RESUMO
BACKGROUND: Quantitative, reverse transcription PCR (qRT-PCR) is currently the gold-standard for SARS-CoV-2 detection and it is also used for detection of other virus. Manual data analysis of a small number of qRT-PCR plates per day is a relatively simple task, but automated, integrative strategies are needed if a laboratory is dealing with hundreds of plates per day, as is being the case in the COVID-19 pandemic. RESULTS: Here we present shinyCurves, an online shiny-based, free software to analyze qRT-PCR amplification data from multi-plate and multi-platform formats. Our shiny application does not require any programming experience and is able to call samples Positive, Negative or Undetermined for viral infection according to a number of user-defined settings, apart from providing a complete set of melting and amplification curve plots for the visual inspection of results. CONCLUSIONS: shinyCurves is a flexible, integrative and user-friendly software that speeds-up the analysis of massive qRT-PCR data from different sources, with the possibility of automatically producing and evaluating melting and amplification curve plots.
Assuntos
COVID-19 , Análise de Dados , Humanos , Pandemias , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2RESUMO
Patients with adenosine deaminase (ADA) deficiency exhibit spontaneous and partial clinical remission associated with somatic reversion of inherited mutations. We report a child with severe combined immunodeficiency (T-B- SCID) due to ADA deficiency diagnosed at the age of 1 month, whose lymphocyte counts including CD4+ and CD8+ T and NK cells began to improve after several months with normalization of ADA activity in Peripheral blood lymphocytes (PBL), as a result of somatic mosaicism caused by monoallelic reversion of the causative mutation in the ADA gene. He was not eligible for haematopoietic stem cell transplantation (HSCT) or gene therapy (GT); therefore he was placed on enzyme replacement therapy (ERT) with bovine PEG-ADA. The follow-up of metabolic and immunologic responses to ERT included gradual improvement in ADA activity in erythrocytes and transient expansion of most lymphocyte subsets, followed by gradual stabilization of CD4+ and CD8+ T (with naïve phenotype) and NK cells, and sustained expansion of TCRγδ+ T cells. This was accompanied by the disappearance of the revertant T cells as shown by DNA sequencing from PBL. Although the patient's clinical condition improved marginally, he later developed a germinal cell tumour and eventually died at the age of 67 months from sepsis. This case adds to our current knowledge of spontaneous reversion of mutations in ADA deficiency and shows that the effects of the ERT may vary among these patients, suggesting that it could depend on the cell and type in which the somatic mosaicism is established upon reversion.
Assuntos
Adenosina Desaminase/metabolismo , Terapia de Reposição de Enzimas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Neoplasias Primárias Desconhecidas/genética , Neoplasias Primárias Desconhecidas/terapia , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Adenosina Desaminase/administração & dosagem , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Bovinos , Contagem de Células , Criança , Pré-Escolar , Análise Mutacional de DNA , Evolução Fatal , Humanos , Imunofenotipagem , Lactente , Células Matadoras Naturais/patologia , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/fisiopatologia , Neoplasias Pulmonares/secundário , Masculino , Mosaicismo/efeitos dos fármacos , Mutação/genética , Neoplasias Primárias Desconhecidas/complicações , Neoplasias Primárias Desconhecidas/patologia , Neoplasias Primárias Desconhecidas/fisiopatologia , Receptores de Antígenos de Linfócitos T/metabolismo , Imunodeficiência Combinada Severa/complicações , Imunodeficiência Combinada Severa/patologia , Imunodeficiência Combinada Severa/fisiopatologia , Choque SépticoRESUMO
In this communication, we report on the genomic surveillance of SARS-CoV-2 using wastewater samples in Jefferson County, KY. In February 2021, we analyzed seven wastewater samples for SARS-CoV-2 genomic surveillance. Variants observed in smaller catchment areas, such as neighborhood manhole locations, were not necessarily consistent when compared to associated variant results in downstream treatment plants, suggesting catchment size or population could impact the ability to detect diversity.
RESUMO
Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA-CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human-mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126-143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26(+) ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans.
