RESUMO
Background: Mycobacterium tuberculosis (M.tb), the causative bacterium of tuberculosis (TB), establishes residence and grows in human alveolar macrophages (AMs). Inter-individual variation in M.tb-human AM interactions can indicate TB risk and the efficacy of therapies and vaccines; however, we currently lack an understanding of the gene and protein expression programs that dictate this variation in the lungs. Results: Herein, we systematically analyze interactions of a virulent M.tb strain H37Rv with freshly isolated human AMs from 28 healthy adult donors, measuring host RNA expression and secreted candidate proteins associated with TB pathogenesis over 72h. A large set of genes possessing highly variable inter-individual expression levels are differentially expressed in response to M.tb infection. Eigengene modules link M.tb growth rate with host transcriptional and protein profiles at 24 and 72h. Systems analysis of differential RNA and protein expression identifies a robust network with IL1B, STAT1, and IDO1 as hub genes associated with M.tb growth. RNA time profiles document stimulation towards an M1-type macrophage gene expression followed by emergence of an M2-type profile. Finally, we replicate these results in a cohort from a TB-endemic region, finding a substantial portion of significant differentially expressed genes overlapping between studies. Conclusions: We observe large inter-individual differences in bacterial uptake and growth, with tenfold variation in M.tb load by 72h.The fine-scale resolution of this work enables the identification of genes and gene networks associated with early M.tb growth dynamics in defined donor clusters, an important step in developing potential biological indicators of individual susceptibility to M.tb infection and response to therapies.
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Recent studies of leukemic tumors in individual extramedullary sites showed they adopt the clinical and metastatic behavior of solid cancers originating in those sites. To elucidate features of leukemic tumors that render them resistant to agents effective against marrow leukemia, we analyzed a series of AML breast tumors by histology, immunohistochemistry, and RNA sequencing. Striking histologic similarities to solid cancers were found: a single-filing architectural pattern virtually identical to that of invasive lobular breast carcinoma and dense desmoplastic keloid-like fibrosis similar to colon, gallbladder, and pancreas carcinomas. Sequencing found 2157 genes significantly downregulated in AML breast tumors compared to normal breast. Comparison to triple-negative breast cancer found 859 genes similarly downregulated. At least 30 of these genes have been associated with poor prognosis in breast cancers. Five were reported in AML marrow studies to correlate with poor prognosis. The findings of this pilot study suggest the seed-and-soil interaction recognized in solid cancer growth may help explain how leukemic cells, in some patients, adopt solid tumor behavior in non-marrow sites. Transformed cells that metastasize from tumor to marrow can impart chemoresistance and be an unrecognized cause of treatment failure and death. Further studies comparing leukemic tumor to simultaneous marrow could potentially identify biomarkers that predict extramedullary resistance and lead to new therapeutic targets. Recognizing the potential for leukemia to adopt solid tumor phenotype, and implementation of body scanning and ablative tumor treatment, could decrease the persistently high rates of marrow resistance and treatment failure.
Assuntos
Mama/patologia , Leucemia Mieloide Aguda/patologia , Sarcoma Mieloide/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Mama/química , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Carcinoma/patologia , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Especificidade de Órgãos , Projetos Piloto , Prognóstico , RNA Mensageiro/análise , RNA Neoplásico/análise , Sarcoma Mieloide/tratamento farmacológico , Sarcoma Mieloide/genética , Sarcoma Mieloide/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The mechanisms underlying abnormal granuloma formation in patients with sarcoidosis are complex and remain poorly understood. A novel in vitro human granuloma model was used to determine the molecular mechanisms of granuloma genesis in patients with sarcoidosis in response to putative disease-causing mycobacterial antigens. Peripheral blood mononuclear cells (PBMCs) from patients with active sarcoidosis and from normal, disease-free control subjects were incubated for 7 days with purified protein derivative-coated polystyrene beads. Molecular responses, as reflected by differential expression of genes, extracellular cytokine patterns, and cell surface receptor expression, were analyzed. Unbiased systems biology approaches were used to identify signaling pathways engaged during granuloma formation. Model findings were compared with human lung and mediastinal lymph node gene expression profiles. Compared with identically treated PBMCs of control subjects (n = 5), purified protein derivative-treated sarcoidosis PBMCs (n = 6) were distinguished by the formation of cellular aggregates resembling granulomas. Ingenuity Pathway Analysis of differential expression gene patterns identified molecular pathways that are primarily regulated by IL-13, which promotes alternatively activated (M2) macrophage polarization. M2 polarization was further demonstrated by immunohistochemistry performed on the in vitro sarcoidosis granuloma-like structures. IL-13-regulated gene pathways were confirmed in human sarcoidosis lung and mediastinal lymph node tissues. The in vitro human sarcoidosis granuloma model provides novel insights into early granuloma formation, particularly IL-13 regulation of molecular networks that regulate M2 macrophage polarization. M2 macrophages are predisposed to aggregation and multinucleated giant cell formation, which are characteristic features of sarcoidosis granulomas. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).
