored by suspension of the drug. Altogether, the results reported here indicate that finasteride treatment, but not its withdrawal, affects T metabolism in the rat corpus cavernosum, and this alteration was linked to mechanisms associated with erectile dysfunction. Data here reported could also suggest that the PFS sexual side effects are more related to dysfunction in a sexual central control rather than peripheral compromised condition.

AAV-mediated gene therapy for rare metabolic disorders: turning a promise into a reality

Gene therapy is emerging as the realistic treatment option for inborn errors of metabolism (IEMs) and, with the promising safety and efficacy evidence from the proof-of-concept studies, adeno-associated virus (AAV) has become the frontrunner among viral vector candidates for these monogenic disorders. Different AAV capsids exhibit specific tissue tropisms, which can considerably increase the efficiency of gene transfer to particular organs. Here, we will discuss two distinct diseases: ornithine transcarbamylase (OTC) deficiency and Niemann–Pick disease type C, in which significant advances have been achieved in AAV-based gene therapy trials.

Diagnostic and Management Issues in Patients with Late-Onset Ornithine Transcarbamylase Deficiency.

Ornithine transcarbamylase deficiency (OTCD) is the most common inherited disorder of the urea cycle and, in general, is transmitted as an X-linked recessive trait. Defects in the OTC gene cause an impairment in ureagenesis, resulting in hyperammonemia, which is a direct cause of brain damage and death. Patients with late-onset OTCD can develop symptoms from infancy to later childhood, adolescence or adulthood. Clinical manifestations of adults with OTCD vary in acuity. Clinical symptoms can be aggravated by metabolic stressors or the presence of a catabolic state, or due to increased demands upon the urea. A prompt diagnosis and relevant biochemical and genetic investigations allow the rapid introduction of the right treatment and prevent long-term complications and mortality. This narrative review outlines challenges in diagnosing and managing patients with late-onset OTCD.

Genetic Therapy Approaches for Ornithine Transcarbamylase Deficiency.

Ornithine transcarbamylase deficiency (OTCD) is the most common urea cycle disorder with high unmet needs, as current dietary and medical treatments may not be sufficient to prevent hyperammonemic episodes, which can cause death or neurological sequelae. To date, liver transplantation is the only curative choice but is not widely available due to donor shortage, the need for life-long immunosuppression and technical challenges. A field of research that has shown a great deal of promise recently is gene therapy, and OTCD has been an essential candidate for different gene therapy modalities, including AAV gene addition, mRNA therapy and genome editing. This review will first summarise the main steps towards clinical translation, highlighting the benefits and challenges of each gene therapy approach, then focus on current clinical trials and finally outline future directions for the development of gene therapy for OTCD.

China nationwide landscape of 16 types inherited metabolic disorders: a retrospective analysis on 372,255 clinical cases

BackgroundInherited metabolic disorders (IMDs) usually occurs at young age and hence it severely threatening the health and life of young people. While so far there lacks a comprehensive study which can reveals China’s nationwide landscape of IMDs. This study aimed to evaluate IMDs incidence and regional distributions in China at a national and province level to guide clinicians and policy makers.MethodsThe retrospective study conducted from January 2012 to March 2021, we analyzed and characterized 372255 cases’ clinical test information and diagnostic data from KingMed Diagnostics Laboratory. The samples were from 32 provincial regions of China, the urine organic acids were detected by gas chromatography-mass spectrometry (GC–MS), amino acids and acylcarnitines in dried blood spots were detected by liquid chromatography-tandem mass spectrometry (LC–MS/MS). We did a statistical analysis of the distribution of the 16 most common IMDs in amino acid disorders and organic acidemias, and then paid special attention to analyze the age and regional distributions of different IMDs. The statistical analyses and visualization analysis were performed with the programming language R (version 4.2.1).ResultsThere were 4911 positive cases diagnosed, which was 1.32% of the total sample during the ten-year study period. Most diseases tended to occur at ages younger than 18 year-old. The Ornithine Transcarbamylase Deficiency tended to progress on male infants who were less than 28 days old. While the peak of the positive case number of Citrin Deficiency disease (CD) was at 1–6 months. Different IMDs’ had different distribution patterns in China’s provinces. Methylmalonic Acidemias and Hyperphenylalaninemia had an imbalanced distribution pattern in China and its positive rate was significantly higher in North China than South China. Conversely, the positive rate of CD was significantly higher in South China than North China.ConclusionsResults of this work, such as the differences in distribution pattern of different diseases in terms of age, region, etc. provide important insights and references for clinicians, researchers and healthcare policy makers. The policy makers could optimize the better health screening programs for covering children and infants in specific ages and regions based on our findings.

