Biomedical subjects
Publications and source records attributed to V E Shih.
We report on a neonate with hyperammonemic coma in whom hyperornithinemia-hyperammonemia-homocitrullinuria syndrome was diagnosed. Appropriate treatment led to rapid clinical and metabolic improvement. The incorporation of 14C-ornithine on cultured fibroblasts confirmed the diagnosis. At the age of 18 months, the patient is in excellent clinical condition.
Prenatal testing of 12 pregnancies at risk for argininosuccinic aciduria due to argininosuccinate lyase (ASAL) deficiency and three pregnancies at risk for citrullinaemia due to argininosuccinate synthatase (ASAS) deficiency was performed by metabolite detection in amniotic fluid and measurement of enzyme activity in uncultured and cultured chorionic tissue and in cultured amniocytes. From our data and those of previous studies, amniotic fluid argininosuccinate measurement alone is clearly a reliable and rapid diagnostic test for both severe and mild ASAL deficiency if maternal ASAL deficiency can be excluded. For prenatal diagnosis of ASAS deficiency, however, both measurement of the amniotic fluid citrulline level and enzyme assay should be employed.
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Cystathionine beta-synthase (CBS) deficiency is an autosomal recessive disorder characterized by homocystinuria and multisystem clinical disease. Patients responsive to pyridoxine usually have a milder clinical phenotype than do nonresponsive patients, and we studied the molecular pathology of this disorder in an attempt to understand the molecular basis of the clinical variation. We previously reported a T833C transition in exon 8 causing a substitution of threonine for isoleucine at codon 278 (I278T). By PCR amplification and sequencing of exon 8 from genomic DNA we have now detected the I278T mutation in 7 of 11 patients with in vivo pyridoxine responsiveness and in 0 of 27 pyridoxine-nonresponsive patients. Two pyridoxine-responsive patients are homozygous and five are heterozygous for I278T. We have now observed the I278T mutation in 41% (9 of 22) of the independent alleles in pyridoxine-responsive patients of varied ethnic backgrounds. In two of the compound heterozygotes we identified a novel mutation (G139R and E144K) in the other allele. The finding that the two patients who are homozygous for I278T have only ectopia lentis and mild bone demineralization suggests that this mutation is associated with both in vivo pyridoxine responsiveness and mild clinical disease. Compound heterozygous patients who have one copy of this missense mutation are likely to retain some degree of pyridoxine responsiveness.
Cystathionine beta-synthase (CBS) deficiency is an autosomal recessive disorder associated with multisystem clinical disease. We analyzed PCR amplified products from patients' RNA and genomic DNA. Direct sequencing of the entire coding region of the CBS gene revealed a G-919 to A transition in exon 8, resulting in replacement of Gly 307 by Ser (G307S) in the protein. The mutation was detected in one allele of patient L171 of French/Scottish ancestry and in both alleles of patient L198 of Irish ancestry. Amplifying and sequencing exon 8 from the genomic DNA showed that both parents of L198 were heterozygotes for G307S. The pathogenicity of the mutation was demonstrated in an expression experiment. The mutant protein was apparently stable in E.coli extracts and lacked catalytic activity. Sequencing of exon 8 revealed the G307S mutation in five additional families. All patients have pyridoxine nonresponsive homocystinuria. We have now observed this mutation in 9 of 52 apparently unrelated alleles of varied ethnic backgrounds. All 9 are from patients with Celtic (Irish/English/Scottish/French) ancestry in either one or both parents. The G307S mutation was detected in 50% (9 of 18) of the Celtic alleles in our series. The second mutation found in exon 8 is the I278T mutation, which was described previously in one allele of a pyridoxine responsive patient. This missense mutation was detected in one allele of a pyridoxine nonresponsive patient and in both alleles of a pyridoxine responsive patient. The latter suggests that I278T is probably associated with pyridoxine responsiveness.
To determine whether the naturally occurring amino acid threonine, a potential precursor for glycine biosynthesis in the spinal cord, has an effect on spasticity in multiple sclerosis, 26 ambulatory patients were entered into a randomized crossover trial. Threonine administered at a total daily dose of 7.5 g reduced signs of spasticity on clinical examination, although no symptomatic improvement could be detected by the examining physician or the patient. In contrast to the side effects of sedation and increased motor weakness associated with antispasticity drugs commonly used for the treatment of multiple sclerosis, no side effects or toxic effects of threonine were identified. Levels of threonine were elevated in serum and cerebrospinal fluid during treatment, but glycine levels did not change. Enhancement by threonine of glycinergic postsynaptic inhibition of the motor reflex arc in the spinal cord may represent a non-sedating, nontoxic approach to the management of spasticity in multiple sclerosis.
