X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: heterozygote has two clonal populations.

Clones of skin fibroblasts cultured from the mother of two sons with X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency (Lesch-Nyhan syndrome) were assayed for activity of this enzyme by measurement of the incorporation of (3)H-guanine into guanylic acid as counts per minute per microgram of protein and by autoradiography. The demonstration of two populations of clones, wild-type clones with normal enzyme activity and mutant clones unable to incorporate (3)H-guanine, is evidence that the locus for hypoxanthineguanine phosphoribosyl transferase on one of the X chromosomes is inactive.

Positive newborn screen in the biochemically normal infant of a mother with treated holocarboxylase synthetase deficiency.

Expanded programmes of newborn screening permit early diagnosis in time to prevent serious complications. These programmes have begun to detect patients who might otherwise remain asymptomatic. An additional confounding variable is the positive screen that results from maternal rather than neonatal disease. This was the case in an infant in whom elevated hydroxyisovalerylcarnitine (C(5)OH) in his newborn screen was the result of placental transfer from his mother, whose holocarboxylase synthetase deficiency was being successfully treated with biotin. The mother had been diagnosed and treated with biotin prenatally. She had no phenotypic feature of holocarboxylase synthetase deficiency, most importantly no episodes ever of acute metabolic acidosis. In the infant a repeat screen was also positive. On day 28 the infant's plasma C(5)OH carnitine was 0.05 mumol/L (normal) and urinary organic acids on day 39 were normal. The mother's excretion of 3-hydroxyisovaleric acid was 109 mmol/mol creatinine. These observations indicate that holocarboxylase synthetase deficiency is one more maternal metabolic disease which may lead to a positive screen in her unaffected newborn infant. They also make the point that holocarboxylase synthetase deficiency in an infant should be detectable in programmes of neonatal screening, which was not clear previously.

Inhibition by propionyl-coenzyme A of N-acetylglutamate synthetase in rat liver mitochondria. A possible explanation for hyperammonemia in propionic and methylmalonic acidemia.

In the search for the mechanism by which hyperammonemia complicates propionic and methylmalonic acidemia the effects of a series of acyl-coenzyme A (CoA) derivatives were studied on the activity of N-acetylglutamate synthetase in rat liver mitochondria using acetyl-CoA as substrate. Propionyl-CoA was found to be a competitive inhibitor. The inhibition constant of 0.71 mM is in the range of concentrations of propionate found in the serum of patients with propionic and methylmalonic acidemia. Propionyl-CoA was also found to be a substrate for N-acetylglutamate synthetase, forming N-propionylglutamate. This compound was a weak activator of rat liver carbamoylphosphate synthetase; the activation constant was 1.1 mM as compared with 0.12 mM for N-acetylglutamate. A decreased level of N-acetylglutamate in liver mitochondria that would follow inhibition of N-acetylglutamate synthetase by propionyl-CoA would be expected to lead to hyperammonemia. Methylmalonyl-CoA, tiglyl-CoA, and isovaleryl-CoA at a concentration of 3 mM caused 30-70% inhibition of N-acetylglutamate synthetase. 3the latter two compounds are readily detoxified by the formation of N-acylglycine conjugates in liver, which may prevent large accumulations and could explain why hyperammonemia is not characteristic of patients with beta-ketothiolase deficiency or isovaleric acidemia in whom these compounds would be expected to be elevated.

Mutant holocarboxylase synthetase: evidence for the enzyme defect in early infantile biotin-responsive multiple carboxylase deficiency.

