1/10. Biochemical and molecular characterization of mutant hexosaminidase a in a Turkish family.BACKGROUND: tay-sachs disease is a form of monosialoganglioside triaose (GM2) gangliosidosis that results from the mutations in the alpha-subunit gene of hexosaminidase a. In the B1 variant, the active site of the alpha-subunit of the enzyme is thought to be affected. In the present study, a patient who had previously been diagnosed as a B1 variant is further analyzed. The patient's parents and brother were also analyzed. methods: Single-stranded conformational polymorphism (SSCP) and dna sequencing analysis were conducted in all cases. In addition, hexosaminidase a (Hex A) was isolated from leukocyte homogenates of the patient's parents and brother using DE 52 ion-exchange chromatography, and thermostability analyses of the isolated enzymes were performed. RESULTS: hexosaminidase a of the parents was found to be more thermostable than normal Hex A. dna sequencing analysis revealed a 12-bp deletion mutation in exon 10 of the Hex A gene. The patient was a homozygote and the parents were heterozygotes for the mutation, which could also be observed at the dna double strands by SSCP analysis. These deleted bases are located within the catalytic domain of the alpha-subunit. CONCLUSIONS:The 12-bp deletion mutation in exon 10 of Hex A is responsible for the increased thermostability of the enzyme. Considering this mutation has previously been found in a Turkish Tay-Sachs patient, the patient in the present study may have another mutation on the Hex B gene that causes decreased thermostability of the enzyme. Thermal inactivation assay may not be sufficient for a correct diagnosis in such unusual cases.- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |
2/10. tay-sachs disease with conspicuous cranial computerized tomographic appearances.An autopsy case of a 3-year-old female infant with tay-sachs disease was presented. A cherry red spot in the fundus and a deficiency of N-acetyl-beta-hexosaminidase a in the white blood cells were revealed soon after admission at the age of one year. Her parents and sister were found to be healthy carriers. The patient showed a typical clinical course with marked cranial swelling. In addition to the marked ballooning of neurons on light microscope, membranous cytoplasmic body (MCB) on electron microscope and abnormal accumulation of g(m2) ganglioside in the cerebral cortex by thin layer chromatography were confirmed in the autopsy specimens. In the late stage of her clinical course, the cranial computerized tomography (CT) demonstrated symmetric and deep-wavy hyperdense cerebral cortical zones, diffuse hypodensity and diminished volume of cerebral white matter, mild to moderate ventricular dilatation, and a small cerebellum and brainstem. These conspicuous appearances of the cranial CT seem to be characteristic of tay-sachs disease in the late stage, and they are derived from abnormal accumulation of g(m2) ganglioside in the cerebral cortex, and diffuse intense demyelination (dysmyelinating demyelination) of the cerebral white matter.- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |
3/10. adult-onset GM2 gangliosidosis diagnosed in a fetus.amniocentesis and subsequent tests are reported on a fetus conceived of a rare mating type: its mother has an intermediate level of beta hexosaminidase a (HEX A), characteristic of carriers of tay-sachs disease (TSD), while the father suffers from an adult-onset GM2 gangliosidosis (AOG) with severe HEX A deficiency. Activity of HEX A in the cultured fetal cells was very low when measured by the heat-inactivation method, thus showing the typical biochemical phenotype of TSD fetuses. However, upon separation of HEX isozymes by ion exchange chromatography, residual HEX A (17 per cent of total HEX) was demonstrated. Also in contrast to TSD fetuses, this fetus' fibroblasts were able to synthesize the precursor of alpha chains of HEX, and ultrastructural examination of its brain revealed few atypical lamellar bodies, unlike those found in TSD fetuses of the same gestational age. It is therefore concluded that the fetus was not affected with TSD, but rather with AOG.- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |
4/10. GM1 gangliosidosis: clinical and laboratory findings in eight families.GM1 Gangliosidosis is an autosomal recessive genetic disorder due to deficiency of the lysosome enzyme beta-galactosidase, with consequent tissue accumulation of glycolipids, oligosaccharides, and especially GM1 ganglioside. In the present paper we report the clinical and laboratory findings obtained for eight families starting from eight index cases exhibiting the childhood form of the disease. The total number of cases in these families may be as high as 14, thus causing GM1 gangliosidosis to be the inborn metabolic error most frequently diagnosed in our service. Hypotonia, neuromotor retardation, hepatosplenomegaly, macrocephaly, and hydrocele are some of the most frequent clinical findings. The disease evolves towards convulsions and bronchopneumonia, leading to patient death generally during the first half of the second year of life. The presence of vacuolated lymphocytes, alterations of the lumbar vertebrae, and cherry spots on the retina were observed in almost all patients. When tested for inborn metabolic errors, all patients gave normal results, a fact that may have confused and delayed diagnosis. Diagnosis was made by urine oligosaccharide chromatography and confirmed by beta-galactoside measurement in peripheral blood leukocytes. This method proved to be accurate also for the detection of heterozygotes, which permitted post-mortem diagnosis in two families. The authors speculate that increased fetal loss and tendency towards macrosomy may be possible characteristics of the disease, suggest that testing for vacuolated lymphocytes be used as a screening method, and propose that urine oligosaccharide chromatography be included in the routine screening for inborn metabolic errors.- - - - - - - - - - ranking = 2keywords = chromatography (Clic here for more details about this article) |
5/10. Thermal activation of hexosaminidase a in a genetic compound with tay-sachs disease.Increase in total hexosaminidase activity has been observed during heat treatment of serum and leukocyte specimens from a 1-year-old boy with cherry-red spot and severe and progressive mental and motor deterioration. The activity increased 40% in the first 40-70 min of incubation at 50 degrees C and pH 4.3, but declined thereafter and was only slightly above the initial activity in the final 2-3 h of incubation. Heat treatment of specimens from family members revealed very reduced rates of inactivation of hexosaminidase in the proband's father and some paternal relatives, whereas those of the mother and some maternal relatives were indistinguishable from those of Tay-Sachs carriers. Mixing experiments with enzyme preparations from the proband, normal controls and patients with tay-sachs disease resulted in additive values and did not support the possibility of inhibitor- or activator-related defect. Fractionation of heat-treated samples by ion exchange chromatography and electrophoresis, as well as examination of the separated fractions for their thermostability, have shown that hexosaminidase a is the activated component and hexosaminidases B, I1 and I2 are not affected. These findings suggest that the patient is a genetic compound and the apparent thermal activation is probably due to formation of hexosaminidase a from altered alpha-subunits produced by the paternal mutant alpha-allele and beta-subunits produced by the normal beta-alleles.- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |
6/10. Chronic GM2 gangliosidosis masquerading as atypical friedreich ataxia: clinical, morphologic, and biochemical studies of nine cases.A progressive spinocerebellar degenerative disorder was characterized in nine patients, aged 11 to 37 years, from four unrelated Ashkenazi Jewish families; affected individuals had markedly deficient beta-hexosaminidase a activity. Symptoms included early onset of cerebellar signs (tremor, incoordination, and dysarthia) and, with maturity, the development of upper and lower motor neuron disorders, marked dysarthia, and ataxia. Three older patients, aged 26, 32, and 37 years, had dementia or recurrent psychotic episodes. Membrane-bound lamellar cytoplasmic inclusions, consistent with lysosomal ganglioside accumulation, were observed in rectal ganglia. The activity of beta-hexosaminidase a was markedly deficient in all sources analyzed. parents had activities consistent with heterozygosity, confirming autosomal-recessive transmission of the beta-hexosaminidase a-deficient gene and the adult variant disorder. Residual beta-hexosaminidase a activity, partially purified by anion-exchange chromatography from cultured skin fibroblasts of the affected individuals, was heat-labile and co-electrophoresed with normal beta-hexosaminidase a. These findings suggest that these patients were allelic for a new beta-hexosaminidase a mutation and may represent a genetic compound of this allele and the allele causing tay-sachs disease.- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |
