RESUMO
Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.
Assuntos
Chaperonas Moleculares/fisiologia , Mutação de Sentido Incorreto , Dobramento de Proteína , Transaminases/genética , Transaminases/metabolismo , Tripsina/metabolismo , Sequência de Aminoácidos , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Ativação Enzimática/genética , Humanos , Ligantes , Mimetismo Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto/fisiologia , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transaminases/químicaRESUMO
A novel splice site mutation (IVS7-1G-->A) in the T-protein gene (aminomethyltransferase, or AMT) of the glycine cleavage enzyme complex was found in a patient with nonketotic hyperglycinemia (NKH). A PCR/restriction enzyme method to detect this mutation was used to screen 100 NKH alleles and identified the mutation in three unrelated families.
Assuntos
Aminoácido Oxirredutases/genética , Proteínas de Transporte/genética , Hidroximetil e Formil Transferases/genética , Hiperglicinemia não Cetótica/genética , Complexos Multienzimáticos/genética , Mutação/genética , Sítios de Splice de RNA/genética , Transferases/genética , Aminometiltransferase , Triagem de Portadores Genéticos , Humanos , Hiperglicinemia não Cetótica/enzimologia , Íntrons/genética , Reação em Cadeia da PolimeraseRESUMO
Following infection of RN2 rat Schwannoma cells with unfiltered JHMV inocula, a cell line with an altered phenotype evolved, which was shown to be a somatic cell hybrid of RN2 and mouse L-2 cells. This cell line, EJ, persistently yields JHMV at titres greater than 10(6) pfu/ml and does not show the suppression of virus production at 39.5 degrees C that is characteristic of a persistently infected RN2 line. Intracellular viral nucleocapsids are demonstrated. Cloning of EJ hybrids yields cell lines that show a variety of responses to infection by JHMV or MHV3.
Assuntos
Vírus da Hepatite Murina/crescimento & desenvolvimento , Neurilemoma/microbiologia , Animais , Antígenos Virais/análise , Linhagem Celular , Fibroblastos/microbiologia , Células Híbridas , Camundongos , Microscopia Eletrônica , Vírus da Hepatite Murina/isolamento & purificação , Ratos , Replicação ViralRESUMO
OBJECTIVE: To identify the molecular basis of arylsulfatase A deficiency in a family at risk for metachromatic leukodystrophy (MLD) and determine the genetic risk in the offspring. METHODS: Mutations in the arylsulfatase A gene were identified by PCR amplification and restriction enzyme digestion. Individuals had previously been tested for arylsulfatase A activity. RESULTS: Assays of arylsulfatase A activity had resulted in ambiguous results for MLD carrier identification. DNA analysis clearly identified two MLD mutations in the family, and an unsuspected arylsulfatase A pseudodeficiency. The DNA information immediately clarified the MLD risk for the family and confirmed that a newborn with low arylsulfatase A activity was unaffected. CONCLUSIONS: The overlap between activities for various combinations of MLD and pseudodeficiency alleles and the variability inherent in the assay of arylsulfatase A complicate the interpretation of activity levels in families at risk for MLD. Use of simple molecular biological tests for pseudodeficiency and the common MLD mutations in combination with the enzyme data can facilitate carrier identification and prenatal diagnosis.
