RESUMO
Bardet-Biedl syndrome is an autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, polydactyly and hypogonadism. Other findings include hypertension, diabetes mellitus and renal and cardiovascular anomalies. We have performed a genome-wide search for linkage in a large inbred Bedouin family. Pairwise analysis established linkage with the locus D16S408 with no recombination and a lod score of 4.2. A multilocus lod score of 5.3 was observed. By demonstrating homozygosity, in all affected individuals, for the same allele of marker D16S408, further support for linkage is found, and the utility of homozygosity mapping using inbred families is demonstrated. In a second family, linkage was excluded at this locus, suggesting non-allelic genetic heterogeneity in this disorder.
Assuntos
Cromossomos Humanos Par 16 , Deficiência Intelectual/genética , Obesidade/genética , Retinose Pigmentar/genética , Mapeamento Cromossômico , Feminino , Ligação Genética , Homozigoto , Humanos , Hipogonadismo/genética , Escore Lod , Masculino , Linhagem , Polidactilia/genéticaRESUMO
Clinically similar asthma patients may develop airway obstruction by different mechanisms. Asthma treatments that specifically interfere with the 5-lipoxygenase (ALOX5) pathway provide a method to identify those patients in whom the products of the ALOX5 pathway (that is, the leukotrienes) contribute to the expression of the asthma phenotype. Failure of an asthma patient to respond to treatment with ALOX5-pathway modifiers indicates that leukotrienes are not critical to the expression of the asthmatic phenotype in that patient. We previously defined a family of DNA sequence variants in the core promoter of the gene ALOX5 (on chromosome 10q11.2) associated with diminished promoter-reporter activity in tissue culture. Because expression of ALOX5 is in part transcriptionally regulated, we reasoned that patients with these sequence variants may have diminished gene transcription, and therefore decreased ALOX5 product production and a diminished clinical response to treatment with a drug targeting this pathway. Such an effect indicates an interaction between gene promoter sequence variants and drug-treatment responses, that is, a pharmacogenetic effect of a promoter sequence on treatment responses.
Assuntos
Antiasmáticos/uso terapêutico , Araquidonato 5-Lipoxigenase/genética , Asma/tratamento farmacológico , Asma/genética , Hidroxiureia/análogos & derivados , Regiões Promotoras Genéticas , Alelos , Asma/enzimologia , Frequência do Gene , Variação Genética , Humanos , Hidroxiureia/uso terapêutico , Inibidores de Lipoxigenase/uso terapêutico , Fenótipo , Resultado do TratamentoRESUMO
Asthma is a chronic disease characterized by increased airway responsiveness and airway inflammation. The functional role of nitric oxide (NO) and the various nitric oxide synthase (NOS) isoforms in human asthma is controversial. To investigate the role of NO in an established model of allergic asthma, mice with targeted deletions of the three known isoforms of NOS (NOS1, 2, and 3) were studied. Although the inducible (NOS2) isoform was significantly upregulated in the lungs of ovalbumin (OVA)-sensitized and -challenged (OVA/OVA) wild-type (WT) mice and was undetectable in similarly treated NOS2-deficient mice, airway responsiveness was not significantly different between these groups. OVA/OVA endothelial (NOS3)-deficient mice were significantly more responsive to methacholine challenge compared with similarly treated NOS1 and NOS1&3-deficient mice. Airway responsiveness in OVA/OVA neuronal (NOS1)-deficient and neuronal/endothelial (NOS1&3) double-deficient mice was significantly less than that observed in similarly treated NOS2 and WT groups. These findings demonstrate an important function for the nNOS isoform in controlling the inducibility of airway hyperresponsiveness in this model of allergic asthma.