Assuntos
Adenosina Desaminase/metabolismo , Substituição de Aminoácidos/genética , Dipeptidil Peptidase 4/metabolismo , Mutação Puntual/genética , Imunodeficiência Combinada Severa/genética , Adenosina Desaminase/química , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Células Cultivadas , Citometria de Fluxo , Deleção de Genes , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Imunodeficiência Combinada Severa/enzimologia , Linfócitos T/enzimologia , Linfócitos T/metabolismoRESUMO
T lymphocytes cultured from a patient (T.D.) with adenosine deaminase (ADA) deficiency expressed ADA activity in the normal range, inconsistent with her severe immunodeficiency, metabolic abnormalities, and with the absence of ADA activity in her B lymphocytes and other nucleated hematopoietic cells. ADA from T.D. T cells had normal Km, heat stability, and sensitivity to ADA inhibitors. Examination of HLA phenotype and polymorphic DNA loci indicated that T.D. was neither chimeric nor a genetic mosaic. Amplified and subcloned ADA cDNA from ADA+ T.D. T cells was shown by allele-specific oligonucleotide hybridization to possess the same mutations (Arg101----Trp, Arg211----His) previously found in the ADA-T.D. B cell line GM 2606 (Akeson, A. L., D. A. Wiginton, M. R. Dusing, J. C. States, and J. J. Hutton. 1988. J. Biol. Chem. 263:16291-16296). Our findings suggest that one of these mutant alleles can be expressed selectively in IL-2-dependent T cells as stable, active enzyme. Cultured T cells from other patients with the Arg211----His mutation did not express significant ADA activity, while some B cell lines from a patient with an Arg101----Gln mutation have been found to express normal ADA activity. We speculate that Arg101 may be at a site that determines degradation of ADA by a protease that is under negative control by IL-2 in T cells, and is variably expressed in B cells. Il-2 might increase ADA expression in T cells of patients who possess mutations of Arg101.
Assuntos
Adenosina Desaminase/deficiência , Adenosina Desaminase/metabolismo , Síndromes de Imunodeficiência/enzimologia , Nucleosídeo Desaminases/deficiência , Nucleosídeo Desaminases/metabolismo , Linfócitos T/enzimologia , Adenosina Desaminase/genética , Antígenos CD/análise , Northern Blotting , Southern Blotting , Células Cultivadas , Expressão Gênica , Células-Tronco Hematopoéticas/enzimologia , Interleucina-2/farmacologia , Mutação , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genética , Linfócitos T/imunologiaRESUMO
We examined the genetic basis for adenosine deaminase (ADA) deficiency in seven patients with late/delayed onset of immunodeficiency, an underdiagnosed and relatively unstudied condition. Deoxyadenosine-mediated metabolic abnormalities were less severe than in the usual, early-onset disorder. Six patients were compound heterozygotes; 7 of 10 mutations found were novel, including one deletion (delta 1019-1020), three missense (Arg156 > His, Arg101 > Leu, Val177 > Met), and three splicing defects (IVS 5, 5'ss T+6 > A; IVS 10, 5'ss G+1 > A; IVS 10, 3'ss G-34 > A). Four of the mutations generated stop signals at codons 131, 321, 334, and 348; transcripts of all but the last, due to delta 1019-1020, were severely reduced. delta 1019-1020 (like delta 955-959, found in one patient and apparently recurrent) is at a short deletional hot spot. Arg156 > His, the product of which had detectable activity, was found in three patients whose second alleles were unlikely to yield active ADA. The oldest patient diagnosed was homozygous for a single base change in intron 10, which activates a cryptic splice acceptor, resulting in a protein with 100 extra amino acids. We speculate that this "macro ADA," as well as the Arg156 > His, Arg101 > Leu, Ser291 > Leu, and delta 1019-1020 products, may contribute to mild phenotype. Tissue-specific variation in splicing efficiency may also ameliorate disease severity in patients with splicing mutations.
