RESUMO
OBJECTIVE: Determine the utility of a clinical calculator to predict the benefit of chemotherapy in stage IA uterine papillary serous cancer (UPSC). PATIENTS AND METHODS: Data were collected from NCDB from years 2010-2014. Based on demographic and surgical characteristics, a clinical score was developed using the random survival forest machine learning algorithm. RESULTS: Of 1,751 patients with stage IA UPSC, 1,012 (58%) received chemotherapy and 739 (42%) did not. Older age (HR 1.06), comorbidities (HR 1.31), larger tumor size (HR 1.27), lymphovascular invasion (HR 1.86), positive peritoneal cytology (HR 2.62), no pelvic lymph node dissection (HR 1.51), and no chemotherapy (HR 2.16) were associated with poorer prognosis. Compared to no chemotherapy, patients who underwent chemotherapy had a 5-year overall survival of 80% vs. 67%. To better delineate those who may derive more benefit from chemotherapy, we designed a clinical calculator capable of dividing patients into low, moderate, and high-risk groups with associated 5-year OS of 86%, 73%, and 53%, respectively. Using the calculator to assess the relative benefit of chemotherapy in each risk group, chemotherapy improved the 5-year OS in the high (42% to 64%; p < 0.001) and moderate risk group (66% to 79%; p < 0.001) but did not benefit the low risk group (84% to 87%; p = 0.29). CONCLUSION: Our results suggest a clinical calculator is useful for counseling and personalizing chemotherapy for stage IA UPSC.
Assuntos
Algoritmos , Cistadenocarcinoma Papilar/tratamento farmacológico , Cistadenocarcinoma Seroso/tratamento farmacológico , Aprendizado de Máquina , Neoplasias Uterinas/tratamento farmacológico , Idoso , Cistadenocarcinoma Papilar/patologia , Cistadenocarcinoma Papilar/cirurgia , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/cirurgia , Feminino , Humanos , Estadiamento de Neoplasias , Nomogramas , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Reprodutibilidade dos Testes , Neoplasias Uterinas/patologia , Neoplasias Uterinas/cirurgiaRESUMO
Disruption of the endothelial barrier in response to Gram positive (G+) bacterial toxins is a major complication of acute lung injury (ALI) and can be further aggravated by antibiotics which stimulate toxin release. The integrity of the pulmonary endothelial barrier is mediated by the balance of disruptive forces such as the small GTPase RhoA, and protective forces including endothelium-derived nitric oxide (NO). How NO protects against the barrier dysfunction is incompletely understood and our goal was to determine whether NO and S-nitrosylation can modulate RhoA activity and whether this mechanism is important for G+ toxin-induced microvascular permeability. We found that the G+ toxin listeriolysin-O (LLO) increased RhoA activity and that NO and S-NO donors inhibit RhoA activity. RhoA was robustly S-nitrosylated as determined by biotin-switch and mercury column analysis. MS revealed that three primary cysteine residues are S-nitrosylated including cys16, cys20 and cys159. Mutation of these residues to serine diminished S-nitrosylation to endogenous NO and mutant RhoA was less sensitive to inhibition by S-NO. G+-toxins stimulated the denitrosylation of RhoA which was not mediated by S-nitrosoglutathione reductase (GSNOR), thioredoxin (TRX) or thiol-dependent enzyme activity but was instead stimulated directly by elevated calcium levels. Calcium-promoted the direct denitrosylation of WT but not mutant RhoA and mutant RhoA adenovirus was more effective than WT in disrupting the barrier integrity of human lung microvascular endothelial cells. In conclusion, we reveal a novel mechanism by which NO and S-nitrosylation reduces RhoA activity which may be of significance in the management of pulmonary endothelial permeability induced by G+-toxins.
