Genotype-Phenotype Correlations in Ornithine Transcarbamylase Deficiency: A Mutation Update

Ornithine transcarbamylase(OTC) deficiency is an X-linked trait that accounts for nearly half of all inherited disorders of the urea cycle.OTC is one of the enzymes common to both the urea cycle and the bacterial arginine biosynthesis pathway; however, the role of OTC has changed over evolution. For animals with a urea cycle, defects in OTC can trigger hyperammonemic episodes that can lead to brain damage and death. This is the fifth mutation update for human OTC with previous updates reported in 1993, 1995, 2002, and 2006. In the2006 update, 341 mutations were reported. This current update contains 417 disease-causing mutations, and also is the first report of this series to incorporate information about natural variation of the OTC gene in the general population through examination of publicly available genomic data and examination of phenotype/genotype correlations from patients participating in the Urea Cycle Disorders Consortium Longitudinal Study and the first to evaluate the suitability of systematic computational approaches to predict severity of disease associated with different types of OTC mutations.     

Reduced Functional Connectivity of Default Mode and Set-Maintenance Networks in Ornithine Transcarbamylase Deficiency

Background and Purpose

Ornithine transcarbamylase deficiency (OTCD) is an X-chromosome linked urea cycle disorder (UCD) that causes hyperammonemic episodes leading to white matter injury and impairments in executive functioning, working memory, and motor planning. This study aims to investigate differences in functional connectivity of two resting-state networks—default mode and set-maintenance—between OTCD patients and healthy controls.

Methods

Sixteen patients with partial OTCD and twenty-two control participants underwent a resting-state scan using 3T fMRI. Combining independent component analysis (ICA) and region-of-interest (ROI) analyses, we identified the nodes that comprised each network in each group, and assessed internodal connectivity.

Results

Group comparisons revealed reduced functional connectivity in the default mode network (DMN) of OTCD patients, particularly between the anterior cingulate cortex/medial prefrontal cortex (ACC/mPFC) node and bilateral inferior parietal lobule (IPL), as well as between the ACC/mPFC node and the posterior cingulate cortex (PCC) node. Patients also showed reduced connectivity in the set-maintenance network, especially between right anterior insula/frontal operculum (aI/fO) node and bilateral superior frontal gyrus (SFG), as well as between the right aI/fO and ACC and between the ACC and right SFG.

Conclusion

Internodal functional connectivity in the DMN and set-maintenance network is reduced in patients with partial OTCD compared to controls, most likely due to hyperammonemia-related white matter damage. Because several of the affected areas are involved in executive functioning, it is postulated that this reduced connectivity is an underlying cause of the deficits OTCD patients display in this cognitive domain.     

Derepression Kinetics of Ornithine Transcarbamylase in Escherichia coli

A mathematical model for the derepression of ornithine transcarbamylase (OTC) in Escherichia coli strain W was derived from a set of 14 assumptions concerning the arginine regulon. The model assumes that active repressor for the arginine regulon is unstable and is only formed when the level of arginyl-tRNA is in excess of the level necessary to maintain protein synthesis for a given cell doubling time. The presence of active repressor was assumed to inhibit the synthesis of messenger RNA coding for the synthesis of the enzymes of the arginine biosynthetic pathway. Numerical estimates of the model's parameters were made and, by simulation on a digital computer, the model was shown to fit kinetic data for derepression of OTC in E. coli W cells in minimal medium growing in flask culture with a doubling time of 60 min and growing in a chemostat with a generation time of 460 min for an assumed OTC-specific mRNA half-life (t_1/2) of 9 min. The model was also shown to predict the increase in the size of bursts of OTC synthesis elicited by addition of arginine to cultures of derepressing E. coli cells with the increase in the delay time before arginine addition. Approximate analytical solutions to the model were obtained for the early phase of derepression and for repression of OTC. These were used to derive graphical methods for determining t_1/2 from repression and derepression transient changes in the OTC level.     

Purification and Characterization of Ornithine Transcarbamylase from Pea (Pisum sativum L.)

Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using δ-N-(phosphonacetyl)-l-ornithine-Sepharose 6B affinity chromatography. The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37°C, pH 8.5. Pea OTC represents approximately 0.05% of the total soluble protein in the leaf. The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the pea OTC is a trimer of identical subunits. The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher. The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.     

A New Mouse Model of Mild Ornithine Transcarbamylase Deficiency (spf-j) Displays Cerebral Amino Acid Perturbations at Baseline and upon Systemic Immune Activation

Ornithine transcarbamylase deficiency (OTCD, OMIM# 311250) is an inherited X-linked urea cycle disorder that is characterized by hyperammonemia and orotic aciduria. In this report, we describe a new animal model of OTCD caused by a spontaneous mutation in the mouse Otc gene (c.240T>A, p.K80N). This transversion in exon 3 of ornithine transcarbamylase leads to normal levels of mRNA with low levels of mature protein and is homologous to a mutation that has also been described in a single patient affected with late-onset OTCD. With higher residual enzyme activity, spf-J were found to have normal plasma ammonia and orotate. Baseline plasma amino acid profiles were consistent with mild OTCD: elevated glutamine, and lower citrulline and arginine. In contrast to WT, spf-J displayed baseline elevations in cerebral amino acids with depletion following immune challenge with polyinosinic:polycytidylic acid. Our results indicate that the mild spf-J mutation constitutes a new mouse model that is suitable for mechanistic studies of mild OTCD and the exploration of cerebral pathophysiology during acute decompensation that characterizes proximal urea cycle dysfunction in humans.     

Analysis of Pyrimidine Synthesis De Novo Intermediates in Urine During Crisis of a Patient with Ornithine Transcarbamylase Deficiency

Analysis of pyrimidine synthesis de novo intermediates and pyrimidine degradation products in urine samples from a decompensated patient with an ornithine transcarbamylase deficiency showed a strikingly aberrant metabolic profile. Strongly elevated levels of N-carbamyl-aspartate, orotate and uracil were present whereas the concentration of uridine was only marginally increased. The level of pyrimidine excretion appeared to be independent of the ammonia levels in blood, which were only mildly increased.     

Structural Genes for Ornithine Transcarbamylase in Salmonella typhimurium and Escherichia coli K-12

Mutations of Salmonella typhimurium affecting the structural gene for ornithine transcarbamylase (argl) have been isolated and mapped. The two ornithine transcarbamylase loci in Escherichia coli K-12 have been demonstrated by F′ episome transfer.     

Mapping the Gene Determining Ornithine Transcarbamylase and Its Operator in Escherichia coli B

The gene determining ornithine transcarbamylase in Escherichia coli B is argI, not argF. The argI gene has been located at 84 min on the linkage map, close to the gene for aspartate transcarbamylase, with neighboring markers in clockwise order fdp pyrB argI valS. A mutation in its operator locus, argO_I, has been mapped between argI and valS, so that this gene, unlike the argCBH portion of the argECBH cluster, appears to be oriented in the same direction as most of the other known E. coli operons.     

Causes and Outcomes of Acute Liver Failure in China

Objectives

No extensive investigation has been performed and thus no representative data are available regarding acute liver failure (ALF) in China. This study aims to investigate the causes and outcomes of ALF in China and establish a prognostic model.

Methods

Patients diagnosed as ALF in seven hospitals in different areas of China from January 2007 to December 2012 were retrospectively selected.

Results

Of the 177 patients included in this study, 112 (63.28%) eventually died. The common causes of ALF were drug toxicity (43.50%), indeterminate etiology (29.38%) and acute viral hepatitis (11.30%). Additionally, traditional Chinese herbs predominated in the causes of drug-induced ALF (30/77). No patients in this study received liver transplantation. In the established model for predicting death in ALF, four variables were finally selected out, including age (P=0.01), the entry hepatic encephalopathy grade (P=0.04), international normalized ratio (P<0.01) and arterial blood ammonia (P=0.02). Using a threshold value of 0.5683, this model had a sensitivity of 95.24% and a specificity of 91.30%.