Assuntos
Asma/imunologia , Óxido Nítrico Sintase/deficiência , Pneumonia/imunologia , Animais , Asma/enzimologia , Asma/etiologia , Líquido da Lavagem Broncoalveolar/citologia , Cálcio/metabolismo , Modelos Animais de Doenças , Marcação de Genes/métodos , Histocitoquímica , Humanos , Isoenzimas/deficiência , Pulmão/enzimologia , Cloreto de Metacolina , Camundongos , Camundongos Knockout , Ovalbumina , PletismografiaRESUMO
We examined the role of the interleukin-8 (IL-8) receptor in a murine model of allergen-induced pulmonary inflammation using mice with a targeted deletion of the murine IL-8 receptor homologue (IL-8r-/-). Wild-type (Wt) and IL-8r-/- mice were systemically immunized to ovalbumin (OVA) and were exposed with either single or multiple challenge of aerosolized phosphate-buffered saline (OVA/PBS) or OVA (OVA/OVA). Analysis of cells recovered from bronchoalveolar lavage (BAL) revealed a diminished recruitment of neutrophils to the airway lumen after single challenge in IL-8r-/- mice compared with Wt mice, whereas multiply challenged IL-8r-/- mice had increased B cells and fewer neutrophils compared with Wt mice. Both Wt and IL-8r-/- OVA/OVA mice recruited similar numbers of eosinophils to the BAL fluid and exhibited comparable degrees of pulmonary inflammation histologically. Both total and OVA-specific IgE levels were greater in multiply challenged IL-8r-/- OVA/OVA mice than in Wt mice. Both the IL-8r-/- OVA/OVA and OVA/PBS mice were significantly less responsive to methacholine than their respective Wt groups, but both Wt and IL-8r mice showed similar degrees of enhancement after multiple allergen challenge. The data demonstrate that the IL-8r modulates IgE production, airway responsiveness, and the composition of the cells (B cells and neutrophils) recruited to the airway lumen in response to antigen.
Assuntos
Alérgenos/imunologia , Antígenos CD/imunologia , Linfócitos B/imunologia , Imunoglobulina E/biossíntese , Pulmão/imunologia , Ovalbumina/imunologia , Receptores de Interleucina/imunologia , Animais , Antígenos CD/genética , Linfócitos B/citologia , Contagem de Células Sanguíneas , Lavagem Broncoalveolar , Broncoconstritores/farmacologia , Citometria de Fluxo , Pulmão/patologia , Linfócitos/citologia , Masculino , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina-8ARESUMO
Cancer cells exhibit dramatic alterations of chromatin organization at cis-regulatory elements, but the molecular basis, extent, and impact of these alterations are still being unraveled. Here, we identify extensive genome-wide modification of sites bearing the active histone mark H3K4me2 in primary human colorectal cancers, as compared with corresponding benign precursor adenomas. Modification of certain colorectal cancer sites highlighted the activity of the transcription factor CNOT3, which is known to control self-renewal of embryonic stem cells (ESC). In primary colorectal cancer cells, we observed a scattered pattern of CNOT3 expression, as might be expected for a tumor-initiating cell marker. Colorectal cancer cells exhibited nuclear and cytoplasmic expression of CNOT3, suggesting possible roles in both transcription and mRNA stability. We found that CNOT3 was bound primarily to genes whose expression was affected by CNOT3 loss, and also at sites modulated in certain types of colorectal cancers. These target genes were implicated in ESC and cancer self-renewal and fell into two distinct groups: those dependent on CNOT3 and MYC for optimal transcription and those repressed by CNOT3 binding and promoter hypermethylation. Silencing CNOT3 in colorectal cancer cells resulted in replication arrest. In clinical specimens, early-stage tumors that included >5% CNOT3+ cells exhibited a correlation to worse clinical outcomes compared with tumors with little to no CNOT3 expression. Together, our findings implicate CNOT3 in the coordination of colonic epithelial cell self-renewal, suggesting this factor as a new biomarker for molecular and prognostic classification of early-stage colorectal cancer. Cancer Res; 77(3); 766-79. ©2016 AACR.