Assuntos
Toxinas Bacterianas/farmacologia , Endotélio Vascular/metabolismo , Proteínas de Choque Térmico/farmacologia , Proteínas Hemolisinas/farmacologia , Compostos Nitrosos/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Pulmão/irrigação sanguínea , Microvasos/metabolismo , Mutação , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Permeabilidade , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
OBJECTIVES: Body size is postulated to modulate type 1 diabetes as either a trigger of islet autoimmunity or an accelerator to clinical onset after seroconversion. As overweight and obesity continue to rise among children, the aim of this study was to determine whether human leukocyte antigen DQ (HLA-DQ) genotypes may be related to body size among children genetically at risk for type 1 diabetes. METHODS: Repeated measures of weight and height were collected from 5969 children 2-4 years of age enrolled in The Environmental Determinants of Diabetes in the Young prospective study. Overweight and obesity was determined by the International Obesity Task Force cutoff values that correspond to body mass index (BMI) of 25 and 30 kg m(-)(2) at age 18. RESULTS: The average BMI was comparable across specific HLA genotypes at every age point. The proportion of overweight was not different by HL A, but percent obesity varied by age with a decreasing trend among DQ2/8 carriers (P for trend=0.0315). A multivariable regression model suggested DQ2/2 was associated with higher obesity risk at age 4 (odds ratio, 2.41; 95% confidence interval, 1.21-4.80) after adjusting for the development of islet autoantibody and/or type 1 diabetes. CONCLUSIONS: The HLA-DQ2/2 genotype may predispose to obesity among 2-4-year-old children with genetic risk for type 1 diabetes.
Assuntos
Autoanticorpos/genética , Autoimunidade/genética , Diabetes Mellitus Tipo 1/genética , Antígenos HLA-DQ/genética , Obesidade Infantil/genética , Idade de Início , Peso ao Nascer , Estatura , Índice de Massa Corporal , Peso Corporal , Pré-Escolar , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/imunologia , Feminino , Finlândia/epidemiologia , Predisposição Genética para Doença , Genótipo , Alemanha/epidemiologia , Humanos , Ilhotas Pancreáticas , Masculino , Programas de Rastreamento , Mães , Obesidade Infantil/epidemiologia , Obesidade Infantil/imunologia , Prevalência , Estudos Prospectivos , Fatores de Risco , Suécia/epidemiologia , Estados Unidos/epidemiologiaRESUMO
AIMS/HYPOTHESIS: Viruses are candidate causative agents in the pathogenesis of autoimmune (type 1) diabetes. We hypothesised that children with a rapid onset of type 1 diabetes may have been exposed to such agents shortly before the initiation of islet autoimmunity, possibly at high dose, and thus study of these children could help identify viruses involved in the development of autoimmune diabetes. METHODS: We used next-generation sequencing to search for viruses in plasma samples and examined the history of infection and fever in children enrolled in The Environmental Determinants of Diabetes in the Young (TEDDY) study who progressed to type 1 diabetes within 6 months from the appearance of islet autoimmunity, and in matched islet-autoantibody-negative controls. RESULTS: Viruses were not detected more frequently in plasma from rapid-onset patients than in controls during the period surrounding seroconversion. In addition, infection histories were found to be similar between children with rapid-onset diabetes and control children, although episodes of fever were reported less frequently in children with rapid-onset diabetes. CONCLUSIONS/INTERPRETATION: These findings do not support the presence of viraemia around the time of seroconversion in young children with rapid-onset type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Vírus/genética , Autoimunidade/genética , Autoimunidade/imunologia , Pré-Escolar , Diabetes Mellitus Tipo 1/virologia , Feminino , Humanos , Lactente , Recém-Nascido , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Masculino , Viroses/genéticaRESUMO
AIMS/HYPOTHESIS: Over 50 regions of the genome have been associated with type 1 diabetes risk, mainly using large case/control collections. In a recent genome-wide association (GWA) study, 18 novel susceptibility loci were identified and replicated, including replication evidence from 2,319 families. Here, we, the Type 1 Diabetes Genetics Consortium (T1DGC), aimed to exclude the possibility that any of the 18 loci were false-positives due to population stratification by significantly increasing the statistical power of our family study. METHODS: We genotyped the most disease-predicting single-nucleotide polymorphisms at the 18 susceptibility loci in 3,108 families and used existing genotype data for 2,319 families from the original study, providing 7,013 parent-child trios for analysis. We tested for association using the transmission disequilibrium test. RESULTS: Seventeen of the 18 susceptibility loci reached nominal levels of significance (p < 0.05) in the expanded family collection, with 14q24.1 just falling short (p = 0.055). When we allowed for multiple testing, ten of the 17 nominally significant loci reached the required level of significance (p < 2.8 × 10(-3)). All susceptibility loci had consistent direction of effects with the original study. CONCLUSIONS/INTERPRETATION: The results for the novel GWA study-identified loci are genuine and not due to population stratification. The next step, namely correlation of the most disease-associated genotypes with phenotypes, such as RNA and protein expression analyses for the candidate genes within or near each of the susceptibility regions, can now proceed.
