RESUMO
Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine spermatogenesis-specific isoform of the E1a subunit of the pyruvate dehydrogenase complex. To begin to delineate the mechanisms regulating its expression in vivo, we have generated transgenic mice lines carrying Pdha-2 promoter deletion constructs. Here we report that transgenic mice harboring a construct containing only 187 bp of promoter and upstream sequences (core promoter) is sufficient for directing the testis-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Like the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. Our studies also show a correlation between CpG methylation within the core promoter and its capacity to regulate transcription. In NIH 3T3 cell lines stably transfected with the Pdha-2 core promoter-CAT construct, high levels of CAT reporter expression are observed, whereas the endogenous Pdha-2 gene is repressed. In these cells, the CpG dinucleotides residing within the transfected promoter are hypomethylated whereas those residing in the endogenous promoter are methylated. Furthermore, promoter activity can be abated by the in vitro methylation of its CpG dinucleotides. DNase I footprint analysis indicates that at least one site for the methylation-mediated repression may occur through the ATF/cyclic AMP response element binding element located within the core promoter. Mutations within this element reduces activity to approximately 50% of the wild-type promoter activity. These results suggest that tissue-specific gene expression may be modulated by other mechanisms in addition to specific transcription factor availability and cooperativity. We propose that methylation may be a mechanism by which repression of the testis-specific Pdha-2 gene is established in somatic tissue.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento/genética , Regiões Promotoras Genéticas/genética , Piruvato Desidrogenase (Lipoamida) , Complexo Piruvato Desidrogenase/genética , Testículo/metabolismo , Células 3T3 , Fatores Ativadores da Transcrição , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Sanguíneas/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: There is a lack of information on prevalence, cause and consequences of slight/mild bilateral sensorineural hearing loss (SNHL) in children. We report the first systematic genetic analysis of the GJB2 gene in a population-derived sample of children with slight/mild bilateral SNHL. METHODS: Hearing tests were conducted in 6240 Australian elementary school children in Grades 1 and 5. 55 children (0.88%) were found to have a slight/mild sensorineural hearing loss. 48 children with slight/mild sensorineural hearing loss and a matched group of 90 children with normal hearing participated in a genetic study investigating mutations in the GJB2 gene, coding for connexin 26, and the presence of the del(GJB6-D13S1830) and del(GJB6-D13S1854) deletions in the GJB6 gene, coding for connexin 30. RESULTS: Four of 48 children with slight/mild sensorineural hearing loss were homozygous for the GJB2 V37I change. The four children with homozygous V37I mutations were all of Asian background and analysis of SNPs in or near the GJB2 gene suggests that the V37I mutation arose from a single mutational event in the Asian population. DISCUSSION: Based on the prevalence of carriers of this change we conclude that V37I can be a causative mutation that is often associated with slight/mild sensorineural hearing loss. No other children in the slight/mild hearing loss group had a hearing loss related to a GJB2 mutation. One child with normal hearing was homozygous for the R127H change and we conclude that this change does not cause hearing loss. Two children of Asian background were carriers of the V37I mutation. Our data indicate that slight/mild sensorineural hearing loss due to the GJB2 V37I mutation is common in people of Asian background.
Assuntos
Conexinas/genética , Perda Auditiva Bilateral/genética , Perda Auditiva Neurossensorial/genética , Mutação , Alelos , Austrália , Criança , Conexina 26 , Análise Mutacional de DNA , Feminino , Testes Genéticos , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Instituições AcadêmicasRESUMO
We have characterized two mouse genes that code for the E1 alpha subunit of pyruvate dehydrogenase (PDH), Pdha-1 and Pdha-2. The coding regions show a high degree of homology with each other and with the human PDH genes, PDAH1 and PDHA2. Conserved regions include mitochondrial import sequences, phosphorylation sites and a putative TPP binding site. The PDH genes have an analogous chromosomal arrangement to PGK genes in that two isoforms code for a functionally and structurally similar product. Pdha-1 codes for a somatic isoform and maps to the X-chromosome. Pdha-2 is located on an autosome, is intronless and only expressed in spermatogenic cells. Comparison of human and mouse PDH and PGK gene sequences shows that the somatic sequences are more conserved relative to the testis-specific isoforms, and that the mouse PDH E1 alpha genes have experienced a faster rate of DNA change compared to their human counterparts.