Assuntos
Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição/metabolismo , Animais , Biomarcadores Tumorais/análise , Imunoprecipitação da Cromatina , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos , TranscriptomaRESUMO
Epstein-Barr virus infection of peripheral B lymphocytes predominantly results in a latent infection, with a concomitant growth transformation of the infected cells. These cells express six nuclear antigens (EBNAs) and three membrane antigens. Transcription of all the EBNA genes is driven by one of two promoters, Cp or Wp, located near the left end of the viral genome, and the activities of these promoters are mutually exclusive. We have previously shown that Wp is exclusively used during the initial stages of B-cell immortalization, followed by a switch to Cp usage. However, several cell lines which appear to have failed to switch from Wp to Cp usage and which exhibit constitutive Wp activity have been identified. In two cases, we have shown that this failure to switch is the result of a deletion of approximately 3.5 kb, spanning Cp. In this paper, we characterize the deletion in one of these cell lines, X50-7, and demonstrate not only that the viral genome in this cell line has sustained a deletion in the region of Cp, but also that there has been a rearrangement into the BamHI C region of viral sequences from the BamHI W and Y fragments.
Assuntos
Linfócitos B/microbiologia , Rearranjo Gênico , Genoma Viral , Herpesvirus Humano 4/genética , Regiões Promotoras Genéticas/genética , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Desoxirribonuclease BamHI/metabolismo , Dados de Sequência MolecularRESUMO
Suppressor of cytokine signaling (SOCS) proteins are involved in the negative regulation of cytokine-induced STAT (signal transducers and activators of transcription) factor signaling. We cloned genomic regions of SOCS1 and SOCS2 genes and mapped these genes to chromosome 16p12-p13.1 and chromosome 12q21.3-q23 regions, respectively, by cytogenetic and radiation hybrid mapping. In addition, we mapped SOCS2 by yeast artificial chromosome (YAC) pool screening to YAC contig WC 12.5 on chromosome 12 with an unambiguous hit to CHLC.ATA19H12 and WI-5940, which is 461.5 cR from the top of the map.
Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 16 , Proteínas de Ligação a DNA , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas/genética , Proteínas Repressoras , Transativadores , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Clonagem Molecular , Citocinas/metabolismo , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de CitocinaRESUMO
Asthma is a common, but heterogeneous disease, characterized by reversible airway obstruction, bronchial hyperresponsiveness (BHR); and is commonly associated with atopy. The messenger molecule nitric oxide (NO), that is formed by neuronal NO synthase (NOS1), is known to have a key role in bronchomotor control in animals. In humans the gene for NOS1 is located on chromosome 12q24, in a region that had been shown in family studies to be linked to the diagnosis of asthma. We identified variants of the NOS1 gene, and assessed whether there was a genetic association between these variants of NOS1 and the diagnosis asthma. A total of 410 Caucasian asthma patients and 228 Caucasian controls were screened for three bi-allelic polymorphisms in the NOS1 gene that had been detected by single-stranded conformational polymorphism (SSCP) analysis and confirmed by sequencing. Allele frequencies of a polymorphism in exon 29 of the NOS1 gene were significantly different between asthmatics and controls (P<0.05). These findings suggest that variants of the NOS1 gene may be one source of genetic risk for asthma.
Assuntos
Asma/genética , Óxido Nítrico Sintase/genética , Éxons , Humanos , Mutação , Óxido Nítrico Sintase Tipo I , Polimorfismo Conformacional de Fita SimplesRESUMO
Transcription of the genes encoding the six viral nuclear antigens present in Epstein-Barr virus latently infected lymphocytes can be initiated from one of two promoters (Cp and Wp) mapping near the left end of the viral genome. These promoters are used in a mutually exclusive manner in clonal cell lines established from either Burkitt lymphoma tumors or in vitro infection of peripheral B lymphocytes. In this paper the role of Cp and Wp during viral latency is investigated. Cp appears to be the promoter normally employed during established latent infection. Analysis of two cell lines that use Wp revealed a deletion spanning Cp in the endogenous viral genomes, suggesting that cell lines exhibiting Wp activity harbor mutated viral genomes with a nonfunctional Cp. However, in contrast to the preferred usage of Cp exhibited by established Epstein-Barr virus-infected cell lines, Wp was shown to be exclusively utilized during the initial stages of viral infection. In addition to Wp activity, Cp usage was apparent by 6 days post-infection. A model is proposed involving B-lymphocyte differentiation-driven promoter switching during the establishment of viral latency.
