Standardization of complementation grouping of peroxisome-deficient disorders and the second Zellweger patient with peroxisomal assembly factor-1 (PAF-1) defect.
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Adrenoleukodystrophy↗
Biomedical subjects
N Shimozawa
Publications and source records attributed to N Shimozawa.
[Clinical and molecular aspects of peroxisome-deficient disorders].
Peroxisome-deficient disorders including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) are characterized by the absence of peroxisomes associated with secondary multiple enzyme deficiencies and by a defect in the neuronal migration. Collaborative complementation studies revealed the presence of at least 9 genetic groups among these disorders. Clinical phenotypes did not correlate with the genetic grouping. Responsible gene for one of these groups (group F) was determined as peroxisome assembly factor-1 (PAF-1) by the functional cloning using peroxisome-deficient CHO cell mutant. A patient of group F was a homozygote with C to T transitions in PAF-1 gene which resulted in a nonsense mutation. These results will pave the way for the elucidation of mechanisms of peroxisome biogenesis and the pathophysiology of neuronal migration.
Adrenoleukodystrophy↗
[Peroxisomal disorders--DNA analysis].
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Adrenoleukodystrophy↗
A human gene responsible for Zellweger syndrome that affects peroxisome assembly.
The primary defect arising from Zellweger syndrome appears to be linked to impaired assembly of peroxisomes. A human complementary DNA has been cloned that complements the disease's symptoms (including defective peroxisome assembly) in fibroblasts from a patient with Zellweger syndrome. The cause of the syndrome in this patient was a point mutation that resulted in the premature termination of peroxisome assembly factor-1. The homozygous patient apparently inherited the mutation from her parents, each of whom was heterozygous for that mutation.
Amino Acid Sequence↗
Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells.
Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1'-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.
Adrenoleukodystrophy↗
Transformation and characterization of mutant human fibroblasts defective in peroxisome assembly.
Human skin fibroblasts deficient in peroxisome biogenesis were transformed by transfecting SV40 ori- DNA with the use of an electroporator, and the biochemical, immunocytochemical, and cytogenetic properties of the transformants were analyzed. Cells (1 x 10(6)) from a patient with Zellweger syndrome and one with neonatal adrenoleukodystrophy were suspended with 2 micrograms of SV40 ori- DNA in PBS; then a high-voltage pulse (2000 V, 30 microseconds) was generated two times. Several colonies expressing large T-antigen were picked up 4 weeks after transfection. Doubling time of the transformants was about half of that and the saturation density was 5 to 10 times greater than that of the parental cells. Biochemical abnormalities including defective lignoceric acid oxidation, dihydroxyacetone phosphate acyltransferase deficiency, and disturbed biosynthesis of peroxisomal beta-oxidation enzymes were preserved in the transformants. Peroxisomes were defective in all colonies, as determined by immunofluorescence staining using anti-catalase IgG. Cell fusion studies confirmed that the transformants belong to the same complementation groups as those of the parental cells. These transformed mutant cell lines are expected to be useful tools for investigating the pathogenesis of inherited diseases related to defects in peroxisome biogenesis.
Acyltransferases↗
Animal cell mutants represent two complementation groups of peroxisome-defective Zellweger syndrome.
Generalized peroxisome-deficient disorders including cerebro-hepato-renal Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease are autosomal recessive diseases, where catalase-containing particles (peroxisomes) are morphologically absent. We previously isolated two Chinese hamster ovary (CHO) cell mutants (Z24 and Z65) that resemble the fibroblasts from patients with such diseases, in their defective peroxisome assembly (Tsukamoto, T., S. Yokota, and Y. Fujiki. 1990. J. Cell Biol. 110:651-660). Here we report isolation by the P9OH/UV method of a peroxisome-deficient CHO mutant, ZP92, of the third complementation group distinct from those of Z24 and Z65. Peroxisomal membrane ghosts were noted by immunochemical staining in all of the CHO mutants. Complementation analysis by cell fusion of the CHO mutants with cultured fibroblasts from patients with generalized peroxisomal disorders revealed that two CHO mutants (Z24 and ZP92) represent the human complementation groups, E (the same as group 1 in the U.S.) and C (the same as group 4), respectively. These CHO cell mutants are an apparently relevant animal cell model for studies on the molecular bases and primary defects of human peroxisome-deficient diseases.
