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Ornithine transcarbamylase deficiency resulting from a C-to-T substitution in exon 5 of the ornithine transcarbamylase gene. 总被引:11,自引:3,他引:8 下载免费PDF全文
A Hata C Setoyama K Shimada E Takeda Y Kuroda I Akaboshi I Matsuda 《American journal of human genetics》1989,45(1):123-127
To define the molecular basis for the TaqI site alteration in the ornithine transcarbamylase (OTC) (E.C.2.1.3.3) gene of a female patient with mild OTC deficiency, we used a combination of genomic amplification followed by direct sequencing and oligodeoxyribonucleotide hybridization. We obtained evidence for a C-to-T substitution in exon 5 (codon 141) of this gene. This mutation generates a stop codon, in place of Arg, at amino acid 109 of the mature OTC protein. The mutation arose, de novo, in a germ cell of one of the parents. 相似文献
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Direct and indirect mutation analyses in patients with ornithine transcarbamylase deficiency. 总被引:1,自引:0,他引:1
Ornithine transcarbamylase (OTC) is one of 5 enzymes in the detoxification of ammonia to urea, and its deficiency, an X-linked disease, is the most common inborn error of urea genesis in humans. Because of the devastating nature of the disease there is a strong demand for reliable and rapid molecular analyses in OTC families in order to offer carrier detection and prenatal diagnosis. This paper presents the efficiency of direct and indirect mutation analyses in 22 OTC families using Southern blotting and polymerase chain reaction (PCR) amplification. For 89% of the mothers with an affected child, at least 1 RFLP of the OTC locus was informative concerning prenatal diagnosis. 100% informativity was reached by using the additional flanking markers 754 and LI.28. In total, 3 deletions (14%) and 1 TaqI site mutation (4.5%) in exon 3 were detected. 13 (60%) of our 22 mothers were found to be carriers, 9 of them being obligate carriers and 4 detected by biochemical testing. 4 mothers were excluded as carriers by DNA analyses, and in 5 mothers the carrier status could not be assessed positively. DNA analyses permitted carrier detection in 32% and carrier exclusion in 55% of 22 female relatives. Prenatal diagnosis was performed in 4 families: in 1 family by direct mutation detection and in 3 families by linkage analyses. It was possible to determine the mutation origin in 6 families, all of them with male probands. In 4 families the mutation had occurred during grandpaternal spermiogenesis, suggesting higher mutation rates in males, but in 2 cases it was the result of an event during maternal oogenesis, proving that new mutations in the OTC gene do also occur in eggs. Our recommended strategy for carrier detection and prenatal diagnosis in OTC deficiency is to examine routinely Southern blots of BamHI, EcoRI, HindIII, MspI, PstI and TaqI digestions using the OTCcDNA probe pH0731 and the flanking markers 754 and LI.28, as well as the TaqI-digested PCR products of exons 3, 5 and 9. 相似文献
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Ornithine transcarbamylase catalyzes the synthesis of citrulline from carbamyl phosphate and ornithine. This enzyme is involved in the biosynthesis of arginine in many organisms and participates in the urea cycle of mammals. The biosynthetic ornithine transcarbamylase has been purified from the filamentous fungus, Neurospora crassa. It was found to be a homotrimer with an apparent subunit molecular weight of 37,000 and a native molecular weight of about 110,000. Its catalytic activity has a pH optimum of 9.5 and Km's of about 5 and 2.5 mM for the substrates, ornithine and carbamyl phosphate, respectively, at pH 9.5. The Km's and pH optimum are much higher than those of previously characterized enzymes from bacteria, other fungi, and mammals. These unusual kinetic properties may be of significance with regard to the regulation of ornithine transcarbamylase in this organism, especially in the avoidance of a futile ornithine cycle. Polyclonal antibodies were raised against the purified enzyme. These antibodies and antibody raised against purified rat liver ornithine transcarbamylase were used to examine the structural similarities of the enzyme from a number of organisms. Cross-reactivity was observed only for mitochondrial ornithine transcarbamylases of related organisms. 相似文献
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New mutation and prenatal diagnosis in ornithine transcarbamylase deficiency. 总被引:12,自引:5,他引:7 下载免费PDF全文
R L Nussbaum B A Boggs A L Beaudet S Doyle J L Potter W E O''''Brien 《American journal of human genetics》1986,38(2):149-158
Ornithine transcarbamylase (OTC) (E.C.2.1.3.3) is an X-linked hepatic enzyme in the urea cycle necessary for ammonia detoxification. Deficiency of OTC results in neonatal hyperammonemia, coma, and death in childhood. Because fibroblasts do not express OTC, prenatal diagnosis in the past has required fetal liver biopsy. Using a complementary DNA (cDNA) for OTC for Southern blot analysis of genomic DNA, we have found probands with complete OTC deficiency from two unrelated families in whom the same TaqI restriction endonuclease site has been altered because of independent, but not necessarily identical, mutations in the OTC gene, suggesting that this site may be a relative hotspot for mutation at a location that is critical for normal gene function. This TaqI alteration has allowed the identification of the individual in each family in whom the mutation originated as well as the exclusion of a recurrence of OTC deficiency in a male fetus at risk for the disease. OTC deficiency joins the growing list of genetic disorders for which Southern blot analysis allows accurate heterozygote detection and prenatal diagnosis in conditions for which they were not previously available. 相似文献
