Menkes' kinky hair disease: further definition of the defect in copper transport.

Duodenal mucosa obtained from two patients with Menkes' syndrome contained abnormally large amounts of copper. The defect in copper absorption in this disease must lie in the process of intracellular handling or of transport across the serosal cell membrane. Fibroblastic cells cultured from the skin of patients and of heterozygous females show intense metachromasia in primary culture which disappears in subculture. These cells may be useful for the study of copper transport in vitro and for the identification of heterozygotes in affected families.

Biochemical basis of prolidase deficiency. Polypeptide and RNA phenotypes and the relation to clinical phenotypes.

Cultured skin fibroblasts or lymphoblastoid cells from eight patients with clinical symptoms of prolidase deficiency were analyzed in terms of enzyme activity, presence of material crossreacting with specific antibodies, biosynthesis of the polypeptide, and mRNA corresponding to the enzyme. There are at least two enzymes that hydrolyze imidodipeptides in these cells and these two enzymes could be separated by an immunochemical procedure. The specific assay for prolidase showed that the enzyme activity was virtually absent in six cell strains and was markedly reduced in two (less than 3% of controls). The activities of the labile enzyme that did not immunoprecipitate with the anti-prolidase antibody were decreased in the cells (30-60% of controls). Cell strains with residual activities of prolidase had immunological polypeptides crossreacting with a Mr 56,000, similar to findings in the normal enzyme. The polypeptide biosynthesis in these cells and the controls was similar. Northern blot analyses revealed the presence of mRNA in the polypeptide-positive cells, yet it was absent in the polypeptide-negative cells. The substrate specificities analyzed in the partially purified enzymes from the polypeptide-positive cell strains differed, presumably due to different mutations. Thus, there seems to be a molecular heterogeneity in prolidase deficiency. There was no apparent relation between the clinical symptoms and the biochemical phenotypes, except that mental retardation was present in the polypeptide-negative patients. The activities of the labile enzyme may not be a major factor in modifying the clinical symptoms.

Developmental variation in copper, zinc and metallothionein mRNA in brindled mutant and nutritionally copper deficient mice.

The concentrations of copper, zinc and metallothionein-I (MT-I) mRNA were determined in the liver, kidney and brain of the brindled mutant mouse from birth until the time of death. Despite accumulation of copper in the kidney of the mutant, MT-I mRNA concentrations were normal. There was no difference between the MT-I mRNA in the brain of mutant and normal in the first 10 days of life, but after day 10 metallothionein mRNA levels were increased in the mutant. The concentration of copper was very low in the liver of the mutant, and on day 6 after birth the metallothionein mRNA was also reduced by about 50%. This reduction was not seen in copper-deficient 6-day-old pups, despite very low hepatic copper levels. This suggests that the lower hepatic MT-I mRNA in the day 6 brindled mouse was not simply due to the reduction in hepatic copper and also that hepatic copper is not regulating metallothionein gene expression the liver of neonatal mice. After day 12 hepatic MT-I mRNA levels were elevated in mutant and in copper deficient mice, both of which die at 14 to 16 days. These increases and the increase in brain MT-I mRNA in older mutant mice are likely to be caused by stress. Overall the results support the conclusions that the brindled mutation does not cause a constitutive activation of the metallothionein genes, and that the differences in metallothionein mRNA between mutant and normal are most probably secondary consequences of the mutation.

Hairs from patients with maple syrup urine disease show a structural defect in the fiber cuticle.

Models for the surface of cuticle cells in hair fibers consist of a monolayer of fatty acids covalently bound to the underlying protein membrane by thioester linkages. The most prominent of these fatty acids is 18-methyleicosanoic acid (C21a), the synthesis of which requires the oxidative decarboxylation of isoleucine. Maple syrup urine disease (MSUD) is caused by an inherited deficiency in the enzyme branched chain 2-oxo acid dehydrogenase, which leads to the accumulation of branched chain alpha-keto-acids derived from the amino acids, leucine, isoleucine, and valine. Transmission electron microscopy studies of developing hair fibers show a structural defect in the fiber shaft in hair from patients with MSUD. This defect is confined to the cuticle of the fiber, where the cuticle membrane directly apposes the intercellular material. Thus, the defect indicates that C21a is located exclusively on the upper surface of fiber cuticle cells. Lipid analysis of MSUD hairs has demonstrated significant changes in the relative abundance of the covalently bound fatty acids and an almost complete absence of C21a, whereas there was little difference in the amino acid composition compared with normal hair. These results provide further evidence for the existence of the surface lipid monolayer and its crucial role in cellular adhesion.