Assuntos
Cerebrosídeo Sulfatase/deficiência , Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/genética , Alelos , Arilsulfatases/química , Arilsulfatases/deficiência , Arilsulfatases/genética , Arilsulfatases/metabolismo , Linhagem Celular , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/metabolismo , Pré-Escolar , Feminino , Fibroblastos/enzimologia , Triagem de Portadores Genéticos , Humanos , Leucócitos/enzimologia , Leucodistrofia Metacromática/enzimologia , Masculino , Linhagem , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVES: To develop a protocol capable of identifying deletions in mitochondrial DNA and use it to identify the breakpoints of a mtDNA deletion in a patient with chronic progressive external ophthalmoplegia (CPEO). DESIGN AND METHODS: Deletions in mtDNA were identified by a combination of long range PCR and Southern blotting. The precise breakpoints were determined by automated DNA sequencing. RESULTS: A series of DNA samples from patients with suspected mitochondrial disease was subjected to a protocol, which combines long range PCR and Southern blotting. We found a unique deletion in a patient with CPEO and we identified the precise location of this deletion through DNA sequencing. CONCLUSIONS: Long range PCR has the advantages of speed, minimal samples requirements, and sensitivity. Southern blotting is better able to evaluate heteroplasmy and detect duplications. We suggest a protocol that enables us to identify precisely the breakpoints in a unique mutation of mtDNA in a patient with CPEO.
Assuntos
Southern Blotting/métodos , DNA Mitocondrial/análise , Oftalmoplegia/genética , Reação em Cadeia da Polimerase/métodos , Deleção de Sequência , Adolescente , Blefaroptose/genética , Humanos , Síndrome de Kearns-Sayre/genética , Masculino , Encefalomiopatias Mitocondriais/genética , Oftalmoplegia Externa Progressiva Crônica/genética , Sensibilidade e EspecificidadeRESUMO
We report two siblings with metachromatic leukodystrophy, one who presented at 7 years of age (juvenile onset) and his sister who presented at 22 years of age (adult onset). They are compound heterozygotes for two novel mutations in the arylsufatase A gene (ARSA). The responsible mutations in this Vietnamese family consist of a missense mutation with 5% enzyme activity (R143G) and a nonsense mutation (W318ter), from which no enzyme activity would be expected. These mutations in the ARSA gene have not been previously reported and may be useful when diagnosing metachromatic leukodystrophy in other affected Vietnamese individuals. The variability in presentation suggests that the genotype alone is not sufficient to determine the onset and course of the disease and modifying genetic and perhaps nongenetic factors likely contribute.
Assuntos
Cerebrosídeo Sulfatase/genética , Códon sem Sentido/genética , Leucodistrofia Metacromática/genética , Mutação de Sentido Incorreto/genética , Adulto , Criança , Feminino , Humanos , Leucodistrofia Metacromática/diagnóstico , Masculino , Linhagem , VietnãAssuntos
Alelos , Cerebrosídeo Sulfatase/genética , Quimera/genética , Leucodistrofia Metacromática/genética , Mutação de Sentido Incorreto/genética , Espermatozoides/fisiologia , Adolescente , Idade de Início , Células Cultivadas , Cerebrosídeo Sulfatase/metabolismo , Criança , Cromossomos Humanos Par 22/genética , Feminino , Fibroblastos , Heterozigoto , Humanos , Cariotipagem , Leucócitos , Leucodistrofia Metacromática/enzimologia , Leucodistrofia Metacromática/patologia , Masculino , Repetições de Microssatélites/genética , Núcleo Familiar , LinhagemRESUMO
The primary hyperoxalurias are diseases of overproduction of oxalate. The immediate precursor of oxalate is glyoxylate. Metabolism of hydroxyproline, derived from collagen turnover or the diet, appears to be a major source of glyoxylate, and a potential target for a therapeutic strategy of substrate depletion.