Biotin-responsive multiple carboxylase deficiency is an inherited disorder of organic acid metabolism in man in which there are deficiencies of propionyl-coenzyme A (CoA), 3-methylcrotonyl-CoA, and pyruvate carboxylases that can be corrected with large doses of biotin. It has been proposed that the basic defect in patients with the early infantile form of the disease is in holocarboxylase synthetase, the enzyme that covalently attaches biotin to the inactive apocarboxylases to form active holocarboxylases. We have developed an assay for holocarboxylase synthetase in extracts of human fibroblasts using as substrate apopropionyl-CoA carboxylase partially purified from livers of biotin-deficient rats. Fibroblasts from the initial patient with the infantile form of biotin-responsive multiple carboxylase deficiency were shown to have abnormal holocarboxylase synthetase activity with a maximum velocity about 30-40% of normal, a Km for ATP of 0.3 mM similar to the normal Km of 0.2 mM, and a highly elevated Km for biotin of 126 ng/ml, about 60 times the normal Km of 2 ng/ml. These results show that the primary defect in this patient is a mutation affecting holocarboxylase synthetase activity, and thus a genetic defect of the metabolism of biotin.

Defective glycine cleavage system in nonketotic hyperglycinemia. Occurrence of a less active glycine decarboxylase and an abnormal aminomethyl carrier protein.

The activities of then glycine cleavage system in the liver and brain of patient with nonketotic hyperglycinemia was extremely low as compared with those of control human liver and brain. The activities of glycine decarboxylase (P-protein) and the aminomethyl carrier protein (H-protein), two of the four protein components of the glycine cleavage system, were considerably reduced in both the liver and brain; the extent of reduction was greater in the H-protein. The activity of the T-protein was normal. Purified H-protein from the patient did not react with lipoamide dehydrogenase, and titration of thiol groups with [2,3-14C]N-ethylmaleimide suggested that this H-protein is devoid of lipoic acid. This structural abnormality in the H-protein is considered to constitute the primary molecular lesion in this patient with non-ketotic hyperglycinemia. Immunochemical studies using an antibody specific for P-protein showed that the patient was due to reduction of the catalytic activity of the protein rather than a decrease in the actual amount of the P-protein. Partial inactivation of P-protein could result secondarily from impaired metabolism of glycine resulting from deficiency in the activity of H-protein. However, the H-protein from the patient could stimulate the P-protein catalyzed exchange of the carboxyl carbon of glycine with 14CO2, although the specific activity of the purified H-protein from the patient was only 4% of that of control human H-protein. The content of H-protein in the liver of the patient was approximately 35% of that of control human liver.

Deficiency of 3-methylglutaconyl-coenzyme A hydratase in two siblings with 3-methylglutaconic aciduria.

We studied two patients with 3-methylglutaconic aciduria in order to determine the molecular defect. A new assay for 3-methylglutaconyl-coenzyme A (CoA) hydratase has been developed in which the substrate, [5-14C]3-methylglutaconyl-CoA, was synthesized using 3-methylcrotonyl-CoA carboxylase purified from bovine kidney. In this assay the products of the reaction are isolated by reverse-phase high performance liquid chromatography and the rates of conversion from substrate are measured. The Michaelis constant for 3-methylglutaconyl-CoA in normal fibroblasts was 6.9 mumol/liter. The mean activity of 3-methylglutaconyl-CoA hydratase in control fibroblasts was 495 pmol/min per mg protein. In the two patients the values were 11 and 17 pmol/min per mg protein, or 2-3% of normal.

Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

An inherited, structurally abnormal and superactive form of the enzyme 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) synthetase (EC 2.7.6.1) has been characterized in fibroblasts cultured from a 14-yr-old male (S.M.) with clinical manifestations of uric acid overproduction present since infancy. PP-ribose-P synthetase from the cells of this child showed four- to fivefold greater than normal resistance to purine nucleotide (ADP and GDP) feedback inhibition of enzyme activity and hyperbolic rather than sigmoidal inorganic phosphate (Pi) activation in incompletely dialyzed extracts. Excessive maximal velocity of the enzyme reaction catalyzed by the mutant enzyme was indicated by: enzyme activities twice those of normal at all concentrations of Pi in chromatographed fibroblast extracts; normal affinity constants for substrates and for the activator, Mg2+; and twofold greater than normal activity per immunoreactive enzyme molecule. The mutant enzyme thus possessed deficient regulatory and superactive catalytic properties, two mechanisms previously demonstrated individually to underlie the excessive PPRribose-P and uric acid synthesis of affected members of families with superactive PP-ribose-P synthetases. Increased PP-ribose-P concentration (4-fold) and generation (2.7-fold) and enhanced rates of PP-ribose-P dependent purine synthetic reactions, including purine synthesis de novo, in S.M. fibroblasts confirmed the functional significance of this patient's mutant enzyme. Diminished stability of the variant PP-ribose-P synthetase was manifested in vitro by increased thermal lability and in vivo by deficiency of enzyme activity at Pi concentrations greater than 0.3 mM in hemolysates and by an accelerated, age-related decrement in enzyme activity in lysates of erythrocytes separated by specific density. Despite the diminished amount of PP-ribose-P synthetase in the S.M. erythrocyte population, S.M. erythrocytes had increased PP-ribose-P concentration and increased rates of incorporation of [14C]adenine and hypoxanthine into acid-soluble nucleotides during incubation at 1 mM Pi. These findings provided further confirmation of the extent to which PP-ribose-P synthesis is modulated in the normal cell at physiological Pi concentration by purine nucleotide inhibition of PP-ribose-P synthetase. The activity and kinetic characteristics of PP-ribose-P synthetase from fibroblasts of the mother of patient S.M. indicated that this woman was a heterozygous carrier of the enzyme defect expressed in hemizygous manner by her son.

Effects of 2'-deoxycoformycin on the metabolism of purines and the survival of malignant cells in a patient with T-cell leukemia.

The in vitro effects of deoxyadenosine and an adenosine deaminase inhibitor, deoxycoformycin, on the synthesis of DNA and the metabolism of purines were investigated in human leukemic T-cells. In the presence of 10 microM deoxycoformycin, the synthesis of DNA was completely inhibited by concentrations of deoxyadenosine of 10 microM or greater. In contrast, the synthesis of DNA in normal bone marrow cells was not inhibited in the presence of up to 20 microM deoxycoformycin and up to 10 microM deoxyadenosine. Following incubation of leukemia T-cells with deoxycoformycin and deoxyadenosine, there was a significant rise in the concentration of deoxyadenosine 5'-triphosphate which was accompanied by reductions in the concentrations of adenosine 5'-triphosphate and guanosine 5'-triphosphate, as revealed by high-pressure liquid chromatographic analysis. The effects of deoxycoformycin on T-cell leukemia were examined in vivo. A patient with acute T-cell leukemia in the terminal stage received five daily injections of 250 micrograms of deoxycoformycin per kg. Among the noted changes, most prominent was the drop in the leukocyte count. Initially, the cell count rose from 7,200 cells/microliters on Day 1 to 120,000 cells/microliters on Day 3. On Day 5, the cell count began to decline and reached a nadir of 600 cells/microliter on Day 10. The leukocyte count remained below 1,000 cells/microliter through Day 12. The reduction in cell count was preceded by a decline in the incorporation of [3H]thymidine in the cells, which dropped to negligible amount by Day 7. The other prominent change was a decrease in adenosine deaminase activity in both red cells and leukemic cells. Adenosine deaminase activity of red cells dropped to 5% on Day 4, and that of leukemic cells dropped to 59% on Day 5. In addition, there were considerable alterations in the concentrations of purine metabolites which were characterized by a progressive reduction in the concentrations of total purine metabolites, especially adenosine 5'-triphosphate, and a transient rise in the concentrations of deoxyadenosine 5'-triphosphate, adenosine 5'-monophosphate, and adenosine 5-diphosphate. These findings suggest that treatment with deoxycoformycin may be of therapeutic value for T-cell leukemia. It may provide opportunities for studying the purine metabolism in T-leukemic cells which could lead to better approaches to treatment.

Successful Domino Liver Transplantation from a Patient with Methylmalonic Acidemia.