Animals↗
Different intracellular localization of peroxisomal proteins in fibroblasts from patients with aberrant peroxisome assembly.
We investigated intracellular localization of peroxisomal proteins in fibroblasts from patients with Zellweger syndrome and neonatal adrenoleukodystrophy in whom peroxisomes were morphologically deficient or severely decreased. Indirect immunofluorescence staining revealed that catalase was mainly detected in the cytosol of fibroblasts from these patients, but a small amount of catalase was detected in granular pattern in a small percentage of cells. Double immunofluorescence staining revealed that catalase-containing particles in these patients also contained acyl-CoA oxidase and nonspecific lipid transfer protein. However, a 70 kD integral membrane protein and 3-ketoacyl-CoA thiolase were detected in all cells in granular pattern. Subcellular fractionation using digitonin after cell labeling revealed that a small amount of acyl-CoA oxidase and about half of thiolase in the precursor form were detected in the particulate fraction. These data suggest that the mechanisms of the transport and processing of catalase, acyl-CoA oxidase and nonspecific lipid transfer protein are different from those of the 70 kD integral membrane protein and 3-ketoacyl-CoA thiolase.
Acyl-CoA Oxidase↗
Effects of sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) on fatty acid oxidation in fibroblasts from patients with peroxisomal diseases.
The effects of sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a potent inhibitor of carnitine palmitoyltransferase I, on fatty acid oxidation were investigated using fibroblasts from control subjects and from patients with peroxisomal disorders. [1-14C]Palmitate oxidation was inhibited by 8% of the control value when 15 microM POCA was added to the medium. The inhibition by POCA was significantly (P less than 0.05) stronger in fibroblasts from patients with Zellweger syndrome or with neonatal adrenoleukodystrophy, in which peroxisomes and peroxisomal beta-oxidation enzymes were absent. However, the inhibition in fibroblasts from patients with X-linked adrenoleukodystrophy, in which a specific defect of peroxisomal lignoceroyl-CoA synthetase was speculated, was similar to that in the controls. [1-14C]Lignocerate oxidation was not influenced by the addition of POCA, in samples from the controls and from the patients. These results indicate that peroxisomes account for a small but demonstrable proportion of palmitate oxidation, and add new evidence to the concept that lignocerate is oxidized exclusively in the peroxisomes. Our findings also support the hypotheses that the activity of palmitoyl-CoA synthetase and the enzymes of beta-oxidation cycle in peroxisomes are normal in patients with X-linked adrenoleukodystrophy and that a specific defect of lignoceroyl-CoA synthetase is responsible for the accumulation of very long chain fatty acids in these patients.
Adrenoleukodystrophy↗
[Clinical, pathological and biochemical abnormality in peroxisomal disorders].
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Brain↗
Gene assignment of Zellweger syndrome to 7q11.23: report of the second case associated with a pericentric inversion of chromosome 7.
The case of a newborn girl with Zellweger syndrome and a pericentric inversion of chromosome 7, 46,XX, inv(7)(p12q11.23), is reported. The diagnosis was confirmed by marked deficiency of peroxisomal beta-oxidation enzymes in hepatic cells from autopsy samples. This is the second case of Zellweger syndrome associated with a rearrangement of chromosome 7, the tentative gene assignment to 7q11 being further supported; the gene is probably confiend to 7q11.23.
Acetyl-CoA C-Acyltransferase↗
Major peroxisomal membrane polypeptides are synthesized in cultured skin fibroblasts from patients with Zellweger syndrome.
