RESUMO
Modifications in RNA are numerous (â¼170) and in higher numbers compared to DNA (â¼5) making the ability to sequence an RNA molecule to identify these modifications highly tenuous using next generation sequencing (NGS). The ability to immobilize an exoribonuclease enzyme, such as XRN1, to a solid support while maintaining its activity and capability to cleave both the canonical and modified ribonucleotides from an intact RNA molecule can be a viable approach for single-molecule RNA sequencing. In this study, we report an enzymatic reactor consisting of covalently attached XRN1 to a solid support as the groundwork for a novel RNA exosequencing technique. The covalent attachment of XRN1 to a plastic solid support was achieved using EDC/NHS coupling chemistry. Studies showed that the solid-phase digestion efficiency of model RNAs was 87.6 ± 2.8%, while the XRN1 solution-phase digestion for the same model was 78.3 ± 4.4%. The ability of immobilized XRN1 to digest methylated RNA containing m6A and m5C ribonucleotides was also demonstrated. The processivity and clipping rate of immobilized XRN1 secured using single-molecule fluorescence measurements of a single RNA transcript demonstrated a clipping rate of 26 ± 5 nt s-1 and a processivity of >10.5 kb at 25°C.
Assuntos
Distrofina/genética , Enzimas Imobilizadas/metabolismo , Exorribonucleases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , RNA/metabolismo , Análise de Sequência de RNA/métodos , Humanos , Clivagem do RNARESUMO
Detection of low-abundance mutations in cell-free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR-LDR-qPCR assay capable of detecting point mutations at a single-molecule resolution in the presence of an excess of wild-type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer-binding regions as well as wild-type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single-well version of the assay detected 2-5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild-type human genomic DNA (hgDNA) from buffy coat. A 12-well (pixel) version of the assay was capable of single-molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000-2,250 GE of wild-type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias/genética , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real , Imagem Individual de Molécula/métodos , Alelos , Substituição de Aminoácidos , Linhagem Celular Tumoral , DNA Tumoral Circulante , Análise Mutacional de DNA/métodos , Genótipo , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
BACKGROUND: Interrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR. METHODS: In this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood. RESULTS: Our results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB, and a non-coding region of chromosome 1 (upstream of EFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27). CONCLUSION: This report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Ácidos Nucleicos Livres/sangue , Metilação de DNA , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Ilhas de CpG , Feminino , Humanos , Células MCF-7 , Reação em Cadeia da Polimerase Multiplex , Proteína Quinase C beta/genética , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
ß-Tryptase is released from mast cells upon degranulation in response to allergic and inflammatory stimuli. Human tryptase is a homotetrameric serine protease with 4 identical active sites directed toward a central pore. These active sites present an optimized scenario for the rational design of bivalent inhibitors, which bridge 2 adjacent active sites. Using (3-[1-acylpiperidin-4-yl]phenyl)methanamine as the pharmacophoric core and a disiloxane linker to span 2 active sites we have successfully produced a novel bivalent tryptase inhibitor, compound 1a, with a comparable profile to previously described inhibitors. Pharmacological properties of compound 1a were studied in a range of in vitro enzymic and cellular screening assays, and in vivo xenograft models. This non-peptide inhibitor of tryptase demonstrated superior activity (IC50 at 100 pmol/L tryptase = 1.82 nmol/L) compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and 1a demonstrated good oral bioavailability and efficacy in HMC-1 xenograft models. Furthermore, compound 1a demonstrated extremely slow off rates and high selectivity against-related proteases. This highly potent, orally bioavailable and selective inhibitor of human tryptase will be an invaluable tool in future studies to explore the therapeutic potential of attenuating the activity of this elusive target.