Assuntos
Regulação da Expressão Gênica , Granuloma/imunologia , Interleucina-13/metabolismo , Leucócitos Mononucleares/imunologia , Pulmão/imunologia , Macrófagos/imunologia , Sarcoidose Pulmonar/imunologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Granuloma/genética , Granuloma/metabolismo , Granuloma/patologia , Humanos , Técnicas In Vitro , Interleucina-13/genética , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Sarcoidose Pulmonar/genética , Sarcoidose Pulmonar/metabolismo , Sarcoidose Pulmonar/patologia , TranscriptomaRESUMO
Human alveolar macrophages (HAM) are primary bacterial niche and immune response cells during Mycobacterium tuberculosis (M.tb) infection, and human blood monocyte-derived macrophages (MDM) are a model for investigating M.tb-macrophage interactions. Here, we use a targeted RNA-Seq method to measure transcriptome-wide changes in RNA expression patterns of freshly obtained HAM (used within 6 h) and 6 day cultured MDM upon M.tb infection over time (2, 24 and 72 h), in both uninfected and infected cells from three donors each. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. AmpliSeqTM yields highly precise and reproducible gene expression profiles (R2 >0.99). Taking advantage of AmpliSeq's reproducibility, we establish well-defined quantitative RNA expression patterns of HAM versus MDM, including significant M.tb-inducible genes, in networks and pathways that differ in part between MDM and HAM. A similar number of expressed genes are detected at all time-points between uninfected MDM and HAM, in common pathways including inflammatory and immune functions, but canonical pathway differences also exist. In particular, at 2 h, multiple genes relevant to the immune response are preferentially expressed in either uninfected HAM or MDM, while the HAM RNA profiles approximate MDM profiles over time in culture, highlighting the unique RNA expression profile of freshly obtained HAM. MDM demonstrate a greater transcriptional response than HAM upon M.tb infection, with 2 to >10 times more genes up- or down-regulated. The results identify key genes involved in cellular responses to M.tb in two different human macrophage types. Follow-up bioinformatics analysis indicates that approximately 30% of response genes have expression quantitative trait loci (eQTLs in GTEx), common DNA variants that can influence host gene expression susceptibility or resistance to M.tb, illustrated with the TREM1 gene cluster and IL-10.
