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1.
PLoS Genet ; 16(8): e1008752, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790717

RESUMO

Myelin is composed of plasma membrane spirally wrapped around axons and compacted into dense sheaths by myelin-associated proteins. Myelin is elaborated by neuroepithelial derived oligodendrocytes in the central nervous system (CNS) and by neural crest derived Schwann cells in the peripheral nervous system (PNS). While some myelin proteins accumulate in only one lineage, myelin basic protein (Mbp) is expressed in both. Overlapping the Mbp gene is Golli, a transcriptional unit that is expressed widely both within and beyond the nervous system. A super-enhancer domain within the Golli/Mbp locus contains multiple enhancers shown previously to drive reporter construct expression specifically in oligodendrocytes or Schwann cells. In order to determine the contribution of each enhancer to the Golli/Mbp expression program, and to reveal if functional interactions occur among them, we derived mouse lines in which they were deleted, either singly or in different combinations, and relative mRNA accumulation was measured at key stages of early development and at maturity. Although super-enhancers have been shown previously to facilitate interaction among their component enhancers, the enhancers investigated here demonstrated largely additive relationships. However, enhancers demonstrating autonomous activity strictly in one lineage, when missing, were found to significantly reduce output in the other, thus revealing cryptic "stealth" activity. Further, in the absence of a key oligodendrocyte enhancer, Golli accumulation was markedly and uniformly attenuated in all cell types investigated. Our observations suggest a model in which enhancer-mediated DNA-looping and potential super-enhancer properties underlie Golli/Mbp regulatory organization.


Assuntos
Elementos Facilitadores Genéticos , Proteína Básica da Mielina/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/metabolismo , Neurogênese , Oligodendroglia/citologia , Oligodendroglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células de Schwann/citologia , Células de Schwann/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo
2.
Arthritis Rheumatol ; 72(2): 242-250, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31449733

RESUMO

OBJECTIVE: Autoantibodies against citrullinated proteins are found in 64-89% of rheumatoid arthritis (RA) patients, with 88-99% specificity. This study was undertaken to create an unbiased, comprehensive profile of serum antibodies against the human proteome, including the citrullinome and the homocitrullinome, in RA patients, using a high-density peptide array. METHODS: Our high-density peptide array, consisting of >4.6 million peptides, contained the entire annotated human proteome. The 20,246 proteins were represented as overlapping 16-mer peptides. In addition to native peptides, citrullinated and homocitrullinated peptides were included, as substitutions for arginine and lysine, and provided a comprehensive screen against all possible epitopes. Twenty-six serum samples (from 8 controls and 18 RA patients) were profiled on the high-density peptide array. Using RA-specific epitopes, we constructed an 8-epitope diagnostic biomarker on a Gyrolab xPlore instrument with a cohort of 92 serum samples (from 29 controls and 63 RA patients). The diagnostic biomarker was further validated with an independent cohort of 181 serum samples (from 54 controls and 127 RA patients). RESULTS: In the initial cohort the diagnostic performance of the 8-epitope biomarker yielded 96.6% specificity and 92.1% sensitivity. The overall diagnostic performance in the validation cohort was 94.4% specificity and 85% sensitivity. In both cohorts, the performance of the 8-epitope diagnostic biomarker compared favorably against the Abnova cyclic citrullinated peptide 2 (CCP2) assay. Using data from the peptide array, we identified novel RA-specific epitopes and formed the basis of a new RA diagnostic assay. CONCLUSION: Comprehensive antibody profiling using a high-density peptide array not only identified novel RA-specific epitopes but also allowed us to construct a novel diagnostic biomarker that is as specific as and more sensitive than the Abnova CCP2 assay.