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A probable sex difference in mutation rates in ornithine transcarbamylase deficiency 总被引:5,自引:5,他引:0
Catherine Bonaïti-Pellié Anna Pelet Hélène Ogier Jean-René Nelson Claude Largillière Jacques Berthelot Jean-Marie Saudubray Arnold Munnich 《Human genetics》1990,84(2):163-166
Summary Ornithine transcarbamylase deficiency is an X-linked disease with possible manifestations in heterozygous females. Using segregation analysis in families from the literature pooled with a French series, the penetrance could be estimated to be 17% in heterozygous females (15% with severe and 2% with milder symptoms). Using these estimates, the proportion of sporadic cases among heterozygous females and hemizygous males could be derived. This proportion is 57% in females. In males, it depends on mutation rate values: assuming equal mutation rates in sperm and eggs, this proportion should be 40%. However, this value can be strongly rejected based on the proportion of isolated cases in male sibships. In both sets of data, segregation analysis provided no evidence for sporadic affected males, suggesting that there are virtually no mutations in eggs. The upper limit of the confidence interval, 0%–16%, can be taken as the maximum prior probability that an affected male occurs as the result of a new mutation in his mother's germ cells. 相似文献
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Ornithine transcarbamylase, an isoelectric point (pI) isozyme in human liver and its deficiency 总被引:2,自引:0,他引:2
The crude extract of human liver ornithine transcarbamylase, obtained from a patient with hyperammonemia due to enzyme deficiency, was studied by the isoelectric focusing method. The activity of ornithine transcarbamylase in the patient at pH 8.0 was only slightly reduced. 相似文献
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We have found in patients with ornithine transcarbamylase (OTC) deficiency from two Spanish families (A and B), replacement by A of G at the 3-end of exon 4 of the OTC gene. The same mutation is found in the spf-ash mouse, a rodent model of mild OTC deficiency, causing a neutral R129H mutation and inefficient splicing at the 5donor site of the exon 4-intron 4 junction, with resultant 4%–7% residual OTC activity. The mutation, detected in our patients using polymerase chain reaction (PCR) amplification of the ten OTC exons, single strand conformation polymorphism (SSCP) analysis and direct sequencing of PCR-amplified exon 4, results in the loss of a unique MspI restriction site which can be used for rapid diagnosis. The mutation was transmitted by the mother in family A and arose de novo in the patient in family B. Residual OTC activity, determined in a male and a female patient, was 1.3% and 3.5% of normal, respectively. Despite this low activity, the surviving patients have developed normally. 相似文献
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Ryszard Slomski Ingrid Braulke Claudia Behrend Elisabeth Schröder Jean-Pierre Colombo Jochen Reiss 《Human genetics》1992,89(6):632-634
Summary A girl with ornithine transcarbamylase (OTC) deficiency was investigated for molecular and cytogenetic abnormalities that might explain this phenotype. Analysis with polymorphic DNA markers indicated that the patient did not inherit paternal alleles of the OTC locus, but that she did inherit the proximal locus DXS7 and the long arm of chromosome X. High-resolution cytogenetic analysis of the patient indicated a deletion of Xp11.4-p21, whereas both parents had normal karytoypes. Since the mother might be heterozygous according to biochemical tests, a second mutation within the maternal OTC gene cannot be excluded. 相似文献
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Akira Hata Toshinobu Matsuura Chiaki Setoyama Kazuniro Shimada Tohru Yokoi Izumi Akaboshi Ichiro Matsuda 《Human genetics》1991,87(1):28-32
We studied two unrelated male probands with mild ornithine transcarbamylase (OTC) (E.C.2.1.3.3) deficiency presenting a similar clinical course. Previous analyses of their liver OTCs also revealed similar properties. To identify the underlying molecular defects, we first cloned the entire coding region of the OTC gene from one proband and found a single base-substitution (C to T) leading to the substitution of tryptophan for arginine at amino acid position 277. Using a genomic amplification technique followed by allele specific oligonucleotide hybridization, we identified the same point mutation in the OTC gene of the other proband. We observed the presence of the mutation among family members in at least three generations, and in one asymptomatic hemizygous sibling in each family. 相似文献
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Mavri-Damelin D Eaton S Damelin LH Rees M Hodgson HJ Selden C 《The international journal of biochemistry & cell biology》2007,39(3):555-564