Biosynthesis of three major peroxisomal membrane polypeptides (70, 26, and 22 kD) were investigated in an attempt to account for the absence of peroxisomes in patients with Zellweger syndrome. Immunoblot analysis using membrane fractions of autopsied livers revealed that 70- and 22-kD polypeptides were deficient in three Japanese patients with this syndrome. However, pulse-labeling and chase experiments using cultured skin fibroblasts demonstrated that 70-, 26, and 22-kD polypeptides were synthesized in these patients at levels similar to those in the controls. No significant difference in the degradation rates of these polypeptides was noted between the patients and the controls. Subcellular fractionation after pulse-chase experiments revealed 70-kD polypeptides present in a membrane fraction of the cells. These observations indicate that at least 70-, 26-, and 22-kD peroxisomal membrane polypeptides are normally synthesized and considered to be transported into the disorganized peroxisomal membrane in patients with Zellweger syndrome. Molecular and functional analyses of these polypeptides are underway to clarify whether they contribute to membrane integrity or to the transport of peroxisomal matrix enzymes.
Cells, Cultured↗
Diagnosis of Zellweger syndrome by rectal biopsy: immunoblot of peroxisomal beta-oxidation enzyme and activity of dihydroxyacetone phosphate acyltransferase in rectal mucosa.
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Acetyl-CoA C-Acyltransferase↗
Molecular analysis of peroxisomal beta-oxidation enzymes in infants with Zellweger syndrome and Zellweger-like syndrome: further heterogeneity of the peroxisomal disorder.
The biosynthesis of enzymes of peroxisomal beta-oxidation was investigated in an attempt to elucidate the mechanism of deficiencies of proteins of these enzymes in 3 infants with Zellweger syndrome and in a baby with Zellweger-like syndrome with clinical and biochemical findings consistent with Zellweger syndrome except that the peroxisomes were detected electronmicroscopically. Enzyme proteins of peroxisomal beta-oxidation, acyl-CoA oxidase, bifunctional protein and 3-ketoacyl-CoA thiolase were hardly detectable, in both syndromes. Total hepatic RNA extracted from the liver of one patient with each syndrome and three controls was translated in a rabbit reticulocyte lysate protein-synthesizing system in the presence of [35S]methionine. Translatable mRNAs for all of the peroxisomal beta-oxidation enzymes were detected in both patients at much the same levels seen in the controls. Pulse labelling and chase experiments of fibroblasts from the control revealed that the 72 kDa subunit of acyl-CoA oxidase was first synthesized, after which the 52 kDa and 21 kDa subunits were processed from the 72 kDa subunit. In the patient with Zellweger syndrome, little of the 52 kDa and 21 kDa subunits of acyl-CoA oxidase were synthesized. The mature form of peroxisomal 3-ketoacyl-CoA thiolase was also not processed from its precursor form, in this patient. We consider that Zellweger-like syndrome is a new variant form of a peroxisomal disorder in which biogenesis of peroxisomes is intact, while in contrast, the biogenesis of peroxisome is considered to be defective in those with Zellweger syndrome. Multiple defects of enzymes of beta-oxidation in Zellweger-like syndrome are assumed to be caused by a defect of transport or localization of these enzymes. Our molecular analyses indicate that the enzymes of peroxisomal beta-oxidation are synthesized in patients with Zellweger and Zellweger-like syndrome but that these enzymes are not processed normally and are degraded rapidly.
Abnormalities, Multiple↗
Immunoblot detection of enzyme proteins of peroxisomal beta-oxidation in fibroblasts, amniocytes, and chorionic villous cells. Possible marker for prenatal diagnosis of Zellweger's syndrome.
The amounts of enzyme proteins of peroxisomal beta-oxidation in fibroblasts and chorionic villous cells from infants with Zellweger syndrome and in fibroblasts, amniocytes, and chorionic villi from healthy controls were measured by immunoblot analysis. Immunoreactive proteins of peroxisomal acyl-CoA oxidase and 3-ketoacyl-CoA thiolase were absent in fibroblasts and chorionic villous cells from the patients, yet these enzyme proteins were present in fibroblasts, cultured amniocytes, and chorionic villi from the normal controls. These results show that immunoblot analysis of peroxisomal beta-oxidation enzymes in amniocytes and chorionic villous cells is of potential value for the prenatal detection of Zellweger syndrome.