Effect of 28-day administration of Psoraleae Fructus water extract on early liver injury in rats

This study aims to investigate the hepatotoxicity of Psoraleae Fructus water extract and the underlying mechanism in rats. Forty-eight rats were randomly assigned into four groups: a blank group and low-(BZGL, 6.25 g·kg~(-1)), medium-(BGZM, 12.5 g·kg~(-1)), and high-dose(BGZH, 25 g·kg~(-1)) Psoraleae Fructus water extract groups. The rats were treated for 28 days, and toxicity and mortality were observed daily. After 28 days, the rats were sacrificed, and the body weight, liver index, and liver-to-brain ratio were calculated. The morphological changes in the liver tissue were observed, and the serum levels of related biochemical indicators were measured. The results showed that compared with the blank group, Psoraleae Fructus water extracts of different doses decreased the body weight, increased the liver index and liver-to-brain ratio, and caused liver hypertrophy and pathological changes. Pathological examination revealed that the rats in Psoraleae Fructus water extract groups had bile duct hyperplasia, inflammatory cell infiltration, and liver cell fibrosis. Compared with the blank group, BGZL elevated the levels of alanine transaminase(ALT), α-glutathione S-transferase(α-GST), and total bile acid(TBA)(P<0.05), and BGZM and BGZH elevated the levels of ALT, TBA, α-GST, γ-glutamyl transferase(γ-GT), purine nucleoside phosphorylase(PNP), ornithine carbamoyltransferase(OCT), and arginase(ArgI)(P<0.05). Compared with the blank group, Psoraleae Fructus water extracts of different doses down-regulated the mRNA and protein levels of bile salt export pump(BSEP) and farnesoid X receptor(FXR) and up-regulated the mRNA and protein levels of tumor necrosis factor-α(TNF-α), nuclear factor kappaB(NF-κB), and cholesterol 7 alpha-hydroxylase(CYP7A1)(P<0.05). The results suggested that Psoraleae Fructus water extract caused toxicity in rats, showing a dose-toxicity relationship. Psoraleae Fructus water extract may cause liver damage, which may be due to its effect on liver bile acid secretion and induction of inflammation.

Phenotypes of undiagnosed adults with actionable OTC and GLA variants

Inherited metabolic disorders (IMDs) are variably expressive, complicating identification of affected individuals. A genotype-first approach can identify individuals at risk for morbidity and mortality from undiagnosed IMDs and can lead to protocols that improve clinical detection, counseling, and management. Using data from 57,340 participants in two hospital biobanks, we assessed the frequency and phenotypes of individuals with pathogenic/likely pathogenic variants (PLPVs) in two IMD genes: GLA, associated with Fabry disease, and OTC, associated with ornithine transcarbamylase deficiency. Approximately 1 in 19,100 participants harbored an undiagnosed PLPV in GLA or OTC. We identified three individuals (2 male, 1 female) with PLPVs in GLA, all of whom were undiagnosed, and three individuals (3 female) with PLPVs in OTC, two of whom were undiagnosed. All three individuals with PLPVs in GLA (100%) had symptoms suggestive of mild Fabry disease, and one individual (14.2%) had an ischemic stroke at age 33, likely indicating the presence of classic disease. No individuals with PLPVs in OTC had documented hyperammonemia despite exposure to catabolic states, but all (100%) had chronic symptoms suggestive of attenuated disease, including mood disorders and migraines. Our findings suggest that GLA and OTC variants identified via a genotype-first approach are of high penetrance and that population screening of these genes can be used to facilitate stepwise phenotyping and appropriate care.

