Inborn Errors Of Pyrimidine Metabolism Research Articles

Fast, Direct Dihydrouracil Quantitation in Human Saliva: Method Development, Validation, and Application.

Background. Salivary metabolomics is garnering increasing attention in the health field because of easy, minimally invasive saliva sampling. Dihydrouracil (DHU) is a metabolite of pyrimidine metabolism present in urine, plasma, and saliva and of fluoropyrimidines-based chemotherapeutics. Its fast quantification would help in the identification of patients with higher risk of fluoropyrimidine-induced toxicity and inborn errors of pyrimidine metabolism. Few studies consider DHU as the main salivary metabolite, but reports of its concentration levels in saliva are scarce. We propose the direct determination of DHU in saliva by reversed-phase high-performance liquid chromatography (RP-HPLC-UV detector) as a simple, rapid procedure for non-invasive screening. Methods. The method used was validated and applied to 176 saliva samples collected from 21 nominally healthy volunteers and 4 saliva samples from metastatic colorectal cancer patients before and after receiving 5-fluorouracil chemotherapy. Results. DHU levels in all samples analyzed were in the μmol L−1 range or below proving that DHU is not the main metabolite in saliva and confirming the results found in the literature with LC-MS/MS instrumentation. Any increase of DHU due to metabolism dysfunctions can be suggestive of disease and easily monitored in saliva using common, low-cost instrumentation available also for population screening.

Quantitation of Pyrimidine in Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Inborn errors of pyrimidine metabolism result from deficiencies in pyrimidine de novo synthesis, degradation, and salvage pathways. Enzymatic deficiencies in pyrimidine catabolism lead to mitochondrial neurogastrointestinal encephalopathy (MNGIE), pyrimidinuria, dihydropyrimidinuria, ureidopropionic aciduria, and other disorders. While MNGIE presents with gastrointestinal dysmotility, cachexia and leukoencephalopathy, pyrimidinuria, and dihydropyrimidinuria may show symptoms of epilepsy, autism, mental retardation, and dysmorphic features. The application of HPLC-MS/MS facilitates rapid screening of pyrimidine metabolites. Here we describe a sensitive and reliable LC-MS/MS method for quantitative determination of uracil, thymine, thymidine, dihydrouracil, and dihydrothymine in urine that are diagnostic biomarkers of MNGIE, pyrimidinuria, and dihydropyrimidinuria.

HEREDITARY OROTIC ACIDURIA WITH UNUSUAL PRESENTATION- A RARE CASE REPORT

Hereditary orotic aciduria (HOA) is an extremely rare inborn error of pyrimidine metabolism. It results from a defect in the uridine-5-monophosphate synthase (UMPS) gene. It can result in megaloblastic anemia, developmental delay and crystalluria. Molecular genetics is the conrm diagnostic modality. Uridine triacetate is the only established treatment. We thus report a case of 13 month male child having developmental delay with regression, dysmorphic facies, nystagmus, skeletal abnormalities and massive hepatosplenomegaly with past history of repeated LRTI. After clinical evaluation, an impression of storage disorder was presumed. Bicytopenia was also obtained in CBC with high MCV. After detailed evaluation with haematological, bone marrow, TMS and GCMS of urine, a diagnosis of hereditary orotic aciduria was established. Although extremely rare, hereditary orotic aciduria should be suspected in any child with megaloblastic bone marrow, immunodeciency or when developmental delay and anemia coexist

A novel stop-gain mutation in DPYS gene causing Dihidropyrimidinase deficiency, a case report

BackgroundDihidropyrimidinase (DHP) deficiency is an inherited inborn error of pyrimidine metabolism with a variable clinical presentation and even asymptomatic subjects. Dihydropyrimidinase is encoded by the DPYS gene, thus pathogenic mutations in this gene can cause DHP deficiency. To date, several variations in the DPYS gene have been reported but only 23 of them have been confirmed to be pathogenic. Therefore, the biochemical, clinical and genetic aspects of this disease are still unclear.Case presentationHere, we report a 22-year-old woman with DHP deficiency. To identify the genetic cause of DHP deficiency in this patient, Whole Exome Sequencing (WES) was performed, which revealed a novel homozygote stop gain mutation (NM_001385: Exon 9, c.1501 A > T, p.K501X) in the DPYS gene. Sanger sequencing was carried out on proband and other family members in order to confirm the identified mutation. According to the homozygote genotype of the patient and heterozygote genotype of her parents, the autosomal recessive pattern of inheritance was confirmed. In addition, bioinformatics analysis of the identified variant using Mutation Taster and T-Coffee Multiple Sequence Alignment showed the pathogenicity of mutation. Moreover, mRNA expression level of DPYS gene in the proband’s liver biopsy showed about 6-fold reduction compared to control, which strongly suggested the pathogenicity of the identified mutation.ConclusionsThis study identified a novel pathogenic stop gain mutation in DPYS gene in a DHP deficient patient. Our findings can improve the knowledge about the genetic basis of the disease and also provide information for accurate genetic counseling for the families at risk of these types of disorders.