Assuntos
Antígenos Virais/genética , Linfócitos B/microbiologia , Herpesvirus Humano 4/genética , Regiões Promotoras Genéticas , Adulto , Sequência de Bases , Southern Blotting , Linhagem Celular , Transformação Celular Viral , Antígenos Nucleares do Vírus Epstein-Barr , Genes Virais , Herpesvirus Humano 4/patogenicidade , Humanos , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Plasmídeos , RNA Viral/genética , RNA Viral/isolamento & purificação , Transcrição Gênica , Transfecção , Proteínas Estruturais Virais/genéticaRESUMO
Several cDNA clones containing the Epstein-Barr virus BamHI fragment H rightward open reading frame 1 (BHRF1) have been recovered from the tightly latent lymphoblastoid cell line IB4. These clones contain the 5' leader exons encoded in the major internal repeat 1 and the viral BamHI fragment Y, identified in the rightwardly transcribed viral mRNAs associated with the latent viral life cycle. In addition, a cDNA clone containing BHRF1 from the Burkitt lymphoma cell line Jijoye was also recovered and exhibits a distinctive splicing pattern. In vitro transcription and translation of BHRF1, followed by immunoprecipitation with Epstein-Barr virus-positive human sera, indicates that this viral antigen is expressed during infection. RNA blot analyses with a wide panel of lymphoblastoid and Burkitt lymphoma cell lines revealed a complex pattern of transcription. Hybridization data obtained with several probes is presented.
Assuntos
Herpesvirus Humano 4/genética , Clonagem Molecular , DNA/genética , Regulação da Expressão Gênica , Genes Virais , Biossíntese de Proteínas , Splicing de RNA , RNA Mensageiro/genética , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
An in vitro system to assess B-cell function in leprosy patients is described. In vitro lymphoproliferation and antibody synthesis by peripheral blood mononuclear cells (PBMC) in response to pokeweed mitogen (PWM) and Formalin-treated Staphylococcus aureus Cowan I (FSA) from 31 leprosy patients and 13 healthy controls were studied. DNA synthesis was induced by both PWM and FSA in PBMC from all of the leprosy patients and control subjects. Lepromatous leprosy (LL) patients' cells showed higher responses to both PWM and FSA. However, these increases were not statistically significant. The levels of secreted IgM, IgG, or IgA were examined in the 7-day culture supernatants of PBMC cultured with or without PWM or FSA using an enzyme-linked immunosorbent assay. Wide individual variations were observed in in vitro antibody synthesis. IgM secretion in PBMC from normal subjects and various groups of leprosy patients in response to PWM and FSA was comparable. In vitro IgG secretion in response to PWM was the highest in cells from LL patients; it was significantly decreased in cells from tuberculoid leprosy (TT) patients (p less than 0.01). The levels in cells from borderline leprosy (BB) patients were intermediate in response to the same mitogen. Cells from leprosy patients as a group showed a higher spontaneous secretion of IgA in comparison with cells from normal subjects. Overall, the in vitro Ig secretion by PBMC in different patient groups appears to reproduce the spectrum of antibody levels observed in patients in vivo. Thus, the present in vitro culture system may help to delineate the mechanisms of B-cell dysregulation in leprosy.