Assuntos
Mastócitos/efeitos dos fármacos , Silanos/química , Silanos/farmacologia , Triptases/antagonistas & inibidores , Animais , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Imuno-Histoquímica , Masculino , Mastócitos/enzimologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Farmacocinética , Silanos/análise , Silanos/farmacocinéticaRESUMO
Proteolysis-Targeting Chimeras (PROTACs) are bifunctional molecules that bring a target protein and an E3 ubiquitin ligase into proximity to append ubiquitin, thus directing target degradation. Although numerous PROTACs have entered clinical trials, their development remains challenging, and their large size can produce poor drug-like properties. To overcome these limitations, we have modified our Coferon platform to generate Combinatorial Ubiquitination REal-time PROteolysis (CURE-PROs). CURE-PROs are small molecule degraders designed to self-assemble through reversible bio-orthogonal linkers to form covalent heterodimers. By modifying known ligands for Cereblon, MDM2, VHL, and BRD with complementary phenylboronic acid and diol/catechol linkers, we have successfully created CURE-PROs that direct degradation of BRD4 both in vitro and in vivo. The combinatorial nature of our platform significantly reduces synthesis time and effort to identify the optimal linker length and E3 ligase partner to each target and is readily amenable to screening for new targets.
Assuntos
Proteínas Nucleares , Fatores de Transcrição , Proteólise , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , LigantesRESUMO
The microtubule-targeting paclitaxel (PTX) and docetaxel (DTX) are widely used chemotherapeutic agents. However, the dysregulation of apoptotic processes, microtubule-binding proteins, and multi-drug resistance efflux and influx proteins can alter the efficacy of taxane drugs. In this review, we have created multi-CpG linear regression models to predict the activities of PTX and DTX drugs through the integration of publicly available pharmacological and genome-wide molecular profiling datasets generated using hundreds of cancer cell lines of diverse tissue of origin. Our findings indicate that linear regression models based on CpG methylation levels can predict PTX and DTX activities (log-fold change in viability relative to DMSO) with high precision. For example, a 287-CpG model predicts PTX activity at R2 of 0.985 among 399 cell lines. Just as precise (R2=0.996) is a 342-CpG model for predicting DTX activity in 390 cell lines. However, our predictive models, which employ a combination of mRNA expression and mutation as input variables, are less accurate compared to the CpG-based models. While a 290 mRNA/mutation model was able to predict PTX activity with R2 of 0.830 (for 546 cell lines), a 236 mRNA/mutation model could calculate DTX activity at R2 of 0.751 (for 531 cell lines). The CpG-based models restricted to lung cancer cell lines were also highly predictive (R2≥0.980) for PTX (74 CpGs, 88 cell lines) and DTX (58 CpGs, 83 cell lines). The underlying molecular biology behind taxane activity/resistance is evident in these models. Indeed, many of the genes represented in PTX or DTX CpG-based models have functionalities related to apoptosis (e.g., ACIN1, TP73, TNFRSF10B, DNASE1, DFFB, CREB1, BNIP3), and mitosis/microtubules (e.g., MAD1L1, ANAPC2, EML4, PARP3, CCT6A, JAKMIP1). Also represented are genes involved in epigenetic regulation (HDAC4, DNMT3B, and histone demethylases KDM4B, KDM4C, KDM2B, and KDM7A), and those that have never been previously linked to taxane activity (DIP2C, PTPRN2, TTC23, SHANK2). In summary, it is possible to accurately predict taxane activity in cell lines based entirely on methylation at multiple CpG sites.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Paclitaxel/metabolismo , Docetaxel/farmacologia , Epigênese Genética , Modelos Lineares , Taxoides/farmacologia , Taxoides/uso terapêutico , Taxoides/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linhagem Celular , RNA Mensageiro , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Nucleares/metabolismo , Chaperonina com TCP-1/metabolismoRESUMO
PROteolysis TArgeting Chimeras (PROTACs) are of significant current interest for the development of probe molecules and drug leads. However, they suffer from certain limitations. PROTACs are rule-breaking molecules with sub-optimal cellular permeability, solubility, and other drug-like properties. In particular, they exhibit an unusual dose-response curve where high concentrations of the bivalent molecule inhibit degradation activity, a phenomenon known as the hook effect. This will likely complicate their use in vivo. In this study, we explore a novel approach to create PROTACs that do not exhibit a hook effect. This is achieved by equipping the target protein and E3 ubiquitin ligase ligands with functionalities that undergo rapid and reversible covalent assembly in cellulo. We report the development of Self-Assembled Proteolysis Targeting Chimeras that mediate the degradation of the Von Hippel-Lindau E3 ubiquitin ligase and do not evince a hook effect.