Assuntos
Glioxilatos/metabolismo , Hidroxiprolina/metabolismo , Hiperoxalúria Primária/terapia , Adulto , Feminino , Humanos , Hiperoxalúria Primária/metabolismo , Masculino , Oxalatos/metabolismoRESUMO
Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme, deficiency of which results in primary hyperoxaluria type 1 (PH1). More than 65 PH1-related mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have generated a spectrum of 15 missense changes including the most common PH1 mutation, G170R, and expressed them on the appropriate background of the major or minor allele, in an Escherichia coli overexpression system and in a rabbit reticulocyte transcription/translation system. We have investigated their effects on enzyme activity, dimerization, aggregation, and turnover. The effect of pyridoxal phosphate (PLP) on dimerization and stability was also investigated. Although all 15 mutant AGTs were expressed as intact proteins in E. coli, only three: G41R and G41V on the major allele, and the common mutation G170R, resulted in significant amounts of enzymatic activity. Dimerization failure was a frequent observation (13/15) except for G41V and D183N. Dimerization was poor with S187F but was substantially improved with PLP. Proteasome-mediated protein degradation was observed for all the mutations except G41R on the major allele, G41V, D183N, G170R, and S218L. Increases in the stability of the mutant enzymes in the presence of PLP were small; however, G41R on the minor allele showed a direct relationship between its half life and the concentration of PLP. The minor allele AGT product and many of the mutants were subject to a limited non-proteasomal proteolytic cleavage when ATP was depleted.
Assuntos
Mutação de Sentido Incorreto , Complexo de Endopeptidases do Proteassoma/metabolismo , Transaminases/metabolismo , Substituição de Aminoácidos , Animais , Dimerização , Estabilidade Enzimática , Escherichia coli , Humanos , Fosfato de Piridoxal/farmacologia , Coelhos , Transaminases/efeitos dos fármacos , Transaminases/genéticaRESUMO
The B1 variant form of Tay-Sachs disease is enzymologically unique in that the causative mutation(s) appear to affect the active site in the alpha subunit of beta-hexosaminidase A without altering its ability to associate with the beta subunit. Most previously reported B1 variant mutations were found in exon 5 within codon 178. The coding sequence of the alpha subunit gene of a patient with the B1 variant form was examined with a combination of reverse transcription of mRNA to cDNA, PCR, and dideoxy sequencing. A double mutation in exon 6 has been identified: a G574----C transversion causing a val192----leu change and a G598----A transition resulting in a val200----met alteration. The amplified cDNAs were otherwise normal throughout their sequence. The 574 and 598 alterations have been confirmed by amplification directly from genomic DNA from the patient and her mother. Transient-expression studies of the two exon 6 mutations (singly or together) in COS-1 cells show that the G574----C change is sufficient to cause the loss of enzyme activity. The biochemical phenotype of the 574 alteration in transfection studies is consistent with that expected for a B1 variant mutation. As such, this mutation differs from previously reported B1 variant mutations, all of which occur in exon 5.
Assuntos
Éxons , Variação Genética , Mutação , Doença de Tay-Sachs/genética , beta-N-Acetil-Hexosaminidases/genética , Animais , Sequência de Bases , Células Cultivadas , DNA/genética , DNA/isolamento & purificação , Fibroblastos/enzimologia , Expressão Gênica , Humanos , Linfócitos/enzimologia , Substâncias Macromoleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento por Restrição , Doença de Tay-Sachs/enzimologia , TransfecçãoRESUMO
We have recently diagnosed aspartylglucosaminuria (AGU) in four members of a Canadian family. AGU is a lysosomal storage disease in which asparagine-linked glycopeptides accumulate to particularly high concentrations in liver, spleen and thyroid of affected individuals. A lesser accumulation of these glycopeptides is seen in the kidney and brain, and they are also excreted in the urine. The altered metabolism in AGU results from a deficiency of the enzyme aspartylglucosaminidase (1-aspartamido-beta-N-acetylglucosamine amidohydrolase), which hydrolyses the asparagine to N-acetylglucosamine linkages of glycoproteins and glycopeptides. We have used human liver as a source of material for the purification of aspartylglucosaminidase. The enzyme has been purified to homogeneity by using heat treatment, (NH4)2SO4 fractionation, and chromatography on concanavalin A-Sepharose, DEAE-Sepharose, sulphopropyl-Sephadex, hydroxyapatite, DEAE-cellulose and Sephadex G-100. Enzyme activity was followed by measuring colorimetrically the N-acetylglucosamine released from aspartylglucosamine at 56 degrees C. The purified enzyme protein ran at a 'native' molecular mass of 56 kDa in SDS/12.5%-PAGE gels, and the enzyme activity could be quantitatively recovered at this molecular mass by using gel slices as enzyme source in the assay. After denaturation by boiling in SDS the 56 kDa protein was lost with the corresponding appearance of polypeptides alpha,beta and beta 1, lacking enzyme activity, at 24.6, 18.4 and 17.4 kDa respectively. Treatment of heat-denatured enzyme with N-glycosidase F resulted in the following decreases in molecular mass; 24.6 to 23 kDa and 18.4 and 17.4 to 15.8 kDa. These studies indicate that human liver aspartylglucosaminidase is composed of two non-identical polypeptides, each of which is glycosylated. The N-termini of alpha,beta and beta 1 were directly accessible for sequencing, and the first 21, 26 and 22 amino acids respectively were identified.