Gyrate atrophy of the choroid and retina (GA) is an autosomal recessive disorder in which a deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT) leads to progressive blindness. Previously, we and others have reported a number of missense mutations and splice defects in the OAT gene associated with GA. In the present case, through sequencing of the PCR amplified cDNA products, we have detected a novel 15-bp deletion within exon 5 of the OAT gene which retains the original reading frame. The deleted PCR product is the only one produced from the patient's mRNA, while mRNA from the patient's mother yields both deleted and normal length PCR products. The alternate, apparently nonexpressing OAT allele in this patient was inherited from the father, who displays only the normal length PCR product. The codon at the deletion joint remains unaltered, predicting the loss of the pentapeptide Tyr-Thr-Val-Lys-Gly without any other amino acid change. The breakpoints are adjacent to or within two copies of a 4-bp direct repeat, which may have implications for the mechanism of deletion.
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Ornithine aminotransferase (OAT), a mitochondrial matrix enzyme, is deficient in patients with gyrate atrophy of the choroid and retina. In human, the OAT structural gene maps to Chromosome (Chr) 10q26 and several OAT-related sequences, some of which are known to be processed pseudogenes, which map to Xp11.3-11.21. Here, we report chromosomal localization in the mouse of the OAT gene and related sequences. Genomic DNA blot analysis of a well-characterized panel of Chinese hamster x mouse somatic cell hybrids using a human OAT probe revealed two murine loci, one on mouse Chr 7 and the other on Chr X. In addition, segregation of restriction fragment length polymorphisms (RFLPs) detected by the OAT probe in recombinant inbred (RI) strains detected a third locus on Chr 3 and positioned the X locus near Cf-8 and Rsvp. Progeny of an intersubspecific backcross were used to map the Chr 7 locus between Tyr and Int-2, near Cyp2e-1.
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A case report of 3-ketothiolase deficiency due to a defect of mitochondrial acetoacetyl-CoA thiolase protein in a Brazilian boy and its biochemical investigation is presented. The child had moderate generalized hypotonia, EEG alterations and crises of metabolic acidosis following infections. Hypotonia and EEG abnormalities disappeared with a low protein diet, and physical and mental development are normal. Urinary organic acid excretion was typical of 3-ketothiolase deficiency, showing consistently high levels of 2-methyl-3-hydroxybutyric acid and tiglylglycine. Activation of acetoacetyl-CoA thiolase activity by potassium (K) ion in cultured fibroblasts was not observed, demonstrating the lack of activity of mitochondrial acetoacetyl-CoA thiolase. In addition, the signal for the mitochondrial acetoacetyl-CoA thiolase protein was undetectable in the immunoblot analysis. In the pulse-chase experiments, the signal for mitochondrial acetoacetyl-CoA thiolase was detected after a 1-h pulse but not after a 24-h chase. These results indicate that the deficiency was caused by an unstable mitochondrial acetoacetyl-CoA thiolase protein.
We have observed that the fragile hair of two untreated patients with argininosuccinic aciduria showed abnormal alternating zones of bright and dark banding by polarizing microscopy. Scanning electron microscopy documented discontinuous grooves with a 50 to 100 microns periodicity. Results of amino acid analysis of the hair were essentially normal. After the patients were treated with a low-protein, arginine-supplemented diet, the hair assumed a normal appearance. Five patients already treated with diet showed no hair abnormalities. The pathogenesis of the hair changes in unknown, but our findings suggest that products generated in the disease can adversely affect metabolically active tissue such as hair.
Inherited disorders in the metabolism of amino acids and organic acids may cause neurological dysfunction and acute metabolic crises. For many of these disorders, early diagnosis and early treatment can greatly improve the outcome. A general description of the clinical manifestations and a discussion of selected techniques and approaches for the laboratory diagnosis are reviewed.