Assuntos
Diabetes Mellitus Tipo 1/genética , Loci Gênicos , Predisposição Genética para Doença , População Branca/genética , Genótipo , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
OBJECTIVE: To examine the relationship between high-risk human leukocyte antigen (HLA) genotypes for type 1 diabetes and birth size in combination with prenatal ch aracteristics in different countries. STUDY DESIGN: Four high-risk HLA genotypes were enrolled in the Environmental determinants of Diabetes in the Young study newborn babies from the general population in Finland, Germany, Sweden and the United States. Stepwise regression analyses were used to adjust for country, parental physical characteristics and environmental factors during pregnancy. RESULT: Regression analyses did not reveal differences in birth size between the four type 1 diabetes high-risk HLA genotypes. Compared with DQ 4/8 in each country, (1) DQ 2/2 children were heavier in the United States (P=0.028) mostly explained however, by parental weight; (2) DQ 2/8 (P=0.023) and DQ 8/8 (P=0.046) children were longer in Sweden independent of parents height and as well as (3) in the United States for DQ 2/8 (P=0.023), but again dependent on parental height. CONCLUSION: Children born with type 1 diabetes high-risk HLA genotypes have comparable birth size. Longitudinal follow-up of these children should reveal whether birth size differences between countries contribute to the risk for islet autoimmunity and type 1 diabetes.
Assuntos
Peso ao Nascer , Estatura , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Genótipo , Antígenos HLA-DQ/genética , Pais , Peso Corporal , Feminino , Finlândia , Alemanha , Humanos , Recém-Nascido , Masculino , Gravidez , Fatores de Risco , Suécia , Estados UnidosRESUMO
The present study was conducted to assess genetic associations for type 1 diabetes (T1D) reported in previous genome-wide association studies (GWAS). A total of 21 previously reported single-nucleotide polymorphisms (SNPs) were genotyped by TaqMan assays in 1434 Caucasian T1D patients and 1864 normal controls from Georgia. Analysis of the samples identified 18 SNPs (PTPN22, INS, IFIH1, SH2B3, ERBB3, CTLA4, C14orf181, CTSH, CLEC16A, CD69, ITPR3, C6orf173, SKAP2, PRKCQ, RNLS, IL27, SIRPG and CTRB2) with putative association.
Assuntos
Diabetes Mellitus Tipo 1/genética , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único/genética , Adolescente , Idade de Início , Alelos , Criança , Mapeamento Cromossômico , Diabetes Mellitus Tipo 1/epidemiologia , Feminino , Predisposição Genética para Doença/genética , Genótipo , Georgia/epidemiologia , Humanos , Masculino , População Branca/genética , Adulto JovemRESUMO
Previous studies suggested that the SUMO4 gene, located in the IDDM5 interval on chromosome 6q25, was associated with type I diabetes (T1D) and several other autoimmune diseases. Subsequent analyses of the SUMO4 variants with T1D suggested that the association was stronger and more consistent in the Asian populations. In addition, considerable heterogeneity has been observed in the Caucasian populations. In this report, a 40-kb genomic interval including the SUMO4 gene was tagged with 15 single-nucleotide polymorphisms. A total of 2317 affected sib-pair families from the Type I Diabetes Genetic Consortium were genotyped using both the Illumina and Sequenom genotyping platforms. In these Caucasian families, we found little evidence supporting an association between SUMO4 and T1D.
Assuntos
Cromossomos Humanos Par 6 , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/análise , Povo Asiático/genética , Diabetes Mellitus Tipo 1/imunologia , Família , Genótipo , Humanos , Irmãos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , População Branca/genéticaRESUMO
Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is the third major locus affecting risk of type I diabetes (T1D), after HLA-DR/DQ and INS. The most associated single-nucleotide polymorphism (SNP), rs2476601, has a C->T variant and results in an arginine (R) to tryptophan (W) amino acid change at position 620. To assess whether this, or other specific variants, are responsible for T1D risk, the Type I Diabetes Genetics Consortium analyzed 28 PTPN22 SNPs in 2295 affected sib-pair (ASP) families. Transmission Disequilibrium Test analyses of haplotypes revealed that all three haplotypes with a T allele at rs2476601 were overtransmitted to affected children, and two of these three haplotypes showed statistically significant overtransmission (P=0.003 to P=5.9E-12). Another haplotype had decreased transmission to affected children (P=3.5E-05). All haplotypes containing the rs2476601 T allele were identical for all SNPs across PTPN22 and only varied at centromeric SNPs. When considering rs2476601 'C' founder chromosomes, a second haplotype (AGGGGC) centromeric of PTPN22 in the C1orf178 region was associated with protection from T1D (odds ratio=0.81, P=0.0005). This novel finding requires replication in independent populations. We conclude the major association of PTPN22 with T1D is likely due to the recognized non-synonymous SNP rs2476601 (R620W).