Assuntos
Linfócitos B/imunologia , Imunoglobulinas/biossíntese , Hanseníase/imunologia , Ativação Linfocitária , Anticorpos Antibacterianos/biossíntese , DNA Bacteriano/biossíntese , Humanos , Imunidade Celular , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Hanseníase Dimorfa/imunologia , Hanseníase Virchowiana/imunologia , Hanseníase Tuberculoide/imunologia , Leucócitos Mononucleares/imunologia , Mycobacterium leprae/genética , Mycobacterium leprae/imunologiaRESUMO
We developed a simple and rapid amplification-refractory mutation system (ARMS) assay for the factor V mutation [R506Q] (factor VLeiden), which results in the autosomal dominant thrombotic tendency, resistance to activated protein C (rAPC). PCR primers within Exon 10 of the factor V gene were designed. A common upstream primer was paired with either a mutant or wild-type-specific downstream primer. The 3'-most nucleotide of the specific primers recognized either the mutant or normal allele, and the 3' penultimate nucleotide was mismatched to enhance specificity of the reaction. The assay was validated using authentic factor VLeiden DNA samples. Seven of 103 hematologically normal children (6.8%) were found to be heterozygotes. Among 27 patients studied by the rAPC assay, ARMS assay and rAPC results were concordant in 26. Among these were a 1-year-old child with a calcified clot in the inferior vena cava. Both the patient and his father were heterozygous for the mutation and both had abnormal rAPC assays. rAPC and factor VLeiden assays were discordant in a young girl with a history of stroke. Biochemical rAPC assay was abnormal, while ARMS assay revealed amplification only with wild-type primers, suggesting a non-[R506Q] mechanism for rAPC. This assay will be a valuable tool for studying subjects with thromboses and their family members.
Assuntos
Fator V/genética , Mutação/genética , Trombose/genética , Primers do DNA/química , Eletroforese em Gel de Ágar , Fibrinolíticos/farmacologia , Doenças Genéticas Inatas/genética , Humanos , Reação em Cadeia da Polimerase , Proteína C/metabolismo , Fatores de Risco , Tromboembolia/genética , Tromboembolia/metabolismo , Tromboembolia/fisiopatologia , Trombose/diagnósticoRESUMO
Arachidonate 5-lipoxygenase-activating protein (ALOX5AP) is an arachidonic acid binding protein that has been shown to be critical in the biosynthesis of leukotrienes. We mapped the ALOX5AP gene to the chromosome 13q12 region by cytogenetic mapping, yeast artificial chromosome (YAC) pool screening, and radiation hybrid mapping. It was mapped to YAC contig WC13.2 by YAC pool screening with an unambiguous hit to WI-4874, which is at 78 cR on the radiation hybrid map, 3.36 cR, by radiation hybrid mapping, from WI-4874.
Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 13/genética , Proteínas de Membrana/genética , Proteínas Ativadoras de 5-Lipoxigenase , Humanos , Hibridização In Situ , Leucotrienos/genética , Mapeamento Físico do Cromossomo/métodosRESUMO
During latent Epstein-Barr virus (EBV) infection of human B lymphocytes, six viral nuclear antigen (EBNAs) are expressed from long primary transcripts by means of alternative splicing and alternative polyadenylylation sites. These transcripts initiate from one of two promoters, Cp or Wp, that function in a mutually exclusive fashion. Wp is exclusively utilized during the initial stages of infection of primary B lymphocytes, followed by a switch to Cp usage. These studies have been extended to show that (i) a mutant EBV strain lacking the gene encoding EBNA 2 fails to switch from Wp to Cp usage in primary B lymphocytes, although the virus contains a functional Cp; (ii) a region from -429 to -245 base pairs upstream of Cp is essential for Cp activity in B lymphocytes, but only in the context of upstream and downstream sequences; (iii) this region contains an EBNA 2-dependent enhancer; and (iv) DNase I protection employing nuclear extracts from B and T lymphocytes revealed a B-cell-specific footprint in the region of the EBNA 2-dependent enhancer. These results support a model for viral promoter switching during the initial stages of infection in which Wp activity leads to the expression of EBNA 2, followed by activation of Cp through the EBNA 2-dependent enhancer.
