AICA-ribosiduria due to ATIC deficiency: Delineation of the phenotype with three novel cases, and long-term update on the first case.

5-Amino-4-imidazolecarboxamide-ribosiduria (AICA)-ribosiduria is an exceedingly rare autosomal recessive condition resulting from the disruption of the bifunctional purine biosynthesis protein PURH (ATIC), which catalyzes the last two steps of de novo purine synthesis. It is characterized biochemically by the accumulation of AICA-riboside in urine. AICA-ribosiduria had been reported in only one individual, 15 years ago. In this article, we report three novel cases of AICA-ribosiduria from two independent families, with two novel pathogenic variants in ATIC. We also provide a clinical update on the first patient. Based on the phenotypic features shared by these four patients, we define AICA-ribosiduria as the syndromic association of severe-to-profound global neurodevelopmental impairment, severe visual impairment due to chorioretinal atrophy, ante-postnatal growth impairment, and severe scoliosis. Dysmorphic features were observed in all four cases, especially neonatal/infancy coarse facies with upturned nose. Early-onset epilepsy is frequent and can be pharmacoresistant. Less frequently observed features are aortic coarctation, chronic hepatic cytolysis, minor genital malformations, and nephrocalcinosis. Alteration of the transformylase activity of ATIC might result in a more severe impairment than the alteration of the cyclohydrolase activity. Data from literature points toward a cytotoxic mechanism of the accumulated AICA-riboside.

Identification of ATIC as a Novel Target for Chemoradiosensitization.

PURPOSE: Mutations in the gene encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final 2 steps of the purine de novo biosynthetic pathway, were identified in a subject referred for radiation sensitivity testing. Functional studies were performed to determine whether ATIC inhibition was radiosensitizing and, if so, to elucidate the mechanism of this effect and determine whether small molecule inhibitors of ATIC could act as effective radiosensitizing agents. METHODS AND MATERIALS: Both small interfering RNA knockdown and small molecule inhibitors were used to inactivate ATIC in cell culture. Clonogenic survival assays, the neutral comet assay, and γH2AX staining were used to assess the effects of ATIC inhibition or depletion on cellular DNA damage responses. RESULTS: Depletion of ATIC or inhibition of its transformylase activity significantly reduced the surviving fraction of cells in clonogenic survival assays in multiple cancer cell lines. In the absence of ionizing radiation exposure, ATIC knockdown or chemical inhibition activated cell cycle checkpoints, shifting cells to the more radiosensitive G2/M phase of the cell cycle, and depleted cellular adenosine triphosphate but did not result in detectable DNA damage. Cells in which ATIC was knocked down or inhibited and then treated with ionizing radiation displayed increased numbers of DNA double-strand breaks and a delay in the repair of those breaks relative to irradiated, but otherwise untreated, controls. Supplementation of culture media with exogenous adenosine triphosphate ameliorated the DNA repair phenotypes. CONCLUSIONS: These findings implicate ATIC as an effective, and previously unrecognized, target for chemoradiosensitization and, more broadly, suggest that purine levels in cells might have an underappreciated role in modulating the efficiency of DNA damage responses that could be exploited in radiosensitizing strategies.

