Molybdenum cofactor biosynthesis in humans. Identification of two complementation groups of cofactor-deficient patients and preliminary characterization of a diffusible molybdopterin precursor.

Molybdenum cofactor deficiency is a devastating disease with affected patients displaying the symptoms of a combined deficiency of sulfite oxidase and xanthine dehydrogenase. Because of the extreme lability of the isolated, functional molybdenum cofactor, direct cofactor replacement therapy is not feasible, and a search for stable biosynthetic intermediates was undertaken. From studies of cocultured fibroblasts from affected individuals, two complementation groups were identified. Coculture of group A and group B cells, without heterokaryon formation, led to the appearance of active sulfite oxidase. Use of conditioned media indicated that a relatively stable, diffusible precursor produced by group B cells could be used to repair sulfite oxidase in group A recipient cells. Although the extremely low levels of precursor produced by group B cells preclude its direct characterization, studies with a heterologous, in vitro reconstitution system suggest that the precursor that accumulates in group B cells is the same as a molybdopterin precursor identified in the Neurospora crassa molybdopterin mutant nit-1, and that a converting enzyme is present in group A cells which catalyzes an activation reaction analogous to that of a converting enzyme identified in the Escherichia coli molybdopterin mutant ChlA1.

Report on a new patient with combined deficiencies of sulphite oxidase and xanthine dehydrogenase due to molybdenum cofactor deficiency.

A newborn infant exhibiting seizures and spastic tetraparesis at the age of 1 week was shown to excrete excessive quantities of sulphite, taurine, S-sulphocysteine and thiosulphate, characteristic of sulphite oxidase deficiency. In addition, increased renal excretion of xanthine and hypoxanthine combined with a low serum and urinary uric acid was consistent with xanthine dehydrogenase deficiency. Both deficiencies could be established at the enzyme level. The primary defect giving rise to the combined abnormalities is the absence of a molybdenum cofactor, a molybdenum-containing pterin being an essential component of both enzymes. The patient developed a severe neurological syndrome, brain atrophy and lens dislocation and died at the age of 22 months. Attempts at treatment, such as oral administration of ammonium molybdate, sodium sulphate, D-penicillamine, 2-mercaptoethane sulphonic acid, pyridoxine and thiamine did not influence the clinical course.

Molybdenum cofactor deficiency in a patient previously characterized as deficient in sulfite oxidase.

The metabolic status of a patient previously characterized as deficient in sulfite oxidase was reexamined applying new methodology which has been developed to distinguish between a defect specific to the sulfite oxidase protein and sulfite oxidase deficiency which arises as a result of molybdenum cofactor deficiency. Urothione, the metabolic degradation product of the molybdenum cofactor, was undetectable in urine samples from the patient. Analysis of molybdenum cofactor levels in fibroblasts by monitoring reconstitution of apo nitrate reductase in extracts of the Neurospora crassa mutant nit-1 revealed that cells from the patient were severely depleted. Quantitation of urinary oxypurines showed that hypoxanthine and xanthine were highly elevated while uric acid remained in the normal range. These results were interpreted to indicate a severe but incomplete deficiency of the molybdenum cofactor. The presence of very low levels of active cofactor, supporting the synthesis of low levels of active sulfite oxidase and xanthine dehydrogenase, could explain the metabolic patterns of sulfur and purine products and the relatively mild clinical symptoms in this individual.

Molybdenum co-factor deficiency: an easily missed inborn error of metabolism.

A female patient is described with combined deficiency of sulphite, zanthine and aldehyde oxidase. She presented at the age of four weeks with intractable seizures. Initially the diagnosis was suspected because of a very low serum urate level (23 mumol/1-1). This condition can be easily missed and it is proposed that measurement of serum urate be included in the metabolic assessment of neonates with unexplained seizures and developmental delay.

Biochemical and genetic comparison of two nitrate reductase-deficient pea mutants disturbed in the cofactor.

Two nitrate reductase (NaR)-deficient mutants of pea (Pisum sativum L.), E1 and A300, both disturbed in the molybdenum cofactor function and isolated, respectively, from cv Rondo and cv Juneau, were tested for allelism and were compared in biochemical and growth characteristics. The F1 plants of the cross E1 X A300 possessed NaR and xanthine dehydrogenase (XDH) activities comparable to those of the wild types, indicating that these mutants belong to different complementation groups, representing two different loci. Therefore, mutant E1 represents, besides mutant A300 and the allelic mutants A317 and A334, a third locus governing NaR and is assigned the gene destignation nar 3. In comparison with the wild types, cytochrome c reductase activity was increased in both mutants. The mutants had different cytochrome c reductase distribution patterns, indicating that mutant A300 could be disturbed in the ability to dimerize NaR apoprotein monomers, and mutant E1 in the catalytic function of the molybdenum cofactor. In growth characteristics studied, A300 did not differ from the wild types, whereas fully grown leaves of mutant E1 became necrotic in soil and in liquid media containing nitrate.