Diagnosis of trace metal deficiency--with emphasis on copper and zinc.

A method of diagnosing trace metal deficiency is proposed. Measurement of an appropriate metalloprotein before and after administration of a physiological replenishment dose of the metal should distinguish low levels due to metal deficiency from those due to other mechanisms. Serum ceruloplasmin is the logical protein for copper assessment; serum alkaline phosphatase and red cell carbonic anhydrase should be considered for zinc.

beta-hydroxyisobutyryl coenzyme A deacylase deficiency: a defect in valine metabolism associated with physical malformations.

An infant, born to parents who were first cousins had multiple physical malformations. An associated biochemical abnormality was suggested by the urinary excretion of cysteine and cysteamine conjugates of methacrylic acid. The coenzyme A (CoA) ester of this compound is an intermediate in the pathway of valine oxidation. Subsequent investigation revealed a deficiency of beta-hydroxyisobutyryl-CoA deacylase, an enzyme unique to valine metabolism. The enzyme defect results in accumulation of methacrylyl-CoA, a highly reactive compound, which readily undergoes addition reactions with free sulfhydryl groups. Tissue damage due to reactions between methacrylyl-CoA and important sulfhydryl-containing enzymes and cofactors may account for the teratogenic effects seen in this patient.

Mental retardation, unusual face, and intrauterine growth retardation: a new recessive syndrome?

We describe two mentally retarded sisters with intrauterine growth retardation and subsequent dwarfism and an unusual and characteristic face together with two other unrelated patients with similar manifestations. These four patients may represent a new autosomal recessive syndrome.

Geleophysic dysplasia.

We describe 2 children with geleophasic dysplasia. Prominent cardiac disease in one of the patients caused death at an early age. The history of consanguinity in one of the families supports autosomal recessive mode of inheritance. Histological and ultrastructural changes suggest that a disturbance in the relations between cell membrane and extracellular matrix may be involved in the pathogenesis.

Regional localisation of a non-specific X-linked mental retardation gene (MRX19) to Xp22.

A gene responsible for a non-specific form of X-linked mental retardation (MRX19) was localised by linkage analysis. Exclusions and regional localisation were made using 21 highly informative PCR-based markers along the X chromosome. Significant lod scores at a recombination fraction of zero were detected with the marker loci DXS207, DXS987 (Zmax = 3.58) and DXS999 (Zmax = 3.28) indicating that this gene is localised to the proximal portion of Xp22. Recombination between MRX19 and the flanking loci KAL and DXS989 was observed. The multipoint CEPH background map, with map distances in cM, is DXS996-1.8-KAL-19.0-DXS207-0.9-[DXS987,DXS443 ]-4.3-DXS999-3.5-DXS365-14.0-DXS989. Two other MRX disorders and two syndromal mental retardations, Coffin-Lowry syndrome and Partington syndrome, have been mapped to this region. There is a possibility that the 3 MRX disorders are the same entity. Most MRX disorders remain clustered around the pericentromeric region.

Early treatment of Menkes disease with parenteral copper-histidine: long-term follow-up of four treated patients.

We report on the long-term clinical course of 4 boys with Menkes disease, treated from early infancy with parenteral copper-histidine, with follow-up over 10-20 years. Three of the 4 had male relatives with a severe clinical course compatible with classical Menkes disease. As a consequence of early treatment, our patients have normal or near-normal intellectual development, but have developed many of the more severe somatic abnormalities of the related disorder, occipital horn syndrome, including severe orthostatic hypotension in 2. In addition, 1 boy developed a previously unreported anomaly, namely, massive splenomegaly and hypersplenism as a consequence of a splenic artery aneurysm. Previously reported molecular studies in 2 of these patients had shown gene defects which would have predicted a truncated and probably nonfunctional gene product. Despite the favorable effects on the neurological symptoms, parenteral copper treatment for Menkes disease should still be regarded as experimental. The development of more effective treatments must await a more precise delineation of the role which the Menkes protein plays in intracellular copper trafficking.