Assuntos
Piruvato Desidrogenase (Lipoamida) , Complexo Piruvato Desidrogenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Fígado/enzimologia , Masculino , Camundongos , Dados de Sequência Molecular , Complexo Piruvato Desidrogenase/isolamento & purificação , Complexo Piruvato Desidrogenase/metabolismo , Homologia de Sequência do Ácido Nucleico , Espermatozoides/enzimologia , Testículo/enzimologiaRESUMO
BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is a complex condition with high heritability. However, both biochemical investigations and association and linkage studies have failed to define fully the underlying genetic factors associated with ADHD. We have identified a family co-segregating an early onset behavioural/developmental condition, with features of ADHD and intellectual disability, with a pericentric inversion of chromosome 3, 46N inv(3)(p14:q21). METHODS: We hypothesised that the inversion breakpoints affect a gene or genes that cause the observed phenotype. Large genomic clones (P1 derived/yeast/bacterial artificial chromosomes) were assembled into contigs across the two inversion breakpoints using molecular and bioinformatic technologies. Restriction fragments crossing the junctions were identified by Southern analysis and these fragments were amplified using inverse PCR. RESULTS: The amplification products were subsequently sequenced to reveal that the breakpoints lay within an intron of the dedicator of cytokinesis 3 (DOCK3) gene at the p arm breakpoint, and an intron of a novel member of the solute carrier family 9 (sodium/hydrogen exchanger) isoform 9 (SLC9A9) at the q arm. Both genes are expressed in the brain, but neither of the genes has previously been implicated in developmental or behavioural disorders. CONCLUSION: These two disrupted genes are candidates for involvement in the pathway leading to the neuropsychological condition in this family.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Proteínas de Transporte , Inversão Cromossômica , Cromossomos Humanos Par 3 , Predisposição Genética para Doença , Fatores de Troca do Nucleotídeo Guanina , Proteínas do Tecido Nervoso , Trocadores de Sódio-Hidrogênio/genética , Adulto , Animais , Transtorno do Deficit de Atenção com Hiperatividade/diagnóstico , Encéfalo/metabolismo , Linhagem Celular , Criança , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/metabolismo , Humanos , Camundongos , Fenótipo , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Mutations in the human gap junction beta-2 gene (GJB2) that encodes connexin-26 have been shown to cause non-syndromic sensorineural hearing loss (NSSNHL) at the DFNB1 locus on 13q11. Functional and genetic data regarding the disease causing potential of one particular GJB2 sequence variant, 101 T-->C (M34T), have proven contradictory. In this study, we found the prevalence of the M34T allele in a cohort of white sib pairs and sporadic cases with NSSNHL from the United Kingdom and Ireland to be 3.179% of chromosomes screened. Significantly, we identified the first M34T/M34T genotype cosegregating in a single family with mid to high frequency NSSNHL. Screening a control population of 630 subjects we identified 25 M34T heterozygotes; however, no M34T homozygotes were detected. Surprisingly, the majority of M34T alleles (88%) were in cis with a 10 bp deletion in the 5' non-coding sequence. This non-coding deletion was also homozygous in the homozygous M34T subjects. Microsatellite analysis of flanking loci in M34T heterozygotes and controls does not define an extensive ancestral haplotype but preliminary data suggest two common alleles in subjects with the M34T allele. In summary, we provide data that support M34T acting as a recessive GJB2 allele associated with mild-moderate prelingual hearing impairment.
Assuntos
Conexinas/genética , Perda Auditiva Neurossensorial/genética , Alelos , Substituição de Aminoácidos , Sequência de Bases , Segregação de Cromossomos , Conexina 26 , DNA/química , DNA/genética , Análise Mutacional de DNA , Saúde da Família , Feminino , Frequência do Gene , Testes Genéticos , Variação Genética , Genótipo , Perda Auditiva Neurossensorial/diagnóstico , Homozigoto , Humanos , Masculino , Mutação , Linhagem , Deleção de SequênciaRESUMO
A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150 000 recombinant-DNA-containing colonies were screened for the presence of the human beta-globin related genes. Five recombinants were isolated containing the human beta-globin locus and encompassing approx. 70 kb of human DNA.