Assuntos
Perfilação da Expressão Gênica , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Humanos , Interleucina-10/genética , Família Multigênica/genética , Análise de Sequência de RNA , Fatores de Tempo , Receptor Gatilho 1 Expresso em Células Mieloides/genéticaRESUMO
Tuberculosis (TB) is a global epidemic caused by the infection of human macrophages with the world's most deadly single bacterial pathogen, Mycobacterium tuberculosis (M.tb). M.tb resides in a phagosomal niche within macrophages, where trace element concentrations impact the immune response, bacterial metal metabolism, and bacterial survival. The manipulation of micronutrients is a critical mechanism of host defense against infection. In particular, the human zinc transporter Zrt-/Irt-like protein 8 (ZIP8), one of 14 ZIP family members, is important in the flux of divalent cations, including zinc, into the cytoplasm of macrophages. It also has been observed to exist on the membrane of cellular organelles, where it can serve as an efflux pump that transports zinc into the cytosol. ZIP8 is highly inducible in response to M.tb infection of macrophages, and we have observed its localization to the M.tb phagosome. The expression, localization, and function of ZIP8 and other divalent cation transporters within macrophages have important implications for TB prevention and dissemination and warrant further study. In particular, given the importance of zinc as an essential nutrient required for humans and M.tb, it is not yet clear whether ZIP-guided zinc transport serves as a host protective factor or, rather, is targeted by M.tb to enable its phagosomal survival.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Fagossomos/metabolismo , Tuberculose/imunologia , Zinco/metabolismo , Citosol/metabolismo , Humanos , Imunidade Inata , Macrófagos/metabolismo , Tuberculose/microbiologiaRESUMO
Many aspects of pathogenic granuloma formation are poorly understood, requiring new relevant laboratory models that represent the complexity (genetics and diversity) of human disease. To address this need, we developed an in vitro model of granuloma formation using human peripheral blood mononuclear cells (PBMCs) derived from patients with active sarcoidosis, latent tuberculosis (TB) infection (LTBI), or normal healthy control subjects. PBMCs were incubated for 7 days with uncoated polystyrene beads or beads coated with purified protein derivative (PPD) or human serum albumin. In response to PPD-coated beads, PBMCs from donors with sarcoidosis and LTBI formed robust multicellular aggregates resembling granulomas, displaying a typical T-helper cell type 1 immune response, as assessed by cytokine analyses. In contrast, minimal PBMC aggregation occurred when control PBMCs were incubated with PPD-coated beads, whereas the response to uncoated beads was negligible in all groups. Sarcoidosis PBMCs responded to human serum albumin-coated beads with modest cellular aggregation and inflammatory cytokine release. Whereas the granuloma-like aggregates formed in response to PPD-coated beads were similar for sarcoidosis and LTBI, molecular profiles differed significantly. mRNA expression patterns revealed distinct pathways engaged in early granuloma formation in sarcoidosis and LTBI, and they resemble molecular patterns reported in diseased human tissues. This novel in vitro human granuloma model is proposed as a tool to investigate mechanisms of early granuloma formation and for preclinical drug discovery research of human granulomatous disorders. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).
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Granuloma do Sistema Respiratório/imunologia , Tuberculose Latente/imunologia , Modelos Imunológicos , Sarcoidose Pulmonar/imunologia , Células Th1/imunologia , Tuberculose Pulmonar/imunologia , Feminino , Granuloma do Sistema Respiratório/patologia , Humanos , Tuberculose Latente/patologia , Masculino , Sarcoidose Pulmonar/patologia , Células Th1/patologia , Tuberculose Pulmonar/patologiaRESUMO
The blood-brain barrier (BBB) expresses numerous membrane transporters that supply needed nutrients to the central nervous system (CNS), consisting mostly of solute carriers (SLC transporters), or remove unwanted substrates via extrusion pumps through the action of ATP binding cassette (ABC) transporters. Previous work has identified many BBB transporters using hybridization arrays or qRT-PCR, using targeted probes. Here we have performed next-generation sequencing of the transcriptome (RNAseq) extracted from cerebral cortex tissues and brain microvessel endothelial cells (BMEC) obtained from two donors. The same RNA samples had previously been measured for transporter expression using qRT-PCR (Geier et al., 2013), yielding similar expression levels for overlapping mRNAs (R=0.66, p<0.001). RNAseq confirms a number of transporters highly enriched in BMECs (e.g., ABCB1, ABCG2, SLCO2B1, and SLC47A1), but also detects novel BMEC transporters. Multiple splice isoforms detected by RNAseq are either robustly enriched or depleted in BMECs, indicating differential RNA processing in the BBB. The Complete RNAseq data are publically available (GSE94064).