Further delineation of the C (trigonocephaly) syndrome.

This communication brings the number of recognized cases of the C (trigonocephaly) syndrome to 11. The pattern of findings includes an anomaly of the anterior cranium and frontal cortex (trigonocephaly), the root of the nose (broad nasal bridge, epicanthus, and short nose), and palate (thick anterior alveolar ridges); abnormalities of the limbs (polysyndactyly, bridged palmar creases, short limbs, and joint dislocations and/or contractures); visceral defects (congenital heart defects, cryptorchidism, and abnormal lobulations of the lungs and kidneys). Auricular, mandibular, skin, and genital abnormalities also occur. Consistent neurological findings are hypotonia, strabismus, and psychomotor retardation; seizures have been reported. Normal chromosomes, normal parents with multiple affected offspring, equal sex ratio of affected individuals, and consanguineous matings all support autosomal recessive inheritance of the C syndrome. In autopsied cases, there has been a suggestion of defective central nervous system myelination. About 1/2 of the case have died within the first year. All survivors have severe to profound mental retardation except for one child who has moderate retardation.

Human dihydropteridine reductase: a method for the measurement of activity in cultured cells, and its application to malignant hyperphenylalaninemia.

1. The kinetic, assay and culture variables involved in the determination of dihydropteridine reductase activity in human cultured cells have been investigated, and a modified assay is described which allows for accurate determination of activity. Unlike previously used assays, this assay utilizes saturating concentrations of substrates and its increased sensitivity allows for excellent replication on a smaller number of cells than was previously required. 2. The modified assay was used to investigate the levels of dihydropteridine reductase in patients with malignant hyperphenylalaninemia; results of assay for the enzyme in cultured cells from affected infants and parents are presented. These are compared with control data for dihydropteridine reductase activity in fibroblasts and continuous lymphoid cells from normal controls and phenylketonuric patients, in normal amniotic cells and in liver obtained at autopsy.

Urinary dihydroxanthopterin in the diagnosis of malignant hyperphenylalaninemia and phenylketonuria.

Assessment of urinary dihydroxanthopterin is proposed as a simple method of recognition of patients with malignant hyperphenylalaninemia (MHPA). High levels of urinary dihydroxanthopterin are found in untreated patients with phenylketonuria (PKU) or with dihydropteridine reductase (DHPR) deficiency. After dietary control of the serum phenylalanine level in PKU, the urinary dihydroxanthopterin falls to near normal levels. In DHPR deficiency urinary dihydroxanthopterin levels are high even when serum phenylalanine levels are in the range achieved on dietary treatment. Low levels would be expected in patients with defects in tetrahydrobiopterin synthesis even before dietary treatment. Confirmation of the diagnosis of different forms of MHPA then requires more detailed studies, but dietary treatment of other PKU patients can proceed with confidence.

A new sulfur amino acid, named hawkinsin, identified in a baby with transient tyrosinemia and her mother.

An unknown compound present in the urine of a girl with prolonged transient tyrosinemia and her mother was isolated and identified as (2-L-cystein-S-yl-1,4-dihydroxycyclohex-5-en-1-yl)-acetic acid (IVa). The new amino acid was named hawkinsin (Haw) and characterized by gas chromatography-mass spectrometry (GC-MS) of its penta-trimethylsilyl (TMS) derivative and of its desulfuration components. Haw was compared with the synthetic reference compound using GC-MS, IR, TLC, PC, ion-exchange chromatogrpahy and high-voltage electrophoresis. IVa and (2,6-bis-L-cystein-S-yl-1,4-dihydroxycyclohexyl-1)-acetic acid were synthesized from 4-quinolacetic acid, the latter was prepared in two different ways. It is postulated that Haw originates from an intermediate in the 4-hydroxy-phenylpuruvate hydroxylase reaction (EC 1.14.2.2), and that mother and child are heterozygous for an inborn error of metabolism characterized by a defect in this hydroxylase system, which is unable to rearrange the intermediate to homogentisic acid.