Assuntos
Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Haplótipos , Polimorfismo de Nucleotídeo Único , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Adulto JovemRESUMO
The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.
Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Haplótipos , Europa (Continente) , Genótipo , Cadeias beta de HLA-DQ , Cadeias HLA-DRB1 , HumanosRESUMO
The long asymptomatic period before the onset of chronic diseases offers good opportunities for disease prevention. Indeed, many chronic diseases may be preventable by avoiding those factors that trigger the disease process (primary prevention) or by use of therapy that modulates the disease process before the onset of clinical symptoms (secondary prevention). Accurate prediction is vital for disease prevention so that therapy can be given to those individuals who are most likely to develop the disease. The utility of predictive markers is dependent on three parameters, which must be carefully assessed: sensitivity, specificity and positive predictive value. Specificity is important if a biomarker is to be used to identify individuals either for counseling or for preventive therapy. However, a reciprocal relationship exists between sensitivity and specificity. Thus, successful biomarkers will be highly specific without sacrificing sensitivity. Unfortunately, biomarkers with ideal specificity and sensitivity are difficult to find for many diseases. One potential solution is to use the combinatorial power of a large number of biomarkers, each of which alone may not offer satisfactory specificity and sensitivity. Recent technological advances in genetics, genomics, proteomics, and bioinformatics offer a great opportunity for biomarker discovery. The newly identified biomarkers have the potential to bring increased accuracy in disease diagnosis and classification, as well as therapeutic monitoring. In this review, we will use type 1 diabetes (T1D) as an example, when appropriate, to discuss pertinent issues related to high throughput biomarker discovery.
Assuntos
Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Genômica , Proteômica , Doença Crônica , Interpretação Estatística de Dados , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/prevenção & controle , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genômica/métodos , Humanos , Modelos Estatísticos , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , Análise Serial de Proteínas , Proteínas/metabolismo , Proteômica/métodos , Sensibilidade e EspecificidadeRESUMO
The pathogenic mechanisms underlying Batten disease are unclear. Patients uniformly possess autoantibodies against glutamic acid decarboxylase (GAD) that are predominantly reactive with a region of GAD (amino acids 1 to 20) distinct from subjects with autoimmune type 1 diabetes or stiff-person syndrome. Batten patients did not possess autoantibodies against other type 1 diabetes-associated autoantigens and human leukocyte antigen genotypes revealed no specific associations with this disease.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Glutamato Descarboxilase/imunologia , Lipofuscinoses Ceroides Neuronais/imunologia , Adolescente , Adulto , Especificidade de Anticorpos , Autoanticorpos/análise , Autoanticorpos/sangue , Doenças Autoimunes/genética , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Feminino , Antígenos HLA/genética , Humanos , Lactente , Insulina/imunologia , Ilhotas Pancreáticas/imunologia , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Chaperonas Moleculares/genética , Lipofuscinoses Ceroides Neuronais/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/imunologia , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Rigidez Muscular Espasmódica/imunologiaRESUMO
METHODS: We examined monocyte prostaglandin synthase 2 (PGS2/COX2) expression in individuals at risk for or with type 1 diabetes including: (i) 58 established type 1 and 2 diabetic patients; (ii) 34 autoantibody positive (AA+) children and adults; (iii) 164 infants and young children with insulin-dependent diabetes mellitus (IDDM) susceptibility human leukocyte antigen (HLA) alleles; and (iv) 37 healthy control individuals, over a 5-yr period. RESULTS: Established type 1 diabetic patients (1 month to 30+ yr post-disease onset) had significantly higher PGS2 expression than healthy controls; by contrast, insulin-treated type 2 diabetic patients had significantly lower PGS2 expression than healthy controls. Longitudinal studies of AA+ subjects at risk for type 1 diabetes indicated that 73% (11/15) of individuals who developed this disease during the study period expressed high levels of PGS2 prior to or after onset. We also found high level PGS2 expression in genetically at-risk infants and young children that correlated with having a first-degree relative with type 1 diabetes, but not with age, gender, or HLA genotype. In this population, high level PGS2 expression coincided with or preceded autoantibody detection in 30% (3/10) of subjects. CONCLUSIONS: These findings suggest that high level monocyte PGS2 expression, although subject to fluctuation, is present in at-risk subjects at an early age and is maintained during progression to and after type 1 diabetes onset.