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas Carreadoras de Solutos/genética , Processamento Alternativo , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Microvasos/metabolismo , Reação em Cadeia da Polimerase , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Carreadoras de Solutos/metabolismoRESUMO
Dementia with Lewy Bodies (DLB) is the second most common neurodegenerative disorder in the elderly. The development and progression of DLB remain unclear. In this study we used next generation sequencing to assess RNA expression profiles and cellular processes associated with DLB in the anterior cingulate cortex, a brain region affected by DLB pathology. The expression measurements were made in autopsy brain tissues from 8 DLB subjects and 10 age-matched controls using AmpliSeq technology with ion torrent sequencing. The analysis of RNA expression profiles revealed 490 differentially expressed genes, among which 367 genes were down-regulated and 123 were up-regulated. Functional enrichment analysis of genes differentially expressed in DLB indicated downregulation of genes associated with myelination, neurogenesis, and regulation of nervous system development. miRNA binding sites enriched in these mRNAs yielded a list of candidate miRNAs participating in DLB pathophysiology. Our study provides a comprehensive picture of gene expression landscape in DLB, identifying key cellular processes associated with DLB pathology.
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Encéfalo/metabolismo , Doença por Corpos de Lewy/genética , Idoso , Encéfalo/patologia , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Giro do Cíngulo/metabolismo , Giro do Cíngulo/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Doença por Corpos de Lewy/patologia , MicroRNAs/genética , Degeneração Neural/genética , Degeneração Neural/patologia , RNA Mensageiro/genética , Análise de Sequência de RNARESUMO
BACKGROUND: We used RNA sequencing to analyze transcript profiles of ten autopsy brain regions from ten subjects. RNA sequencing techniques were designed to detect both coding and non-coding RNA, splice isoform composition, and allelic expression. Brain regions were selected from five subjects with a documented history of smoking and five non-smokers. Paired-end RNA sequencing was performed on SOLiD instruments to a depth of >40 million reads, using linearly amplified, ribosomally depleted RNA. Sequencing libraries were prepared with both poly-dT and random hexamer primers to detect all RNA classes, including long non-coding (lncRNA), intronic and intergenic transcripts, and transcripts lacking poly-A tails, providing additional data not previously available. The study was designed to generate a database of the complete transcriptomes in brain region for gene network analyses and discovery of regulatory variants. RESULTS: Of 20,318 protein coding and 18,080 lncRNA genes annotated from GENCODE and lncipedia, 12 thousand protein coding and 2 thousand lncRNA transcripts were detectable at a conservative threshold. Of the aligned reads, 52 % were exonic, 34 % intronic and 14 % intergenic. A majority of protein coding genes (65 %) was expressed in all regions, whereas ncRNAs displayed a more restricted distribution. Profiles of RNA isoforms varied across brain regions and subjects at multiple gene loci, with neurexin 3 (NRXN3) a prominent example. Allelic RNA ratios deviating from unity were identified in > 400 genes, detectable in both protein-coding and non-coding genes, indicating the presence of cis-acting regulatory variants. Mathematical modeling was used to identify RNAs stably expressed in all brain regions (serving as potential markers for normalizing expression levels), linked to basic cellular functions. An initial analysis of differential expression analysis between smokers and nonsmokers implicated a number of genes, several previously associated with nicotine exposure. CONCLUSIONS: RNA sequencing identifies distinct and consistent differences in gene expression between brain regions, with non-coding RNA displaying greater diversity between brain regions than mRNAs. Numerous RNAs exhibit robust allele selective expression, proving a means for discovery of cis-acting regulatory factors with potential clinical relevance.
Assuntos
Alelos , Encéfalo/metabolismo , Perfilação da Expressão Gênica , Isoformas de RNA/genética , RNA não Traduzido/genética , Análise de Sequência de RNA , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Fumar/genéticaRESUMO
mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs) in lymphoblast cell lines (LCL) and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T) in ABCB1 (MDR1) on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq) in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs) affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.