Assuntos
Antígenos Virais/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Regiões Promotoras Genéticas , Infecções Tumorais por Vírus/genética , Linfócitos B/microbiologia , Sequência de Bases , Análise Mutacional de DNA , DNA Viral/genética , Elementos Facilitadores Genéticos , Antígenos Nucleares do Vírus Epstein-Barr , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/fisiologiaRESUMO
BACKGROUND: Polymorphisms of the beta(2) adrenoceptor influence receptor function in vitro and asthma phenotypes in vivo. However, their importance in determining responses to inhaled beta agonist treatment has not been clearly defined. METHODS: In a retrospective analysis of previously published data we have examined relationships between polymorphisms at codons 16 and 27 of the beta(2) adrenoceptor and clinical outcomes in a randomised, placebo controlled, crossover trial of regularly scheduled salbutamol and salmeterol in 115 patients with mild to moderate asthma. Genotyping was obtained for positions 16 and 27 in 108 and 107 patients, respectively. For position 16, 17 patients (16%) were homozygous Arg-Arg, 40 (37%) were heterozygous Arg-Gly, and 51 (47%) were homozygous Gly-Gly. RESULTS: Within the homozygous Arg-16 group major exacerbations were more frequent during salbutamol treatment than with placebo (1.91 (95% CI 1.07 to 3.12) per year versus 0.81 (95% CI 0.28 to 1.66) per year; p = 0.005). No significant treatment related differences occurred for heterozygous Arg-Gly patients (salbutamol 0.11 (95% CI 0.01 to 0.40), placebo 0.54 (95% CI 0.26 to 1.00) exacerbations per year) or homozygous Gly-16 patients (salbutamol 0.38 (95% CI 0.17 to 0.73), placebo 0.30 (95% CI 0.12 to 0.61) exacerbations per year). No adverse changes occurred for any position 16 subgroup with salmeterol. There was no significant relationship between position 27 genotypes and treatment related outcomes. CONCLUSION: Homozygous Arg-16 patients are susceptible to clinically important increases in asthma exacerbations during chronic dosing with the short acting beta(2) agonist salbutamol.
Assuntos
Agonistas Adrenérgicos beta/uso terapêutico , Albuterol/análogos & derivados , Albuterol/uso terapêutico , Asma/tratamento farmacológico , Asma/genética , Polimorfismo Genético , Receptores Adrenérgicos beta 2/genética , Adolescente , Adulto , Estudos Cross-Over , Método Duplo-Cego , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Xinafoato de Salmeterol , Resultado do TratamentoRESUMO
An increased concentration of nitric oxide (NO) in exhaled air (FENO) is now recognized as a critical component of the asthmatic phenotype. When we identified patients with asthma on the basis of a standard case definition alone, we found that they were remarkably heterogeneous with respect to their FENO. However, when we included genotype at a prominent asthma candidate gene (i.e., NOS1) in the case definition, and determined the number of AAT repeats in intron 20, we identified a remarkably homogeneous cohort of patients with respect to FENO. Both mean FENO (p = 0.00008) and variability around the mean (p = 0.000002) were significantly lower in asthmatic individuals with a high number (> or = 12) of AAT repeats at this locus than in those with fewer repeats. These data provide a biologically tenable link between genotype at a candidate gene in a region of linkage, NOS1, and an important component of the asthmatic phenotype, FENO. We show that addition of NOS1 genotype to the case definition of asthma allows the identification of a uniform cohort of patients, with respect to FENO, that would have been indistinguishable by other physiologic criteria. Our isolation of this homogeneous cohort of patients ties together the well-established associations among asthma, increased concentrations of NO in the exhaled air of asthmatic individuals, and variations of trinucleotide repeat sequences as identified in several neurologic conditions.