Assuntos
Anticorpos Anti-Proteína Citrulinada/sangue , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Epitopos/sangue , Estudos de Coortes , Humanos
3.
Twin Res Hum Genet ; 22(5): 277-282, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31608842

RESUMO

A high prevalence of asthma has been documented among the inhabitants of Tristan da Cunha, an isolated island in the South Atlantic. The population derives from just 28 founders. We performed lung function testing, including methacholine inhalation challenge, allergen skin prick testing, and collected DNA from essentially all of the current island population (269 individuals), and genotyped a panel of 43 single-nucleotide polymorphisms (SNPs) reported as associated with asthma and atopy. We carried out a mixed-model association analysis using the known pedigree. There were 96 individuals diagnosed as asthmatic (36%), and heritability estimates were similar to those from nonisolated population samples (multifactorial threshold model, h2 = 48%). The first component from a genetic principal components analysis using the entire SNP panel was nonlinearly associated with asthma, with the maximum risk to those intermediate to reference (Human Genome Diversity Project) European and African samples means. The single most strongly associated SNP was rs2786098 (p = 5.5 × 10-5), known to regulate the gene DENND1B. This explained approximately one-third of the trait heritability, with an allelic odds ratio for the A allele of 2.6. Among A/A carriers, 10 out of 12 individuals were asthmatic. The rs2786098*A variant was initially reported to decrease the risk of childhood (atopic) asthma in European but slightly increase the risk in African-descended populations, and does significantly alter Th2 cell function. Despite an absence of overall association with this variant in recent asthma genome wide association studies meta-analyses, an effect may exist on the particular genetic background of the Tristan da Cunha population.


Assuntos
Asma/genética , Asma/imunologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/imunologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Linhagem , Consanguinidade , Feminino , Estudo de Associação Genômica Ampla , Humanos , Ilhas , Masculino
4.
BMC Dev Biol ; 19(1): 19, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31590629

RESUMO

BACKGROUND: Mouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. O-Fucose at this site has been shown by X-ray crystallography to be recognized by both DLL4 and JAG1 Notch ligands. We previously showed that a Notch1 Thr466Ala mutant exhibits very little ligand-induced NOTCH1 signaling in a reporter assay, whereas a Thr466Ser mutation enables the transfer of O-fucose and reverts the NOTCH1 signaling defect. We subsequently generated a mutant mouse with the Thr466Ala mutation termed Notch1[12f](Notch1tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile. RESULTS: We now report that after backcrossing to C57BL/6 J mice for 11-15 generations, few homozygous Notch1[12f/12f] embryos were born. Timed mating showed that embryonic lethality occurred by embryonic day (E) ~E11.5, somewhat delayed compared to mice lacking Notch1 or Pofut1 (the O-fucosyltransferase that adds O-fucose to Notch receptors), which die at ~E9.5. The phenotype of C57BL/6 J Notch1[12f/12f] embryos was milder than mutants affected by loss of a canonical Notch pathway member, but disorganized vasculogenesis in the yolk sac, delayed somitogenesis and development were characteristic. In situ hybridization of Notch target genes Uncx4.1 and Dll3 or western blot analysis of NOTCH1 cleavage did not reveal significant differences at E9.5. However, qRT-PCR of head cDNA showed increased expression of Dll3, Uncx4.1 and Notch1 in E9.5 Notch1[12f/12f] embryos. Sequencing of cDNA from Notch1[12f/12f] embryo heads and Southern analysis showed that the Notch1[12f] locus was intact following backcrossing. We therefore looked for evidence of modifying gene(s) by crossing C57BL/6 J Notch1 [12f/+] mice to 129S2/SvPasCrl mice. Intercrosses of the F1 progeny gave viable F2 Notch1[12f/12f] mice. CONCLUSION: We conclude that the 129S2/SvPasCrl genome contains a dominant modifying gene that rescues the functions of NOTCH1[12f] in signaling. Identification of the modifying gene has the potential to illuminate novel factor(s) that promote Notch signaling when an O-fucose glycan is absent from EGF12 of NOTCH1.