Assuntos
Diabetes Mellitus Tipo 1/enzimologia , Isoenzimas/sangue , Monócitos/enzimologia , Prostaglandina-Endoperóxido Sintases/sangue , Adulto , Autoanticorpos/sangue , Pré-Escolar , Ciclo-Oxigenase 2 , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 2/enzimologia , Feminino , Predisposição Genética para Doença , Genótipo , Antígenos HLA/genética , Humanos , Lactente , Masculino , Proteínas de MembranaRESUMO
Gene expression analysis using high-throughput microarray technology has become a powerful approach to study systems biology. The exponential growth in microarray experiments has spawned a number of investigations into the reliability and reproducibility of this type of data. However, the sample size requirements necessary to obtain statistically significant results has not had as much attention. We report here statistical methods for the determination of the sufficient number of subjects necessary to minimize the false discovery rate while maintaining high power to detect differentially expressed genes. Two experimental designs were considered: 1) a comparison between two groups at a single time point, and 2) a comparison of two experimental groups with sequential time points. Computer programs are available for the methods discussed in this paper and are adaptable to more complicated situations.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reprodutibilidade dos Testes , Projetos de Pesquisa , Tamanho da Amostra , Estatística como Assunto , Fatores de TempoRESUMO
Genes within the class II region of the major histocompatibility complex (MHC), including genes involved in antigen processing and presentation, have been reported to be associated with several autoimmune diseases. We report here that the LMP/TAP gene region is significantly associated with vitiligo, a disorder in which biochemical defects and/or autoimmune destruction cause melanocyte loss and resulting skin depigmentation. Case/control analyses revealed genetic association of vitiligo in Caucasian patients with an early age of onset with the transporter associated with antigen processing-1 (TAP1) gene. A family-based association method revealed biased transmission of specific alleles from heterozygous parents to affected offspring for the TAP1 gene, as well as for the closely linked LMP2 and LMP7 genes encoding subunits of the immunoproteasome. No association with vitiligo was found for the MECL1 gene, which encodes a third immunoproteasome subunit and is unlinked to the MHC class II region. These results suggest a possible role for the MHC class I antigen processing and/or presentation pathway in the antimelanocyte autoimmune response involved in vitiligo pathogenesis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Cisteína Endopeptidases/genética , Genes MHC da Classe II/imunologia , Complexos Multienzimáticos , Vitiligo/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Alelos , Apresentação de Antígeno/genética , Canadá , Estudos de Casos e Controles , Ligação Genética , Predisposição Genética para Doença , Heterozigoto , Humanos , Polimorfismo de Nucleotídeo Único , Complexo de Endopeptidases do Proteassoma , Estados Unidos , População BrancaRESUMO
Over the last few years, there has been a dramatic increase in the use of cDNA microarrays to monitor gene expression changes in biological systems. Data from these experiments are usually transformed into expression ratios between experimental samples and a common reference sample for subsequent data analysis. The accuracy of this critical transformation depends on two major parameters: the signal intensities and the normalization of the experiment vs. reference signal intensities. Here we describe and validate a new model for microarray signal intensity that has one multiplicative variation and one additive background variation. Using replicative experiments and simulated data, we found that the signal intensity is the most critical parameter that influences the performance of normalization, accuracy of ratio estimates, reproducibility, specificity, and sensitivity of microarray experiments. Therefore, we developed a statistical procedure to flag spots with weak signal intensity based on the standard deviation (delta(ij)) of background differences between a spot and the neighboring spots, i.e., a spot is considered as too weak if the signal is weaker than cdelta(ij). Our studies suggest that normalization and ratio estimates were unacceptable when this threshold (c) is small. We further showed that when a reasonable compromise of c (c = 6) is applied, normalization using trimmed mean of log ratios performed slightly better than global intensity and mean of ratios. These studies suggest that decreasing the background noise is critical to improve the quality of microarray experiments.