Liver transplantation has been reported in patients with methylmalonic acidemia (MMA), but long-term outcome is controversial. Many patients with other approved indications for liver transplantation die before donor grafts are available. A 28-year-old man with MMA underwent cadaveric liver transplantation. His liver was used as a domino graft for a 61-year-old man with primary sclerosing cholangitis, who had low priority on the transplant waiting list. Surgical outcome was successful, and after transplantation both patients have excellent graft function. The patient with MMA showed substantial decrease in methylmalonate in urine (from 5,277 ± 1,968 preoperatively to 1,068 ± 384 mmol/mol creatinine) and plasma (from 445.9 ± 257.0 to 333.3 ± 117.7 µmol/l) over >1-year follow-up, while dietary protein intake increased from 0.6 to 1.36 ± 0.33 g/kg/day. The domino recipient maintained near-normal levels of plasma amino acids but did develop elevated methylmalonate in blood and urine while receiving an unrestricted diet (peak plasma methylmalonate 119 µmol/l and urine methylmalonate 84-209 mmol/mol creatinine, with 1.0-1.9 g/kg/day protein). Neither patient demonstrated any apparent symptoms of MMA or metabolic decompensation during the postoperative period or following discharge. CONCLUSION: Liver transplantation substantially corrects methylmalonate metabolism in MMA and greatly attenuates the disease. In this single patient experience, a liver from a patient with MMA functioned well as domino graft although it did result in subclinical methylmalonic acidemia and aciduria in the recipient. Patients with MMA can be considered as domino liver donors for patients who might otherwise spend long times waiting for liver transplantation.

Neurologic nonmetabolic presentation of propionic acidemia.

BACKGROUND: Patients with propionic acidemia usually present in the neonatal period with life-threatening ketoacidosis, often complicated by hyperammonemia. It was thought that the neurologic abnormalities seen in this disease were exclusively the consequences of these acute crises. Experience with 2 patients with propionic acidemia indicates that this disease may present first with prominent neurologic disease without the life-threatening episodes of ketoacidosis that usually serve as the alerting signals for a diagnosis of an organic acidemia. OBJECTIVE: To examine the clinical and metabolic aspects of 2 patients with a phenotype that suggested disease of the basal ganglia. DESIGN: Examination of patterns of organic acids of the urine and enzyme assay for propionyl-CoA carboxylase in fibroblasts and lymphocytes. SETTING: Referral population to a biochemical genetics laboratory. PATIENTS: Two patients whose prominent features were hypotonia followed by spastic quadriparesis and choreoathetosis. Both had seizures. One patient was mildly mentally retarded but grew normally physically. The other had profound mental retardation and failure to thrive; he also self-mutilated his lower lip. Self-injurious behavior has not been reported in this disease. MAIN OUTCOME MEASURES: Clinical description, blood ammonia levels, organic acid levels in the urine, and enzyme activity. RESULTS: Excretion of metabolites, including methylcitrate, was typical. Residual activity of propionyl-CoA carboxylase approximated 5% of the control in each patient. CONCLUSIONS: Propionic acidemia can present as a pure neurologic disease without acute episodes of massive ketoacidosis. Hyperammonemia may occur after infancy in some patients, presenting as Reye syndrome.

Medium-chain acyl coenzyme A dehydrogenase deficiency: occurrence in an infant and his father.

BACKGROUND: Autosomal recessive inborn errors of metabolism often present as life-threatening disease in infancy and have adverse effects on the nervous system. Parents are usually heterozygotes. This is true of most disorders of fatty acid oxidation, which are rare and present with hypoketotic hypoglycemia. However, the gene for medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency is common in white people, raising the possibility that a parent may be homozygous. OBJECTIVE: To document the occurrence of MCAD deficiency in a 12-month-old boy and his father, both of whom were homozygous for the A985G mutation. DESIGN: Clinical observations and definitive biochemical testing. SETTING: Children's hospital and university laboratory. PARTICIPANTS: One child and one adult. INTERVENTIONS: Diagnosis and treatment. MAIN OUTCOME MEASURES: Clinical outcome; analysis results of plasma and urine for carnitine and organic acids. RESULTS: An infant admitted with an acute illness requiring intensive care was found to have carnitine deficiency and dicarboxylic aciduria; MCAD deficiency was diagnosed by assay of his DNA for the common mutation. Test results of the father revealed him also to be homozygous. CONCLUSION: In MCAD deficiency, as opposed to the usual rare autosomal recessive metabolic disease, a parent may also be an affected homozygote.