A model for hyperphenylalaninaemia due to tetrahydrobiopterin deficiency.

A model for tetrahydrobiopterin deficiency in mice is described. Elevated levels of phenylalanine produced in the model were shown to be dramatically reduced after injection of tetrahydrobiopterin. A comparison of several reduced pterins for their efficacy in the system is described. The unnatural S isomer of tetrahydrobiopterin was shown to be active in the system.

Combined xanthine and sulphite oxidase defect due to a deficiency of molybdenum cofactor.

Increased urinary excretion of xanthine, hypoxanthine, sulphite, thiosulphate and decreased serum uric acid were observed in an infant with profound failure to thrive. Other clinical findings included refractory seizures, spastic quadriplegia and profound psychomotor retardation. The patient died at 20 months of age. There were no detectable activities for xanthine oxidase and sulphite oxidase in the postmortem liver. Urothione, which is the metabolic excretory product of the molybdenum cofactor for molybdoenzymes was not present in the urine. A deficiency of the molybdenum cofactor which is common to both xanthine and sulphite oxidase is presumed to be the metabolic defect responsible for the absent activities of both enzymes.

Absence of hepatic molybdenum cofactor. An inborn error of metabolism associated with lens dislocation.

There are many causes of lens dislocation in man. Amongst these are two inborn errors of sulfur amino acid metabolism, viz., homocystinuria and sulfite oxidase deficiency. To date nine patients have been found in whom a combined deficiency of sulfite oxidase and xanthine dehydrogenase was observed. This inherited disease is due to a defective synthesis of molybdenum cofactor, an essential component for the assembly of both enzymes. The main clinical symptoms of these patients were: facial dysmorphic features, severe feeding difficulties, mental retardation, abnormal muscle tone, severe seizures and myoclonia. Four out of nine patients had dislocated eye lenses. The main biochemical findings included hypouricemia, xanthinuria, an increased excretion of sulfite, thiosulfate, S-sulfocysteine, taurine and a decreased excretion of inorganic sulfate. The prognosis of the disease is poor; various attempts at treatment were not successful so far. Prenatal diagnosis by assay of sulfite oxidase in cultured amniotic fluid cells and by direct measurement of amniotic fluid S-sulfocysteine is possible.

Differential diagnosis of tetrahydrobiopterin deficiency.

Six hundred and seventy-three children (483 newborns and 190 older selected children) were screened for tetrahydrobiopterin (BH4) deficiency by HPLC of urine pterins and BH4 load test. One patient with GTP cyclohydrolase I deficiency, 36 patients with dihydrobiopterin synthetase (DHBS) deficiency (of which six were in the newborn and 30 in the older children) and 14 with dihydropteridine reductase deficiency (DHPR) were found. All 37 patients with defective BH4 biosynthesis responded to a BH4 load by lowering of the elevated serum phenylalanine concentration but four of 14 patients with DHPR deficiency did not. Measurement of DHPR activity in blood spots on Guthrie cards is recommended. Since subvariants of patients with BH4 deficiency exist, homovanillic acid, 5-hydroxyindole acetic acid, pterins, phenylalanine, and tyrosine in cerebrospinal fluid should be measured for diagnosis and the control of therapy. The activity of the phosphate-eliminating enzyme (a key enzyme in BH4 biosynthesis and part of "DHBS") was measured in human liver and activities of approx. 1 n U (mg protein)-1 were found. In the liver biopsy of a patient with DHBS deficiency no activity (less than 3% of controls) was demonstrated.

Clinical role of pteridine therapy in tetrahydrobiopterin deficiency.

In most patients with deficiency of tetrahydrobiopterin (BH4) continuous administration of BH4 or of a synthetic analogue such as 6-methyltetrahydropterin (6-MPH4) lowers plasma phenylalanine concentrations to the therapeutic range. The effective dose of BH4 varies from 1 to 2 mg kg-1 daily in patients with defective biopterin synthesis, to 5 mg kg-1 or more in patients with dihydropteridine reductase (DHPR) deficiency. The cost of 2 mg kg-1 day-1 of BH4 is comparable to the cost of a low phenylalanine diet. Higher doses of pterins given orally (20 mg kg-1) raise the levels of tetrahydropterin in cerebrospinal fluid (CSF) to normal in patients with defective biopterin synthesis in whom initial concentration of biopterin species are low. In some, but not all, such patients pterin therapy also raises CSF amine metabolite concentrations and ameliorates symptoms. High dose therapy does not appear to be effective in raising CSF pterin levels in patients with DHPR deficiency who already accumulate dihydrobiopterin (BH2) in CSF. Central folate deficiency is an additional cause of neurological deterioration in patients with DHPR deficiency who require supplementation with folate as folinic acid. It is suggested that the accumulation of BH2 in such patients competitively interferes with folate metabolism.