Conclusions

Traditional Chinese medicine was a major cause of ALF in China. The spontaneous mortality of ALF was high, whereas the rate of liver transplantation was significantly low. The established prognostic model of ALF had superior sensitivity and specificity.     

Acute Liver Failure with Thyrotoxicosis Treated with Liver Transplantation

ObjectiveWe describe a young woman with previously undiagnosed thyrotoxicosis who presented with acute liver failure (ALF).MethodsWe present a case report and review the relevant literature.ResultsAn extensive evaluation excluded possible causes of ALF other than thyrotoxicosis. The management of thyrotoxicosis posed several unique challenges in the setting of ALF, particularly because we did not want to use potentially hepatotoxic thionamides. The patient was treated with prednisone and propranolol and was started on potassium iodide when she was listed for liver transplantation. She underwent an uncomplicated liver transplant and subsequent thyroidectomy and is doing well.ConclusionThis well-characterized case describes thyrotoxicosis as a possible cause of ALF after thoroughly excluding other possible causes and illustrates the challenges of simultaneously managing both disorders. To our knowledge, this is the first report of ALF possibly resulting from untreated thyrotoxicosis that was successfully treated with liver transplantation. (Endocr Pract. 2013;19:e57-e60)     

Melatonin Protects against Apoptosis-Inducing Factor (AIF)-Dependent Cell Death during Acetaminophen-Induced Acute Liver Failure

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure and is primarily caused by cytochrome P450 (CYP) 2E1-driven conversion of APAP into hepatotoxic metabolites. Several reports showed that melatonin attenuated APAP-induced acute liver failure. Nevertheless, the exact mechanism remains obscure. In the present study, we investigated the effects of melatonin on apoptosis-inducing factor (AIF)-dependent cell death in APAP-induced acute liver failure. Mice were intraperitoneally (i.p.) injected with different doses of melatonin (1.25, 5, 20 mg/kg) 30 min before APAP (300 mg/kg, i.p.). As expected, melatonin significantly alleviated APAP-induced cell death, as determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Further analysis showed that melatonin significantly attenuated APAP-induced activation of the serine/threonine kinase receptor interacting protein 1 (RIP1). In addition, melatonin inhibited APAP-induced hepatic c-Jun N-terminal kinase (JNK) phosphorylation and mitochondrial Bax translocation. Correspondingly, melatonin inhibited APAP-induced translocation of AIF from mitochondria to nuclei. Interestingly, no changes were induced by melatonin on hepatic CYP2E1 expression. In addition, melatonin had little effect on APAP-induced hepatic glutathione (GSH) depletion. In conclusion, melatonin protects against AIF-dependent cell death during APAP-induced acute liver failure through its direct inhibition of hepatic RIP1 and subsequent JNK phosphorylation and mitochondrial Bax translocation.     

Alginate Microencapsulated Hepatocytes Optimised for Transplantation in Acute Liver Failure

MethodsHuman hepatocyte microbeads (HMBs) were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs) after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs) produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality.ResultsThe optimised HMBs were of uniform size (583.5±3.3 µm) and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001). 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×10⁶ cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01). Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function.ConclusionAn optimised protocol to produce GMP grade alginate-encapsulated human hepatocytes has been established. Transplantation of microbeads provided effective metabolic function in ALF. These high quality HMBs should be suitable for use in clinical transplantation.     