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Alelos , Polirribossomos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA não Traduzido/genética , Transcriptoma , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Arilamina N-Acetiltransferase/genética , Células CHO , Linhagem Celular , Cricetulus , Células HeLa , Humanos , Isoenzimas/genética , Polimorfismo de Nucleotídeo Único , Isoformas de RNA/genética , Receptores Opioides mu/genéticaRESUMO
BACKGROUND: Cholesteryl ester transfer protein (CETP) is involved in reverse cholesterol transport by exchanging cholesteryl esters for triglycerides between high-density lipoprotein and low-density lipoprotein particles, effectively decreasing high-density lipoprotein cholesterol levels. Variants within a large haplotype block upstream of CETP (rs247616, rs173539) have been shown to be significantly associated with reduced expression; however, the underlying mechanism has not been identified. METHODS: We analyzed the linkage structure of our top candidate single-nucleotide polymorphism (SNP), rs247616, and assessed each SNP of the haplotype block for potential interactions with transcription factor binding sites. We then used a reporter gene assay to assess the effect of three SNPs (rs247616, rs173539, and rs1723150) on expression in vitro. RESULTS: Several variants in the upstream haplotype, including rs247616, rs173539, and rs1723150, disrupt or generate transcription factor binding sites. In reporter gene assays, rs247616 and rs173539 were found to significantly affected expression in HepG2 cells, whereas rs17231506 had no effect. rs247616 decreased expression by 1.7-fold (P<0.0001), whereas rs173539 increased expression by 2.2-fold (P=0.0006). CONCLUSION: SNPs rs247616 and rs173539 are in high linkage disequilibrium (R=0.96, D'=1.00) and have the potential to regulate CETP expression. Although opposing effects suggest that regulation of CETP expression could vary between tissues, the minor allele of rs247616 and SNPs in high linkage with it were found to be associated with reduced expression across all tissues.
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Proteínas de Transferência de Ésteres de Colesterol/genética , Regulação da Expressão Gênica , Polimorfismo de Nucleotídeo Único/genética , Sítios de Ligação , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Elementos Facilitadores Genéticos/genética , Perfilação da Expressão Gênica , Genes Reporter , Ligação Genética , Células Hep G2 , Humanos , Luciferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
CYP2D6 metabolizes nearly 25% of clinically used drugs. Genetic polymorphisms cause large inter-individual variability in CYP2D6 enzyme activity and are currently used as biomarker to predict CYP2D6 metabolizer phenotype. Previously, we had identified a region 115 kb downstream of CYP2D6 as enhancer for CYP2D6, containing two completely linked single nucleotide polymorphisms (SNPs), rs133333 and rs5758550, associated with enhanced transcription. However, the enhancer effect on CYP2D6 expression, and the causative variant, remained to be ascertained. To characterize the CYP2D6 enhancer element, we applied chromatin conformation capture combined with the next-generation sequencing (4C assays) and chromatin immunoprecipitation with P300 antibody, in HepG2 and human primary culture hepatocytes. The results confirmed the role of the previously identified enhancer region in CYP2D6 expression, expanding the number of candidate variants to three highly linked SNPs (rs133333, rs5758550 and rs4822082). Among these, only rs5758550 demonstrated regulating enhancer activity in a reporter gene assay. Use of clustered regularly interspaced short palindromic repeats mediated genome editing in HepG2 cells targeting suspected enhancer regions decreased CYP2D6 mRNA expression by 70%, only upon deletion of the rs5758550 region. These results demonstrate robust effects of both the enhancer element and SNP rs5758550 on CYP2D6 expression, supporting consideration of rs5758550 for CYP2D6 genotyping panels to yield more accurate phenotype prediction.
Assuntos
Citocromo P-450 CYP2D6/genética , Elementos Facilitadores Genéticos , Polimorfismo de Nucleotídeo Único , Adulto , Feminino , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Transcrição GênicaRESUMO
Cholesteryl ester transfer protein (CETP) plays an important role in reverse cholesterol transport, with decreased CETP activity increasing HDL levels. Formation of an alternative splice form lacking exon 9 (Δ9-CETP) has been associated with two single nucleotide polymorphisms (SNPs) in high linkage disequilibrium with each other, namely rs9930761 T>C located in intron 8 in a putative splicing branch site and rs5883 C>T in a possible exonic splicing enhancer (ESE) site in exon 9. To assess the relative effect of rs9930761 and rs5883 on splicing, mini-gene constructs spanning CETP exons 8 to 10, carrying all four possible allele combinations, were transfected into HEK293 and HepG2 cells. The minor T allele of rs5883 enhanced splicing significantly in both cell lines whereas the minor C allele of rs9930761 did not. In combination, the two alleles did not yield greater splicing than the rs5883 T allele alone in HepG2 cells. These results indicate that the genetic effect on CETP splicing is largely attributable to rs5883. We also confirm that Δ9-CETP protein is expressed in the liver but fails to circulate in the blood.