Amnion↗
Origin and clinical significance of subcortical components in short-latency somatosensory evoked potentials in children.
P4 (P14 in adults), N' (N18) and N1' (the negative wave after cortical N1, obtained until late infancy) of short-latency somatosensory evoked potentials (S-SEPs) by median nerve stimulation were studied in 13 selected patients. Of these, 12 patients were suggested to have brain-stem lesions responsible especially for the cardinal symptoms and at the same time showed abnormal S-SEPs characteristically between P3 (P13) and N1 (N20). Another patient showed no cortical response with multilocular cystic encephalopathy. Each wave component could be identified by the same methodology as in controls from neonates to adults. In the normal subject the wave forms of P4 and N' were slightly influenced by sleep level. The abnormal S-SEP findings could be classified by an increase and decrease in the wave amplitudes or by a shortening and prolongation in the peak latencies. Upon these classifications we tried to delineate the pathophysiology underlying abnormalities of S-SEPs between P3 and N1. In addition, a negative wave after N1, compatible with N1', was identified in a case without cortical response. Evidence suggests that the origins of P4 and N' may include not only the lemniscal pathway but partly also the extra-lemniscal synapses in the brain-stem. The neural source of N1' seems to be partly in the subcortical structures. As for the clinical significance of S-SEPs in children, the features of P4 and N' are influenced both by the distribution of the affected lesions and by the underlying pathophysiology.
Adolescent↗
Zellweger-like syndrome with detectable hepatic peroxisomes: a variant form of peroxisomal disorder.
A male infant with typical clinical and biochemical findings of Zellweger syndrome, but in whom hepatic peroxisomes were detected by electron microscopy, had profound hypotonia, hepatomegaly, typical facial appearance including large fontanelle and frontal bossing, convulsions, panaminoaciduria, and hyperammonemia. He died of liver failure at age 5 months. There were increased levels of very long chain fatty acids and trihydroxycoprostanic acid in serum, and increased excretion of dicarboxylic acids and tyrosine metabolites in the urine. Levels of peroxisomal enzymes, acyl coenzyme A oxidase, bifunctional protein, 3-ketoacyl coenzyme A thiolase, and dihydroxyacetone phosphate acyltransferase in the liver tissue from the patient were all deficient, findings consistent with Zellweger syndrome. However, immunocytochemical study and electron microscopic examination of the liver at autopsy revealed that hepatic peroxisomes were present at a level similar to that in a control subject. These observations suggest further heterogeneity in Zellweger syndrome and a different pathogenesis in this variant case.
Bile Acids and Salts↗
Biochemical and morphologic aspects of peroxisomes in the human rectal mucosa: diagnosis of Zellweger syndrome simplified by rectal biopsy.
Rectal mucosa biopsy specimens from five controls and three infants with Zellweger syndrome were investigated morphologically and biochemically to elucidate peroxisomal structure and functions in the human rectal mucosa and to develop a simple method for an early postnatal diagnosis of peroxisomal deficiency disorders. After the alkaline 3,3'-diaminobenzidine reaction, peroxisomes of the rectal mucosa from the controls could be identified, electron microscopically, but not light microscopically. However, they were strongly stained using an immunoenzyme technique applied to semi-thin Epon sections and then were clearly visible under the light microscope. However, no positive granules were observed in the specimens of infants with Zellweger syndrome, using either of the two staining techniques. On immunoblot analysis, immunoreactive proteins of peroxisomal acyl-CoA oxidase and 3-ketoacyl-CoA thiolase were present in rectal mucosae from the controls; however, these enzyme proteins were not detected in tissues from the patients. Activity of dihydroxyacetone phosphate acyltransferase was detectable in rectal mucosae from the controls, whereas in those from infants with Zellweger syndrome, the activity of this enzyme was greatly reduced. These observations indicate that the peroxisomal structure and multiple functions are present in the rectal mucosa and that rectal biopsy is of potential value for the early and less invasive detection of Zellweger syndrome and other peroxisomal disorders.
Biopsy↗