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.

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.

Myoadenylate deaminase deficiency: a new disease of muscle.

Five cases of a new disease presented with muscular weakness or cramping after exercise; three of the cases also had an elevated serum creatine phosphokinase. Muscle biopsies were histologically normal but lacked adenylate deaminase by stain and solution assay, while the erythrocyte isozyme was normal. A clinical diagnostic test has been developed, and the human enzyme was separated by acrylamide-gel electrophoresis.

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.

Disruption of the purine nucleotide cycle. A potential explanation for muscle dysfunction in myoadenylate deaminase deficiency.

A patient with symptoms of easy fatigability, postexercise myalgias, and delayed recovery of muscle strength after activity is described. Skeletal muscle from this patient had <1.0% normal myoadenylate deaminase activity and NH(3) was not released from muscle after ischemic exercise. In association with this enzyme deficiency, exercise led to a >90% reduction in muscle content of adenine nucleotides. No inosine monophosphate accumulated after exercise and total purine content of the muscle fell to 21% of control. Repletion of the adenine nucleotide pool in this patient was delayed compared to controls, and ATP content had only returned to 68% of control at 165 min after exercise. These studies demonstrate that disruption of the purine nucleotide cycle as a consequence of myoadenylate deaminase deficiency results in marked alterations in ATP content of muscle, and potentially, these changes in ATP content could account for muscle dysfunction in this patient.

Myoadenylate deaminase deficiency. Functional and metabolic abnormalities associated with disruption of the purine nucleotide cycle.

To assess the role of the purine nucleotide cycle in human skeletal muscle function, we evaluated 10 patients with AMP deaminase deficiency (myoadenylate deaminase deficiency; MDD). 4 MDD and 19 non-MDD controls participated in an exercise protocol. The latter group was composed of a patient cohort (n = 8) exhibiting a constellation of symptoms similar to those of the MDD patients, i.e., postexertional aches, cramps, and pains; as well as a cohort of normal, unconditioned volunteers (n = 11). The individuals with MDD fatigued after performing only 28% as much work as their non-MDD counterparts. Muscle biopsies were obtained from the four MDD patients and the eight non-MDD patients at rest and following exercise to the point of fatigue. Creatine phosphate content fell to a comparable extent in the MDD (69%) and non-MDD (52%) patients at the onset of fatigue. Following exercise the 34% decrease in ATP content of muscle from the non-MDD subjects was significantly greater than the 6% decrease in ATP noted in muscle from the MDD patients (P = 0.048). Only one of four MDD patients had a measurable drop in ATP compared with seven of eight non-MDD patients. At end-exercise the muscle content of inosine 5'-monophosphate (IMP), a product of AMP deaminase, was 13-fold greater in the non-MDD patients than that observed in the MDD group (P = 0.008). Adenosine content of muscle from the MDD patients increased 16-fold following exercise, while there was only a twofold increase in adenosine content of muscle from the non-MDD patients (P = 0.028). Those non-MDD patients in whom the decrease in ATP content following exercise was measurable exhibited a stoichiometric increase in IMP, and total purine content of the muscle did not change significantly. The one MDD patient in whom the decrease in ATP was measurable, did not exhibit a stoichiometric increase in IMP. Although the adenosine content increased 13-fold in this patient, only 48% of the ATP catabolized could be accounted for by the combined increases of adenosine, inosine, hypoxanthine, and IMP. Studies performed in vitro with muscle samples from seven MDD and seven non-MDD subjects demonstrated that ATP catabolism was associated with a fivefold greater increase in IMP in non-MDD muscle. There were significant increases in AMP and ADP content of the muscle from MDD patients following ATP catabolism in vitro, while there was no detectable increase in AMP or ADP in non-MDD muscle. Adenosine content of MDD muscle increased following ATP catabolism, but there was no detectable increase in adenosine content of non-MDD muscle following ATP catabolism in vitro. These studies demonstrate that AMP deaminase deficiency leads to reduced entry of adenine nucleotides into the purine nucleotide cycle during exercise. We postulate that the resultant disruption of the purine nucleotide cycle accounts for the muscle dysfunction observed in these patients.

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.

Muscle adenylate deaminase deficiency. Report of six new cases.

We describe six adult patients (five men and one woman) out of 364 whose muscle biopsy specimens disclosed muscle adenylate deaminase deficiency. Two men had an associated dermatomyositis and another man had an associated progressive systemic sclerosis. Although the patients were different clinically, all complained of muscular weakness or poor exercise tolerance. The occurrence of muscle adenylate deaminase deficiency in both sexes suggests a possible autosomal mode of inheritance.

Exercising muscle does not produce hypoxanthine in adenylate deaminase deficiency.

The failure of forearm exercise to increase plasma hypoxanthine in subjects with adenylate deaminase deficiency confirms this enzyme's role in hypoxanthine production by normal forearm exercise. The conversion of adenosine monophosphate (AMP) to hypoxanthine may reflect an alternative method of adenosine triphosphate (ATP) regeneration in working muscle.