Assuntos
Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Mutação , Imunodeficiência Combinada Severa/genética , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , Desoxiadenosinas/metabolismo , Feminino , Variação Genética , Heterozigoto , Homozigoto , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Splicing de RNA , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Linfócitos T/enzimologiaRESUMO
When cells in G2 phase are challenged with DNA damage, several key mitotic regulators such as Cdk1/Cyclin B, Aurora A and Plk1 are inhibited to prevent entry into mitosis. Here we have studied how inhibition of Plk1 is established after DNA damage. Using a Förster resonance energy transfer (FRET)-based biosensor for Plk1 activity, we show that inhibition of Plk1 after DNA damage occurs with relatively slow kinetics and is entirely dependent on loss of Plk1-T210 phosphorylation. As T210 is phosphorylated by the kinase Aurora A in conjunction with its co-factor Bora, we investigated how they are affected by DNA damage. Interestingly, we find that the interaction between Bora and Plk1 remains intact during the early phases of the DNA damage response (DDR), whereas Plk1 activity is already inhibited at this stage. Expression of an Aurora A mutant that is refractory to inhibition by the DDR failed to prevent inhibition of Plk1 and loss of T210 phosphorylation, suggesting that inhibition of Plk1 may be established by perturbing recruitment of Aurora A by Bora. Indeed, expression of a fusion in which Aurora A was directly coupled to Bora prevented DNA damage-induced inhibition of Plk1 activity, as well as inhibition of T210 phosphorylation. Taken together, these data demonstrate that DNA damage affects the function of Aurora A at multiple levels: both by direct inhibition of Aurora A activity, as well as by perturbing the interaction with its co-activator Bora. We propose that the DDR targets recruitment of Aurora A to the Plk1/Bora complex to prevent activation of Plk1 during DNA damage in G2.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Aurora Quinase A/genética , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Humanos , Fosforilação , Ligação Proteica , Quinase 1 Polo-LikeRESUMO
ALDH isozymes have been characterized in terms of substrate and coenzyme specificity, heat stability, tissue distribution and electrophoretic properties. The activity of the isozymes has also been examined in rodent-human somatic cell hybrids in order to map the structural genes to specific chromosomes and to study the control of gene expression. One isozyme, designated ALDH3, which is very active against benzaldehyde, was found to show variable expression in hybrids made between rat hepatoma cells and human fibroblasts or fetal liver. Segregation analysis of these hybrids indicates that the structural locus for human ALDH3 may be on chromosome 17. The expression of rodent ALDH3 in these hybrids was extremely variable and not correlated with the appearance of the human enzyme. In hybrids expressing human and rodent ALDH3 no heteromeric isozymes were observed. The human "cytosolic" ALDH1 and "mitochondrial" ALDH2 isozymes did not appear to be expressed in any of the somatic cell hybrids examined.
Assuntos
Aldeído Desidrogenase/análise , Isoenzimas/análise , Aldeído Desidrogenase/genética , Animais , Mapeamento Cromossômico , Variação Genética , Humanos , Ratos , Especificidade da EspécieRESUMO
This is an electrophoretic study of ALDH isozymes in post-mortem tissue extracts. Three different electrophoretic variants of the isozyme ALDH3 were found in the 100 individuals examined. One liver sample showed lack of ALDH1 activity, but it remains unknown whether this is due to genetic mechanisms. The other two isozymes--ALDH2 and ALDH4--did not show any variations.
Assuntos
Aldeído Desidrogenase/análise , Isoenzimas/análise , Fígado/enzimologia , Estômago/enzimologia , Aldeído Desidrogenase/genética , Costa Rica , Eletroforese , Feminino , Humanos , Isoenzimas/genética , Masculino , FenótipoRESUMO
The amino acid profile was studied in individual random samples of urine from 1147 normal schoolchildren and 1074 exceptional children: 628 with mental retardation, 332 with hearing and speech defects and 114 with visual defects as well as in 673 patients with mental disorders. Laboratory procedures included chemical tests and one-dimension paper- electro- and column-chromatography. Phenylketonuria was found in a mentally retarded girl and in one of her brothers; iminoglycinuria in a mentally retarded boy and heterozygote cystinuria in a man with manic-depressive psychosis. The percentage of high excretors of beta-aminoisobutyric acid (B-AIB) in the controls (4.88%) was similar to previous findings in the Caucasian race. The children with hearing and speech defects showed a number of high excretors of B-AIB significantly lower (X2 = 5.32; p less than 0.025) and the children with visual defects a number of hyperglycinurias significantly higher (X2 = 9.19; p less than 0.05). Previous non-consistent findings on the excess of high excretors of B-AIB in Down's syndrome were not confirmed in this study. These results suggest a relationship between transport defects in the plasma membrane and pathological disorders in some of the cases screened.
