Urea Cycle Disorders (UCD)

The UCDC is an NIH-funded 16-site research consortium within the Rare Disease Clinical Research Network to investigate inborn errors of the urea cycle. These rare genetic disorders result from defects in any of the eight genes associated with this important metabolic cycle and have a combined prevalence of about 1 in 30,000. Urea cycle disorders (UCDs) lead to the accumulation of ammonia in the blood and brain and resultant episodes of metabolic encephalopathy, with a great risk of morbidity and mortality. The focus of the UCDC is to perform a longitudinal natural history study and intervention studies of these disorders and to develop and test new diagnostic and therapeutic approaches. Children’s National serves as the leadership hub of the consortium, which is led by Dr. Gropman. The UCDC is supported by funding from the NIH and the Kettering and O’Malley Family Foundations. In the past decade, the consortium successfully brought to market three new drugs to treat hyperammonemia and currently follows more than 700 individuals with these disorders.

Advanced neuroimaging technology, using diffusion tensor imaging, volumetric averaging, fMRI, and magnetic resonance spectroscopy, allows non-invasive investigations of the brain in complex conditions such as hyperammonemia in UCDs. Andrea Gropman, M.D., and her team, including John VanMeter, Ph.D. (Georgetown University), and Matthew Whitehead, M.D., and Stanley Fricke, Ph.D., have been using these methods to identify biomarkers that reflect the downstream impact of UCDs on cognition. Previous imaging research performed as part of the UCDC identified specific biomarkers of neurologic injury in ornithine transcarbamylase deficiency (OTCD). Specifically, the study showed that elevations in brain glutamine, a storage depot for ammonia, may persist and be associated with alterations in mental status and cognition even in the presence of normal plasma ammonia and normal or only slightly elevated plasma glutamine.

In addition, another small biomarker, myoinositol, may be associated with cognitive reserve in patients who have had hyperammonemia. Female carriers of OTCD, an X-linked UCD, who are expected to have milder symptoms, demonstrate challenges in executive function and working memory, although they may function well with simple tasks. This was shown by performance on a number of cognitive tests that target frontal lobe function and by activation and resting state studies on fMRI. While characterization of mutations can be achieved in most cases, this information does not necessarily predict the severity of the underlying neurological compromise in patients. The clinical phenotype varies from one patient to another and results in significant outcome heterogeneity. The group’s neuroimaging studies revealed affected cognitive domains, which include nonverbal learning, fine motor processing, reaction time, visual memory, attention, and executive function. Deficits in these capacities may be seen in symptomatic patients, as well as in asymptomatic carriers with normal IQ, and correlate with variances in brain structure and function in these patients.

These studies allow the team to begin to understand the brain pathophysiology in hyperammonemia and correlate the results with different variables, including treatment modalities. Current studies are aimed at understanding the chronology of recovery from hyperammonemia using neuroimaging biomarkers and studying the brain effects of other UCDs besides OTCD. Dr. Gropman’s group is also exploring the use of optical imaging as a totally non-invasive technique to target the very young and more cognitively challenged patients with OTCD

In an NIH-funded project, Mendel Tuchman, M.D., and Nicholas Ah Mew, M.D., demonstrated that an oral medication, N-carbamylglutamate, can correct the biochemical defect in patients with a UCD known as N-acetylglutamate synthase (NAGS) deficiency, thereby normalizing ammonia levels and restoring normal urea production. Results from this study led to the discovery of the first regulatory mutation in the NAGS gene. Subsequent clinical studies showed that N-carbamylglutamate can reduce ammonia levels and improve urea production in patients with other forms of hyperammonemia, such as carbamyl phosphate synthetase (CPS1) deficiency, and propionic and methylmalonic acidemia. The success of this translational work has led to an NIH-funded groundbreaking randomized, double blind, placebo-controlled clinical trial of N-carbamylglutamate in patients with the aforementioned disorders who present with acute hyperammonemia. Results from this trial should be available within the next year and could potentially be used to expand the clinical indications for this drug.

In a project funded by the Patient-Centered Outcomes Research Institute (PCORI), Nicholas Ah Mew, M.D., and Robert McCarter, Jr., Sc.D., are conducting a study to compare the outcome of liver transplantation with conservative management in urea cycle disorders. This work is being done in collaboration with the School of Public Health at GW, the National Urea Cycle Disorders Foundation and the Emmes Corporation’s Studies of Pediatric Liver Transplantation. The results of the study should be available at the end of 2018 and will greatly help future UCD patients and providers through the difficult process of deciding whether or not to proceed with liver transplantation.