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Processamento Alternativo , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Variação Genética , Alelos , Proteínas de Transferência de Ésteres de Colesterol/química , Elementos Facilitadores Genéticos , Éxons , Células HEK293 , Células Hep G2 , Humanos , Íntrons , Fígado/metabolismo , Polimorfismo de Nucleotídeo Único , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Measuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals. RESULTS: We find that RNA-Seq allelic ratios from standard alignment methods correlate poorly with SNaPshot, but applying alternative alignment strategies and correcting for observed biases significantly improves correlations. Deploying these methods on a transcriptome-wide basis in nine brain regions from a single individual, we identified genes with AEI across all regions (SLC1A3, NHP2L1) and many others with region-specific AEI. In dorsolateral prefrontal cortex (DLPFC) tissues from 14 individuals, we found evidence for frequent regulatory variants affecting RNA expression in tens to hundreds of genes, depending on stringency for assigning AEI. Further, we find that the extent and variability of RNA editing is similar across brain regions and across individuals. CONCLUSIONS: These results identify critical factors affecting allelic ratios measured by RNA-Seq and provide a foundation for using this technology to screen allelic RNA expression on a transcriptome-wide basis. Using this technology as a screening tool reveals tens to hundreds of genes harboring frequent functional variants affecting RNA expression in the human brain. With respect to RNA editing, the similarities within and between individuals leads us to conclude that this post-transcriptional process is under heavy regulatory influence to maintain an optimal degree of editing for normal biological function.
Assuntos
Alelos , Encéfalo/metabolismo , Perfilação da Expressão Gênica , RNA/genética , Análise de Sequência de RNA , Adulto , DNA Complementar/biossíntese , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Córtex Pré-Frontal/metabolismo , Edição de RNA/genética , Adulto JovemRESUMO
BACKGROUND: Membrane transporters control the influx and efflux of endogenous and xenobiotic substrates, including nutrients and drugs, across cellular membranes. OBJECTIVE: Whole transcriptome sequencing enables simultaneous analysis of overall and allele-specific mRNA expression, and the detection of multiple RNA isoforms. METHODS: Here we characterize variation in RNA transcripts emanating from gene loci encoding transporters based on RNAseq data from 10 human brains (including cocaine overdose and normal brain tissues) and 12 normal livers. RESULTS: mRNA expression was detected in 65% of transporter genes in either tissue, with many genes generating multiple mRNA transcripts. Single-nucleotide polymorphisms within transporters with previous evidence for pharmacogenomics impact were detected. We also identified noncoding RNAs in the vicinity of transporter genes with potential regulatory functions. CONCLUSION: The results obtained with RNAseq provide detailed information on transporter mRNA expression at the molecular level, affording new avenues for the study of membrane transport, with relevance to drug efficacy and toxicity.
Assuntos
Fígado/metabolismo , Proteínas de Membrana Transportadoras/genética , Córtex Pré-Frontal/metabolismo , RNA Mensageiro/isolamento & purificação , Autopsia , Feminino , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , Transcriptoma/genéticaRESUMO
BACKGROUND: The 5-hydroxytryptamine 2A receptor, encoded by HTR2A, is a major postsynaptic target for serotonin in the human brain and a therapeutic drug target. Despite hundreds of genetic associations investigating HTR2A polymorphisms in neuropsychiatric disorders and therapies, the role of genetic HTR2A variability in health and disease remains uncertain. METHODS: To discover and characterize regulatory HTR2A variants, we sequenced whole transcriptomes from 10 human brain regions with massively parallel RNA sequencing and measured allelic expression of multiple HTR2A messenger (m)RNA transcript variants. Following discovery of functional variants, we further characterized their impact on genetic expression in vitro. RESULTS: Three polymorphisms modulate the use of novel alternative exons and untranslated regions (UTRs), changing expression of RNA and protein. The frequent promoter variant rs6311, widely implicated in human neuropsychiatric disorders, decreases usage of an upstream transcription start site encoding a longer 5'UTR with greater translation efficiency. rs76665058, located in an extended 3'UTR and unique to individuals of African descent, modulates allelic HTR2A mRNA expression. The third single nucleotide polymorphism, unannotated and present in only a single subject, directs alternative splicing of exon 2. Targeted analysis of HTR2A in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study reveals associations between functional variants and depression severity or citalopram response. CONCLUSIONS: Regulatory polymorphisms modulate HTR2A mRNA expression in an isoform-specific manner, directing the usage of novel untranslated regions and alternative exons. These results provide a foundation for delineating the role of HTR2A and serotonin signaling in central nervous system disorders.