Bifunctional enzyme ATIC promotes propagation of hepatocellular carcinoma by regulating AMPK-mTOR-S6 K1 signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the cancer types with poor prognosis. To effectively treat HCC, new molecular targets and therapeutic approaches must be identified. 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate (IMP) cyclohydrolase (ATIC), a bifunctional protein enzyme, catalyzes the last two steps of the de novo purine biosynthetic pathway. Whether ATIC contributes to cancer development remains unclear. METHODS: ATIC mRNA levels in different types of human HCC samples or normal tissues were determined from Gene Expression across Normal and Tumor tissue (GENT) database. The expression level of ATIC in human HCC samples or cell lines were examined by RT-PCR and western blot. Overall survival and disease-free survival of HCC patients in the ATIC low and ATIC high groups were determined by Kaplan-Meier analysis. Effects of ATIC knockdown by lentivirus infection were evaluated on cell-proliferation, cell-apoptosis, colony formation and migration. The mechanisms involved in HCC cells growth, apoptosis and migration were analyzed by western blot and Compound C (C-C) rescue assays. RESULTS: Here, we first demonstrated that expression of ATIC is aberrantly up-regulated in HCC tissues and high level of ATIC is correlated with poor survival in HCC patients. Knockdown of ATIC expression resulted in a dramatic decrease in proliferation, colony formation and migration of HCC cells. We also identified ATIC as a novel regulator of adenosine monophosphate-activated protein kinase (AMPK) and its downstream signaling mammalian target of rapamycin (mTOR). ATIC suppresses AMPK activation, thus activates mTOR-S6 K1-S6 signaling and supports growth and motility activity of HCC cells. CONCLUSION: Taken together, our results indicate that ATIC acts as an oncogenic gene that promotes survival, proliferation and migration by targeting AMPK-mTOR-S6 K1 signaling.

Adenylate deaminase deficiency in a mutant murine T cell lymphoma cell line.

From a population of wild type S49 cells, a clone, DTB6, was isolated in a single step from selective medium containing thymidine and dibutyryl cyclic AMP that exhibited a 60% deficiency in AMP deaminase (AMP-D) activity. The AMP-D deficiency conferred to the DTB6 cells a striking susceptibility to killing by low concentrations of either adenine or adenosine, the latter in the presence of an inhibitor of adenosine deaminase activity. This growth supersensitivity of DTB6 cells toward adenine could be ameliorated by the addition of hypoxanthine to the culture medium. Immunoprecipitation of AMP-D from wild type and mutant cells revealed that the DTB6 cell line contained markedly diminished amounts of the AMP-D isozyme which reacts with antisera to the predominant isoform expressed in adult kidney. The quantities of the AMP-D isozyme immunoprecipitated by antisera raised to the predominant isoform prepared from adult heart were equivalent in the two cell lines. Although Northern blot analyses revealed no alterations in mRNA sizes or levels encoded by either of the AMP-D genes, Southern blots of genomic DNA hybridized to a cDNA specific for the ampd2 gene revealed the presence of a new BamHI restriction fragment in the DNA of DTB6 cells. These data suggested that a point mutation has occurred in the ampd2 gene of DTB6 cells which encodes the AMP-D isozyme recognized by the kidney antisera. The DTB6 cells also possessed a virtual complete deficiency in thymidine kinase activity. The two enzyme deficiencies were distinguishable. The ability to isolate single step mutants with two seemingly independent biochemical abnormalities raises the speculation that there may be some link between cellular functions responsible for purine nucleotide and thymidine metabolism.

Medical implications of the lactate and ammonia relationship in anaerobic exercise.

The genesis of the modern ischemic forearm exercise test (IFET) employing the measurement of lactate and ammonia as countervailing metabolites is briefly reviewed, along with the application of the lactate ammonia exercise ratio in the diagnosis of myoadenylate deaminase deficiency and disorders of glycolysis and glycogenolysis. Two cases are presented to illustrate the response patterns elicited, their reproducibility, the types of parameters that can be quantified, and the role the IFET may play in the differential diagnosis of fitness failures. The role of the ammonia measurement is emphasized here because the lactate response is more familiar. The roles of hypoxanthine responses and of muscle ammonia measurements in the evaluation of cases are examined, and some pertinent and recently introduced analytic methods are cited. The applications of newer approaches, such as aspiration biopsy and N-14 NMR spectroscopy, are discussed, along with an example of the new clinical defects in enzymes and membrane carriers that should be anticipated during the utilization of the IFET.

Metabolic causes of myoglobinuria.