Stability of protein and mRNA in human postmortem liver--analysis by two-dimensional gel electrophoresis.

The method of two-dimensional polyacrylamide gel electrophoresis has been used to investigate the post mortem stability of protein and mRNA in human liver. The electrophoretic mobility of proteins and of the in vitro translation products of the mRNA were found to be essentially unaffected by incubation of the liver at 37 degrees C for up to 2 h or at 4 degrees C for up to 16 h. This study indicates that the major protein and mRNA species in liver are stable enough following death to allow meaningful studies on tissue collected under standard autopsy conditions.

New metabolites in isovaleric acidemia.

Two metabolites, 4-hydroxyisovaleric acid and mesaconic acid, have been identified and quantified in the urine of a patient with isovaleric acidemia. These compounds do not appear to have been reported previously as being components of human metabolism. In addition, large quantities of 3-methylbutyrolactone, the lactone of 4-hydroxyisovaleric acid, were observed in the volatile profile obtained by headspace chromatography. The demonstration of 4-hydroxyisovaleric acid supports the contention that urinary methylsuccinic acid seen in patients with isovaleric acidemia has arisen by omega-oxidation of isovaleric acid. The identification of mesaconic acid may indicate that the methylsuccinic acid formed in these patients is subject to further metabolism.

Analysis of the distribution of Cu, Fe and Zn and other elements in brindled mouse kidney using a scanning proton microprobe.

An analysis of the distribution of trace metals in the kidney cortex in normal and brindled male mice has been carried out with a scanning proton microprobe. Enzyme histochemical staining techniques were used to distinguish between proximal and distal tubules. Average copper levels were increased in brindled kidney tissue sections, with the above-normal Cu accumulation found to occur entirely within the proximal tubules. Therefore, the proximal tubule is now regarded as the location where the defect in Cu transport in brindled mice is manifested the most clearly. The distribution of Fe was found to be non-uniform with some tubule cross-sections exhibiting high concentrations in both genotypes. The distribution of Zn was found to be uniform, and the concentration was similar for each genotype.

Albumin has no role in the uptake of copper by human fibroblasts.

The mechanism of copper uptake by cells has been the subject of controversy for some time. This paper examines the possibility of a role for albumin in the uptake of copper by fibroblasts. Although the cells could accumulate copper from a copper-albumin complex, there was no evidence for either copper-albumin or albumin receptors on the cell surface. The possibility of a surface exchange mechanism for copper was examined. While copper uptake showed saturation with increasing concentrations of labelled copper-albumin, adding unlabelled copper to the incubation medium did not inhibit uptake. Adding albumin or histidine to the copper-albumin complex resulted in an inhibition of copper uptake. The results can only be explained by the cell taking up free copper from the incubation medium, with the albumin then releasing its copper to maintain the equilibrium between free and bound metal. Since, in vivo there is essentially no free copper in serum, it is concluded that albumin is most unlikely to play a role in the uptake of copper by fibroblasts.

The effect of D-penicillamine on metallothionein mRNA levels and copper distribution in mouse hepatocytes.

Penicillamine increases the levels of metallothionein (MT) mRNA in a time and concentration dependent manner without altering either the rate of copper uptake or the amount of copper within the cell. The effect is dependent on the presence of intracellular copper, however, since depletion of copper by chelators blocks the effect, and does not alter the ability of dexamethasone to stimulate mRNA production. Penicillamine did not alter the distribution of 64Cu in the hepatocytes, as measured by fast protein liquid chromatography (FPLC), although the pattern may be affected by the amount of MT present. The data indicates that penicillamine removes copper from some intermediary ligand, thereby making it available to induce metallothionein. It is possible that this is part of the therapeutic action of the chelator in the treatment of Wilson's disease.