A possible cell source for a bio-artificial liver is the human hepatblastoma-derived cell line HepG2 as it confers many hepatocyte functions, however, the urea cycle is not maintained resulting in the lack of ammonia detoxification via this cycle. We investigated urea cycle activity in HepG2 cells at both a molecular and biochemical level to determine the causes for the lack of urea cycle expression, and subsequently addressed reinstatement of the cycle by gene transfer. Metabolic labelling studies showed that urea production from 15N-ammonium chloride was not detectable in HepG2 conditioned medium, nor could 14C-labelled urea cycle intermediates be detected. Gene expression data from HepG2 cells revealed that although expression of three urea cycle genes Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase was evident, Ornithine Transcarbamylase and Arginase I expression were completely absent. These results were confirmed by Western blot for arginase I, where no protein was detected. Radiolabelled enzyme assays showed that Ornithine Transcarbamylase functional activity was missing but that Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase were functionally expressed at levels comparable to cultured primary human hepatocytes. To restore the urea cycle, HepG2 cells were transfected with full length Ornithine Transcarbamylase and Arginase I cDNA constructs under a CMV promoter. Co-transfected HepG2 cells displayed complete urea cycle activity, producing both labelled urea and urea cycle intermediates. This strategy could provide a cell source capable of urea synthesis, and hence ammonia detoxificatory function, which would be useful in a bio-artificial liver. 相似文献
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The application of new diagnostic techniques has led to improvement in carrier detection and prenatal diagnosis in ornithine transcarbamylase deficiency. Progress has also been made towards somatic gene therapy. 相似文献
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Ornithine transcarbamylase of rat liver has been purified to homogeneity. The purified enzyme of specific activity 870 to 920 focuses as a single protein at pH 7.2. At pH 7.7, the Km for carbamyl phosphate is 0.026 mM, and the Km for ornithine is 0.04 mM. The inhibition constants of a number of amino acids that act as competitive inhibitors of the enzyme are reported. The native enzyme of Mr = 112,000 is composed of three subunits of Mr = 39,600 +/- 1,000. Chemical evidence indicates that the subunits are identical in amino acid composition and amino acid sequence. The amino acid sequence of the NH2-terminal region of ornithine transcarbamylase is Ser-Gln-Val-Gln-Leu-Lys-Gly-Ser-Asp-Leu-Leu-Thr-Leu-Lys-Asn-(Phe)-X-Thr-X-Glu-Ile-Gln-Tyr-Met-. 相似文献
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The R40H mutation in a late onset type of human ornithine transcarbamylase deficiency in male patients 总被引:2,自引:0,他引:2
Atsushi Nishiyori M. Yoshino Hirohisa Kato Toshinobu Matsuura Ryuuji Hoshide Ichiro Matsuda Tateo Kuno Sumio Miyazaki Shin-ichi Hirose Ryu-ichi Kuromaru Masataka Mori 《Human genetics》1997,99(2):171-176
Ornithine transcarbamylase (OTC) deficiency is an X-linked trait and is one of the most frequent of the inherited urea cycle
enzyme deficiencies. Most male patients with OTC deficiency develop a hyperammonemic crisis and die in the neonatal period
or in early infancy. In contrast to those patients, in some male patients the disease first becomes overt in adolescence or
during the reproductive age period. In the present report, we describe six such male patients who first developed clinical
signs at ages ranging from 6 to 58 years, all of whom came from a limited area of the northern part of Kyushu Island in southern
Japan. The mutation analysis disclosed a R40H mutation in exon 2 of the OTC gene in each of these patients. Transmission of
this mutant gene through paternal lineage as well as through maternal lineage was documented in one family. The levels of
mRNA of the mutant OTC gene expressed in transfected Cos 1 cells and in the liver tissue obtained by biopsy in one patient
were both similar to those of the wild-type gene. The activity of the mutant OTC was, however, decreased to a level of 28%
of the wild-type OTC, and the levels of the mutant OTC protein expressed in Cos 1 cells were decreased, as assessed by western
blot analysis. Apparent K
m values of the mutant enzyme for ornithine (1.1 mM) and carbamylophosphate (2.0 mM) were similar to those of the wild-type
enzyme. Both enzymes gave similar pH-dependency profiles, giving a maximal activity at pH 7.8–7.9. Activity of wild-type OTC
expressed in Cos 1 cells did not change after five cycles of freezing and thawing, whereas that of the mutant OTC decreased
to 17% by this treatment. These results suggest that deficiency is due to inactivation of the mutant OTC under certain conditions.
Received: 15 May 1996 相似文献
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Cossu A Orrù S Jacomelli G Carcassi C Contu L Sestini S Corradi MR Pompucci G Carcassi A Micheli V 《Biochimica et biophysica acta》2006,1762(1):29-33
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch-Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch-Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C-->T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT(Sardinia). The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency. 相似文献