Assuntos
Globinas/genética , Bacteriófago lambda/genética , Clonagem Molecular , DNA Recombinante , Genes , Vetores Genéticos , HumanosRESUMO
OBJECTIVE: To define the spectrum of clinical and biochemical features in 51 children with isolated complex I deficiency. BACKGROUND: Mitochondrial respiratory chain defects are one of the most commonly diagnosed inborn errors of metabolism. Until recently there have been technical problems with the diagnosis of respiratory chain complex I defects, and there is a lack of information about this underreported cause of respiratory chain dysfunction. METHODS: A retrospective review of clinical features and laboratory findings was undertaken in all diagnosed patients who had samples referred over a 22-year period. RESULTS: Presentations were heterogeneous, ranging from severe multisystem disease with neonatal death to isolated myopathy. Classic indicators of respiratory chain disease were not present in 16 of 42 patients in whom blood lactate levels were normal on at least one occasion, and in 23 of 37 patients in whom muscle morphology was normal or nonspecific. Ragged red fibers were present in only five patients. Tissue specificity was observed in 19 of 41 patients in whom multiple tissues were examined, thus the diagnosis may be missed if the affected tissue is not analyzed. Nine patients had only skin fibroblasts available, the diagnosis being based on enzyme assay and functional tests. Modes of inheritance include autosomal recessive (suggested in five consanguineous families), maternal (mitochondrial DNA point mutations in eight patients), and possibly X-linked (slight male predominance of 30:21). Recurrence risk was estimated as 20 to 25%. CONCLUSION: Heterogeneous clinical features, tissue specificity, and absence of lactic acidosis or abnormal mitochondrial morphology in many patients have resulted in underdiagnosis of respiratory chain complex I deficiency.
Assuntos
Transporte de Elétrons , Miopatias Mitocondriais/diagnóstico , NADH NADPH Oxirredutases/deficiência , Adolescente , Criança , Pré-Escolar , Feminino , Fibroblastos/patologia , Humanos , Lactente , Recém-Nascido , Fígado/patologia , Masculino , Mitocôndrias Musculares/patologia , Miopatias Mitocondriais/patologiaRESUMO
Inborn errors of the pyruvate dehydrogenase complex (PDC) are associated with lactic acidosis, neuroanatomic defects, developmental delay, and early death. PDC deficiency is a clinically heterogeneous disorder, with most mutations located in the coding region of the X-linked alpha subunit of the first catalytic component, pyruvate dehydrogenase (E1). Treatment of E1 deficiency hs included cofactor replacement, activation of PDC with dichloroacetate, and ketogenic diets. In this report, we describe the outcome of ketogenic diet treatment in seven boys with E1 deficiency. These patients were divided into two groups based on their mutations (R349H, three patients; and R234G, four patients, two sibling pairs). All seven patients received ketogenic diets with varying degrees of carbohydrate restriction. Clinical outcome was compared within each group and between siblings as related to the intensity and duration of dietary intervention. Subjects who either had the diet initiated earlier in life or who were placed on greater carbohydrate restriction had increased longevity and improved mental development. Based on the improved outcomes of patients with identical mutations, it appears that a nearly carbohydrate-free diet initiated shortly after birth may be useful in the treatment of E1 deficiency.
Assuntos
Corpos Cetônicos/biossíntese , Doença da Deficiência do Complexo de Piruvato Desidrogenase/dietoterapia , Doença da Deficiência do Complexo de Piruvato Desidrogenase/metabolismo , Desenvolvimento Infantil , Carboidratos da Dieta/administração & dosagem , Humanos , Recém-Nascido , Longevidade , Masculino , Mutação , Doença da Deficiência do Complexo de Piruvato Desidrogenase/genética , Resultado do TratamentoRESUMO
Three monoclonal antibodies (42 IgG, 47 IgG, 56 IgG) towards factor-VIII:C (VIII:C) have been produced. In ELISA for VIII:C-antigen (VIII:CAg), 47 IgG showed higher affinity for VIII:CAg than 42 IgG and 56 IgG. In solid phase immunoisolation of iodinated VIII:C diluted in EDTA buffer, the three monoclonals, like human VIII:C inhibitors, bound the 77/80 kD-light chain of VIII:C. In the absence of EDTA, 56 IgG bound the heavy chain-light chain complex of VIII:C, while 47 IgG was only able to bind the light chain. When coupled on Sepharose, 56 IgG adsorbed coagulation active VIII:C, while 47 IgG was only able to adsorb coagulation inactive VIII:CAg. In coagulation assay 56 IgG inhibited with 20 BU/mg while 42 IgG and 47 IgG inhibited with 4 BU/mg. A mixture of 42 IgG and 56 IgG showed a synergistic effect and inhibited with 50 BU/mg total IgG. In radioimmunoassay a human VIII:C inhibitor was able to inhibit the VIII:C binding of 42 IgG and 56 IgG but not of 47 IgG. The monoclonals did not inhibit each other. On the contrary, 56 IgG increased the binding of 42 IgG to VIII:C.