To evaluate the proportion of cases of myoglobinuria that can be ascribed to specific metabolic defects, we have studied eight enzymes--phosphorylase, phosphorylase kinase, phosphofructokinase (PFK), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGAM), lactate dehydrogenase (LDH), carnitine palmitoyltransferase (CPT), and myoadenylate deaminase (MAD)--in muscle biopsy specimens from 77 consecutive patients with myoglobinuria (documented in 44, suspected in 33). Enzyme defects were found in 36 patients: CPT deficiency in 17, phosphorylase deficiency in 10, phosphorylase kinase deficiency in 4, MAD deficiency in 3, PGK deficiency in 1, and a combined defect of CPT and MAD in 1. Exercise was the main precipitating factor, both in patients with and in those without detectable enzymopathies. Thirty patients had specific enzymopathies without myoglobinuria: 14 had phosphorylase deficiency, 9 had MAD deficiency, 3 had phosphorylase kinase deficiency, 3 had PFK deficiency, and 1 had PGAM deficiency. Systematic biochemical evaluation of muscle biopsy specimens revealed specific enzymopathies in about half of the patients with idiopathic myoglobinuria. The rest may have blocks of metabolic pathways not yet studied routinely, such as beta oxidation, or genetic defects of the sarcolemma, such as Becker's muscular dystrophy.

Metabolism of D-ribose administered continuously to healthy persons and to patients with myoadenylate deaminase deficiency.

D-ribose was administered orally or intravenously over at least 5 h to eight healthy volunteers and five patients with myoadenylate deaminase deficiency. Intravenous administration rates were 83, 167, and 222 mg/kg/h, which were well tolerated but oral administration of more than 200 mg/kg/h caused diarrhea. The average steady state serum ribose level ranged between 4.8 mg/100 ml (83 mg/kg/h, oral administration) and 81.7 mg/100 ml (222 mg/kg/h, intravenous administration). Serum glucose level decreased during ribose administration. The intestinal absorption rate of orally administered ribose was 87.8%-99.8% of the intake at doses up to 200 mg/kg/h without first pass effect. Urinary losses were 23% of the intravenously administered dose at 222 mg/kg/h. Ribose appeared to be excreted by glomerular filtration without active reabsorption; a renal threshold could not be demonstrated. The amount of ribose transported back from the tubular lumen depended on the serum ribose level. There was no difference in ribose turnover in healthy subjects and patients with MAD deficiency.

The deamination of adenosine and adenosine monophosphate in Plasmodium falciparum-infected human erythrocytes: in vitro use of 2'deoxycoformycin and AMP deaminase-deficient red cells.

The role of enzymatic deamination of adenosine monophosphate (AMP) and adenosine in the in vitro growth of the malaria parasite Plasmodium falciparum was investigated by means of human red cells deficient in AMP deaminase to which the adenosine deaminase inhibitor 2'-deoxycoformycin was added. Malaria parasites grew normally in red cells lacking one or both of these enzyme activities. As a further probe of adenosine triphosphate (ATP) catabolism, both infected and uninfected RBCs were incubated with NaF (with and without 2'-deoxycoformycin) and the purine nucleotide/nucleoside content was analyzed by high-performance liquid chromatography (HPLC). Uninfected RBCs lacking either AMP or adenosine deaminase were able to bypass the enzyme block and degrade ATP to hypoxanthine. Uninfected RBCs with both deaminases blocked were unable to produce significant quantities of hypoxanthine. On the other hand, infected RBCs were able to bypass blockade of both deaminases and produce hypoxanthine and adenosine. These findings establish that deamination of adenosine and/or AMP are not essential for plasmodial growth. However, further work will be required to elucidate the pathways that permit the parasites to bypass these catabolic steps.

AMP deaminase and thymidine kinase deficiencies in a mutant mouse S49 cell clone.