Deficiency of perforin and hCNT1, a novel inborn error of pyrimidine metabolism, associated with a rapidly developing lethal phenotype due to multi-organ failure

Pyrimidine nucleotides are essential for a vast number of cellular processes and dysregulation of pyrimidine metabolism has been associated with a variety of clinical abnormalities. Inborn errors of pyrimidine metabolism affecting enzymes in the pyrimidine de novo and degradation pathway have been identified but no patients have been described with a deficiency in proteins affecting the cellular import of ribonucleosides. In this manuscript, we report the elucidation of the genetic basis of the observed uridine-cytidineuria in a patient presenting with fever, hepatosplenomegaly, persistent lactate acidosis, severely disturbed liver enzymes and ultimately multi-organ failure. Sequence analysis of genes encoding proteins directly involved in the metabolism of uridine and cytidine showed two variants c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in SLC28A1, encoding concentrative nucleotide transporter 1 (hCNT1). Functional analysis showed that these variants affected the three-dimensional structure of hCNT1, altered glycosylation and decreased the half-life of the mutant proteins which resulted in impaired transport activity. Co-transfection of both variants, mimicking the trans disposition of c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in the patient, significantly impaired hCNT1 biological function. Whole genome sequencing identified two pathogenic variants c.50delT; p.(Leu17Argfs*34) and c.853_855del; p.(Lys285del) in the PRF1 gene, indicating that our patient was also suffering from Familial Hemophagocytic Lymphohistiocytosis type 2. The identification of two co-existing monogenic defects might have resulted in a blended phenotype. Thus, the clinical presentation of isolated hCNT1 deficiency remains to be established.

Inborn errors of pyrimidine metabolism: clinical update and therapy.

Inborn errors involving enzymes essential for pyrimidine nucleotide metabolism have provided new insights into their fundamental physiological roles as vital constituents of nucleic acids as well as substrates of lipid and carbohydrate metabolism and in oxidative phosphorylation. Genetic aberrations of pyrimidine pathways lead to diverse clinical manifestations including neurological, immunological, haematological, renal impairments, adverse reactions to analogue therapy and association with malignancies. Maintenance of cellular nucleotides depends on the three aspects of metabolism of pyrimidines: de novo synthesis, catabolism and recycling of these metabolites. Of the ten recognised disorders of pyrimidine metabolism treatment is currently restricted to only two disorders: hereditary orotic aciduria (oral uridine therapy) and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; allogeneic hematopoetic stem cell transplant and enzyme replacement). The ubiquitous role that pyrimidine metabolism plays in human life highlights the importance of improving diagnostic evaluation in suggestive clinical settings, which will contribute to the elucidation of new defects, future development of novel drugs and therapeutic strategies. Limited awareness of the expanding phenotypic spectrum, with relatively recent descriptions of newer disorders, compounded by considerable genetic heterogeneity has often contributed to the delays in the diagnosis of this group of disorders. The lack of an easily recognisable, easily measurable end product, akin to uric acid in purine metabolism, has contributed to the under-recognition of these disorders.This review describes the currently known inborn errors of pyrimidine metabolism, their variable phenotypic presentations, established diagnostic methodology and recognised treatment options.

Exploring the structural features of Aspartate Trans Carbamoylase (TtATCase) from Thermus thermophilus HB8 through in silico approaches: a potential drug target for inborn error of pyrimidine metabolism

Enzymes involved in the pyrimidine biosynthesis pathway have become an important target for the pharmacological intervention. One among those enzymes, Aspartate Trans Carbamoylase (ATCase), catalyses the condensation of aspartate and carbamoyl phosphate to form N-carbamoyl-l-aspartate and inorganic phosphate. The present study provides the molecular insights into the enzyme ATCase. The three-dimensional structure of ATCase from Thermus thermophilus HB8 was modeled based on the crystal structure of ATCase in Pyrococcus abyssi (PDB ID:1ML4). Molecular dynamics simulation was performed to identify the conformational stability of TtATCase with and without its ligand complexes. Based on the pharmacokinetic properties and the glide-docking scores of ligands from four databases (Maybridge, Binding, Asinex and Technology for Organic Synthesis (TOS laboratory) for the screening of ligands, we identified four potential ligand molecules for TtATCase. From the molecular docking results, we proposed that the residues Thr53, Arg104, and Gln219 are consistently involved in strong hydrogen-bonding interactions and play a vital role in the TtATCase activity. From the results of molecular dynamics simulation, the ligand molecules are found to bind appropriately to the target enzyme. However, the structure of TtATCase needs to be determined experimentally to confirm this.