Assuntos
Quimera de Direcionamento de Proteólise , Ubiquitina-Proteína Ligases , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Proteínas/metabolismo , LigantesRESUMO
During disease progression the cells that comprise solid malignancies undergo significant changes in gene copy number and chromosome structure. Colorectal cancer provides an excellent model to study this process. To indentify and characterize chromosomal abnormalities in colorectal cancer, we performed a statistical analysis of 299 expression and 130 SNP arrays profiled at different stages of the disease, including normal tissue, adenoma, stages 1-4 adenocarcinoma, and metastasis. We identified broad (> 1/2 chromosomal arm) and focal (< 1/2 chromosomal arm) events. Broad amplifications were noted on chromosomes 7, 8q, 13q, 20, and X and broad deletions on chromosomes 4, 8p, 14q, 15q, 17p, 18, 20p, and 22q. Focal events (gains or losses) were identified in regions containing known cancer pathway genes, such as VEGFA, MYC, MET, FGF6, FGF23, LYN, MMP9, MYBL2, AURKA, UBE2C, and PTEN. Other focal events encompassed potential new candidate tumor suppressors (losses) and oncogenes (gains), including CCDC68, CSMD1, POLR1D, and PMEPA1. From the expression data, we identified genes whose expression levels reflected their copy number changes and used this relationship to impute copy number changes to samples without accompanying SNP data. This analysis provided the statistical power to show that deletions of 8p, 4p, and 15q are associated with survival and disease progression, and that samples with simultaneous deletions in 18q, 8p, 4p, and 15q have a particularly poor prognosis. Annotation analysis reveals that the oxidative phosphorylation pathway shows a strong tendency for decreased expression in the samples characterized by poor prognosis.
Assuntos
Instabilidade Cromossômica/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Genoma Humano/genética , Deleção Cromossômica , Progressão da Doença , Fator de Crescimento de Fibroblastos 23 , Dosagem de Genes/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Fosforilação , Polimorfismo de Nucleotídeo Único/genética , RNA não Traduzido/genética , Taxa de SobrevidaRESUMO
A circulating tumor cell (CTC) selection microfluidic device was integrated to an electrokinetic enrichment device for preconcentrating CTCs directly from whole blood to allow for the detection of mutations contained within the genomic DNA of the CTCs. Molecular profiling of CTCs can provide important clinical information that cannot be garnered simply by enumerating the selected CTCs. We evaluated our approach using SW620 and HT29 cells (colorectal cancer cell lines) seeded into whole blood as a model system. Because SW620 and HT29 cells overexpress the integral membrane protein EpCAM, they could be immunospecifically selected using a microfluidic device containing anti-EpCAM antibodies immobilized to the walls of a selection bed. The microfluidic device was operated at an optimized flow rate of 2 mm s(-1), which allowed for the ability to process 1 mL of whole blood in <40 min. The selected CTCs were then enzymatically released from the antibody selection surface and hydrodynamically transported through a pair of Pt electrodes for conductivity-based enumeration. The efficiency of CTC selection was found to be 96% ± 4%. Following enumeration, the CTCs were hydrodynamically transported at a flow rate of 1 µL min(-1) to an on-chip electromanipulation unit, where they were electrophoretically withdrawn from the bulk hydrodynamic flow and directed into a receiving reservoir. Using an electric field of 100 V cm(-1), the negatively charged CTCs were enriched into an anodic receiving reservoir to a final volume of 2 µL, providing an enrichment factor of 500. The collected CTCs could then be searched for point mutations using a PCR/LDR/capillary electrophoresis assay. The DNA extracted from the CTCs was subjected to a primary polymerase chain reaction (PCR) with the amplicons used for a ligase detection reaction (LDR) to probe for KRAS oncogenic point mutations. Point mutations in codon 12 of the KRAS gene were successfully detected in the SW620 CTCs for samples containing <10 CTCs in 1 mL of whole blood. However, the HT29 cells did not contain these mutations, consistent with their known genotype.
