Inborn Errors of Bile Acid Metabolism.

Inborn errors of bile acid metabolism are rare causes of neonatal cholestasis and liver disease in older children and adults. The diagnosis should be considered in the context of hyperbilirubinemia with normal serum bile acids and made by urinary liquid secondary ionization mass spectrometry or DNA testing. Cholic acid is an effective treatment of most single-enzyme defects and patients with Zellweger spectrum disorder with liver disease.

Dietary management of urea cycle disorders: European practice.

BACKGROUND: There is no published data comparing dietary management of urea cycle disorders (UCD) in different countries. METHODS: Cross-sectional data from 41 European Inherited Metabolic Disorder (IMD) centres (17 UK, 6 France, 5 Germany, 4 Belgium, 4 Portugal, 2 Netherlands, 1 Denmark, 1 Italy, 1 Sweden) was collected by questionnaire describing management of patients with UCD on prescribed protein restricted diets. RESULTS: Data for 464 patients: N-acetylglutamate synthase (NAGS) deficiency, n=10; carbamoyl phosphate synthetase (CPS1) deficiency, n=29; ornithine transcarbamoylase (OTC) deficiency, n=214; citrullinaemia, n=108; argininosuccinic aciduria (ASA), n=80; arginase deficiency, n=23 was reported. The majority of patients (70%; n=327) were aged 0-16y and 30% (n=137) >16y. Prescribed median protein intake/kg body weight decreased with age with little variation between disorders. The UK tended to give more total protein than other European countries particularly in infancy. Supplements of essential amino acids (EAA) were prescribed for 38% [n=174] of the patients overall, but were given more commonly in arginase deficiency (74%), CPS (48%) and citrullinaemia (46%). Patients in Germany (64%), Portugal (67%) and Sweden (100%) were the most frequent users of EAA. Only 18% [n=84] of patients were prescribed tube feeds, most commonly for CPS (41%); and 21% [n=97] were prescribed oral energy supplements. CONCLUSIONS: Dietary treatment for UCD varies significantly between different conditions, and between and within European IMD centres. Further studies examining the outcome of treatment compared with the type of dietary therapy and nutritional support received are required.

A novel biochemically salvageable animal model of hyperammonemia devoid of N-acetylglutamate synthase.

All knockout mouse models of urea cycle disorders die in the neonatal period or shortly thereafter. Since N-acetylglutamate synthase (NAGS) deficiency in humans can be effectively treated with N-carbamyl-l-glutamate (NCG), we sought to develop a mouse model of this disorder that could be rescued by biochemical intervention, reared to adulthood, reproduce, and become a novel animal model for hyperammonemia. Founder NAGS knockout heterozygous mice were obtained from the trans-NIH Knock-Out Mouse Project. Genotyping of the mice was performed by PCR and confirmed by Western blotting of liver and intestine. NCG and L-citrulline (Cit) were used to rescue the NAGS knockout homozygous (Nags(-/-)) pups and the rescued animals were characterized. We observed an 85% survival rate of Nags(-/-) mice when they were given intraperitoneal injections with NCG and Cit during the newborn period until weaning and supplemented subsequently with both compounds in their drinking water. This regimen has allowed for normal development, apparent health, and reproduction. Interruption of this rescue intervention resulted in the development of severe hyperammonemia and death within 48 h. In addition to hyperammonemia, interruption of rescue supplementation was associated with elevated plasma glutamine, glutamate, and lysine, and reduced citrulline, arginine, ornithine and proline levels. We conclude that NAGS deprived mouse model has been developed which can be rescued by NCG and Cit and reared to reproduction and beyond. This biochemically salvageable mouse model recapitulates the clinical phenotype of proximal urea cycle disorders and can be used as a reliable model of induced hyperammonemia by manipulating the administration of the rescue compounds.

N-acetylglutamate synthase: structure, function and defects.

N-acetylglutamate (NAG) is a unique enzyme cofactor, essential for liver ureagenesis in mammals while it is the first committed substrate for de novo arginine biosynthesis in microorganisms and plants. The enzyme that produces NAG from glutamate and CoA, NAG synthase (NAGS), is allosterically inhibited by arginine in microorganisms and plants and activated in mammals. This transition of the allosteric effect occurred when tetrapods moved from sea to land. The first mammalian NAGS gene (from mouse) was cloned in 2002 and revealed significant differences from the NAGS ortholog in microorganisms. Almost all NAGS genes possess a C-terminus transferase domain in which the catalytic activity resides and an N-terminus kinase domain where arginine binds. The three-dimensional structure of NAGS shows two distinctly folded domains. The kinase domain binds arginine while the acetyltransferase domain contains the catalytic site. NAGS deficiency in humans leads to hyperammonemia and can be primary, due to mutations in the NAGS gene or secondary due to other mitochondrial aberrations that interfere with the normal function of the same enzyme. For either condition, N-carbamylglutamate (NCG), a stable functional analog of NAG, was found to either restore or improve the deficient urea-cycle function.