A Neonate with Recurrent Vomiting and Generalized Hypotonia Diagnosed with a Deficiency of Dihydropyrimidine Dehydrogenase

Deficiency of dihydropyrimidine dehydrogenase (DPD) is a rare inborn error of pyrimidine metabolism. To date, only about 50 patients are known worldwide. The clinical picture is varied and is not yet fully described. Most patients are diagnosed at the age of 1–3 years. We present a patient diagnosed 8 weeks postpartum. The female patient presented in the first 3 days after birth with agitation, choking, and vomiting. Six weeks later, the patient presented again with vomiting and insufficient weight gain. Metabolic screening of urine showed a strongly increased excretion of uracil and thymine, with no other abnormalities. This suggested a deficiency of DPD which was confirmed by enzyme analysis in peripheral blood mononucleair (PBM) cells (patient: activity <0.01 nmol/mg/h; controls: 9.9 ± 2.8 nmol/mg/h). The patient was homozygous for the IVS14+1G>A mutation. MRI of the brain showed some cerebral atrophy; myelinization appeared normal. Many patients with DPD-deficiency suffer from convulsions and mental retardation, some show microcephaly, feeding difficulties, autism, and hypertonia. Our patient showed feeding difficulties and in the second half-year she developed slight motor retardation and generalized hypotonia. Further observation of the development of the patient may shed more light on the relationship between clinical symptoms and DPD deficiency. DPD deficiency may present in newborns with vomiting and hypotonia as the main symptoms.

Dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil associated toxicity.

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is responsible for the breakdown of the widely used chemotherapeutic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. Because 5FU has a relative narrow therapeutic index, toxicity increases as the dose is escalated. Since more than 80% of 5FU is degraded by DPD a partial DPD deficiency is increasingly recognized as an important pharmacogenetic syndrome resulting in severe 5FU associated toxicity. Thus, patients with a low DPD activity and those heterozygous for a mutant DPD allele might be at risk for developing severe toxicity after the administration of 5FU. To evaluate the importance of this specific type of inborn error of pyrimidine metabolism in the aetiology of 5FU toxicity we have determined the DPD activity in peripheral blood mononuclear cells (PBM cells) of 37 cancer patients suffering from severe grade 3 and 4 toxicity after the administration of 5FU. In addition, the prevalence of mutant DPD alleles in patients with a low DPD activity has been investigated.

Defects in Pyrimidine Degradation Identified by HPLC-Electrospray Tandem Mass Spectrometry of Urine Specimens or Urine-soaked Filter Paper Strips

Urinary concentrations of thymine, uracil, and their degradation products are useful indicators of deficiencies of enzymes of the pyrimidine degradation pathway. We describe a rapid, specific method to measure these concentrations to detect inborn errors of pyrimidine metabolism. We used urine or urine-soaked filter-paper strips as samples and measured thymine, uracil, and their degradation products dihydrothymine, dihydrouracil, N:-carbamyl-ss-aminoisobutyric acid, and N:-carbamyl-ss-alanine. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. All pyrimidine degradation products could be measured in one analytical run of 15 min. Detection limits were 0.4-4 micromol/L. The intraassay imprecision (CV) of urine samples with added compounds was 1.3-12% for liquid urines and 1. 0-10% for filter-paper extracts of the urines. The interassay imprecision (CV) was 3-11% (100-200 micromol/L). Recoveries were 89-99% at 100-200 micromol/L and 95-106% at 1 mmol/L in liquid urines, and 93-103% at 100-200 micromol/L and 100-106% at 1 mmol/L in filter-paper samples. Correct identifications of deficiencies of the pyrimidine-degrading enzymes were readily made with urine samples from patients with known defects. HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders of the pyrimidine degradation pathway, and filter-paper samples allow easy collection, transport, and storage of urine samples.

Adenine phosphoribosyl transferase: assay using high performance liquid chromatography

Adenine phosphoribosyl transferase (APRT) (EC 2.4.2.7) is the enzyme which catalyses the reaction of the purine base adenine with S-phosphoribosyl-l-pyrophosphate (PP-ribose-P) to form the mononu~Ieotide adenosine monophosphate. Human APRT is a cytoplasmic enzyme present in all tissues with the highest specific activities being found in nucleated red cells. Severe homozygous deficiency of APRT is associated with nephrolithiasis and passage of urinary calculi composed of 2,8-dihydroxyadenine [1,2], suggesting that the enzyme has an important salvage function in man. Partial APRT deficiency [3,4] with 25-40% residual enzyme activity in red cell lysates is a relatively common heterozygous trait occurring in about 1% of the population [5] and is not associated with disease. Elevated levels of red cell APRT activity are found in the neonatal period [6] and in some patients with megalobtastic anaemia [7], as a direct consequence of reticulocytosis. They are also a characteristic finding in patients with Lesch-Nyhan syndrome [8] and the X-linked form of severe gout and hyperuricaemia associated with hypoxanthine-guanine phosphoribosyl transferase deficiency 191, where substrate stabilisation of PP-ribose-P may be important. Elevated erythrocyte APRT levels have also recently been reported in patients with hereditary erotic aciduria [lo], an inborn error of pyrimidine metabolism. Current methods for the quantitative assay of the enzyme are mainly radiochemical [ll-131. In this paper we describe an assay for red cell APRT activity utilising high performance liquid chromatography which is suitable for use in clinical laboratories,