Assuntos
Anticorpos Monoclonais/imunologia , Fator VIII/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Epitopos , Fator VIII/análise , Humanos , Peso Molecular , RadioimunoensaioRESUMO
Most patients with mitochondrial disorders are diagnosed by finding a respiratory chain enzyme defect or a mutation in the mitochondrial DNA (mtDNA). The provision of accurate genetic counseling and reproductive options to these families is complicated by the unique genetic features of mtDNA that distinguish it from Mendelian genetics. These include maternal inheritance, heteroplasmy, the threshold effect, the mitochondrial bottleneck, tissue variation, and selection. Although we still have much to learn about mtDNA genetics, it is now possible to provide useful guidance to families with an mtDNA mutation or a respiratory chain enzyme defect. We describe a range of current reproductive options that may be considered for prevention of transmission of mtDNA mutations, including the use of donor oocytes, prenatal diagnosis (by chorionic villus sampling or amniocentesis), and preimplantation genetic diagnosis, plus possible future options such as nuclear transfer and cytoplasmic transfer. For common mtDNA mutations associated with mitochondrial cytopathies (such as NARP, Leigh Disease, MELAS, MERRF, Leber's Hereditary Optic Neuropathy, CPEO, Kearns-Sayre syndrome, and Pearson syndrome), we summarize the available data on recurrence risk and discuss the relative advantages and disadvantages of reproductive options.
Assuntos
DNA Mitocondrial/genética , Aconselhamento Genético , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Mutação , Diagnóstico Pré-Natal , Feminino , Humanos , Masculino , GravidezRESUMO
We discuss the definition of mutations in osteogenesis imperfecta (OI) using a chemical cleavage method for detecting mismatched bases in patient mRNA: control cDNA heteroduplexes. The method is based on the increased chemical modification of cytosines (Cs) by hydroxylamine and thymines (Ts) by osmium tetroxide when they are not paired with their complementary base. The DNA is then cleaved at the modified base with piperidine and the use of radioactively labeled DNA probes allows the position of the mismatched base to be determined by electrophoresis of the cleavage-product. The precise mutations are then determined by specific amplification and sequencing of the region containing the mismatched base. In perinatally lethal OI (OI type II) mismatches have been detected in all 17 cases studied; 12 of these have been fully characterized. In 7 of these 12 cases the mismatches were point mutations in the genes for pro alpha 1(I) or pro alpha 2(I) which resulted in glycine substitutions in the triple helical region of the protein. Sequence variation was detected in addition to the glycine substitutions in 2 cases. In 2 cases the RNA mismatch resulted from changes in the amino acid sequence of the C-propeptide domain. In the 3 remaining cases the mismatch resulted from silent nucleotide sequence variants. In the less severe forms of OI we have studied, mismatches have been detected and characterized in 8 of 12 cases. In 4 of these 8 cases the mismatch resulted from presumably neutral sequence variation and in the other 4 cases mutations have been defined.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Colágeno/genética , Mutação , Osteogênese Imperfeita/genética , RNA Mensageiro/genética , Células Cultivadas , Sondas de DNA , Feminino , Fibroblastos , Humanos , Ácidos Nucleicos Heteroduplexes , Reação em Cadeia da Polimerase , Polimorfismo Genético , Gravidez , Diagnóstico Pré-Natal , Análise de Sequência de RNARESUMO
Analysis of the expression of the testis-specific isoform of the mouse pyruvate dehydrogenase E1 alpha subunit gene (Pdha-2) during various stages of spermatogenesis has shown that a 2.0-kb Pdha-2 mRNA is initially transcribed in meiotic prophase. The initial appearance of Pdha-2 mRNA precedes that of Pgk-2 and corresponds to the appearance of Ldh-3 mRNA. A second Pdha-2 1.7-kb transcript is present in post-meiotic round spermatids. Polysomal analysis of purified spermatogenic cell populations demonstrates that the 2.0-kb mRNA species is translated in diploid, pachytene spermatocytes and the 1.7-kb mRNA species is translated in round spermatids, although a large proportion is present on the nonpolysomal fraction and may be stored for use in later stages of spermiogenesis.