Short-chain organic acidemia and Reye's syndrome.

Short-chain fatty acids were determined prior to therapy in seven patients with Reye's syndrome. Elevated concentrations of propionate, butyrate, and isobutyrate were found in all patients. Isovalerate concentrations were high in three patients. In view of the fact that the administration of certain short-chain fatty acids to experimental animals results in coma, electroencephalographic changes, and fatty accumulation in the viscera, the elevations of short-chain fatty acid concentrations observed in the present study suggest that these fatty acids may play a role in the clinical manifestations of Reye's syndrome.

Autosomal dominant striatonigral degeneration. A clinical, pathologic, and biochemical study of a new genetic disorder.

An autosomal dominant striatonigral degeneration is present in a family of Portuguese ancestry numbering in excess of 329 persons in eight generations. The illness begins in the second, third, or fourth decade, and progresses for about 15 years with parkinsonian rigidity, spasticity, spastic dysarthria, and abnormalities of eye movement. Neuropathologic findings are severe neuronal loss and astrocytic gliosis in the corpus striatum and substantia nigra, with a moderate neuronal loss in the dentate nucleus of the cerebellum and nucleus ruber of the midbrain. This is a new genetic entity, distinct from other autosomal dominant neurologic disorders such as nigrospinodentatal degeneration, olivopontocerebellar degeneration, dystonia musculorum deformans, Machado's disease, and Huntington's disease.

Joseph disease: protein patterns in fibroblasts and brain.

The separation of brain and fibroblast proteins was analyzed on two-dimensional acrylamide gels. Proteins were examined from skin fibroblast cultures and brain homogenates from the frontal cerebral cortex, putamen, and cerebellum. Protein species from skin fibroblast cultures of controls and patients with Joseph disease or Huntington disease were not significantly different. The proteins from homogenates of the cerebral cortex, putamen, and cerebellum from controls differed from those of one Joseph disease patient. Two major classes of proteins were increased in the patient's putamen and cerebellum. Proteins of 40,000 and 50,000 daltons--including the glial filamentous acidic protein complex (molecular weight 50,000), and two proteins which migrated near actin--were increased in the cerebellum. The glial filamentous acidic protein complex increased 3.7-fold in the putamen of the patient. These protein changes probably represent gliosis, but may also be an expression of the primary genetic mutation.

Joseph disease and Huntington disease: protein patterns in fibroblasts and brain.

Proteins were separated on two-dimensional acrylamide gels obtained from brain samples of patients with Joseph disease, Huntington disease (HD) and multiple sclerosis. Similar protein separations were made from cultured skin fibroblasts of Joseph disease patients. Two major classes of proteins, one with a MW of 50,000 probably representing the glial filamentous acidic protein, or another class with a MW of 40,000 (proteins Jc, Jd, L1 and L2) were increased in the cerebellum of six Joseph disease patients. The same protein species were abnormally increased in HD brains, mainly in the basal ganglia and frontal cortex. These identical classes of protein changes were present in two nosologically separate autosomal dominant neurological disorders, Joseph disease (a spinocerebellar degeneration) and HD (a basal ganglia and cerebral cortical degeneration) and may reflect a biochemical correlation of gliosis and neuronal disease. However, these changes may be evidence that the two diseases are allelic mutations of the same gene. The dominantly inherited spinocerebellar degenerations may result from a primary deficit of glial-neuronal interaction, resulting in neuronal loss but with a compensatory increase in the number of glial cells attempting to provide additional trophic-metabolic support.