Assuntos
Substituição de Aminoácidos , Embrião de Mamíferos/anatomia & histologia , Genes Modificadores , Endogamia/métodos , Receptor Notch1/genética , Alanina/metabolismo , Animais , Desenvolvimento Embrionário , Feminino , Fucose/metabolismo , Genoma , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Domínios Proteicos , Receptor Notch1/química , Receptor Notch1/metabolismo , Treonina/metabolismo
5.
Oncotarget ; 10(19): 1760-1774, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30956756

RESUMO

The development of cancer is driven by the accumulation of many oncogenesis-related genetic alterations and tumorigenesis is triggered by complex networks of involved genes rather than independent actions. To explore the epistasis existing among oncogenesis-related genes in lung cancer development, we conducted pairwise genetic interaction analyses among 35,031 SNPs from 2027 oncogenesis-related genes. The genotypes from three independent genome-wide association studies including a total of 24,037 lung cancer patients and 20,401 healthy controls with Caucasian ancestry were analyzed in the study. Using a two-stage study design including discovery and replication studies, and stringent Bonferroni correction for multiple statistical analysis, we identified significant genetic interactions between SNPs in RGL1:RAD51B (OR=0.44, p value=3.27x10-11 in overall lung cancer and OR=0.41, p value=9.71x10-11 in non-small cell lung cancer), SYNE1:RNF43 (OR=0.73, p value=1.01x10-12 in adenocarcinoma) and FHIT:TSPAN8 (OR=1.82, p value=7.62x10-11 in squamous cell carcinoma) in our analysis. None of these genes have been identified from previous main effect association studies in lung cancer. Further eQTL gene expression analysis in lung tissues provided information supporting the functional role of the identified epistasis in lung tumorigenesis. Gene set enrichment analysis revealed potential pathways and gene networks underlying molecular mechanisms in overall lung cancer as well as histology subtypes development. Our results provide evidence that genetic interactions between oncogenesis-related genes play an important role in lung tumorigenesis and epistasis analysis, combined with functional annotation, provides a valuable tool for uncovering functional novel susceptibility genes that contribute to lung cancer development by interacting with other modifier genes.

6.
J Clin Med ; 8(2)2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795559

RESUMO

A number of genome-wide association studies (GWASs) and meta-analyses of genetic variants have been performed in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. We reinterpreted previous studies using false-positive report probability (FPRP) and Bayesian false discovery probability (BFDP). This study searched publications in PubMed and Excerpta Medica Database (EMBASE) up to February 2018. Identification of noteworthy associations were analyzed using FPRP and BFDP, and data (i.e., odds ratio (OR), 95% confidence interval (CI), p-value) related to significant associations were separately extracted. Using filtered gene variants, gene ontology (GO) enrichment analysis and protein⁻protein interaction (PPI) networks were performed. Overall, 241 articles were identified, and 7 were selected for analysis. Single nucleotide polymorphisms (SNPs) discovered by GWASs were shown to be noteworthy, whereas only 27% of significant results from meta-analyses of observational studies were noteworthy. Eighty-five percent of SNPs with borderline p-values (5.0 × 10-8 < p < 0.05) in GWASs were found to be noteworthy. No overlapping SNPs were found between PR3-ANCA and MPO-ANCA vasculitis. GO analysis revealed immune-related GO terms, including "antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class II", "interferon-gamma-mediated (IFN-γ) signaling pathway". By using FPRP and BFDP, network analysis of noteworthy genetic variants discovered genetic risk factors associated with the IFN-γ pathway as novel mechanisms potentially implicated in the complex pathogenesis of ANCA-associated vasculitis.

8.
CRISPR J ; 1: 223-229, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-31021258

RESUMO

Precise genome editing using CRISPR typically requires delivery of guide RNAs, Cas9 endonuclease, and DNA repair templates. Both microinjection and electroporation effectively deliver these components into mouse zygotes provided the DNA template is an oligonucleotide of only a few hundred base pairs. However, electroporation completely fails with longer double-stranded DNAs leaving microinjection as the only delivery option. Here, we overcome this limitation by first injecting all CRISPR components, including long plasmid-sized DNA templates, into the sub-zona pellucida space. There they are retained, supporting subsequent electroporation. We show that this simple and well-tolerated method achieves intracellular reagent concentrations sufficient to effect precise gene edits.