恩替卡韦治疗乙型肝炎相关急性肝衰竭疗效和安全性研究

目的:通过临床观察,研究乙型肝炎相关急性肝衰竭的综合治疗中恩替卡韦抗病毒治疗的有效性和安全性.方法:本研究前瞻性选取了近18个月住院的15例感染乙型肝炎病毒而明确诊断急性肝衰竭的患者,明确诊断后第一时间给予每日服用恩替卡韦0.5mg抗病毒治疗,定期观察生化及病毒学指标,并予以随访.结果:15名患者中除5例病情恶化死亡,2例行原位肝移植外,其余8例服用恩替卡韦患者痊愈出院并完整随访.所有病人在开始恩替卡韦抗病毒治疗后2个月内生化指标完全正常,HBVDNA低于检测下限,耐受性良好.6个月内有7位患者都发生了血清学转换,一位患者HBsAg始终阳性.结论:通过临床观察进一步证实,对乙肝相关急性肝衰竭患者尽早使用恩替卡韦抗病毒治疗,可以缩短病程,改善预后,甚至避免肝移植.     

恩替卡韦改善了乙型肝炎病毒所致急性肝衰竭的预后

目的：阐明乙型肝炎相关急性肝衰竭的预后因素和恩替卡韦的疗效。方法：本研究回顾性选取了47名因感染乙型肝炎病毒而明确诊断急性肝衰竭的患者,其中5名短期内行肝移植手术被排除,其余42名患者的数据被总结分析。结果：42名患者中12名患者服用了恩替卡韦,在诊断之初,服用恩替卡韦组和未服用恩替卡韦组的一般情况基本相当。使用与未使用恩替卡韦者的生存率分别为67%和23%,此外,年龄〉45岁和没有服用恩替卡韦是不良预后的两个独立相关因素。在年龄〉45岁的患者中使用恩替卡韦和未使用恩替卡韦者的存活率分别为50%和8%。结论：本研究证实了恩替卡韦对乙肝相关急性肝衰竭的治疗作用,我们建议所有乙型肝炎病毒引起的急性肝衰竭患者均应尽早开始恩替卡韦抗病毒治疗。     

Ornithine Decarboxylase Induction by Glucocorticoids in Brain and Liver of Adrenalectomized Rats

Abstract: Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute S.C. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [³H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.     

Phosphatase of Regenerating Liver 2 (PRL2) Deficiency Impairs Kit Signaling and Spermatogenesis

The Phosphatase of Regenerating Liver (PRL) proteins promote cell signaling and are oncogenic when overexpressed. However, our understanding of PRL function came primarily from studies with cultured cell lines aberrantly or ectopically expressing PRLs. To define the physiological roles of the PRLs, we generated PRL2 knock-out mice to study the effects of PRL deletion in a genetically controlled, organismal model. PRL2-deficient male mice exhibit testicular hypotrophy and impaired spermatogenesis, leading to decreased reproductive capacity. Mechanistically, PRL2 deficiency results in elevated PTEN level in the testis, which attenuates the Kit-PI3K-Akt pathway, resulting in increased germ cell apoptosis. Conversely, increased PRL2 expression in GC-1 cells reduces PTEN level and promotes Akt activation. Our analyses of PRL2-deficient animals suggest that PRL2 is required for spermatogenesis during testis development. The study also reveals that PRL2 promotes Kit-mediated PI3K/Akt signaling by reducing the level of PTEN that normally antagonizes the pathway. Given the strong cancer susceptibility to subtle variations in PTEN level, the ability of PRL2 to repress PTEN expression qualifies it as an oncogene and a novel target for developing anti-cancer agents.     

Biallelic Mutations in NBAS Cause Recurrent Acute Liver Failure with Onset in Infancy

Acute liver failure (ALF) in infancy and childhood is a life-threatening emergency. Few conditions are known to cause recurrent acute liver failure (RALF), and in about 50% of cases, the underlying molecular cause remains unresolved. Exome sequencing in five unrelated individuals with fever-dependent RALF revealed biallelic mutations in NBAS. Subsequent Sanger sequencing of NBAS in 15 additional unrelated individuals with RALF or ALF identified compound heterozygous mutations in an additional six individuals from five families. Immunoblot analysis of mutant fibroblasts showed reduced protein levels of NBAS and its proposed interaction partner p31, both involved in retrograde transport between endoplasmic reticulum and Golgi. We recommend NBAS analysis in individuals with acute infantile liver failure, especially if triggered by fever.