Assuntos
Aspartilglucosilaminase/isolamento & purificação , Fígado/enzimologia , Sequência de Aminoácidos , Aspartilglucosilaminase/química , Aspartilglucosilaminase/metabolismo , Dextranos , Eletroforese em Gel de Poliacrilamida , Etanolaminas , Glicosídeo Hidrolases/metabolismo , Humanos , Hidrolases/isolamento & purificação , Substâncias Macromoleculares , Dados de Sequência Molecular , Sefarose , Dodecilsulfato de SódioRESUMO
Modification of mammalian DNA by the methylation of cytosine in CpG dinucleotides is a complex phenomenon involved in a number of cellular and developmental processes. In particular, the characteristic hypermutability of CpGs may be a major contributor of point mutations leading to human genetic disease. We have hypothesized that DNA methylation contributes to mutations in the gene causing neurofibromatosis type 1 (NF1), one of the most common genetic disorders in humans and a disease where up to half of all cases are the result of sporadic germline mutations, usually in the paternally-derived allele. We have used two experimental approaches to analyze patterns of DNA methylation at CpG dinucleotides in the NF1 gene region. Southern analyses using isoschizomeric restriction pairs have revealed DNA methylation in areas flanking the NF1 gene region, while PCR-methylation assays have shown that methylation occurs both on genomic sequences flanking the NF1 gene and within the coding region of the gene itself. We suggest that methylated CpG dinucleotides within and around the highly mutable NF1 gene serve as a reservoir within which C-->T transitions contribute to the high frequency of spontaneous germline mutations associated with the disease.
Assuntos
Cromossomos Humanos Par 17 , DNA/química , DNA/genética , Genes da Neurofibromatose 1 , Neurofibromatose 1/genética , Sequência de Bases , Ligação Genética , Marcadores Genéticos , Humanos , Metilação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
Screening a DNA bank from 50 patients with enzymatic confirmation of their diagnosis of nonketotic hyperglycinemia gave allele frequencies of 5% for R515S of P-protein (glycine decarboxylase) and 7% for R320H of T-protein (aminomethyltransferase). In a previous report we found that 3% of the same patient alleles were positive for T-protein IVS7-1G>A. In total, testing for these three mutations identified 15% of alleles and positive results (one or two mutations) were found in 11 of the 50 patients. In addition, a novel point mutation in T-protein, N145I, was found in a single case and a PCR/restriction enzyme assay was developed for its detection.