From a mutagenized population of wild type S49 cells, a clone was isolated in a single step that possessed functional and biochemical deficiencies in both AMP deaminase and thymidine kinase activities. This mutant cell line, DTB6, was selected in semi-solid medium containing 1mM thymidine and 1mM dibutyryl cyclic AMP. In comparative growth rate experiments, DTB6 cells were considerably less sensitive than parental cells to the growth inhibitory effects of thymidine. In contrast, DTB6 cells were much more sensitive to the cytotoxic effects of adenine and adenosine. The supersensitivity of DTB6 cells toward adenine could be ameliorated by the addition of hypoxanthine to the culture medium. The growth phenotype of the mutant cells could be attributed to deficiencies in two enzyme activities. First, DTB6 cells possessed a 60-70% deficiency in AMP deaminase activity, although the residual activity appeared kinetically similar to the wild type enzyme. Second, DTB6 cells possessed a virtual complete deficiency in thymidine kinase activity. Both enzyme deficiencies behaved in a recessive fashion in intraspecies hybrids. Revertants of DTB6 cells possessed wild type levels of AMP deaminase activity but remained deficient in thymidine kinase activity, while another revertant of DTB6 cells expressed 11% of the wild type thymidine kinase level but did not perceptibly change its AMP deaminase activity. The ability to isolate single step mutants with two seemingly independent biochemical abnormalities raises the speculation that there may be some link between cellular functions responsible for purine nucleotide and thymidine metabolism.

Myoadenylate deaminase deficiency: a clinical, genetic, and biochemical study in nine families.

The clinical significance of myoadenylate deaminase (MAD) deficiency and its mode of inheritance is still questioned. There were 36 relatives of 9 unrelated MAD deficient patients who were examined with the aid of a standardized ischemic forearm test: 8 new cases of MAD deficiency were detected, 5 of which were confirmed histochemically and biochemically. Obligate heterozygotes showed a normal ammonia production and MAD staining, but the mean activity of the enzyme was significantly less than in a group of controls. The results obtained from the family study strongly suggest an autosomal recessive mode of inheritance. However, only 2 of the 8 newly found MAD deficient individuals complained of exertional myalgia, whereas the remaining 6 were without any symptoms or complaints. This finding casts doubt on the clinical significance of MAD deficiency and the relationship of the deficiency state with exertional myalgia.

Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells.

A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles between pyrimidine deoxyribosides and their 5'-phosphates. Such cycles were suggested earlier to set the rates of cellular import and export of deoxyribosides, thereby influencing pool sizes (V. Bianchi, E. Pontis, and P. Reichard, Proc. Natl. Acad. Sci. USA 83:986-990, 1986). While normal V79 cells derived more than 80% of their dTTP from CDP reduction via deamination of dCMP, the mutant cells had to rely completely on UDP reduction for de novo synthesis of dTTP, which became limiting for DNA synthesis. Because of the allosteric properties of ribonucleotide reductase, CDP reduction was not diminished, leading to a large expansion of the dCTP pool. The increase of this pool was kept in check by a shift in the balance of the deoxycytidine/dCMP cycle towards the deoxynucleoside, leading to massive excretion of deoxycytidine. In contrast, the balance of the deoxyuridine/dUMP cycle was shifted towards the nucleotide, facilitating import of extracellular deoxynucleosides.

Myoadenylate deaminase deficiency.

Myoadenylate deaminase (MAD) is the rate-limiting enzyme in the purine nucleotide cycle which is biochemically linked to glycolysis and the citric cycle and thereby providing energy during intense muscular activity. In muscle fibers, myoadenylate deaminase operates at considerably higher activity levels than in other organs. First detected using enzyme-histochemical methods, it now appears that deficiency of myoadenylate deaminase is one of the most frequent enzyme defects in muscle. The primary defect may occur as an isolated nosological entity or not infrequently it is also associated with a large spectrum of different neuromuscular conditions. It seems to be the primary unassociated MAD deficiency that has recently become amenable to successful treatment with D-ribose in high doses. Secondary MAD deficiency may occur in muscle fibers and muscles that have undergone structural damage as seen, for instance, in polymyositis, muscular dystrophy, and denervation. The wealth of biochemical, morphological, and clinical data that has accumulated since the discovery of MAD deficiency during the past decade provides nosological significance of MAD deficiency as a real entity.