Assuntos
Contagem de Células/instrumentação , Separação Celular/instrumentação , Eletricidade , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Condutividade Elétrica , Eletroforese Capilar , Células HT29 , Humanos , Hidrodinâmica , Ligases/metabolismo , Reação em Cadeia da Polimerase , Propriedades de SuperfícieRESUMO
If properly translated to clinical use, our knowledge about biomarkers may lead to a more effective way of combating colorectal cancer (CRC). Biomarkers are biomolecular, genetic, or cytogenetic attributes indicative of the disease's progression, predisposition, prognosis, or therapeutic options. For CRC, these include chromosomal instability, mutations in KRAS and TP53, loss of 18q, and elevated level of carcinoembryonic antigen (CEA), which are all associated with poor prognosis. The prognostic significance of 18q loss can be attributed to reduced expression of SMAD4, or DCC, although the chromosomal arm is actually heavily populated by genes whose downregulation correlate to worse survival. Potentially, identification of prognostic biomarkers can help the oncologist decide whether adjuvant chemotherapy is necessary after surgery. Testing for therapeutic biomarkers can be necessary if targeted therapeutics are being considered. The identification of highly penetrant predisposition markers (such as mutations in APC and MLH1) can be a lifesaver for carrier individuals, who would then have to undergo colonoscopy at an earlier age. Even sporadic CRCs may have some hereditary components, according to recent studies. Genome-wide association studies (using SNP arrays) showed that polymorphisms of certain genes can have subtle influence on CRC predisposition. Our own SNP array-based analysis suggested that long stretches of germline homozygosity (autozygosity), indicative of consanguinity, may also factor in CRC predisposition.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Neoplasias do Colo/terapia , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Progressão da Doença , Estudo de Associação Genômica Ampla , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , PrognósticoRESUMO
Our understanding of metastatic prostate cancer (mPrCa) has dramatically advanced during the genomics era. Nonetheless, many aspects of the disease may still be uncovered through reanalysis of public datasets. We integrated the expression datasets for 209 PrCa tissues (metastasis, primary, normal) with expression, gene dependency (GD) (from CRISPR/cas9 screen), and drug viability data for hundreds of cancer lines (including PrCa). Comparative statistical and pathways analyses and functional annotations (available inhibitors, protein localization) revealed relevant pathways and potential (and previously reported) protein markers for minimally invasive mPrCa diagnostics. The transition from localized to mPrCa involved the upregulation of DNA replication, mitosis, and PLK1-mediated events. Genes highly upregulated in mPrCa and with very high average GD (~1) are potential therapeutic targets. We showed that fostamatinib (which can target PLK1 and other over-expressed serine/threonine kinases such as AURKA, MELK, NEK2, and TTK) is more active against cancer lines with more pronounced signatures of invasion (e.g., extracellular matrix organization/degradation). Furthermore, we identified surface-bound (e.g., ADAM15, CD276, ABCC5, CD36, NRP1, SCARB1) and likely secreted proteins (e.g., APLN, ANGPT2, CTHRC1, ADAM12) that are potential mPrCa diagnostic markers. Overall, we demonstrated that comprehensive analyses of public genomics data could reveal potentially clinically relevant information regarding mPrCa.