Assuntos
Isoenzimas/genética , Complexo Piruvato Desidrogenase/genética , RNA Mensageiro/metabolismo , Espermatogênese/genética , Testículo/metabolismo , Animais , Sequência de Bases , Primers do DNA , L-Lactato Desidrogenase/genética , Masculino , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Espermatozoides/enzimologiaRESUMO
Osmium tetroxide and hydroxylamine are used to detect mutations in DNA and RNA after hybridization of mutant and wild-type DNA. Mismatched T and C bases, respectively, are modified by these reagents and the DNA strand cleaved at the mismatched bases by subsequent treatment with piperidine. This allows detection and location of the mutation. Although most T.G mismatches have been reported to be reactive to osmium tetroxide, some have been reported to be unreactive. The aim of this study was to collect and analyze the reactive and unreactive T.G mismatches. We have collected sequence contexts of all reactive and unreactive T.G mismatches for analysis. This involves 10 unreactive T.G mismatches (plus one T.C) and 19 reactive T.G mismatches. Sequence effects of bases surrounding these mismatches must influence this reactivity. There must be many types of such sequence effects. We postulate that because of the dominance of 5' G bases near the T of unreactive T.G mismatches and the absence of 5' G bases in reactive T.G mismatches that the stacking of the 5' G on the mismatched T is the reason for this lack of reactivity in the majority of the cases studied here.
Assuntos
Guanina , Tetróxido de Ósmio/química , Timina , Sequência de Bases , Hidroxilamina , Hidroxilaminas/química , Ácidos Nucleicos Heteroduplexes , OligodesoxirribonucleotídeosRESUMO
Brain malformations and neurological dysfunctions are often seen in pyruvate dehydrogenase (PDH) deficient patients. To understand these clinical presentations, we have analyzed the localization and developmental expression of PDH in the embryonic mouse nervous system. Immunostaining was performed to localize PDH E1 alpha protein. PDH activities were measured before and after activation. PDH E1 alpha mRNA levels were quantitated by reverse transcriptase-polymerase chain reaction. Abundant PDH E1 alpha protein was localized in the central nervous system and other neural tissues in embryos at embryonic day (E) 11 onwards. The PDH activity was very low in E9 brain and it increased continuously until the end of gestation. The proportion of active form of PDH increased significantly in E15 brain. Analysis of the PDH E1 alpha mRNA showed that only the X-linked form of the gene was transcribed. The overall mRNA level of E9 brain was approximately 93% of the adult value. It decreased gradually during embryogenesis. A large increase took place at the end of gestation. The mRNA level of PDH was approximately 100 times higher than that of the acetoacetyl-CoA thiolase gene. These results suggest that PDH E1 alpha transcripts of E9 brain are not translated at a high level. The appearance of PDH activity and its increase during E11 and E15 are mainly due to increased levels of translation and activation of PDH. Increased PDH activity at the end of gestation is attributed to an increase in transcription. Our data to a large extent explain pathological presentations in PDH E1 alpha deficient patients with congenital brain disorders.
Assuntos
Encéfalo/enzimologia , Embrião de Mamíferos/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Acetil-CoA C-Acetiltransferase/genética , Actinas/genética , Animais , Sequência de Bases , Encéfalo/embriologia , DNA/metabolismo , Feminino , Masculino , Camundongos , Sondas Moleculares/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA/metabolismo , Distribuição Tecidual , Transcrição GênicaRESUMO
BACKGROUND: The most common form of nonsyndromic neurosensory autosomal recessive deafness, DFNB1, is caused by mutations in the connexin 26 gene (GJB2) on chromosome 13. One mutation, in which one guanosine (G) residue is deleted from a run of 6 Gs (35delG), is found in 40% to 70% of DFNB1 cases and has an expected population frequency of one in 40 to one in 100. METHODS AND RESULTS: Polymerase chain reaction (PCR)-based tests for the 35delG mutation were developed. They are based on mismatched PCR primers that produce novel EcoRII or DdeI restriction enzyme sites depending on the number of Gs at the 35delG locus. An EcoRII site is generated in the wild-type sequence (6 Gs), but not when the 35delG mutation is present. Alternatively, a DdeI site can be generated so that this enzyme cuts the PCR product when the 35delG mutation is present, but not the wild-type sequence. CONCLUSIONS: These tests enable a quick and reliable screen for the common 35delG mutation.