Assuntos
Aminoácido Oxirredutases/genética , Glicina/sangue , Hidroximetil e Formil Transferases/deficiência , Hidroximetil e Formil Transferases/genética , Hiperglicinemia não Cetótica/genética , Mutação , Aminometiltransferase , Primers do DNA/química , Éxons , Frequência do Gene , Glicina/metabolismo , Glicina Desidrogenase (Descarboxilante) , Heterozigoto , Humanos , Hiperglicinemia não Cetótica/enzimologia , Iminas , Recém-Nascido , Cetose , Fígado/enzimologia , Reação em Cadeia da Polimerase , Diagnóstico Pré-NatalRESUMO
This report is concerned with neurofibromatosis type 1 (NF-1, 17q11.2) in south-western Ontario, an ethnically diverse population derived from multiple immigrations. The population incidence, prevalence, and mutation rates for this disease are similar in most racial groups of this population and are also comparable to earlier reports. NF-1 is one of the most common single gene disorders in this population. The occurrence of the disease is not affected by the birth order or sex of the transmitting parent. The severe manifestation of this disease is statistically related to paternal transmission. Five polymorphic DNA probes (pA1041, pHHH202, pTH1719, NF1, pEW206, pEW207) were evaluated in relation to segregation of NF-1 using appropriate restriction enzymes. The observed heterozygosity was found to be relatively high, ranging from 25% to 55% for all the probes on 17q and flanking the NF-1 gene. We recommend the use of pHHH202/pTH1719 and pEW206 in any linkage analysis for detection of the presence of the NF-1 mutation. For informative families the degree of certainty is as high as 99.5%. Some future modifications may include the use of NF-1 exon specific probes and primers that remain to be evaluated for heterogeneity and heterozygosity among populations.
Assuntos
Neurofibromatose 1/genética , Sondas de DNA , Feminino , Ligação Genética , Genética Populacional , Heterozigoto , Humanos , Masculino , Neurofibromatose 1/epidemiologia , Hibridização de Ácido Nucleico , Ontário/epidemiologia , Fenótipo , Polimorfismo de Fragmento de RestriçãoRESUMO
We describe three novel deletions in the human AGT gene in three patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme, alanine glyoxylate aminotransferase (AGT; EC 2.6.1.44). A deletion of 4 nucleotides in the exon 6/intron 6 splice junction (679-IVS6+2delAAgt) is expected to cause missplicing. It would also code for a K227E missense alteration in any mRNA successfully spliced. A 2-bp deletion in exon 11 (1125-1126del CG, cDNA) results in a frameshift. A deletion of at least 5-6 kb, EX1 EX5del, spanned exons 1-5 and contiguous upstream sequence. All three deletions are heterozygous with previously documented missense mutations; the intron 6 deletion with F152I, the exon 11 deletion with G82E, and EX1 EX5del with the common mistargeting mutation, G170R.
Assuntos
Hiperoxalúria Primária/genética , Transaminases/genética , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , Análise Mutacional de DNA , Saúde da Família , Feminino , Humanos , Hiperoxalúria Primária/enzimologia , Lactente , Masculino , Dados de Sequência Molecular , Mutação , Polimorfismo Genético , Deleção de Sequência , Transaminases/efeitos dos fármacos , Transaminases/metabolismoRESUMO
The investigation of 14 unrelated patients with nonketotic hyperglycinemia led to the identification of mutations in 4 cases. Patients were initially categorized into probable P- or T-protein defects of the glycine cleavage enzyme complex, by the use of the glycine exchange assay without supplemental H-protein, then screened for mutations in the P-protein and T-protein genes, respectively.