Myoadenylate deaminase deficiency: absence of correlation with exercise intolerance in 452 muscle biopsies.

A histochemical assay was routinely performed of myoadenylate deaminase (MAD) in muscle biopsy specimens. MAD was absent in 13 cases, i.e. 2.9% of the specimens. In 10 cases the deficiency was confirmed biochemically. The diagnoses in the 13 patients were: polyneuropathy (n = 5), infantile spinal muscular atrophy (n = 3), congenital myopathy with type 2 fibre atrophy, facioscapulohumeral myopathy, polymyositis, myotonic dystrophy and hyperornithinaemia with gyrate atrophy of the retina. In contrast, 35 unrelated patients presenting with exercise-related muscle cramps or pains showed normal histochemical MAD activity. The biopsy specimens in all of these patients were essentially normal and in none of them was the diagnosis of a neuromuscular disease made. The results failed to confirm the association of MAD deficiency with aches, cramps and pains or exertional myalgia.

Myoadenylate deaminase deficiency and forearm ischemic exercise testing.

Myoadenylate deaminase (MADA) deficiency has been associated with symptoms of postexertional aches, cramps, weakness, and skeletal muscle dysfunction. Measurement of plasma lactate and ammonia concentrations after forearm ischemic exercise has been suggested as a screening test for this disorder. We performed forearm ischemic tests on 3 patients with histochemically defined MADA deficiency and 13 healthy control subjects, in a standardized fashion. Our results demonstrated that subject effort and/or performance during the exercise portion of testing is a critical variable. In addition to lactate and ammonia, plasma purine compounds (adenosine, inosine, and hypoxanthine) were measured. The finding of decreased purine release after exercise in MADA-deficient patients compared with that in normal individuals increases the specificity of the test and supports the hypothesis that disordered purine metabolism occurs in MADA deficiency.

McArdle's disease with myoadenylate deaminase deficiency: observations in a combined enzyme deficiency.

Exercise and work potential of a patient with coexistent myophosphorylase and myoadenylate deaminase (AMPDA) deficiency was compared with that of three patients with myophosphorylase deficiency alone. The patient with the combined defect failed to produce an abnormal rise in serum ammonia or hypoxanthine as seen in the other patients after forearm exercise. Maximum oxygen consumption and work rates during cycle ergometer testing were similar in all patients, but well below controls. The occurrence of two defects involving short-term energy metabolism in muscle presents an opportunity to define further the metabolic role of AMPDA.

AMP deaminase deficiency: study of the human skeletal muscle purine metabolism during ischaemic isometric exercise.

Muscle biopsies were taken from 10 control subjects and five AMP deaminase (AMPD) deficient individuals before and after an ischaemic isometric exercise test and analysed for purine nucleotide, NAD+, creatine phosphate (CP) and lactate content. The decrease of ATP induced by the exercise test was significantly lower in the AMPD deficient patients than in the controls, but the decrease of creatine phosphate and the increase of lactate did not differ. There were no significant differences in the exertional performance level between patients and controls and no evidence was obtained of an increased energy expenditure per unit of performance in AMPD deficiency. The AMPD deficient individuals were equally capable of maintaining a high adenylate energy charge (EC) as the control subjects, which indicates a normal regulation of the balance between ATP consumption and ATP regeneration. ATP, ADP and total adenine nucleotide (TAN) but not AMP, were significantly elevated in the AMPD deficient patients as compared with the controls before as well as after the exercise test. This underlines the role of AMPD activity in the adenine nucleotide catabolism of skeletal muscle.