Anatomo-pathological findings in a case of combined deficiency of sulphite oxidase and xanthine oxidase with a defect of molybdenum cofactor.

A case of combined deficiency of sulphite-oxidase and xanthine-oxidase with a defect of the molybdenum cofactor, which is vital to the activity of sulphite-, xanthine- and aldehyde-oxidase, is reported here. Seven cases of combined deficiencies have been described with regard to both clinical and laboratory findings. The clinical, laboratory and anatomo-pathological features and, in particular, the central nervous system lesions of the present case correspond exactly to those in the case described Rosenblum in which an isolated deficiency in sulphite-oxidase was present. As the cerebral alterations in the present case are comparable to those described in Rosenblum's case, they probably result from the defect in sulphite-oxidase activity.

Tetrahydrobiopterin deficiencies: preliminary analysis from an international survey.

Tetrahydrobiopterin deficiency is a rare cause of hyperphenylalaninemic syndromes. The natural history of the disease is characterized by progressive neurologic illness unresponsive to a phenylalanine-restricted diet. Fifty patients have been reported. From the documented cases, the following statements can be made: (1) An incidence of 2% among hyperphenylalaninemic babies can be reasonably estimated. (2) Most patients have high neonatal blood phenylalanine concentrations, but some have only mild elevations. (3) Among the available diagnostic tests, measurement of urine pteridines should be proposed in all hyperphenylalaninemic babies, (4) The tolerance to dietary phenylalanine is generally high. (5) The results of neurotransmitter replacement therapy are encouraging, but treatment should be started within the first month and requires a strict follow-up protocol. Consequently, in every newborn infant with positive Guthrie test results, a rapid investigation of BH4 metabolism should be accomplished in order to differentiate between phenylalanine-hydroxylase deficiencies (phenylketonuria, mild hyperphenylalaninemia, transient hyperphenylalaninemia) and BH4 deficiencies.

Blood spots on Guthrie cards can be used for inherited tetrahydrobiopterin deficiency screening in hyperphenylalaninaemic infants.

We describe a method of screening for dihydropteridine reductase deficiency and dihydrobiopterin synthesis deficiency--the two inherited defects that cause tetrahydrobiopterin deficiency--using blood spots on Guthrie cards. Dihydropteridine reductase deficiency may be identified positively, and a biopterin value of less than 6.0 micrograms/l in the presence of hyperphenylalaninaemia indicates further investigation for dihydrobiopterin synthesis deficiency.

A genetic analysis of the pteridine biosynthetic enzyme, guanosine triphosphate cyclohydrolase, in Drosophila melanogaster.

Strains with mutant eye color were surveyed for levels of GTP cyclohydrolase (GTP CH), the first enzyme acting in the biosynthesis of pteridines, the pigments causing red eye color in Drosophila. Six strains were found to have reduced GTP CH activity. In five of the six strains, the reduction of activity is apparent only in the adult head of homozygous mutants. We show that mutations in Punch (2-97, Pu) have severe effects on GTP CH activity. In most cases, the reduction of activity is apparent in all tissues and stages that express the enzyme. The activity of GTP CH is shown to be closely correlated with the number of Pu+ genes in the genome. One ethyl methanesulfonate (EMS)-induced Pu mutant has a GTP CH enzyme that is unstable when compared with the wild-type enzyme. Mutations in Pu fall into three general classes. The largest class has a recessive lethal and eye color phenotype, 50% or higher GTP CH activity in heterozygotes, and equivalent defects in all tissues. A second class is dominant in eye color phenotype and recessive lethal, with less than 50% GTP CH activity in heterozygotes. The third class is homozygous viable and has severe reduction of activity in the adult head, but no or less severe loss in other tissues.

Diagnostic and therapeutic aspects of dihydrobiopterin deficiency.

The first Scandinavian hyperphenylalaninaemic patient with a cofactor deficiency is described. By neonatal screening the Guthrie test showed a serum phenylalanine of 302 mumol/1 (5 mg/dl), which at age 6 weeks had fallen to high normal values. At age 5 1/2 months the serum phenylalanine was around 2000 mumol/1 and the child presented with severe neurological symptoms. The diagnosis of defect dihydrobiopterin biosynthesis was made by high performance liquid chromatography of the urine. Loading tests followed by daily treatment of the missing cofactor was able to keep the serum phenylalanine in the normal level. Because of persisting, yet diminishing neurological symptoms neurotransmitter treatment was started. Breast feeding as the cause of the low neonatal levels of serum phenylalanine and the late start of clinical symptoms is proposed and the importance of screening all hyperphenylalaninaemic newborns for defect biopterin metabolism is stressed.