Syndrome of mild mental retardation, spastic gait, and skeletal malformations in a family with partial deficiency of hypoxanthine-guanine phosphoribosyltransferase.

A syndrome has been observed in a kindred with deficient activity of hypoxanthine-guanine phosphoribosyltransferase in which affected hemizygotes have had mild mental retardation, a spastic gait, pyramidal tract signs, shortness of stature, proximally placed thumbs and clinodactyly of the fifth fingers. Activity of the enzyme was virtually zero in lysates of erythrocytes or hair roots, but in intact fibroblasts, the level of activity was 7.5% of normal, placing this variant in a group distinct from any previously studied. Kinetic studies revealed a Michaelis constant for hypoxanthine that was also different from other enzymes studied. These observations indicate the presence in this kindred of a distinct variant of hypoxanthine-guanine phosphoribosyltransferase.

Clinical and metabolic abnormalities in a boy with dietary deficiency of biotin.

Dietary deficiency of biotin was documented in an 11-year-old retarded boy as a consequence of a dietary prescription containing raw eggs. Clinical manifestations were alopecia totalis and an erythematous, exfoliative dermatosis. Metabolic characteristics included increased excretion of 3-methylcrotonylglycine, 3-hydroxyisovaleric acid, 3-hydroxypropionic acid, methylcitric acid, and lactic acid, as well as a propensity for the development of ketosis. The activities of propionyl coenzyme A carboxylase and 3-methylcrotonyl coenzyme A carboxylase in extracts of leukocytes were deficient. Treatment with biotin and the removal of raw eggs, which contain the biotin-binding protein, avidin, from the diet led to the reversal of all of the clinical and metabolic manifestations observed.

Multiple carboxylase deficiency: clinical and biochemical improvement following neonatal biotin treatment.

Multiple carboxylase deficiency is characterized by deficient activities of three biotin-dependent enzymes, propionyl coenzyme A carboxylase, pyruvate carboxylase, and beta-methylcrotonyl coenzyme A carboxylase. A newborn infant was seen with metabolic ketoacidosis, hyperammonemia, organic aciduria, seizures, and coma. Multiple carboxylase deficiency was subsequently confirmed by enzyme activity determinations in his peripheral blood leukocytes and cultured skin fibroblasts. The infant's neurologic and metabolic status improved markedly within a few days of administration of pharmacologic doses of oral biotin. His EEG, which was distinctly abnormal, became normal; his extensive computed tomography scan changes resolved, with the exception of ventricular dilation, over the next two months. After two weeks of biotin treatment the excretion of abnormal organic acid metabolites was reduced and his carboxylase activities increased to the normal range. However, the activities of these enzymes increased only to 30% to 55% of normal in fibroblasts incubated in supplemental biotin. This partial correction of enzyme activity differs from that observed in other individuals with multiple carboxylase deficiency and suggests biochemical heterogeneity in this disorder. Prompt diagnosis and intervention can avert some of the pathologic complications of this biotin-responsive condition.

Synergistic activity of purine metabolism inhibitors in cultured human tumor cells.

Cytotoxic effects of 3-deazaguanosine (3-DGUO) result from the inhibition of DNA synthesis and incorporation of the drug into DNA. Synergistic antiproliferative effects of a combination of 3-deazaguanosine and 2-beta-D-ribofuranosylthiazole-4-carboxamide, a potent inhibitor of inosine monophosphate dehydrogenase, was observed in human tumor cells. Inosine reversed the antiproliferative effects of the 3-deazaguanosine but not 2-beta-D-ribofuranosylthiazole-4-carboxamide. 3-Deazaguanosine monophosphate was shown to inhibit the activity of the de novo purine synthesis enzyme, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide transformylase. The data suggested a cytotoxic effect of 3-DGUO associated with the inhibition of de novo purine synthesis by drug nucleotides, an effect which may account for the synergistic action noted.