Assuntos
Córtex Cerebral/metabolismo , Regulação da Expressão Gênica/genética , Variação Genética/genética , Regiões Promotoras Genéticas/genética , Receptor 5-HT2A de Serotonina/biossíntese , Alelos , Ensaios Clínicos como Assunto , Depressão/tratamento farmacológico , Depressão/genética , Éxons/genética , Humanos , Metilação , Polimorfismo de Nucleotídeo Único/genética , Isoformas de Proteínas/biossíntese , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Transcriptoma/genética , Regiões não Traduzidas/genéticaRESUMO
BACKGROUND: Sulfamethoxazole (SMX) is a commonly used antibiotic for prevention of infectious diseases associated with HIV/AIDS and immune-compromised states. SMX-induced hypersensitivity is an idiosyncratic cutaneous drug reaction with genetic components. Here, we tested association of candidate genes involved in SMX bioactivation and antioxidant defense with SMX-induced hypersensitivity. RESULTS: Seventy seven single nucleotide polymorphisms (SNPs) from 14 candidate genes were genotyped and assessed for association with SMX-induced hypersensitivity, in a cohort of 171 HIV/AIDS patients. SNP rs761142 T > G, in glutamate cysteine ligase catalytic subunit (GCLC), was significantly associated with SMX-induced hypersensitivity, with an adjusted p value of 0.045. This result was replicated in a second cohort of 249 patients (p = 0.025). In the combined cohort, heterozygous and homozygous carriers of the minor G allele were at increased risk of developing hypersensitivity (GT vs TT, odds ratio = 2.2, 95% CL 1.4-3.7, p = 0.0014; GG vs TT, odds ratio = 3.3, 95% CL 1.6 - 6.8, p = 0.0010). Each minor allele copy increased risk of developing hypersensitivity 1.9 fold (95% CL 1.4 - 2.6, p = 0.00012). Moreover, in 91 human livers and 84 B-lymphocytes samples, SNP rs761142 homozygous G allele carriers expressed significantly less GCLC mRNA than homozygous TT carriers (p < 0.05). CONCLUSIONS: rs761142 in GCLC was found to be associated with reduced GCLC mRNA expression and with SMX-induced hypersensitivity in HIV/AIDS patients. Catalyzing a critical step in glutathione biosynthesis, GCLC may play a broad role in idiosyncratic drug reactions.
Assuntos
Domínio Catalítico , Glutamato-Cisteína Ligase/genética , Infecções por HIV/complicações , Hipersensibilidade/etiologia , Hipersensibilidade/genética , Polimorfismo de Nucleotídeo Único , Sulfametoxazol/efeitos adversos , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Adulto , Antibacterianos/efeitos adversos , Antibacterianos/metabolismo , Antibacterianos/uso terapêutico , Feminino , Técnicas de Genotipagem , Glutamato-Cisteína Ligase/química , Glutationa/biossíntese , Glutationa/metabolismo , Humanos , Hipersensibilidade/metabolismo , Inativação Metabólica , Masculino , Sulfametoxazol/metabolismo , Sulfametoxazol/uso terapêutico , Combinação Trimetoprima e Sulfametoxazol/efeitos adversos , Combinação Trimetoprima e Sulfametoxazol/metabolismo , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5), allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10)) and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8)) (in high linkage disequilibrium with allele frequencies 6-7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28) and rs5883 p = 8.6 × 10(-10), adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex-dependent CETP splicing effects on cardiovascular risk by a mechanism independent of circulating HDL-C levels.