Expression of enzymes involved in the urea cycle and muscle and mammary gland development of Holstein × Gyr heifers in a rotational grazing system supplemented with increasing protein levels

Studies evaluating the crude protein (CP) supplementation strategies across the year for grazing cattle and its association with the enzymes involved in the urea cycle and muscle and mammary gland developments are scarce. Thus, we aimed to evaluate the effect of supplementation with different levels of CP on the expression of genes involved in the urea cycle and muscle and mammary gland development of Holstein × Gyr crossbreed heifers grazing intensively managed Brachiaria decumbens throughout the year. Thirty-eight heifers with average initial BW of 172.5 ± 11.15 kg (mean ± SE) and 8.2 ± 0.54 mo of age were randomly assigned to 1 of 4 treatments: 3 protein supplements (SUP) fed at 5g/kg of body weight, plus a control group (CON, non-supplemented animals). The supplement CP levels evaluated were: 12, 24, and 36%. The study was divided into 4 seasons: rainy, dry, rainy-dry transition (RDT), and dry-rainy transition (DRT). On the penultimate day of each season, ultrasound images of the carcass and mammary gland were taken. Five animals from each treatment were randomly chosen on the last day of each season, and liver and muscle tissue biopsies were performed. The target genes were the mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) in the muscle samples. Carbamoyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), arginosuccinate lyase (ASL), and arginase (ARG) were evaluated in the liver samples. Data were analyzed using PROC GLIMMIX of the SAS with repeated measures. We observed a greater rib eye area (cm2) and fat thickness (mm) in SUP animals than in non-supplemented animals. However, we did not observe differences among SUP levels for both variables. No effects of supplementation were detected on mammary gland development. Nevertheless, seasonal effects were observed, where the RDT and dry season had the most and least accumulated fat in the mammary gland. In muscle, we observed greater expression of AMPK in non-supplemented animals than SUP animals. On the other hand, no differences were observed in gene expression between SUP and non-supplemented animals and among SUP animals for mTOR. Season affected both AMPK and mTOR; heifers had a greater AMPK gene expression on rainy than RDT. For mTOR, we observed greater gene expression in RDT and DRT than in rainy. No differences were observed among RDT, dry, and DRT, and between dry and rainy seasons for mTOR. We observed greater CPS, ASL, and ARG gene expression in SUP animals than in non-supplemented animals. Among SUP animals, supplement CP linearly affected CPS. In conclusion, the supplementation strategy did not affect mammary gland development and mTOR expression in muscle tissue. However, we observed a seasonal effect on mammary gland development and AMPK and mTOR expression. The CP supplementation increased the rib eye area and fat thickness, directly affecting AMPK expression in the muscle. Moreover, the CP supplementation increased urea cycle enzyme expression, indicating greater urea production in the liver.

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.

Difficulties in diagnostics of urea synthesis cycle disturbance

Introduction. Hereditary metabolic diseases include a large group of diseases caused by genetic mutations with a high potential risk of transmitting this disorder to future offspring. Manifesting at any age and accompanied by recurrent and progressive clinical symptoms, some of these mutations become incompatible with life, while most lead to gross violations of the normal physiological process of child development. The paper presents a clinical case of hereditary urea synthesis disorder caused by deficiency of ornithine transcarbamylase of mitochondrial matrix in liver (hyperammonemia type 2) as a result of mutation of the OTC gene (MIM 300461). The disease recessive, linked with the X-chromosome and is associated with a high risk of mortality due to accumulation of toxic concentrations of ammonia. Materials and methods. The boy E., born in 2015. Introduction of protein-based complimentary foods and increase in its portion volume is associated with decrease in appetite, regurgitation, nausea, vomiting, flatulence, dyspepsia appear, and sharp weakness progresses. There is a significant increase in the blood concentration of intracellular liver enzymes. Physical development is slowed due to poor weight gain, and neuropsychiatric development lags significantly. Repeatedly examined in the hospital at the place of residence. Results obtained. A total of 47 genes with mutations causing hereditary diseases with predominant liver damage were studied by mass parallel sequencing. Substitution of c.523G>A (p.Asn175Asp) in the OTC gene in hemizygous state was detected. Clinical diagnosis was established: urea cycle metabolism disorder, hyperammonemia, ornithine transcarbamylase deficiency, E 72.2. Dietary recommendations were given. Carbaglu, Recordati, France at the rate of 100 mg/kg/24 h was prescribed on vital signs. Against the background of the therapy and dietary recommendations the condition of the child had a pronounced positive trend. Conclusions. Since the true incidence of this pathology in Russia has not been established (many cases of metabolic disorders of the urea cycle remain undiagnosed), the presented clinical case will allow clinicians, using modern possibilities of structural and functional analysis of the human genome, to make a timely diagnosis and prescribe adequate therapy, thereby not only prolonging the life of a young patient but also significantly improving its quality.