In addition to ammonia removal, a new therapeutic paradigm for treating hyperammonemia directly protects the brain. Ljubica Caldovic, Ph.D., Mendel Tuchman, M.D., and Hiroki Morizono, Ph.D., are screening chemicals as potential drugs that can protect the brain from the toxic effects of ammonia. In a project previously funded by NIH and now by industry, the team developed a zebrafish model of hyperammonemia and is using it to screen thousands of chemicals for their ability to prolong survival of zebrafish larvae in water containing high ammonia concentrations. Several chemicals that affect neurotransmission were already documented in this screen to protect zebrafish from high ammonia exposure. 

Dr. Caldovic received a pilot award to investigate whether chemicals affecting different neurotransmission systems act synergistically to provide more effective neuroprotection. Two pairs of chemicals were more effective at protecting zebrafish than either chemical alone. The team has also identified early behavioral markers of hyperammonemia in mice and has shown that changes in behavior coincide with the onset of abnormal neuronal electrical activity in mice experiencing hyperammonemia. This will allow the team to test efficacy of the lead compounds in a mouse model of inducible hyperammonemia. Those confirmed to be both effective and nontoxic will proceed to clinical trials.

Hiroki Morizono, Ph.D., and Mark L. Batshaw, M.D., along with their long-term collaborators at the University of Pennsylvania, James Wilson, M.D., Ph.D., and Lili Wang, Ph.D., have been investigating the efficacy of adeno-associated virus (AAV)–based gene therapy for the treatment of OTC deficiency (the most common urea cycle disorder) in mouse models. The virus is used to deliver a functional copy of the OTC gene to the liver. At the inception of this project, it took more than two weeks for AAV gene delivery to reach protective levels of OTC gene expression, which is a problem for this neonatal-onset, potentially fatal disorder. Continual optimizations of the vector reduced this time from days to hours, resulting in a clinical candidate vector design. 

The team’s industry partner has begun enrolling adult patients with a milder form of OTC deficiency to participate in a clinical trial using this AAV vector. The preclinical program has also successfully demonstrated that CRISPR/Cas9-based gene editing can correct specific mutations in OTC-deficient mice at levels that protect against a hyperammonemic episode. Ljubica Caldovic, Ph.D., Dr. Morizono and Lisa Tuchman, M.D., have also shown that AAV-based gene delivery of the NAGS gene successfully rescues NAGS-deficient mice so they do not require N-carbamylglutamate supplementation for survival.

In another project funded by the NIH, Ljubica Caldovic, Ph.D., continued to investigate the structural biology of NAGS and CPS1 proteins of the urea cycle. 

Dr. Caldovic’s group compared biophysical properties of bacterial and mammalian NAGS proteins, and found, with high resolution crystal structures, that some NAGS proteins are ensembles of different oligomers while others are not. This result provides rationale for selecting dog, Tasmanian devil, American bison and domestic yak NAGS proteins as good candidates for structural studies of the whole protein.

In a project funded by the NIH, Mendel Tuchman, M.D., Ljubica Caldovic, Ph.D., and Hiroki Morizono, Ph.D., created a mouse model with complete NAGS deficiency that can be rescued by supplementation of N-carbamylglutamate and L-citrulline. This is the only mouse model of a urea cycle defect that can be rescued to reach adulthood and reproduce. The team has now used this model and adeno-associated virus (AAV)-based gene transfer to investigate the function of NAGS in vivo. Delivery of E354A arginine-insensitive NAGS via AAV-based gene transfer system rescued the NAGS deficient mice but they had elevated plasma ammonia.

Dr. Caldovic’s team also carried out biochemical and biophysical characterization of an arginine-insensitive NAGS protein that was found in a patient with NAGS deficiency. This shows that binding of arginine to NAGS is important for its function and efficient ureagenesis. Dr. Caldovic’s laboratory is studying conserved DNA sequences upstream of the NAGS gene and in its first intron that seem to regulate its expression and where mutations (missed by clinical testing) can lead to hyperammonemia. The lab used computational methods and reporter gene assays to show that five sequence variants found outside of the NAGS coding region affect expression of the NAGS gene and lead to NAGS deficiency.