10.
Circ Cardiovasc Genet ; 10(2)2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28420666

RESUMO

BACKGROUND: Available data suggests that double mutations in patients with hypertrophic cardiomyopathy are not rare and are associated with a more severe phenotype. Most of this data, however, is based on noncontemporary variant classification. METHODS AND RESULTS: Clinical data of all hypertrophic cardiomyopathy patients with 2 rare genetic variants were retrospectively reviewed and compared with a group of patients with a single disease-causing variant. Furthermore, a literature search was performed for all studies with information on prevalence and outcome of patients with double mutations. Classification of genetic variants was reanalyzed according to current guidelines. In our cohort (n=1411), 9% of gene-positive patients had 2 rare variants in sarcomeric genes but only in 1 case (0.4%) were both variants classified as pathogenic. Patients with 2 rare variants had a trend toward younger age at presentation when compared with patients with a single mutation. All other clinical variables were similar. In data pooled from cohort studies in the literature, 8% of gene-positive patients were published to have double mutations. However, after reanalysis of reported variants, this prevalence diminished to 0.4%. All patients with 2 radical mutations in MYBPC3 in the literature had severe disease with death or heart transplant during the first year of life. Data on other specific genotype-phenotype correlations were scarce. CONCLUSIONS: Double mutations in patients with hypertrophic cardiomyopathy are much less common than previously estimated. With the exception of double radical MYBPC3 mutations, there is little data to guide clinical decision making in cases with double mutations.


Assuntos
Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Mutação , Adolescente , Adulto , Idade de Início , Cardiomiopatia Hipertrófica/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência
11.
Arthritis Rheumatol ; 69(5): 1054-1066, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28029757

RESUMO

OBJECTIVE: To identify risk alleles relevant to the causal and biologic mechanisms of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). METHODS: A genome-wide association study and subsequent replication study were conducted in a total cohort of 1,986 cases of AAV (patients with granulomatosis with polyangiitis [Wegener's] [GPA] or microscopic polyangiitis [MPA]) and 4,723 healthy controls. Meta-analysis of these data sets and functional annotation of identified risk loci were performed, and candidate disease variants with unknown functional effects were investigated for their impact on gene expression and/or protein function. RESULTS: Among the genome-wide significant associations identified, the largest effect on risk of AAV came from the single-nucleotide polymorphism variants rs141530233 and rs1042169 at the HLA-DPB1 locus (odds ratio [OR] 2.99 and OR 2.82, respectively) which, together with a third variant, rs386699872, constitute a triallelic risk haplotype associated with reduced expression of the HLA-DPB1 gene and HLA-DP protein in B cells and monocytes and with increased frequency of complementary proteinase 3 (PR3)-reactive T cells relative to that in carriers of the protective haplotype. Significant associations were also observed at the SERPINA1 and PTPN22 loci, the peak signals arising from functionally relevant missense variants, and at PRTN3, in which the top-scoring variant correlated with increased PRTN3 expression in neutrophils. Effects of individual loci on AAV risk differed between patients with GPA and those with MPA or between patients with PR3-ANCAs and those with myeloperoxidase-ANCAs, but the collective population attributable fraction for these variants was substantive, at 77%. CONCLUSION: This study reveals the association of susceptibility to GPA and MPA with functional gene variants that explain much of the genetic etiology of AAV, could influence and possibly be predictors of the clinical presentation, and appear to alter immune cell proteins and responses likely to be key factors in the pathogenesis of AAV.


Assuntos
Granulomatose com Poliangiite/genética , Cadeias beta de HLA-DP/genética , Poliangiite Microscópica/genética , Mieloblastina/genética , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Linfócitos T/metabolismo , alfa 1-Antitripsina/genética , Adulto , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Autoantígenos/imunologia , Linfócitos B/metabolismo , Estudos de Casos e Controles , Feminino , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Antígenos HLA-DP/metabolismo , Cadeias beta de HLA-DP/metabolismo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Mieloblastina/imunologia , Neutrófilos/metabolismo , Razão de Chances , Peroxidase/imunologia , Polimorfismo de Nucleotídeo Único , Linfócitos T/imunologia
12.
Can J Cardiol ; 32(12): 1552-1560, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27523271