Assuntos
Aminoácidos/urina , Deficiência Intelectual/urina , Transtornos Mentais/urina , Alanina/urina , Erros Inatos do Metabolismo dos Aminoácidos/urina , Ácidos Aminoisobutíricos/urina , Criança , Costa Rica , Creatinina/urina , Feminino , Glicina/urina , Transtornos da Audição/urina , Humanos , Masculino , Fenilalanina/urina , Distúrbios da Fala/urinaRESUMO
Adenosine deaminase (ADA) deficiency was the first known cause of primary immunodeficiency. Over the past 25 years the basis for immune deficiency has largely been established. Now it appears that ADA deficiency may also cause hepatic toxicity, raising new questions about its pathogenesis. The ADA gene has been sequenced and the ADA three-dimensional structure solved. The relationship between genotype and phenotype is being analysed, and ADA deficiency has become a focus for novel approaches to enzyme replacement and gene therapy.
Assuntos
Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Animais , Humanos , Erros Inatos do Metabolismo da Purina-Pirimidina/enzimologia , Erros Inatos do Metabolismo da Purina-Pirimidina/genéticaRESUMO
We have isolated a cDNA clone that codes for human cytochrome b5 reductase. The cDNA was used to analyse, by Southern-blot hybridization, DNA isolated from a panel of 11 independent human-rodent somatic cell hybrids. The results indicate that cytochrome b5 reductase is encoded by a single gene located on human chromosome 22.
Assuntos
Mapeamento Cromossômico , Redutases do Citocromo/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Cromossomos Humanos Par 22 , Clonagem Molecular , Citocromo-B(5) Redutase , Humanos , Dados de Sequência MolecularRESUMO
We have recently isolated a cloned cDNA that codes for a human orthologue of the major phenobarbital-inducible cytochrome P450IIB subfamily of rodents. The cloned human cDNA was used to analyse, by Southern blot hybridization, DNA extracted from a panel of 9 independent human-rodent somatic cell hybrids. The results indicate that all members of the P450IIB gene subfamily of man are located on chromosome 19. Evidence from hybrids containing different regions of human chromosome 19 localizes this cytochrome P-450 gene subfamily further to the long arm of chromosome 19 in the region cen-q13.3. We propose the designation CYP2B for this locus.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 19 , Sistema Enzimático do Citocromo P-450/genética , Animais , Southern Blotting , Cricetinae , Cricetulus , DNA/análise , Sondas de DNA , Humanos , Células Híbridas , Camundongos , Hibridização de Ácido Nucleico , RatosRESUMO
The biochemical properties of ALDH isozymes have been examined in human tissues and one set, designated ALDH3, has been studied in detail. These components occur at highest levels in lung and stomach, but were not expressed in fetal tissues, or in blood, hair roots and fibroblasts. The ALDH3 isozymes show optimal activity with benzaldehyde and can use either NAD or NADP as cofactor. Antiserum against a partially purified ALDH3, from stomach, selectively precipitates this isozyme from human tissues and selectively recognizes an homologous component in the rat. Human and rodent ALDH3 were not immunoprecipitated by anti-ALDH1 or anti-ALDH2 antisera. High levels of expression were found in human-rodent hybrids, constructed using rat hepatoma cells, and these hybrids were used to assign the human ALDH3 gene to chromosome 17.
Assuntos
Aldeído Desidrogenase/genética , Cromossomos Humanos 16-18 , Isoenzimas/genética , Animais , Mapeamento Cromossômico , Eletroforese em Gel de Amido , Feto , Humanos , Células Híbridas , Fígado/enzimologia , Pulmão/enzimologia , Testes de Precipitina , Ratos , Especificidade da Espécie , Estômago/enzimologia , Especificidade por Substrato , Distribuição TecidualRESUMO
We have isolated and sequenced a cDNA clone (pB8) that codes for a novel member of the cytochrome P450IIC sub-family of man. Analysis, by Southern blot hybridization, of DNA isolated from a panel of nine independent human-rodent somatic cell hybrids demonstrated that the corresponding gene (CYP2C) is located on human chromosome 10. Northern blot hybridization of RNA isolated from human livers revealed a 10-fold inter-individual variation in the expression of the gene.