Assuntos
Doenças Cardiovasculares/genética , Proteínas de Transferência de Ésteres de Colesterol/genética , HDL-Colesterol/sangue , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Splicing de RNA/genética , Caracteres Sexuais , Adulto , Alelos , Atenolol/uso terapêutico , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/epidemiologia , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Feminino , Haplótipos , Humanos , Desequilíbrio de Ligação/genética , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Ensaios Clínicos Controlados Aleatórios como Assunto , Verapamil/uso terapêuticoRESUMO
The dopamine receptor D2 (encoded by DRD2) is implicated in susceptibility to mental disorders and cocaine abuse, but mechanisms responsible for this relationship remain uncertain. DRD2 mRNA exists in two main splice isoforms with distinct functions: D2 long (D2L) and D2 short (D2S, lacking exon 6), expressed mainly postsynaptically and presynaptically, respectively. Two intronic single-nucleotide polymorphisms (SNPs rs2283265 (intron 5) and rs1076560 (intron 6)) in high linkage disequilibrium (LD) with each other have been reported to alter D2S/D2L splicing and several behavioral traits in human subjects, such as memory processing. To assess the role of DRD2 variants in cocaine abuse, we measured levels of D2S and D2L mRNA in human brain autopsy tissues (prefrontal cortex and putamen) obtained from cocaine abusers and controls, and genotyped a panel of DRD2 SNPs (119 abusers and 95 controls). Robust effects of rs2283265 and rs1076560 on reducing formation of D2S relative to D2L were confirmed. The minor alleles of rs2283265/rs1076560 were considerably more frequent in Caucasians (18%) compared with African Americans (7%). Also, in Caucasians, rs2283265/rs1076560 minor alleles were significantly overrepresented in cocaine abusers compared with controls (rs2283265: 25 to 9%, respectively; p=0.001; OR=3.4 (1.7-7.1)). Several SNPs previously implicated in diverse clinical association studies are in high LD with rs2283265/rs1076560 and could have served as surrogate markers. Our results confirm the role of rs2283265/rs1076560 in D2 alternative splicing and support a strong role in susceptibility to cocaine abuse.
Assuntos
Processamento Alternativo/genética , Transtornos Relacionados ao Uso de Cocaína/genética , Predisposição Genética para Doença , Íntrons/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de Dopamina D2/genética , Adulto , Encéfalo/metabolismo , Encéfalo/patologia , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Transtornos Relacionados ao Uso de Cocaína/patologia , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Dopamina D2/biossíntese , Adulto JovemRESUMO
CHRNA5, encoding the nicotinic α5 subunit, is implicated in multiple disorders, including nicotine addiction and lung cancer. Previous studies demonstrate significant associations between promoter polymorphisms and CHRNA5 mRNA expression, but the responsible sequence variants remain uncertain. To search for cis-regulatory variants, we measured allele-specific mRNA expression of CHRNA5 in human prefrontal cortex autopsy tissues and scanned the CHRNA5 locus for regulatory variants. A cluster of six frequent single-nucleotide polymorphisms (rs1979905, rs1979906, rs1979907, rs880395, rs905740, and rs7164030), in complete linkage disequilibrium (LD), fully account for a >2.5-fold allelic expression difference and a fourfold increase in overall CHRNA5 mRNA expression. This proposed enhancer region resides more than 13 kilobases upstream of the CHRNA5 transcription start site. The same upstream variants failed to affect CHRNA5 mRNA expression in peripheral blood lymphocytes, indicating tissue-specific gene regulation. Other promoter polymorphisms were also correlated with overall CHRNA5 mRNA expression in the brain, but were inconsistent with allelic mRNA expression ratios, a robust and proximate measure of cis-regulatory variants. The enhancer region and the nonsynonymous polymorphism rs16969968 generate three main haplotypes that alter the risk of developing nicotine dependence. Ethnic differences in LD across the CHRNA5 locus require consideration of upstream enhancer variants when testing clinical associations.