Mendel Tuchman

Cross-sectional multicenter study of patients with urea cycle disorders in the United States.

Inherited urea cycle disorders comprise eight disorders (UCD), each caused by a deficiency of one of the proteins that is essential for ureagenesis. We report on a cross-sectional investigation to determine clinical and laboratory characteristics of patients with UCD in the United States. The data used for the analysis was collected at the time of enrollment of individuals with inherited UCD into a longitudinal observation study. The study has been conducted by the Urea Cycle Disorders Consortium within the Rare Diseases Clinical Research Network (RDCRN) funded by the National Institutes of Health. One-hundred eighty-three patients were enrolled into the study. Ornithine transcarbamylase (OTC) deficiency was the most frequent disorder (55%), followed by argininosuccinic aciduria (16%) and citrullinemia (14%). Seventy-nine percent of the participants were white (16% Latinos), and 6% were African American. Intellectual and developmental disabilities were reported in 39% with learning disabilities (35%) and half had abnormal neurological examination. Sixty-three percent were on a protein restricted diet, 37% were on Na-phenylbutyrate and 5% were on Na-benzoate. Forty-five percent of OTC deficient patients were on L-citrulline, while most patients with citrullinemia (58%) and argininosuccinic aciduria (79%) were on L-arginine. Plasma levels of branched-chain amino acids were reduced in patients treated with ammonia scavenger drugs. Plasma glutamine levels were higher in proximal UCD and in neonatal type disease. The RDCRN allows comprehensive analyses of rare inherited UCD, their frequencies and current medical practices.

N-carbamylglutamate Markedly Enhances Ureagenesis in N-acetylglutamate Deficiency and Propionic Acidemia as Measured by Isotopic Incorporation and Blood Biomarkers

N-acetylglutamate (NAG) is an endogenous essential cofactor for conversion of ammonia to urea in the liver. Deficiency of NAG causes hyperammonemia and occurs because of inherited deficiency of its producing enzyme, NAG synthase (NAGS), or interference with its function by short fatty acid derivatives. N-carbamylglutamate (NCG) can ameliorate hyperammonemia from NAGS deficiency and propionic and methylmalonic acidemia. We developed a stable isotope 13C tracer method to measure ureagenesis and to evaluate the effect of NCG in humans. Seventeen healthy adults were investigated for the incorporation of 13C label into urea. [13C]urea appeared in the blood within minutes, reaching maximum by 100 min, whereas breath 13CO2 reached a maximum by 60 min. A patient with NAGS deficiency showed very little urea labeling before treatment with NCG and normal labeling thereafter. Correspondingly, plasma levels of ammonia and glutamine decreased markedly and urea tripled after NCG treatment. Similarly, in a patient with propionic acidemia, NCG treatment resulted in a marked increase in urea labeling and decrease in glutamine, alanine, and glycine. These results provide a reliable method for measuring the effect of NCG on nitrogen metabolism and strongly suggest that NCG could be an effective treatment for inherited and secondary NAGS deficiency.

1H MRS identifies symptomatic and asymptomatic subjects with partial ornithine transcarbamylase deficiency.

OBJECTIVE To evaluate brain metabolism in subjects with partial ornithine transcarbamylase deficiency (OTCD) utilizing (1)H MRS. METHODS Single-voxel (1)H MRS was performed on 25 medically-stable adults with partial OTCD, and 22 similarly aged controls. Metabolite concentrations from frontal and parietal white matter (FWM, PWM), frontal gray matter (FGM), posterior cingulate gray matter (PCGM), and thalamus (tha) were compared with controls and IQ, plasma ammonia, glutamine, and disease severity. RESULTS Cases ranged from 19 to 59 years; average 34 years; controls ranged from 18 to 59 years; average 33 years. IQ scores were lower in cases (full scale 111 vs. 126; performance IQ 106 vs. 117). Decreased myoinositol (mI) in FWM (p=0.005), PWM (p<0.001), PCGM (p=0.003), and tha (p=0.004), identified subjects with OTCD, including asymptomatic heterozygotes. Glutamine (gln) was increased in FWM (p<0.001), PWM (p<0.001), FGM (p=0.002), and PCGM (p=0.001). Disease severity was inversely correlated with [mI] in PWM (r=-0.403; p=0.046) and directly correlated with [gln] in PCGM (r=0.548; p=0.005). N-Acetylaspartate (NAA) was elevated in PWM (p=0.002); choline was decreased in FWM (p=0.001) and tha (p=0.002). There was an inverse relationship between [mI] and [gln] in cases only. Total buffering capacity (measured by [mI/mI+gln] ratio, a measure of total osmolar capacity) was inversely correlated with disease severity in FWM (r=-0.479; p=0.018), PWM (r=-0.458; p=0.021), PCGM (r=-0.567; p=0.003), and tha (r=-0.345; p=0.037). CONCLUSION Brain metabolism is impaired in partial OTCD. Depletion of mI and total buffering capacity are inversely correlated with disease severity, and serve as biomarkers.

