A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Adult patient diagnosed with NADSYN1 associated congenital NAD deficiency and analysis of NAD levels to be published in: European Journal of Medical Genetics.

INTRODUCTION: Nicotinamide adenine dinucleotide (NAD) is an essential cosubstrate/coenzyme in multiple cellular redox processes and a substrate in several non-redox reactions. NADSYN1 encodes NAD synthetase 1, an enzyme in the NAD de novo synthesis pathway and the Preiss-Handler pathway, and biallelic pathogenic variants causes NAD deficiency associated with vertebral, cardiac, renal and limb defects. Szot et al. and Kortbawi et al. have reported a total of seven patients with NADSYN1 associated congenital NAD deficiency disorder with the oldest patient being seven years old. PATIENT DATA: We present a male patient age 30 with a height of 130 cm and numerous skeletal malformations including segmentation defects of the spine, rib anomalies and unequal leg length as well as bilateral ptosis, cleft palate and asymmetric dysmorphic facial features. The patient underwent surgery for an aortic stenosis due to a bicuspid valve. No malformations of the kidneys or urinary tract were identified. RESULTS: Trio exome sequencing revealed a homozygous missense variant in NADSYN1 c.1717G > A (p.Ala573Thr). Both parents were unaffected carriers of the variant. Analysis of NAD levels showed that the patient had a lower NAD pool compared to his unaffected siblings. The NAD pool rose approximately 25% after supplementation with nicotinamide, a NAD precursor for the salvage pathway. CONCLUSION: The variant was previously reported in four patients and functional analyses by Szot et al. support the pathogenicity of the variant. We report an adult patient with NADSYN1 associated congenital NAD deficiency disorder and expand the phenotypic spectrum. We also present analysis of the NAD levels before and after supplementation with nicotinamide.

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.

Characterization of carbamoyl phosphate synthetase of Streptomyces spp.

Carbamoyl phosphate synthetase (CPS) activity in Streptomyces lividans was repressed (70%) by addition of arginine and uracil in the growth medium. Enzyme activity was also inhibited by UMP and activated by ornithine and IMP. Pattern of inhibition and activation was similar irrespective of whether the cells were grown in medium supplemented with arginine or with uracil. A mutant of S. coelicolor with dual auxotrophy for arginine and uracil possessed only about 20% of CPS activity compared to the wild-type strain. An activity staining protocol has been developed for CPS enzyme. Using this method a single CPS band has been observed in the crude extracts of Escherichia coli as well as in S. lividans. Taken together, our results supported the conclusion that Streptomyces species might possess a single CPS enzyme unlike other gram-positive bacteria, which show the presence of two pathway-specific isozymes (Bacillus) or none (Lactobacillus and Leuconostoc).

Cloning and transcriptional analysis of the ADE6 gene of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae gene, ADE6, encoding 5'-phosphoribosylformyl glycinamidine synthetase (EC 6.3.5.3) has been cloned by complementation of an ade6 auxotroph. Transformation of ade6 mutants with ADE6-carrying centromeric plasmids restored normal, adenine-independent growth behavior in the recipients. Strains containing a disrupted ade6 allele were constructed and behaved as stable adenine auxotrophs. Southern transfer and genetic analyses of strains carrying a disrupted ade6 allele demonstrated that the cloned gene was ADE6 and not a suppressor. The cloned ADE6 DNA was mapped on the RAD2-proximal fragment of chromosome VII by hybridization on yeast chromosomes separated by pulsed-field gel electrophoresis. Northern-blot hybridization experiments show that the ADE6 region produces two different mRNA species of approx. 5 and 2 kb. Disappearance of the larger, but not the smaller, transcript is associated with ade6 mutations. A threefold repression in the amount of the 5-kb ADE6 mRNA is observed when growth medium is supplemented with exogenous adenine.