Measuring in vivo ureagenesis with stable isotopes.

Stable isotopes have been an invaluable adjunct to biomedical research for more than 70years. Indeed, the isotopic approach has revolutionized our understanding of metabolism, revealing it to be an intensely dynamic process characterized by an unending cycle of synthesis and degradation. Isotopic studies have taught us that the urea cycle is intrinsic to such dynamism, since it affords a capacious mechanism by which to eliminate waste nitrogen when rates of protein degradation (or dietary protein intake) are especially high. Isotopes have enabled an appreciation of the degree to which ureagenesis is compromised in patients with urea cycle defects. Indeed, isotopic studies of urea cycle flux correlate well with the severity of cognitive impairment in these patients. Finally, the use of isotopes affords an ideal tool with which to gauge the efficacy of therapeutic interventions to augment residual flux through the cycle.

Favourable long-term outcome after immediate treatment of neonatal hyperammonemia due to N-acetylglutamate synthase deficiency.

INTRODUCTION: N-Acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder, which may present in the neonatal period with severe hyperammonemia and marked neurological impairment. CASE REPORT: We report on a Turkish family with a patient who died due to hyperammonemia in the neonatal period. Reduced activity of NAGS and carbamyl phosphate synthetase were found at autopsy. A second child who developed hyperammonemia on the second day of life was immediately treated with arginine hydrochloride, sodium benzoate and protein restriction. After NAGS deficiency was suspected by enzyme analysis, sodium benzoate was replaced by N-carbamylglutamate (NCG). A third child who developed slight hyperammonemia on the third day of life was treated with NCG before enzyme analysis confirmed reduced NAGS activity. Neither of the patients developed hyperammonemia in the following years. After the human NAGS gene was identified, mutation analysis revealed that the older sibling on NCG therapy was homozygous for a 971G>A (W324X) mutation. The parents and the younger sibling were heterozygous. Therapy was continued in the older sibling until now without any adverse effects and favourable neurodevelopment outcome. In the younger sibling, therapy was stopped without any deterioration of urea cycle function. CONCLUSION: NAGS deficiency can be successfully treated with NCG and arginine hydrochloride with favourable outcome. Molecular diagnostic rather than enzyme analysis should be used in patients with suspected NAGS deficiency.

Polar body-based preimplantation genetic diagnosis for N-acetylglutamate synthase deficiency.

OBJECTIVE: We describe a sensitive and highly reliable preimplantation genetic diagnosis (PGD) assay for N-acetylglutamate synthetase (NAGS) deficiency using polar body (PB) analysis in conjunction with multiple markers flanking the gene. This rare autosomal recessive mitochondrial disorder is characterized by hyperammonemia, uncontrollable movements, developmental delay, visual impairment, failure to thrive and vomiting and is caused by mutations in the NAGS gene located on chromosome 17q21.31. METHODS: For a family with an affected child we have developed a multiplex fluorescent PCR protocol that included detection of the specific familial mutation (2729insC) in conjunction with the analysis of five informative polymorphic markers flanking the gene: D17S902, D17S965, D17S1861, D17S791 and D17S1868. Following successful amplification in single-cell fibroblasts, this protocol was used in the couple carriers of NAGS mutation. RESULTS: Of 18 retrieved eggs, 16 were at the M2 stage and 9 fertilized. 12 polar body 1s (PB1) were heterozygotes, 1 homozygote wild-type, 1 total amplification failure, and two showed inconclusive results. Three oocytes that had heterozygote PB1s showed mutant polar body 2 (PB2) indicating a wild-type oocyte. Despite the fact that the specific 2729insC mutation did not amplify in the PGD cycle, analysis of linked markers in PBs was sufficient to ensure an accurate diagnosis in 5 out of 9 oocytes. This cycle resulted in the transfer of 3 embryos originating from oocytes diagnosed as wild-type by PB analysis, with the subsequent birth of healthy twin girls. Postnatal genetic testing revealed that both girls harbored the healthy maternal allele and carried the mutant paternal allele. CONCLUSIONS: Our multiplex-nested PCR protocol based on several linked microsatellite markers offers an efficient and accurate method for PGD for NAGS syndrome even when the mutation is not amplified.

Carglumic acid: a second look. Confirmed progress in a rare urea cycle disorder.

(1) N-acetylglutamate synthase deficiency is a rare congenital disorder that causes hyperammonaemic comas, resulting in severe neurological morbidity and usually leading to death during childhood. (2) Carglumic acid is the first drug to be used for replacement therapy. Data available in 2003 showed beneficial effects on growth and psychomotor development. (3) In 2007, about 20 patients treated with carglumic acid for N-acetyglutamate synthase deficiency, for at least 5 years in half of cases, were all still alive. Their development was normal when treatment was initiated before complications occurred. (4) No serious adverse effects have been observed. (5) In practice, although this treatment has to continue for life, carglumic acid represents a major advance for patients with N-acetylglutamate synthase deficiency.

