Radical S-Adenosylmethionine Enzymes in Human Health and Disease.

Radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Of approximately 114,000 of these enzymes, 8 are known to be present in humans: MOCS1, molybdenum cofactor biosynthesis; LIAS, lipoic acid biosynthesis; CDK5RAP1, 2-methylthio-N(6)-isopentenyladenosine biosynthesis; CDKAL1, methylthio-N(6)-threonylcarbamoyladenosine biosynthesis; TYW1, wybutosine biosynthesis; ELP3, 5-methoxycarbonylmethyl uridine; and RSAD1 and viperin, both of unknown function. Aberrations in the genes encoding these proteins result in a variety of diseases. In this review, we summarize the biochemical characterization of these 8 radical S-adenosylmethionine enzymes and, in the context of human health, describe the deleterious effects that result from such genetic mutations.

Chronological changes of the amplitude-integrated EEG in a neonate with molybdenum cofactor deficiency.

Molybdenum cofactor (Moco) deficiency is a rare neurometabolic disorder, characterized by neurological impairment and refractive seizures, due to toxic accumulation of sulfite in the brain. Earlier it was suggested that in Moco-deficient humans maternal clearance of neurotoxic metabolites prevents prenatal brain damage. However, limited data are available about the time profile in which neurophysiologic deterioration occurs after birth. The amplitude-integrated electroencephalography (aEEG) is a bedside method in neonates to monitor cerebral recovery after hypoxic-ischemic insults, detect epileptic activity, and evaluate antiepileptic drug treatment. We describe a chronological series of changes in aEEG tracings in a neonate with Moco deficiency. He presented with myoclonic spasms and hypertonicity a few hours after birth, however, the aEEG pattern was still normal. Within 2 days, the aEEG rapidly changed into a burst suppression pattern with repetitive seizures. After antiepileptic treatment, the aEEG remained abnormal. In this patient, the normal aEEG pattern at birth may have been due to maternal clearance of sulfite in utero. After birth, accumulation of sulfite causes progressive brain damage, reflected by the progressive depression of the aEEG tracings. This is in agreement with the results from a Moco-deficient mouse model, suggesting that maternal sulfite clearance suppresses prenatal brain damage. To our knowledge, this is the first case report describing the chronological changes in the aEEG pattern in a Moco-deficient patient. Insight into the time profile in which neurologic deterioration in Moco-deficient humans occurs is essential, especially when potential treatment strategies are being evaluated.

Hyperphosphatasia with neurologic deficit: a pyridoxine-responsive seizure disorder?

This report describes the case of a 4 1/2-year-old female with developmental delay and tonic-clonic seizures, persistently elevated serum alkaline phosphatase activity, and low serum pyridoxal 5'-phosphate. Born at term to consanguineous parents, she was dysmorphic and delayed at 5 months. At 11 months, seizures and microcephaly were evident but skeletal and cerebral imaging, karyotyping, and genetic metabolic tests were unremarkable. Serum alkaline phosphatase activity, however, was elevated (1.3 +/- 0.6 times greater than the upper limit of normal) on seven occasions between 5 months and 4(1/2) years of age. Hyperphosphatasia with neurologic deficit (MIM #239300), a rare autosomal recessive disorder, was diagnosed. The low serum levels of pyridoxal 5'-phosphate (6 nmol/L; normal >20 nmol/L) prompted a pyridoxine challenge. A clinically significant but paradoxical response was observed. On electroencephalography, diffuse delta slow waves (1-2 Hz) were observed, suggestive of stage 3 or 4 slow-wave sleep. With daily administration of 100 mg pyridoxine and withdrawal of phenobarbital, seizures were not evident. We suggest that serum alkaline phosphatase should be measured in cases of seizures with paradoxical electroencephalographic response to pyridoxine. Conversely, pyridoxine challenge should be considered in cases of hyperphosphatasia with seizures and neurologic deficit.

Isolated sulfite oxidase deficiency: identification of 12 novel SUOX mutations in 10 patients.

We report twelve novel mutations in patients with isolated sulfite oxidase deficiency. The mutations are in SUOX, the gene that encodes the molybdohemoprotein sulfite oxidase. These include two frameshift mutations, a four-basepair deletion (562del4) and a single-basepair insertion (113insC), both resulting in premature termination. Nonsense mutations predicting Y343X and Q364X substitutions were identified in a homozygous state in three patients, the latter in two sibs. The remaining eight are missense mutations generating single amino acid substitutions. From the position of the substituted residues, seven of these mutations are considered to be causative of the enzyme deficiency: I201L, R211Q, G305S, R309H, K322R, Q339R, and W393R. The eighth, a C>T transition, predicts an R319C substitution, which could affect the binding of the molybdenum cofactor and thus severely reduce sulfite oxidase activity. This mutation, however, is downstream of a frameshift mutation and is therefore not the causative mutation in this individual.

The copper-iron connection: hereditary aceruloplasminemia.

Hereditary aceruloplasminemia is an autosomal recessive disorder of iron homeostasis due to loss-of-function mutations in the ceruloplasmin gene. Affected individuals may present in adulthood with evidence of hepatic iron overload, diabetes, peripheral retinal degeneration, dystonia, dementia, or dysarthria. Laboratory studies demonstrate microcytic anemia, elevated serum ferritin, and a complete absence of serum ceruloplasmin ferroxidase activity. Consistent with the observed neurologic findings, magnetic resonance imaging reveals iron accumulation within the basal ganglia. Histologic studies detect abundant iron in hepatocytes, reticuloendothelial cells of the liver and spleen, beta cells of the pancreas, and astrocytes and neurons throughout the central nervous system. Characterization of this disorder reveals an essential role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores and provides new insights into the mechanisms of human iron metabolism.