Assuntos
Asma/fisiopatologia , Proteínas do Tecido Nervoso/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico/fisiologia , Adulto , Asma/genética , Sequência de Bases , Estudos de Coortes , DNA/genética , Feminino , Frequência do Gene/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Óxido Nítrico Sintase Tipo I , Fenótipo , Estatísticas não ParamétricasRESUMO
Recent studies have suggested that two polymorphisms of the beta(2)-adrenergic receptor (beta(2)AR) gene at codons 16 (arginine to glycine) and 27 (glutamine to glutamate) affect an individual's airway responsiveness, or response to acute or chronic beta(2)-agonist therapy but are not risk factors for asthma. We hypothesize that there is an interaction effect on asthma between the beta(2)AR gene polymorphisms and cigarette smoking. A case-control study was conducted in 128 asthma cases and 136 control individuals identified from 10,014 studied subjects in rural Anqing, China. Allele-specific polymerase chain reaction (PCR) was used to genotype beta(2)AR gene polymorphisms. Multiple logistic regression was used to adjust for potential confounding factors. We found a marginally significant interaction between cigarette smoking and beta(2)AR-16 genotype after adjusting for important confounding factors (p = 0.06). Specifically, we found that compared with never-smoking Gly-16 homozygotes, those ever-smokers who are Arg-16 homozygotes had a significantly increased risk of asthma (odds ratio [OR] = 7.81; 95% confidence interval [CI]: 2.07 to 29.5). This association showed a clear dose-response relationship with the number of cigarettes smoked. However, there was no significant association of asthma with polymorphisms of the beta(2)AR at position 27 (OR = 1.38; 95% CI: 0.69 to 2.73). Our study suggests a gene-environment interaction between the Arg-16 genotype and ever cigarette smoking with respect to the susceptibility of an individual to asthma.
Assuntos
Asma/etiologia , Predisposição Genética para Doença/genética , Polimorfismo Genético/genética , Receptores Adrenérgicos beta 2/genética , Fumar/efeitos adversos , Adulto , Asma/diagnóstico , Asma/epidemiologia , Estudos de Casos e Controles , China/epidemiologia , Fatores de Confusão Epidemiológicos , Feminino , Volume Expiratório Forçado , Genótipo , Homozigoto , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prevalência , Fatores de Risco , Distribuição por Sexo , Inquéritos e QuestionáriosRESUMO
The association of subclasses of Alu repetitive elements with various classes of trinucleotide and tetranucleotide microsatellites was characterized as a first step toward advancing our understanding of the evolution of microsatellite repeats. In addition, information regarding the association of specific classes of microsatellites with families of Alu elements was used to facilitate the development of genetic markers. Sequences containing Alu repeats were eliminated because unique primers could not be designed. Various classes of microsatellites are associated with different classes of Alu repeats. Very abundant and poly(A)-rich microsatellite classes (ATA, AATA) are frequently associated with an evolutionarily older subclass of Alu repeats, AluSx, whereas most of GATA and CA microsatellites are associated with a recent Alu subfamily, AluY. Our observations support all three possible mechanisms for the association of Alu repeats to microsatellites. Primers designed using a set of sequences from a particular microsatellite class showed higher homology with more sequences of that class than probes designed for other classes. We developed an efficient method of prescreening GGAA and ATA microsatellite clones for Alu repeats with probes designed in this study. We also showed that Alu probes labeled in a single reaction (multiplex labeling) could be used efficiently for prescreening of GGAA clones. Sequencing of these prescreened GGAA microsatellites revealed only 5% Alu repeats. Prescreening with primers designed for ATA microsatellite class resulted in the reduction of the loss of markers from approximately 50% to 10%. The new Alu probes that were designed have also proved to be useful in Alu-Alu fingerprinting.