Assuntos
Análise Mutacional de DNA , Hiperglicinemia não Cetótica/genética , Metiltransferases/genética , Sequência de Aminoácidos , Primers do DNA/química , Éxons , Feminino , Glicina/metabolismo , Homocisteína S-Metiltransferase , Humanos , Hiperglicinemia não Cetótica/enzimologia , Recém-Nascido , Íntrons , Fígado/enzimologia , Masculino , Metiltransferases/metabolismo , Dados de Sequência Molecular , LinhagemRESUMO
JHM virus, when inoculated into neonatal rats, can cause either a rapidly fatal acute encephalomyelitis or, after longer incubation periods, a paralytic disease. The cerebrospinal fluid (CSF) and serum anti-JHM virus IgG concentrations present in rats prior to onset of clinical symptoms or during the acute and paralytic phases of disease were compared. High CSF/serum ratios, indicative of local antibody production in the CNS, were noted only where disease was demonstrable suggesting that local antibody production accompanied the infection but did not prevent the neurological disease. Among animals in which neurologic symptoms had not become manifest, only those with elevated CSF/serum ratios were found to have histological CNS lesions. Immunofluorescent microscopy indicated that viral antigens were present in both glia and neurons. Antigen-positive cells were frequently present in histologically normal CNS tissue, while regions of necrosis were antigen negative. Testing for the presence of viral RNA with JHM cDNA probes revealed that the virus was rapidly disseminated throughout the CNS, presumably establishing centers of infection prior to the development of recognizable tissue damage. Viral RNA was also detected in the CNS following recovery from paralysis and as late as 5 months postinfection, where no disease occurred. These findings indicate that, although infection by JHM virus can spread rapidly throughout the CNS, formation of lesions during chronic disease is a slower process. The current data and previous observations suggest that JHM virus can remain in a latent state for periods of at least several months in rats without apparent neurologic disease despite the absence of any known provirus phase in the replicative strategy of coronaviruses.
Assuntos
Infecções por Coronaviridae/microbiologia , Coronaviridae/patogenicidade , Doenças Desmielinizantes/microbiologia , Animais , Anticorpos Antivirais/análise , Formação de Anticorpos , Antígenos Virais/análise , Encéfalo/microbiologia , Encéfalo/patologia , Infecções por Coronaviridae/patologia , DNA/isolamento & purificação , DNA Viral/genética , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Imunofluorescência , Ratos , Ratos Endogâmicos , Ratos Endogâmicos WFRESUMO
Neurofibromatosis type 1 (NF1) is a common, autosomal dominant genetic disorder with a variety of highly variable symptoms including cutaneous manifestations (such as café au lait spots), Lisch nodules, plexiform neurofibromas, skeletal abnormalities, an increased risk for malignancy, and the development of learning disabilities. The wide clinical variability of expression of the disease phenotype and high (spontaneous) mutation rate of the NF1 gene indicate that careful clinical examination of patients and family members is necessary to provide an accurate diagnosis of the disease. Since very few NF1 mutations have been identified, and with the apparent lack of a predominant mutation in this large, highly mutable gene, molecular diagnosis of NF1 will continue to be based on haplotypes using linkage analysis. Here we report our experiences while providing a molecular diagnostic service for NF1 in the ethnically diverse region of south-western Ontario. Molecular diagnoses with at least one informative probe/enzyme combination are reported for 19 families including two families requesting prenatal diagnosis for NF1. We have augmented the classical Southern based approach to linkage analysis with the use of PCR based assays for molecular linkage. Furthermore, criteria have been established in our laboratory for executing molecular linkage based on heterozygosity values, recombination fractions, and the use of intragenic probes/markers.
Assuntos
Amostra da Vilosidade Coriônica , Cromossomos Humanos Par 17 , Genes da Neurofibromatose 1/genética , Ligação Genética , Neurofibromatose 1/genética , Alelos , Sequência de Bases , Southern Blotting , DNA/análise , DNA/química , Feminino , Doenças Fetais/diagnóstico , Doenças Fetais/genética , Frequência do Gene , Marcadores Genéticos , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Neurofibromatose 1/diagnóstico , Ontário , Linhagem , Reação em Cadeia da Polimerase , GravidezRESUMO
Permanent lymphoblastoid cell lines are important in the molecular analysis and characterization of human genetic disorders, when immortalized cells must be banked for future diagnostic or research purposes. However, routine methods for transformation using Epstein-Barr virus (EBV) require blood volumes that may be difficult to collect from clinically compromised neonates and small children. Here we report a modified transformation procedure utilizing blood samples of small volume (less than 1.0 ml), which we have found to be particularly useful for the immortalization of lymphocytes destined for future molecular genetic studies.