Assuntos
Modelos Estatísticos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Teorema de Bayes , Simulação por Computador , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Camundongos , Camundongos Endogâmicos NOD/genética , Análise de Sequência com Séries de Oligonucleotídeos/normas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We report here the cloning and characterization of a novel gene belonging to the tubby superfamily proteins (TUSP) in mouse and human. The mouse Tusp cDNA is 9120 bp in length and encodes a deduced protein of 1547 amino acids, while the human TUSP gene is 11,127 bp and encodes a deduced protein of 1544 amino acids. The human and mouse genes are 87% identical for their nucleotide sequences and 85% identical for their amino acid sequences. The protein sequences of these genes are 40-48% identical to other tubby family proteins at the C-terminal conserved 'tubby domain'. In addition, the TUSP proteins contain a tubby signature motif (FXGRVTQ), two bipartite nuclear localization signals (NLSs) at the C-terminal, two proline-rich regions, one WD40 repeat region and one suppressor of cytokines signaling domain. Transfection assay with green fluorescent protein-tagged TUSP expression constructs showed that the complete TUSP protein and the N-terminal portion of TUSP are localized in the cytoplasm but the C-terminal portion with the two NLSs produced distinct dots or spots localized in the cytoplasm. Northern blotting analysis showed that the major transcript with the complete coding sequence is expressed mainly in the brain, skeletal muscle, testis and kidney. Radiation hybrid mapping localized the mouse gene to chromosome 17q13 and the human TUSP gene to chromosome 6q25-q26 near the type 1 diabetes gene IDDM5. However, association analysis in diabetic families with a polymorphic microsatellite marker did not show any evidence for association between TUSP and type 1 diabetes. The precise biological function of the tubby superfamily genes is still unknown; the highly conserved tubby domain in different species, however, suggests that these proteins must have fundamental biological functions in a wide range of multi-cellular organisms.
Assuntos
Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Sequência de Aminoácidos , Animais , Northern Blotting , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 6/genética , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/química , DNA Complementar/genética , Diabetes Mellitus/genética , Repetições de Dinucleotídeos/genética , Feminino , Expressão Gênica , Genes/genética , Proteínas de Fluorescência Verde , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The autoimmune regulator (AIRE) protein is a putative transcription regulator with two plant homeodomain-type zinc fingers, a putative DNA-binding domain (SAND), and four nuclear receptor binding LXXLL motifs. We have shown here that in vitro, recombinant AIRE can form homodimers and homotetramers that were also detected in thymic protein extracts. Recombinant AIRE also oligomerizes spontaneously upon phosphorylation by cAMP dependent protein kinase A or protein kinase C. Similarly, thymic AIRE protein is phosphorylated at the tyrosine and serine/threonine residues. AIRE dimers and tetramers, but not the monomers, can bind to G-doublets with the ATTGGTTA motif and the TTATTA-box. Competition assays revealed that sequences with one TTATTA motif and two tandem repeats of ATTGGTTA had the highest binding affinity. These findings demonstrate that AIRE is an important DNA binding molecule involved in immune regulation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Timo/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Dedos de Zinco , Proteína AIRERESUMO
Despite extensive studies on HLA polymorphism, there have been few, if any, studies on allelic forms or mutations in proteins that serve as autoantigens. The present experiments were designed to look for alterations in the coding and promoter regions of the autoantigen IA-2 in type one (insulin-dependent) diabetic patients with autoantibodies to IA-2 as compared with siblings without diabetes or autoantibodies to IA-2. Genomic DNA was used as a template and was amplified by polymerase chain reaction, with pairs of primers encompassing the promoter region and the 23 exons of the coding region of IA-2. A total of nine nucleotide changes were found in the coding region of the six type 1 diabetic patients; four were silent and five were missense changes, but all occurred in the extracellular domain of IA-2 to which autoantibodies are not directed. Few, if any, changes were found in the 5' upstream (-706 to +135) promoter region. The results of the experiments support the null hypothesis that differences among individuals in the nucleotide and amino acid sequences of the promoter and coding regions of IA-2, respectively, do not account for why some individuals develop autoantibodies to IA-2 and others do not.