RESUMO

More than a century after German physician Paul Ehrlich put forth the idea of a "magic bullet" that could seek out and neutralize disease-causing agents in the body, the first monoclonal antibodies (mAbs) to lower low-density lipoprotein cholesterol (LDL-C) have been introduced into clinical use. This novel class of lipid-lowering agents targets proprotein convertase subtilisin/kexin type 9 (PCSK9), an enzyme that chaperones hepatic LDL receptors (LDLRs) toward intracellular degradation, reducing LDLR-mediated endocytosis and the clearance of LDL-C from the circulation. These new drugs represent the first mAb therapy intended for long-term cardiovascular use. We review the development of mAbs, the selection of PCSK9 as a target, and the current safety and efficacy data and regulatory status of these new therapeutic agents. We also provide guidance to clinicians regarding the potential role for these new agents in clinical practice.


Assuntos
Anticorpos Monoclonais/farmacologia , Hipercolesterolemia , Pró-Proteína Convertase 9 , Anticolesterolemiantes/farmacologia , Controle de Medicamentos e Entorpecentes , Humanos , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/metabolismo , Terapia de Alvo Molecular/métodos , Guias de Prática Clínica como Assunto , Pró-Proteína Convertase 9/antagonistas & inibidores , Pró-Proteína Convertase 9/metabolismo , Resultado do Tratamento
13.
Clin Immunol ; 168: 25-29, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27109640

RESUMO

Primary Sjögren's syndrome (pSS) has a strong female bias. We evaluated an X chromosome dose effect by analyzing 47,XXY (Klinefelter's syndrome, 1 in 500 live male births) among subjects with pSS. 47,XXY was determined by examination of fluorescence intensity of single nucleotide polymorphisms from the X and Y chromosomes. Among 136 pSS men there were 4 with 47,XXY. This was significantly different from healthy controls (1 of 1254 had 47,XXY, p=0.0012 by Fisher's exact test) as well men with rheumatoid arthritis (0 of 363 with 47,XXY), but not different compared to men with systemic lupus erythematosus (SLE) (4 of 136 versus 8 of 306, Fisher's exact test p=NS). These results are consistent with the hypothesis that the number of X chromosomes is critical for the female bias of pSS, a property that may be shared with SLE but not RA.


Assuntos
Artrite Reumatoide/genética , Síndrome de Klinefelter/genética , Lúpus Eritematoso Sistêmico/genética , Síndrome de Sjogren/genética , Cromossomos Humanos X/genética , Cromossomos Humanos Y/genética , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único
14.
Nat Commun ; 6: 8019, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26394269

RESUMO

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist.


Assuntos
Estudo de Associação Genômica Ampla , Cirrose Hepática Biliar/genética , Humanos
15.
J Rheumatol ; 42(9): 1587-94, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26178283

RESUMO

OBJECTIVE: To describe the expression and diagnostic use of 14-3-3η autoantibodies in early rheumatoid arthritis (RA). METHODS: 14-3-3η autoantibody levels were measured using an electrochemiluminescent multiplexed assay in 500 subjects (114 disease-modifying antirheumatic drug-naive patients with early RA, 135 with established RA, 55 healthy, 70 autoimmune, and 126 other non-RA arthropathy controls). 14-3-3η protein levels were determined in an earlier analysis. Two-tailed Student t tests and Mann-Whitney U tests compared differences among groups. Receiver-operator characteristic (ROC) curves were generated and diagnostic performance was estimated by area under the curve (AUC), as well as specificity, sensitivity, and likelihood ratios (LR) for optimal cutoffs. RESULTS: Median serum 14-3-3η autoantibody concentrations were significantly higher (p < 0.0001) in patients with early RA (525 U/ml) when compared with healthy controls (235 U/ml), disease controls (274 U/ml), autoimmune disease controls (274 U/ml), patients with osteoarthritis (259 U/ml), and all controls (265 U/ml). ROC curve analysis comparing early RA with healthy controls demonstrated a significant (p < 0.0001) AUC of 0.90 (95% CI 0.85-0.95). At an optimal cutoff of ≥ 380 U/ml, the ROC curve yielded a sensitivity of 73%, a specificity of 91%, and a positive LR of 8.0. Adding 14-3-3η autoantibodies to 14-3-3η protein positivity enhanced the identification of patients with early RA from 59% to 90%; addition of 14-3-3η autoantibodies to anticitrullinated protein antibodies (ACPA) and/or rheumatoid factor (RF) increased identification from 72% to 92%. Seventy-two percent of RF- and ACPA-seronegative patients were positive for 14-3-3η autoantibodies. CONCLUSION: 14-3-3η autoantibodies, alone and in combination with the 14-3-3η protein, RF, and/or ACPA identified most patients with early RA.