7/10. Hereditary heat-labile hexosaminidase b: its implication for recognizing Tay-Sachs genotypes.Two pairs of alleles, at the two loci of hexosaminidase (HEX), were found to segregate in an Arab inbred family: the normal and the mutant Tay-Sachs (TSD) alleles of HEX A, and the normal and a mutant allele of HEX B. Since the mutant HEX B is heat labile, no reliable identification of TSD genotypes can be obtained in its presence, as long as the proportions of HEX A and B are estimated by the routinely used heat-inactivation method. The genotypes may be correctly identified in such cases by separation of the two isoenzymes on ion-exchange chromatography, estimating their individual activities, and calculating the ratio between them. Of the nine genotype combinations possible with these two pairs of alleles, five have been identified in the reported family by this procedure.- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |
8/10. Extraction and purification of gangliosides from plasma and fibroblasts before analysis by thin layer chromatography.A procedure to extract and purify gangliosides from small volumes of plasma (0.6 mL), cerebrospinal fluid (1 mL) and fibroblasts is described. gangliosides were extracted with chloroform/methanol and purified by means of reversed phase chromatography and gel filtration before analysis by thin layer chromatography. The procedure proved to be useful in confirming deficiency of lysosomal enzyme activity affecting ganglioside breakdown. The new procedure also appeared to be useful to monitor ganglioside catabolism in cultured fibroblasts loaded with ganglioside.- - - - - - - - - - ranking = 6keywords = chromatography (Clic here for more details about this article) |
9/10. Two new mutations in a late infantile Tay-Sachs patient are both in exon 1 of the beta-hexosaminidase alpha subunit gene.We have identified two new point mutations in the beta-hexosaminidase alpha subunit (HEX A) gene in a non-Jewish tay-sachs disease patient with an unusual late infantile onset disease phenotype. The patient was a compound heterozygote with each allele of the HEX A gene containing a different mutation in exon 1. One of these is a T to C transition in the initiation codon, expected to produce no alpha subunit and therefore a classical infantile phenotype. The unusual clinical aspects and later onset in the patient must therefore be a result of residual hexosaminidase a activity associated with a mutant alpha subunit containing the second mutation, substitution of serine for proline at amino acid 25 owing to a C to T change at nucleotide 73. Western blotting and DE-52 ion exchange chromatography have been used to examine the behaviour of this mutant alpha subunit.- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |
10/10. Naturally occurring GM2 gangliosidosis in two Muntjak deer with pathological and biochemical features of human classical tay-sachs disease (type B GM2 gangliosidosis).Two juvenile sibling male Muntjak deer (Muntiacus muntjak) with histories of depression, ataxia, circling and visual deficits were studied. cerebrospinal fluid analyses revealed vacuolated macrophages that contained long parallel needle-like intracytoplasmic inclusions. light microscopically, nerve cell bodies throughout the brain, ganglion cells within the retina and neurons in the myenteric plexuses were variably swollen and had pale granular to finely vacuolated eosinophilic cytoplasm. Neuronal cytoplasm stained specifically with sudan black and Luxolfast blue stains. Within the brain there were occasional axonal spheroids, foci of astrogliosis and scattered microglial cells with abundant pale foamy cytoplasm. Electron microscopy of the brain and retina revealed numerous neurons and ganglion cells, respectively, with multiple membrane-bound structures that contained compact electron-dense membranous whorls and fewer parallel membranous stacks. Thin layer chromatography of total lipid extracts of the cerebral cortex of both cases revealed massive accumulation of G(M2) ganglioside. Crude kidney extracts of the two affected deer were able to hydrolyze 4-methylumbelliferyl beta-GlcNAc, but not 4-methylumbelliferyl beta-GlcNAc-6-sulfate, indicating the defect of beta-hexosaminidase a. Cellogel electrophoresis of the kidney extracts also revealed the deficiency of beta-hexosaminidase a in the two deer. It is concluded that these two deer had the biochemical lesion identical to that of human type B G(M2) gangliosidosis (classical tay-sachs disease).- - - - - - - - - - ranking = 1keywords = chromatography (Clic here for more details about this article) |
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