A 5-year-old Asian-Indian female presented with bilateral cobblestone-like peripheral lesions, a single area of chorioretinal atrophy, infero-nasal to the disc, in her right eye and a non-recordable single flash ERG. Serum ornithine level was assayed at 841 mumol/l, ten times normal levels, and a diagnosis of gyrate atrophy, due to ornithine aminotransferase deficiency was made. The patient was refractory to any form of therapy and her clinical lesions spread rapidly in both eyes, showing both centrifugal and centripetal spread in her right eye. A rapid deterioration in her psychophysical tests was also seen over the 28 months follow-up. The presence of cobblestone-like peripheral lesions in a child should alert the clinician to the possibility of gyrate atrophy, and the rapid spread of the chorioretinal lesions coupled with non-recordable single flash ERGs, in a young patient may suggest a much poorer prognosis.
Gyrate atrophy (GA) is an autosomal recessive eye disease characterized by progressive loss of vision due to chorioretinal degeneration. It is associated with a deficiency of the mitochondrial enzyme ornithine aminotransferase (OATase) with consequent hyperornithinemia. Although the clinical phenotype is largely confined to the eye, OATase deficiency is a systemic disorder. A step toward delineation of the enzyme defect in GA at the molecular level has been made by cloning and characterizing the cDNA and structural gene for OATase. The structural gene for OATase maps to chromosome 10 (10q26) and OATase-related sequences map to the X chromosome (Xp11.2). A diverse number of mutations at the OATase locus in GA patients of varied ethnic origins have been defined employing polymerase chain reaction and other molecular biological techniques. The majority of these mutations are of the missense type although a splicing mutation in one patient has recently been identified. The functional consequences of some of these mutations have been tested and confirmed in a eukaryotic expression system. These mutations demonstrate the allelic heterogeneity, which extends to both pyridoxine responsive and non-responsive forms of GA, reflecting the clinical and biochemical heterogeneity observed in this disease. The molecular studies in addition to providing information on the structure/function of the enzyme will facilitate understanding of the retinal pathophysiology in this disorder.
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A 39-year-old man and his 42-year-old sister, both vegetarians, had episodic confusion for many years, but their mental function was normal between those episodes. They were recently diagnosed with hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome. Hyperammonemia was documented during an episode of confusion in the male sibling but not in his sister. Both had elevated plasma ornithine, glutamine, and alanine levels and persistently low plasma lysine levels. Homocitrulline was present in their urine, and orotic aciduria and orotidinuria developed in the male sibling following ingestion of allopurinol. Studies on their cultured skin fibroblasts showed deficient metabolism of ornithine, indicating a defect in ornithine transport across the mitochondrial membrane. During therapy with citrulline and phenylbutyrate sodium, plasma ornithine levels increased in both patients, while plasma levels of glutamine and alanine decreased to normal. Since therapy started, their clinical conditions have also improved, and no recurrent neurologic dysfunction has occurred during a follow-up period of 20 months.
Glutamate stimulates resting ventilation by altering neural excitability centrally. Hypoxia increases central ventilatory drive through peripheral chemoreceptor stimulation and may also alter cerebral perfusion and glutamate metabolism locally. Therefore the effect of hypoxia and peripheral chemodenervation on cerebrospinal fluid (CSF) transfer rate of in vivo tracer amidated central nervous system glutamate was studied in intact and chemodenervated pentobarbital-anesthetized dogs during normoxia and after 1 h of hypoxia induced with 10 or 12% O2 in N2 breathing at constant expired ventilation and arterial CO2 tension. Chemodenervation was performed by bilateral sectioning of the carotid body nerves and cervical vagi. CSF transfer rates of radiotracer 13NH4+ and [13N]glutamine synthesized via the reaction, glutamate + NH4(+)----glutamine, in brain glia were measured during normoxia and after 1 h of hypoxia. At normoxia, maximal glial glutamine efflux rate jm = 103.3 +/- 11.2 (SE) mumol.l-1.min-1 in all animals. After 1 h of hypoxia in intact animals, jm = 78.4 +/- 10.0 mumol.l-1.min-1. In denervated animals, jm was decreased to 46.3 +/- 4.3 mumol.l-1.min-1. During hypoxia, mean cerebral cortical glutamate concentration was higher in denervated animals (9.98 +/- 1.43 mumol/g brain tissue) than in intact animals (7.63 +/- 1.82 mumol/g brain tissue) and corresponding medullary glutamate concentration tended to be higher in denervated animals. There were no differences between mean glutamine and gamma-aminobutyric acid concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)