Citrulline in the management of patients with urea cycle disorders

BackgroundTreatment recommendations for urea cycle disorders (UCDs) include supplementation with amino acids involved in the urea cycle (arginine and/or citrulline, depending on the enzyme deficiency), to maximize ammonia excretion through the urea cycle, but limited data are available regarding the use of citrulline. This study retrospectively reviewed clinical and biological data from patients with UCDs treated with citrulline and/or arginine at a reference center since 1990. The aim was to describe the prescription, impact, and safety of these therapies. Data collection included patient background, treatment details, changes in biochemical parameters (plasma ammonia and amino acids concentrations), decompensations, and patient outcomes.ResultsOverall, 79 patients (median age at diagnosis, 0.9 months) received citrulline and/or arginine in combination with a restricted protein diet, most with ornithine transcarbamylase (n = 57, 73%) or carbamoyl phosphate synthetase 1 (n = 15, 19%) deficiencies. Most patients also received ammonium scavengers. Median follow-up was 9.5 years and median exposure to first treatment with arginine + citrulline, citrulline monotherapy, or arginine monotherapy was 5.5, 2.5, or 0.3 years, respectively. During follow-up, arginine or citrulline was administered at least once (as monotherapy or in combination) in the same proportion of patients (86.1%); the overall median duration of exposure was 5.9 years for arginine + citrulline, 3.1 years for citrulline monotherapy, and 0.6 years for arginine monotherapy. The most common switch was from monotherapy to combination therapy (41 of 75 switches, 54.7%). During treatment, mean ammonia concentrations were 35.9 µmol/L with citrulline, 49.8 µmol/L with arginine, and 53.0 µmol/L with arginine + citrulline. Mean plasma arginine concentrations increased significantly from the beginning to the end of citrulline treatment periods (from 67.6 µmol/L to 84.9 µmol/L, P < 0.05). At last evaluation, mean height and weight for age were normal and most patients showed normal or adapted behavior (98.7%) and normal social life (79.0%). Two patients (2.5%) experienced three treatment-related gastrointestinal adverse reactions.ConclusionsThis study underlines the importance of citrulline supplementation, either alone or together with arginine, in the management of patients with UCDs. When a monotherapy is considered, citrulline would be the preferred option in terms of increasing plasma arginine concentrations.

Lipid nanoparticle-targeted mRNA formulation as a treatment for ornithine-transcarbamylase deficiency model mice

Ornithine transcarbamylase (OTC) plays a significant role in the urea cycle, a metabolic pathway functioning in the liver to detoxify ammonia. OTC deficiency (OTCD) is the most prevalent urea cycle disorder. Here, we show that intravenously delivered human OTC (hOTC) mRNA by lipid nanoparticles (LNP) was an effective treatment for OTCD by restoring the urea cycle. We observed a homotrimer conformation of hOTC proteins produced by the mRNA-LNP in cells by cryo-electron microscopy. The immunohistochemistry revealed the mitochondria localization of produced hOTC proteins in hepatocytes in mice. In livers of mice intravenously injected with hOTC-mRNA/LNP at 1.0mg/kg, the delivered hOTC mRNA levels steeply decreased with a half-life (t1/2) of 7.1 h, whereas the produced hOTC protein levels retained for 5days and then declined with a t1/2 of 2.2days. In OTCD model mice (high-protein diet-fed Otcspf-ash hemizygous males), a single dose of hOTC-mRNA/LNP at 3.0mg/kg ameliorated hyperammonemia and weight loss with prolonged survival rate (22days) compared with that of untreated mice (11days). Weekly repeated doses at 0.3 and 1.0mg/kg were well tolerated in wild-type mice and showed a dose-dependent amelioration of survival rate in OTCD mice, thus, showing the therapeutic potential of LNP-formulated hOTC mRNA for OTCD.

Clinical utility of reporting research genetic predisposition testing results to Total Cancer Care participants.