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.

Familial myoadenylate deaminase deficiency and exertional myalgia.

In 14 members of four families with a hereditary syndrome of exertional myalgia, five of eight muscle biopsies from symptomatic individuals showed histochemical and biochemical absence of myoadenylate deaminase (MADA). In the others, MADA biochemical activity was normal in two and reduced but not absent (intermediate level) in one. Asymptomatic relatives had normal histochemical MADA activity, but three had intermediate biochemical levels. In a survey of 302 routine muscle biopsies, 3 of 36 patient with myalgia had absence of MADA. Three of 266 biopsied for other conditions were MADA-deficient. Despite some inconsistencies, MADA deficiency seems to be relevant to this clinical syndrome.

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 in a patient with facial and limb girdle myopathy.

Absence of AMP-deaminase was demonstrated by histochemical and biochemical methods in a muscle biopsy of a 25-year-old woman with facial and limb girdle myopathy. Venous ammonia failed to rise after ischaemic exercise. This patient further contributes to the variety of clinical pictures associated with AMP-deaminase deficiency. Whereas AMP-deaminase has been shown to play an essential role in the regulation of adenine nucleotide metabolism in the liver, its physiological function in muscle remains uncertain.

Myoadenylate deaminase deficiency--muscle biopsy and muscle culture in a patient with gout.

AMP deaminase activity was undetectable by a sensitive spectrophotometric assay in the muscle biopsy of a 37-year-old man with gout and exercise-related cramps and myalgia. Venous ammonia failed to rise after ischemic exercise, but the diagnostic value of this test is uncertain because changes of plasma ammonia after exercise varied greatly in different normal individuals. In the patient, AMP deaminase activity was normal not only in erythrocytes, leukocytes and cultured fibroblasts but also in muscle cultures. Presence of AMP deaminase in muscle cultures was probably due to the expression of a fetal isoenzyme under separate genetic control from adult muscle AMP deaminase.

Myoadenylate deaminase deficiency or not? Observations on two brothers with exercise-induced muscle pain.

This paper describes 2 brothers with increasingly severe exercise-induced muscle pain and stiffness, beginning in adolescence. Histochemical studies showed that myoadenylate deaminase activity was absent in the propositus, but present in his younger brother. Biochemical examination of muscle homogenates confirmed these findings, with enzyme activity approximately 60% of the mean control value in the younger sibling. Red cell adenylate deaminase activity was normal in both cases. The possible relationship between the clinical and biochemical findings in these patients is discussed.

Myoadenylate deaminase deficiency in children.

Myoadenylate deaminase (MADA) is an enzyme which participates in the purine nucleotide cycle necessary for energy production in human skeletal muscle. Approximately 35 patients with deficiency of this enzyme have been reported; one-half experienced their initial difficulties in childhood. Children with "primary" MADA deficiency typically have symptoms including muscle cramps, stiffness, and post-exercise myalgia and weakness. In "secondary" MADA deficiency, the clinical findings have been variable with delayed motor development, hypotonia, cardiomyopathy, delayed speech development, and generalized weakness. In most cases creatine kinase determinations, nerve conduction velocity studies, and routine muscle histopathology have been normal. Diagnosis has been established by demonstrating an absence of MADA activity by either direct muscle enzyme assay or histochemical staining. In this report we describe a 12-year-old boy with primary MADA deficiency and contrast his symptoms with those of previously described pediatric patients.

Myoadenylate deaminase deficiency: diagnosis by forearm ischemic exercise testing.

These results indicate that measuring venous ammonia concentrations after forearm ischemic exercise is an effective means of screening for MADA deficiency but that submaximal exercise performance, whether due to weakness, pain or poor effort, can provide false positive results. Measurements of purine compounds released after exercise may increase the specificity of forearm ischemic exercise testing for MADA deficiency. The low level of purines released after exercise in MADA-deficient subjects supports the hypothesis that disordered purine metabolisms occurs when MADA activity is absent.

Myoadenylate deaminase deficiency: an enzyme defect in search of a disease.

The frequency of MAD deficiency in cases with exercise intolerance compared with the frequency in series of consecutive muscle biopsies suggests a relation between the deficiency and exercise intolerance. Deficiency cases can be presumed by an impaired NH3 production during ischaemic exercise. The ischaemic exercise test also gives information concerning the familial character of the deficiency.

Skeletal muscle adenosine, inosine and hypoxanthine release following ischaemic forearm exercise in myoadenylate deaminase deficiency and McArdle's disease.

Plasma adenosine, inosine and hypoxanthine concentrations were assayed in 7 controls, 5 MADD patients and 6 McArdle patients before and after ischaemic forearm exercise. The MADD patients showed a significantly lower increase in plasma inosine and hypoxanthine following exercise as compared to the controls. In the McArdle patients the increase in plasma inosine and hypoxanthine after exercise did not differ significantly from the control values. The plasma adenosine increase was very low in the three test groups and there were no significant differences.

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.