Assuntos
Proteínas 14-3-3/imunologia , Artrite Reumatoide/diagnóstico , Autoanticorpos/sangue , Adulto , Idoso , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Biomarcadores/sangue , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/sangue , Osteoartrite/imunologia , Sensibilidade e Especificidade
16.
PLoS Genet ; 11(1): e1004944, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25615622

RESUMO

The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.


Assuntos
Adenosina Trifosfatases/biossíntese , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Proteínas Cromossômicas não Histona/genética , Flores/genética , Proteínas do Grupo Polycomb/genética , Fatores de Transcrição/biossíntese , Adenosina Trifosfatases/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Histonas/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plântula/genética , Plântula/crescimento & desenvolvimento , Fatores de Transcrição/genética
17.
Ann Rheum Dis ; 74(3): e19, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24448344

RESUMO

OBJECTIVE: To evaluate the contribution of the SPP1 rs11439060 and rs9138 polymorphisms, previously reported as autoimmune risk variants, in the rheumatoid arthritis (RA) genetic background according to anti-citrullinated protein antibodies (ACPAs) status of RA individuals. METHODS: We analysed a total of 11,715 RA cases and 26,493 controls from nine independent cohorts; all individuals were genotyped or had imputed genotypes for SPP1 rs11439060 and rs9138. The effect of the SPP1 rs11439060 and rs9138 risk-allele combination on osteopontin (OPN) expression in macrophages and OPN serum levels was investigated. RESULTS: We provide evidence for a distinct contribution of SPP1 to RA susceptibility according to ACPA status: the combination of ≥3 SPP1 rs11439060 and rs9138 common alleles was associated mainly with ACPA negativity (p=1.29×10(-5), ORACPA-negative 1.257 (1.135 to 1.394)) and less with ACPA positivity (p=0.0148, ORACPA-positive 1.072 (1.014 to 1.134)). The ORs between these subgroups (ie, ACPA-positive and ACPA-negative) significantly differed (p=7.33×10(-3)). Expression quantitative trait locus analysis revealed an association of the SPP1 risk-allele combination with decreased SPP1 expression in peripheral macrophages from 599 individuals. To corroborate these findings, we found an association of the SPP1 risk-allele combination and low serum level of secreted OPN (p=0.0157), as well as serum level of secreted OPN correlated positively with ACPA production (p=0.005; r=0.483). CONCLUSIONS: We demonstrate a significant contribution of the combination of SPP1 rs11439060 and rs9138 frequent alleles to risk of RA, the magnitude of the association being greater in patients negative for ACPAs.


Assuntos
Artrite Reumatoide/genética , Autoanticorpos/imunologia , Citrulina/imunologia , Osteopontina/genética , Peptídeos/imunologia , Alelos , Artrite Reumatoide/imunologia , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Haplótipos , Humanos , Macrófagos/metabolismo , Masculino , Osteopontina/metabolismo , Polimorfismo de Nucleotídeo Único
18.
Clin Rev Allergy Immunol ; 48(2-3): 176-81, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24744192