10602 Background: The American College of Medical Genomics and Genetics (ACMG) has identified genes with highly penetrant phenotypes with treatment, prevention, or early disease identification strategies called secondary findings (SF) genes. ACMG recommends clinical laboratories report pathogenic/likely pathogenic (P/LP) SF variants identified during genomic testing. Cancer patients may undergo germline sequencing as part of research studies that can identify valuable SF variants. The objective of this institutional sub-study is to evaluate the feasibility of using ACMG SF genes to assess hereditary disease risk in a cancer population undergoing research sequencing. Methods: Between June 2017 and January 2021, patients unselected for family history, disease site, or stage enrolled in the prospective, observational Total Cancer Care study. Patients underwent research-grade germline whole exome sequencing, with bioinformatic analysis in a CLIA certified laboratory to identify P/LP variants in any ACMG SF v2.0 genes. P/LP variants were reported to treating oncologists with recommendations for genetic counseling referral. We assessed if patients with P/LP variants met NCCN guideline-directed germline testing criteria to quantify benefit from reporting research results. Results: We enrolled 786 patients, which included those with gynecologic (182, 23%), colorectal (124, 16%), lung/bronchus (83, 11%), and head/neck (82, 10%) cancers. The median age was 61 (IQR 52-68) years, 57% were female, 96% were non-Hispanic White, 51% had stage III/IV disease, and 64% resided in an Appalachian community. We identified 57 patients (7%) with cancer predisposition and 14 (2%) with other hereditary disease variants. 2021 NCCN guideline-directed testing would have missed 59% (42/71) of these patients. Incremental high penetrance cancer gene variants included: BRCA1 – endometrial cancer (n = 1); BRCA2– cholangiocarcinoma (n = 1), colon neuroendocrine tumor (n = 1), and lung (n = 2), endometrial (n = 1), and non-epithelial ovarian (n = 1) cancers; MSH6 – duodenal cancer (n = 1); SDHAF2 – non-melanoma skin cancer (n = 1); SDHB – head/neck cancer (n = 1); and TP53 – CNS tumor (n = 1) and head/neck cancer (n = 1). We identified patients at risk for other hereditary diseases including hypertrophic/dilated cardiomyopathy (n = 1), malignant hyperthermia (n = 3), arrhythmogenic right ventricular cardiomyopathy (n = 1), Romano-Ward long QT / Brugada syndromes (n = 4), familial hypercholesterolemia (n = 4), and ornithine transcarbamylase deficiency (n = 1). Conclusions: Reporting germline variants identified during research sequencing is feasible and can benefit cancer patients and their relatives. Though not traditionally assessed, non-cancer predisposition genetic variants may have important intervention opportunities and unexpected patterns may have oncologic implications that require further study.

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

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. We retrospectively reviewed our anesthesia-related experience in all cases of liver transplantation for ornithine transcarbamylase deficiency in our center. 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. 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.

Investigating the changes of some enzymes and metabolites of the Urea cycle in patients with type 2 diabetes treated with squalene

Background: Type 2 diabetes mellitus is a chronic disease that diminishes the body’s ability to regulate glucose levels due to the lack of insulin produced. In recent studies, squalene has been reported to have beneficial effects for diabetic patients, especially within the liver where the urea cycle takes place.Objective: Our main goal was to evaluate the molecular effects of different doses of squalene on the enzymes, intermediates, and molecules of the urea cycle, in order to determine if squalene has beneficial effects among groups of people with type 2 diabetes mellitus. The enzymes and molecules that are being studied are ornithine transcarbamylase (OTC), arginosuccinate synthetase (ASS), arginase, carbamoyl-phosphate synthetase 1 (CSP1), urea, aspartate, and ammonium ion (NH4+).Methods: In this study, healthy volunteers were categorized as the healthy control (group 1) and volunteers with type 2 diabetes mellitus were selected. The patients with diabetes were divided up into 4 groups. Group 2 consists of the patients that will not be treated with squalene. Groups 3, 4, 5 were treated with 200, 400, 600 mg, respectively. The patients were treated with their respective amounts every 14 days for the duration of 84 days. The enzymes and molecules were measured on days 1, 14, 28, 56, and 84.Results: The squalene-treated diabetic groups were compared to group 2, who was not treated with any squalene to determine the differences between the parameters. Throughout the 84 days, it was observed that NH4+ or ammonium molecules decreased in all treated diabetic patients with high statistical difference (P &lt; 0.05). For the majority of the diabetic patients treated with squalene, there was also a decrease in aspartate. The other parameters did not have consistent significant differences (P &gt; 0.05).Conclusion: Based on the findings of this study, the addition of various doses of squalene to a diabetic patient’s diet decreased the amount of ammonium and aspartate in the body. As ammonium is the direct product of the urea cycle, it is evident that squalene does play a key role in reducing the amount of ammonium in a diabetic patient to a healthier level.Keywords: Diabetes mellitus, urea cycle, enzyme, metabolite, squalene.

A case with late-onset Ornithine Transcarbamylase Deficiency as a possible cause of osteoporosis

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