Assuntos
Mapeamento Cromossômico , Genoma Humano , Repetições de Microssatélites/genética , Sequências Repetitivas de Ácido Nucleico , Animais , Cromossomos Artificiais de Levedura , Humanos , CamundongosRESUMO
Thiamine-responsive megaloblastic anemia, also known as "TRMA" or "Rogers syndrome," is an early-onset autosomal recessive disorder defined by the occurrence of megaloblastic anemia, diabetes mellitus, and sensorineural deafness, responding in varying degrees to thiamine treatment. On the basis of a linkage analysis of affected families of Alaskan and of Italian origin, we found, using homozygosity mapping, that the TRMA-syndrome gene maps to a region on chromosome 1q23.2-23.3 (maximum LOD score of 3.7 for D1S1679). By use of additional consanguineous kindreds of Israeli-Arab origin, the putative disease-gene interval also has been confirmed and narrowed, suggesting genetic homogeneity. Linkage analysis generated the highest combined LOD-score value, 8.1 at a recombination fraction of 0, with marker D1S2799. Haplotype analysis and recombination events narrowed the TRMA locus to a 16-cM region between markers D1S194 and D1S2786. Several heterozygote parents had diabetes mellitus, deafness, or megaloblastic anemia, which raised the possibility that mutations at this locus predispose carriers in general to these manifestations. Characterization of the metabolic defect of TRMA may shed light on the role of thiamine deficiency in such common diseases.
Assuntos
Anemia Megaloblástica/genética , Cromossomos Humanos Par 1/genética , Diabetes Mellitus Tipo 1/genética , Genes Recessivos , Perda Auditiva Neurossensorial/genética , Tiamina/uso terapêutico , Alaska , Anemia Megaloblástica/tratamento farmacológico , Anemia Megaloblástica/etnologia , Árabes , Mapeamento Cromossômico , Consanguinidade , Diabetes Mellitus Tipo 1/etnologia , Feminino , Haplótipos/genética , Perda Auditiva Neurossensorial/etnologia , Homozigoto , Humanos , Israel/etnologia , Itália/etnologia , Escore Lod , Masculino , Repetições de Microssatélites , Linhagem , Federação Russa/etnologia , SíndromeRESUMO
We examined the influence of two common polymorphic forms of the beta(2)-adrenergic receptor (beta(2)AR): the Gly16 and Glu27 alleles, on acute and long-term beta(2)AR desensitization in human airway smooth muscle (HASM) cells. In cells from 15 individuals, considered without respect to genotype, pretreatment with Isoproterenol (ISO) at 10(-7) M for 1 h or 24 h caused approximately 25% and 64% decreases in the ability of subsequent ISO (10(-6) M) stimulation to reduce HASM cell stiffness as measured by magnetic twisting cytometry. Similar results were obtained with ISO-induced cyclic adenosine monophosphate (cAMP) as the outcome indicator. Data were then stratified post hoc by genotype. Cells containing at least one Glu27 allele (equivalent to presence of the Gly16Glu27 haplotype) showed significantly greater acute desensitization than did cells with no Glu27 allele, whether ISO-induced cell stiffness (34% versus 19%, p < 0.03) or cAMP formation (58% versus 11%, p < 0.02) was measured. Likewise, cells with any Glu27 allele showed greater long-term desensitization of cell stiffness and cAMP formation responses than did cells without the Glu27 allele. The distribution of genotypes limited direct conclusions about the influence of the Gly16 allele. However, presence of the Gly16Gln27 haplotype was associated with less acute and long-term desensitization of ISO-induced cAMP formation than was seen in cells without the Gly16Gln27 haplotype (14% versus 47%, p < 0.09 for short-term desensitization; 32% versus 84%, p < 0.01 for long-term desensitization), suggesting that the influence of Glu27 is not through its association with Gly16. The Glu27 allele was in strong linkage disequilibrium with the Arg19 allele, a polymorphic form of the beta(2)AR upstream peptide of the 5'-leader cistron of the beta(2)AR, and this polymorphism in the beta(2)AR 5'-flanking region may explain the effects of the Glu27 allele. Cells with any Arg19 allele showed significantly greater acute and long-term desensitization of ISO-induced cAMP formation than did cells without the Arg19 allele (54% versus 2%, p < 0.01 for short-term desensitization; 73% versus 35%, p < 0.05 for long-term desensitization). Similar results were obtained for ISO-induced changes in cell stiffness. Thus, the presence of the Glu27 allele is associated with increased acute and long-term desensitization in HASM.