RESUMO

Primary biliary cirrhosis is characterised by a progressive and destructive lymphocytic cholangitis, targeting small intra-hepatic bile ducts. In association with the histologic liver injury, patients characteristically express highly specific auto-antibodies that recognise a conserved epitope of the pyruvate dehydrogenase complex found on the inner membrane of the mitochondria. Family studies demonstrate a clear increased incidence and prevalence of associated autoimmune diseases; and historically, a clear HLA association with disease has been evident. With the use of a high-throughput whole-genome array technology, significant insights into the non-HLA loci associated with risk for disease development have been made. These studies, which have primarily incorporated genome-wide association screens and targeted analysis of immune genes, have highlighted the integral roles for immune cell development and function in disease risk. This has revealed the IL-12/JAK-STAT signalling pathway as a key etiologic factor. In conjunction with a better understanding of environmental triggers, such work lays the foundation for better disease insights mechanistically and, hopefully, therapeutically. Obstacles to uncovering all the associated genetic risk and the correlation between genotype and phenotype remain to be circumvented, as do better appreciation of the processes that underpin not only disease initiation but also presentation and outcome.


Assuntos
Predisposição Genética para Doença , Cirrose Hepática Biliar/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Estudo de Associação Genômica Ampla , Antígenos HLA/genética , Antígenos HLA/imunologia , Humanos , Cirrose Hepática Biliar/imunologia , Fatores de Risco
19.
Cell Rep ; 9(4): 1417-29, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25453756

RESUMO

A large portion of common variant loci associated with genetic risk for schizophrenia reside within noncoding sequence of unknown function. Here, we demonstrate promoter and enhancer enrichment in schizophrenia variants associated with expression quantitative trait loci (eQTL). The enrichment is greater when functional annotations derived from the human brain are used relative to peripheral tissues. Regulatory trait concordance analysis ranked genes within schizophrenia genome-wide significant loci for a potential functional role, based on colocalization of a risk SNP, eQTL, and regulatory element sequence. We identified potential physical interactions of noncontiguous proximal and distal regulatory elements. This was verified in prefrontal cortex and -induced pluripotent stem cell-derived neurons for the L-type calcium channel (CACNA1C) risk locus. Our findings point to a functional link between schizophrenia-associated noncoding SNPs and 3D genome architecture associated with chromosomal loopings and transcriptional regulation in the brain.


Assuntos
DNA Intergênico/genética , Polimorfismo de Nucleotídeo Único/genética , Esquizofrenia/genética , Artrite Reumatoide/genética , Canais de Cálcio Tipo L/genética , Bases de Dados Genéticas , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Anotação de Sequência Molecular , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Fatores de Risco
20.
PLoS One ; 9(10): e111229, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25340707

RESUMO

TNFα is one of the initial and important mediators to activate downstream signaling pathways by binding to trimerized TNFα receptors (TNFR), and thus is an ideal drug target for cancer therapy. Taking advantage of intrinsic homotimerization of the globular domain of adiponectin (gAD), we have developed a novel TNFα antagonist, the trimerized fusion protein named sTNFRII-gAD. However, our previously-used CHO expression system yielded less than 10 mg/L of sTNFRII-gAD. To produce large quantities of sTNFRII-gAD efficiently, we used a modified CHO-S cell expression system, which is based on a pMH3 vector with non-coding GC-rich DNA fragments for high-level gene expression. We obtained stable clones that produced 75 mg/L of sTNFRII-gAD in the 96-well plate, adapted the clones to 40 ml suspension serum-free batch culture, then optimized the culturing conditions to scale up the fed-batch culture in a 3 L shake-flask and finally in a 5 L AP30 bioreactor. We achieved a final yield of 52 mg/L of sTNFRII-gAD. The trimerized sTNFRII-gAD exhibited the higher affinity to TNFα with a dissociation constant (Kd) of 5.63 nM than the dimerized sTNFRII-Fc with a Kd of 13.4 nM, and further displayed the higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in a L929 cytotoxicity assay. Therefore, the strategy employed in this study may provide an efficient avenue for large-scale production of other recombinant proteins by use of the modified CHO-S cell expression system.


Assuntos
Adiponectina/biossíntese , Receptores Tipo II do Fator de Necrose Tumoral/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Bioensaio , Células CHO , Cricetinae , Cricetulus , Meios de Cultura/química , Enzimas de Restrição do DNA , Perfilação da Expressão Gênica , Humanos , Plasmídeos , Multimerização Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Ressonância de Plasmônio de Superfície , Fator de Necrose Tumoral alfa/antagonistas & inibidores
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