Crystal structure of N-acetylornithine transcarbamylase from Xanthomonas campestris: a novel enzyme in a new arginine biosynthetic pathway found in several eubacteria.

We have identified in Xanthomonas campestris a novel N-acetylornithine transcarbamylase that replaces ornithine transcarbamylase in the canonic arginine biosynthetic pathway of several Eubacteria. The crystal structures of the protein in the presence and absence of the reaction product, N-acetylcitrulline, were determined. This new family of transcarbamylases lacks the DxxSMG motif that is characteristic of all ornithine transcarbamylases (OTCases) and contains a novel proline-rich loop that forms part of the active site. The specificity for N-acetylornithine is conferred by hydrogen bonding with residues in the proline-rich loop via water molecules and by hydrophobic interactions with residues from the adjacent 80s, 120s, and proline-rich loops. This novel protein structure provides a starting point for rational design of specific analogs that may be useful in combating human and plant pathogens that utilize acetylornithine transcarbamylase rather than ornithine transcarbamylase.

Augmenting ureagenesis in patients with partial carbamyl phosphate synthetase 1 deficiency with N-carbamyl-L-glutamate.

Identical studies using stable isotopes were performed before and after a 3-day trial of oral N-carbamyl-l-glutamate (NCG) in 5 subjects with late-onset carbamyl phosphate synthetase deficiency. NCG augmented ureagenesis and decreased plasma ammonia in 4 of 5 subjects. There was marked improvement in nitrogen metabolism with long-term NCG administration in 1 subject.

Structure of N-acetyl-l-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor l-arginine bound

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when L-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by L-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.

The clinically variable R40H mutant ornithine carbamoyltransferase shows cytosolic degradation of the precursor protein in CHO cells

Ornithine carbamoyltransferase (OCT) deficiency is now frequently found in adults with hyperammonaemia affected by mutations that cause partial deficiency of this urea cycle enzyme. One of these mutations (R40H) has occurred in several families and has been found also in asymptomatic relatives. To better understand the phenotypic heterogeneity of this recurrent mutation, we investigated the biological properties of the mutant protein. Using 35S labelling, the import and processing of the R40H mutant OCT protein was investigated in intact CHO cells and in isolated rat liver mitochondria and compared to the wild type and R141Q mutant that causes complete enzyme deficiency. The R40H OCT protein seems to be imported and processed by the mitochondria in a manner similar to that of wild type. However, it is consistently degraded to a smaller fragment in the intact cells, unlike the wild type and R141Q mutant. The mature form of the enzyme is not susceptible to degradation. These data, obtained in CHO cells, suggest that deficiency in OCT enzymatic function conferred by the R40H mutation is likely caused by enhanced degradation of the preprotein in the cytosol. We propose therefore that variation in the rate of OCT turnover is responsible for the heterogeneity of the clinical phenotype in these patients.