Contrasting features of urea cycle disorders in human patients and knockout mouse models.

The urea cycle exists for the removal of excess nitrogen from the body. Six separate enzymes comprise the urea cycle, and a deficiency in any one of them causes a urea cycle disorder (UCD) in humans. Arginase is the only urea cycle enzyme with an alternate isoform, though no known human disorder currently exists due to a deficiency in the second isoform. While all of the UCDs usually present with hyperammonemia in the first few days to months of life, most disorders are distinguished by a characteristic profile of plasma amino acid alterations that can be utilized for diagnosis. While enzyme assay is possible, an analysis of the underlying mutation is preferable for an accurate diagnosis. Mouse models for each of the urea cycle disorders exist (with the exception of NAGS deficiency), and for almost all of them, their clinical and biochemical phenotypes rather closely resemble the phenotypes seen in human patients. Consequently, all of the current mouse models are highly useful for future research into novel pharmacological and dietary treatments and gene therapy protocols for the management of urea cycle disorders.

Deficiency of the carnitine transporter (OCTN2) with partial N-acetylglutamate synthase (NAGS) deficiency.

A patient with recurrent episodes of hyperammonaemia (highest ammonia level recorded 229 micromol/L, normal 9-33) leading to altered levels of consciousness was diagnosed with partial N-acetylglutamate synthase (NAGS) deficiency (9% residual activity) at age 5 years and was treated with ammonia-conjugating agents (Ucephan 250 mg/kg per day and later sodium phenylbutyrate 200-250 mg/kg per day) for 15 years. A chronically low serum carnitine level (pretreatment plasma free carnitine 4 nmol/L, normal 37 +/- 8 nmol/L; total carnitine 8 nmol/L, normal 46 +/- 10) was assumed to be secondary and was treated with supplemental carnitine (30-50 mg/kg per day). Hypoglycaemia (blood sugar 35 mg/dl, normal 70-100), cardiomegaly, and fatty liver were also noted at diagnosis. The patient died unexpectedly at age 20 years. In retrospect, it was learned that the patient had stopped his carnitine without medical consultation several weeks prior to his death. Additional molecular investigations identified two mutations (R254X and IVS3 + 1G > A) in the patient's OCTN2 (SLC22A5) gene, consistent with a diagnosis of primary carnitine deficiency due to carnitine transporter defect. R245X is a founder mutation in Southern Chinese populations. It is unknown whether the original NAGS deficiency was primary or secondary, but molecular analysis of the NAGS gene failed to identify mutations. Urea cycle enzyme expression may be affected by fatty acid suppression of an AP-1 binding site in the promoter enhancer region of the urea cycle gene. Regardless, it is clear that the NAGS abnormality has led to delay of recognition of the OCTN2 defect, and modified the clinical course in this patient.

A trial with N-carbamylglutamate may not detect all patients with NAGS deficiency and neonatal onset.

N-acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder. An effective treatment, N-carbamoyl-L-glutamic acid (NCGA), is now available, increasing the importance of identifying and treating these patients early. We describe a case with genetically verified NAGS deficiency and neonatal onset of severe hyperammonaemia. The ammonia levels increased above 1400 micromol/L. The patient did not respond to NCGA treatment during the first 15 h, indicating that a delayed response or no response cannot be used as a safe indicator for excluding NAGS deficiency in the acute situation. Hence, conventional treatment should not be delayed by a diagnostic procedure, such as a loading test. Furthermore, at 3 years of age this patient has normal psychomotor development, underlining the possibility of a favourable outcome despite markedly elevated ammonia levels, coma, and seizures in the neonatal period. Including NCGA early in the treatment of patients with hyperammonaemia may be of clinical importance. In order to detect patients with NAGS deficiency and neonatal onset and to optimize care, it is important to use the available treatment strategies to reduce plasma ammonia concentrations without delay. We propose the use of combined symptomatic treatment, i.e. glucose infusion, sodium benzoate, arginine or citrulline, and when indicated haemodialysis, as well as NCGA treatment in all neonates presenting with severe hyperammonaemia. The treatment should be continued until laboratory investigations are complete or indicate another disorder.

Late onset N-acetylglutamate synthase deficiency caused by hypomorphic alleles.

N-acetylglutamate (NAG) is a unique cofactor that is essential for the conversion of ammonia to urea in the liver. N-acetylglutamate synthase (NAGS) catalyzes the formation of NAG. Deficiency of NAGS causes a block in ureagenesis resulting in hyperammonemia. Although a number of mutations have been identified in the NAGS gene, their effects on NAGS enzymatic activity have not been examined. We describe here three mutations in two families with NAGS deficiency. Studies of the purified recombinant mutant proteins revealed deleterious effects on NAGS affinity for substrates, and on the rate of catalysis. These studies provide a better understanding of the function of NAGS, and the mechanisms for deleterious effect of mutations causing inherited NAGS deficiency.