Assuntos
Mapeamento Cromossômico , Sistema Enzimático do Citocromo P-450/genética , Genes , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA/análise , Humanos , Fígado/análise , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/análiseRESUMO
Adenosine deaminase (ADA) deficiency causes lymphopenia and immunodeficiency due to toxic effects of its substrates. Most patients are infants with severe combined immunodeficiency disease (SCID), but others are diagnosed later in childhood (delayed onset) or as adults (late onset); healthy individuals with "partial" ADA deficiency have been identified. More than 50 ADA mutations are known; most patients are heteroallelic, and most alleles are rare. To analyze the relationship of genotype to phenotype, we quantitated the expression of 29 amino acid sequence-altering alleles in the ADA-deleted Escherichia coli strain SO3834. Expressed ADA activity of wild-type and mutant alleles ranged over five orders of magnitude. The 26 disease-associated alleles expressed 0.001%-0.6% of wild-type activity, versus 5%-28% for 3 alleles from "partials." We related these data to the clinical phenotypes and erythrocyte deoxyadenosine nucleotide (dAXP) levels of 52 patients (49 immunodeficient and 3 with partial deficiency) who had 43 genotypes derived from 42 different mutations, including 28 of the expressed alleles. We reduced this complexity to 13 "genotype categories," ranked according to the potential of their constituent alleles to provide ADA activity. Of 31 SCID patients, 28 fell into 3 genotype categories that could express <=0.05% of wild-type ADA activity. Only 2 of 21 patients with delayed, late-onset, or partial phenotypes had one of these "severe" genotypes. Among 37 patients for whom pretreatment metabolic data were available, we found a strong inverse correlation between red-cell dAXP level and total ADA activity expressed by each patient's alleles in SO3834. Our system provides a quantitative framework and ranking system for relating genotype to phenotype.
Assuntos
Adenosina Desaminase/deficiência , Mutação , Erros Inatos do Metabolismo da Purina-Pirimidina/genética , Imunodeficiência Combinada Severa/genética , Nucleotídeos de Adenina/análise , Adenosina Desaminase/genética , Adolescente , Adulto , Idade de Início , Alelos , Criança , Pré-Escolar , Eritrócitos/química , Expressão Gênica , Heterogeneidade Genética , Genótipo , Humanos , Lactente , Fenótipo , Padrões de ReferênciaRESUMO
We have isolated and sequenced cDNA clones that code for rat and human NADPH-dependent cytochrome P-450 reductase. The cDNA coding for the human protein was used to analyse, by Southern blot hybridization, DNA isolated from a panel of 8 independent human-rodent somatic cell hybrids. The results indicate that cytochrome P-450 reductase is encoded by a single gene (POR) located on human chromosome 7(pter-q22). Analysis of human metaphase chromosomes by hybridization in situ confirmed the results and refined the localization to 7q11.2. Northern blot hybridization revealed that in human liver the expression of the gene varies by less than 3-fold between different individuals.
Assuntos
Cromossomos Humanos Par 7 , DNA/genética , NADPH-Ferri-Hemoproteína Redutase/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RatosRESUMO
Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes, with delayed or late onset and gradual decline in immune function, also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. We have characterized the mutations responsible for ADA deficiency in siblings with striking disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more mildly affected sib (RG) than in her younger, more severely affected sister (EG). Cultured T cells, fibroblasts, and B lymphoblasts of RG had detectable residual ADA activity, while cells of EG did not. ADA mRNA was undetectable by northern analysis in these cells of both patients. Both sibs were found to be compound heterozygotes for the following novel splicing defects: (1) a G+1-->A substitution at the 5' splice site of IVS 2 and (2) a complex 17-bp rearrangement of the 3' splice site of IVS 8, which inserted a run of seven purines into the polypyrimidine tract and altered the reading frame of exon 9. PCR-amplified ADA cDNA clones with premature translation stop codons arising from aberrant pre-mRNA splicing were identified, which were consistent with these mutations. However, some cDNA clones from T cells of both patients and from fibroblasts and Epstein-Barr virus (EBV)-transformed B cells of RG, were normally spliced at both the exon 2/3 and exon 8/9 junctions. A normal coding sequence was documented for clones from both sibs. The normal cDNA clones did not appear to arise from either contamination or PCR artifact, and mosaicism seems unlikely to have been involved. These findings suggest (1) that a low level of normal pre-mRNA splicing may occur despite mutation of the invariant first nucleotide of the 5' splice donor sequence and (2) that differences in efficiency of such splicing may account for the difference in residual ADA activity, immune dysfunction, and clinical severity in these siblings.