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

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

The Clinical and Translational Science Institute at Children's National: Improving Health through pediatric Research

at Children’s National (CTSI-CN) was designed to develop a uniquely pediatric perspective on the task of improving human health through research. Th is important initiative was based on several important insights regarding the pressing need to translate basic discovery into clinically eff ective preventive, diagnostic, and therapeutic pediatric interventions. First, we appreciated that the origins of adult morbidity and mortality arise from both genetic predisposition and environmental circumstances that occur much earlier in life, anytime from the pre-conception period through adolescence. Th is is obviously true for single gene disorders such as cystic fi brosis, Duchenne muscular dystrophy or the many inborn errors of metabolism. But it is also a useful perspective when considering much more common and complex conditions such as cardiovascular disease, asthma, obesity/metabolic syndrome, mild-to-moderate developmental disability, and even HPVassociated malignancies. Second, research to more defi nitively elucidate etiologic mechanisms may best be undertaken early in life. Th e complex impact of comorbid conditions, multiple treatments, and disease progression itself are much less pronounced in children and adolescents than in adults. Th is makes it possible to more readily identify the comparative contributions of genetic susceptibility, for example, and specifi c environmental insults. Th ird, we were especially concerned with the persistent and unacceptable burden of health disparities and recognized that these must be addressed in childhood to assure optimal health and development for all. Both health disparities in childhood itself and those arising later in adulthood can be investigated by carefully designed research and ameliorated through targeted intervention, both typically undertaken in collaboration with the communities we serve. Fourth, we understood that a variety of factors have historically disadvantaged research conducted to benefi t children. Th ese include, for example, the epidemiologic reality that childhood illness is much less common than adult illness (although predispositions and precursors of disease are universal) and is distributed across a highly diverse set of conditions. Th ese facts limit the market for drug and device development, thus serving as a barrier to pediatric research. Th e simple fact that children range in size from premature infants of less than 1 kg in weight to morbidly obese adolescents of over 150 kg is a tremendous challenge for pediatric research. Of course, the evolving psychological and cognitive abilities of children impose additional demands with respect to their participation in research. For all these reasons, we appreciated the importance of pediatric research eff orts in facilities specifi cally designed to meet the medical needs of children. Finally, pediatric research provides the unique opportunity to investigate rare diseases that typically manifest early in life, often with devastating consequences. Perhaps best represented by the inborn errors of metabolism, rigorous investigation of such diseases require not only multidisciplinary pediatric expertise, but also collaborative networks of investigators to accrue a sufficient number of representative patients for meaningful statistical analysis. Moreover, research on the pathophysiology and treatment of single gene disorders provides invaluable insights into normal physiology and the role of these genes in more complex and common disease and thus contributes to improving the general health of the public at large. In spite of our focus on supporting pediatric research, the CTSI-CN was also committed to investigating adult conditions, with a special focus on those which have their origins in childhood, as discussed above. Increasingly, of course, this also includes previously fatal diagnoses such as cystic fi brosis, muscular dystrophy, and certain congenital heart defects for which new treatments have meant improving prognoses and survival into adulthood. In July 2010, Children’s National Medical Center and its partnering institution, Th e George Washington (GW) University, were funded by a Clinical and Translational Science Award to participate together in a newly established CTSI-CN. In addition to a well-established program of mechanistic T1 research, strengths at GW included its creative but rigorous educational programs, with nationally and internationally recognized expertise in distance education, and a preeminent group in health policy. Th e CTSI-CN at GW launched a master’s degree in clinical and translational science in less than 1 year and is now developing an innovative doctoral degree in Clinical and Translational Practice for clinical practitioners committed to substantial involvement in such research. Additional CTSI-CN expertise in evaluation was provided by collaborators at RTI International, thereby, assuring a degree of separation between those who would be implementing the CTSI-CN and those who would be measuring and analyzing its progress. Below we briefl y highlight what we see as the early notable accomplishments and strengths of the CTSI-CN. Figure 1 illustrates the range of representative topics of research supported by the CTSI-CN and the position of the projects within the continuum from disease prevention to treatment and across T1 through T4 translation. Given our understanding of the importance of pediatric research for improving the health of the population at large, it is not surprising that many of our investigators are conducting research to prevent disease before it manifests with irreversible consequences. Th ere is increasing National Institute of Health (NIH) and industry emphasis on the discovery, development, and approval of novel therapeutics based