Simultaneous occurrence of xanthine oxidase and sulfite oxidase deficiency. A molybdenum dependent inborn error of metabolism?

In a 3-week old female child with clinical features including neurologic abnormalities and lens dislocation, xanthinuria co-existed with increased excretion of sulfur compounds (sulfite, S-sulfocysteine, taurine and thio-sulfate). Low xanthine oxidase and absent sulfite oxidase activities were found on liver biopsy. No abnormality was detected in either parent. Both the above enzymes are molybdenum-flavoproteins. Normal serum molybdenum concentration seemed to rule out dietary deficiency or impaired absorption. A defect in the incorporation of the metal into flavoproteins is postulated in this case.

Dominant mutants of ceruloplasmin impair the copper loading machinery in aceruloplasminemia.

The multicopper oxidase ceruloplasmin plays a key role in iron homeostasis, and its ferroxidase activity is required to stabilize cell surface ferroportin, the only known mammalian iron exporter. Missense mutations causing the rare autosomal neurodegenerative disease aceruloplasminemia were investigated by testing their ability to prevent ferroportin degradation in rat glioma C6 cells silenced for endogenous ceruloplasmin. Most of the mutants did not complement (i.e. did not stabilize ferroportin) because of the irreversible loss of copper binding ability. Mutant R701W, which was found in a heterozygous very young patient with severe neurological problems, was unable to complement per se but did so in the presence of copper-glutathione or when the yeast copper ATPase Ccc2p was co-expressed, indicating that the protein was structurally able to bind copper but that metal loading involving the mammalian copper ATPase ATP7B was impaired. Notably, R701W exerted a dominant negative effect on wild type, and it induced the subcellular relocalization of ATP7B. Our results constitute the first evidence of "functional silencing" of ATP7B as a novel molecular defect in aceruloplasminemia. The possibility to reverse the deleterious effects of some aceruloplasminemia mutations may disclose new possible therapeutic strategies.

Congenital copper deficiency: copper therapy and dopamine-beta-hydroxylase activity in the mottled (brindled) mouse.

The mottled (Mo) mouse is an animal model of the human congenital copper (Cu) deficiency disorder, Menkes' kinky hair syndrome. Intraperitoneal Cu chloride injections have been shown to produce clinical and morphological improvements in this mutant mouse. Cu injections (10 micrograms/g) on postnatal days 7 and 10 are shown to increase endogenous activity of the Cu-dependent enzyme dopamine-beta-hydroxylase in the brains of Mo mice. The present study provides insight into the long-term neurochemical changes resulting from a possible treatment regimen for Menkes' kinky hair syndrome.

ATP6H, a subunit of vacuolar ATPase involved in metal transport: evaluation in canine copper toxicosis.

Copper toxicosis (CT), resulting in liver disease, occurs commonly in Bedlington terriers. Canine CT is of particular interest because identification of the causative gene may lead to the discovery of another important gene in the copper transport pathway possibly related to human copper diseases not yet identified. Homologs of the copper transporting ATPase ATP7B, defective in Wilson disease, and the copper chaperone ATOX1 were potential candidates, but both have been excluded. The CT locus in Bedlington terriers has been mapped to canine chromosome region CFA10q26, which has a syntenic human chromosome region, HAS2p13-21. The gene ATP6H, for human vacuolar proton-ATPase subunit M9.2, is associated with copper and iron transport in yeast and has been mapped to HAS2p21 and suggested as a candidate gene for CT. We cloned canine ATP6H, which encodes a predicted protein with 99% amino acid sequence identity to the orthologous human protein. Canine ATP6H shows a conserved potential metal binding site, CSVCC, and a glycosylation site, NET. The canine ATP6H is organized into four exons, with a 246-bp open reading frame. Sequence analysis of the coding regions showed no mutations in ATP6H from genomic DNA of an affected dog. We have also identified two, apparently non-transcribed canine ATP6H pseudogenes. Mapping of the true ATP6H gene and a marker closely linked to the CT locus on a canine radiation hybrid panel indicted lack of close physical association. We have therefore excluded canine ATP6H as a candidate gene for canine copper toxicosis, indicating that some other unidentified gene is responsible for this copper storage disease.

Menkes' kinky hair syndrome: a genetic disease involving copper.

The kinky hair syndrome (KHS) is an X-linked defect of copper transport in man. An animal model is available in mutants at the X-linked mottled locus in mice. The defect does not involve the uptake of copper from the intestinal lumen but rather the transport of copper from intestinal cells. The reduced activity of several copper-dependent enzymes and the lower copper content of serum, liver, and probably brain account for the manifestations of the disorder which are evident at, or shortly after, birth. Intrauterine involvement is likely but prenatal diagnosis is not yet possible. Although the delivery of iron to the erythropoietic system, and its utilization, are impaired in nutritionally induced copper deficiency, as is neutrophil production, these processes appear normal in KHS. thus, adequate copper to carry them out is available in KHS. While there may be more than one transport system for copper (only one of which is affected in KHS) it is also possible that the hematopoietic tissue in KHS, like the intestinal cells, has abnormally high afficity for copper. The presence of multiple alleles at the KHS locus (and at other genetic loci) in man, which cause different degrees of reduction in copper transport, could account for variations in the susceptibility to copper deficiency observed in infant populations.