Assuntos
Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Mutação Puntual , Splicing de RNA , Imunodeficiência Combinada Severa/genética , Adenosina Desaminase/metabolismo , Sequência de Bases , Linhagem Celular , Células Cultivadas , Pré-Escolar , DNA/química , DNA/genética , Primers do DNA , Éxons , Feminino , Fibroblastos/enzimologia , Triagem de Portadores Genéticos , Humanos , Lactente , Dados de Sequência Molecular , Núcleo Familiar , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Imunodeficiência Combinada Severa/enzimologia , Linfócitos T/enzimologiaRESUMO
Three new missense mutations (H15D, A83D, and A179D) and a new splicing defect (573 + IG-->A) in the 5' splice site of intron 5 were among six mutant adenosine deaminase (ADA) alleles found in three unrelated patients with severe combined immunodeficiency disease, the most common phenotype associated with ADA deficiency. When expressed in vitro, the H15D, A83D, and A179D proteins lacked detectable ADA activity. The splicing defect caused skipping of exon 5, resulting in premature termination of translation and a reduced level of mRNA. H15D is the first naturally occurring mutation of a residue that coordinates directly with the enzyme-associated zinc ion. Molecular modeling based on the atomic coordinates of murine ADA suggests that the D15 mutation would create a cavity or gap between the zinc ion and the side chain carboxylate of D15. This could alter the ability of zinc to activate a water molecule postulated to play a role in the catalytic mechanism. A83 and A179 are not directly involved in the active site, but are conserved residues located respectively in alpha helix 4 and beta strand 4 of the alpha/beta barrel. Replacement of these small hydrophobic Ala residues with the charged, more bulky Asp side chain may distort ADA structure and affect enzyme stability or folding.
Assuntos
Adenosina Desaminase/genética , Alanina/metabolismo , Histidina/metabolismo , Mutação , Imunodeficiência Combinada Severa/genética , Adenosina Desaminase/metabolismo , Sequência de Bases , Sítios de Ligação , População Negra/genética , Sequência Conservada , Humanos , Lactente , Íntrons , Masculino , Modelos Químicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Splicing de RNA , População Branca/genética , Zinco/metabolismoRESUMO
We report three novel adenosine deaminase (ADA) mutations with interesting implications. A Somali child with severe combined immunodeficiency disease (SCID) had reduced ADA mRNA in T cells and was homozygous for the nonsense mutation Q3X. Unexpectedly, her healthy father was a compound ADA heterozygote whose second allele carried a 'partial' mutation, R142Q, due to a G-->A transition of a CpG dinucleotide. A C-->T transition of the same CpG produced a nonsense mutation, R142X, in two homozygous Canadian Mennonite infants with SCID. The severe and healthy phenotypes associated with R142X and R142Q, the high frequency of 'partial' ADA mutations arising from CpGs in healthy individuals of African descent and the presence of CAA (glutamine) at codon 142 in murine ADA, suggest selection for replacement of this CpG hotspot by CpA during ADA evolution. R142X, located within a purine-rich segment at nt 62/116 of exon 5, caused skipping of the exon, possibly by disrupting a splicing enhancer. Absence of exon 5 in T cell ADA mRNA and low ADA activity in T cells and erythrocytes obtained at age 18-22 months from one of the Mennonite children, indicate limited expression of a normal ADA cDNA from retrovirally transduced CD34+ umbilical cord leukocytes infused shortly after birth in an attempt at stem cell gene therapy.