RESUMO
BACKGROUND: MGMT (O6-methylguanine-DNA methyltransferase) is primarily responsible for limiting the activity of some widely used chemotherapeutic agents, including temozolomide (TMZ) and carmustine (BCNU). The gene encoding this protein is epigenetically regulated, and assessment of methylation at its promoter region is used to predict glioma patients' response to TMZ. METHODS: In this report, we employed a bioinformatic approach to elucidate MGMT's epigenetic regulation. Integrated for the analysis were genome-wide methylation and transcription datasets for > 8,600 human tissue (representing 31 distinct cancer types) and 500 human cancer cell line samples. Also crucial to the interpretation of results were publicly available data from the ENCODE Project: tracks for histone modifications (via ChIP-seq) and DNase I hypersensitivity (via DNaseseq), as well as methylation and transcription data for representative cell lines (HeLa-S3, HMEC, K562). RESULTS AND DISCUSSION: We were able to validate (perhaps more comprehensively) the contrasting influences of CpG methylation at promoter region and at gene body on MGMT transcription. While the MGMT promoter is populated by CpG sites whose methylation levels displayed high negative correlation (R) with MGMT mRNA counts, the gene body harbors CpG sites exhibiting high positive R values. The promoter CpG sites with very high negative R's across cancer types include cg12981137, cg12434587, and cg00618725. Among the notable gene body CpG sites (high positive R's across cancer types) are cg00198994 (Intron 1), cg04473030 (Intron 2), and cg07367735 (Intron 4). For certain cancer types, such as melanoma, gene body methylation appears to be a better predictor of MGMT transcription (compared to promoter methylation). In general, the CpG methylation v. MGMT expression R values are higher in cell lines relative to tissues. Also, these correlations are noticeably more prominent in certain cancer types such as colorectal, adrenocortical, esophageal, skin, and head and neck cancers, as well as glioblastoma. As expected, hypomethylation at the promoter region is associated with more open chromatin, and enrichment of histone marks H3K4m1, H3K4m2, H3K4m3, and H3K9ac. CONCLUSION: Overall, our analysis illustrated the contrasting influence of promoter and gene body methylation on MGMT expression. These observations may help improve diagnostic assays for MGMT.
Assuntos
Carmustina/farmacologia , Metilação de DNA/fisiologia , Neoplasias , O(6)-Metilguanina-DNA Metiltransferase/genética , Temozolomida/farmacologia , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Biologia Computacional/métodos , Ilhas de CpG/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Código das Histonas , Humanos , Neoplasias/classificação , Neoplasias/metabolismo , Neoplasias/patologia , Regiões Promotoras GenéticasRESUMO
Epigenetic modulation of gene expression is essential for tissue-specific development and maintenance in mammalian cells. Disruption of epigenetic processes, and the subsequent alteration of gene functions, can result in inappropriate activation or inhibition of various cellular signaling pathways, leading to cancer. Recent advancements in the understanding of the role of epigenetics in cancer initiation and progression have uncovered functions for DNA methylation, histone modifications, nucleosome positioning, and non-coding RNAs. Epigenetic therapies have shown some promise for hematological malignancies, and a wide range of epigenetic-based drugs are undergoing clinical trials. However, in a dynamic survival strategy, cancer cells exploit their heterogeneous population which frequently results in the rapid acquisition of therapy resistance. Here, we describe novel approaches in drug discovery targeting the epigenome, highlighting recent advances the selective degradation of target proteins using Proteolysis Targeting Chimera (PROTAC) to address drug resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Terapia de Alvo Molecular , Neoplasias , Proteólise , Descoberta de Drogas/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética , Epigenoma/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Individuals are frequently observed to have long segments of uninterrupted sequences of homozygous markers. One of the major mechanisms that gives rise to such long homozygous segments is consanguineous marriages, where parents pass shared chromosomal segments to their child. Such chromosomal segments are also known as autozygous segments. The clinical evidence that progeny from inbred individuals may have reduced health and fitness because of homozygosity of recessive alleles is well known. As the length of such homozygous segments depends on the degree of parental consanguinity, it would be logical to observe shorter homozygous segments in more outbred populations. However, a recent study identified long homozygous regions, thus likely to be autozygous segments in the HapMap populations. While an abundance of homozygous segments may significantly reduce the ability to fine map disease genes using association studies, detecting tracts of extended homozygosity related to disease status seems the natural next step in genome-wide association studies beyond allele, genotype and haplotype association analyses. In this study, we propose a new algorithm to map disease-related segments based on autozygosity using case-control data. The underlying rationale for the proposed method is that shared autozygosity regions that differ between diseased and healthy individuals may harbor mutations underlying diseases. Specifically, our algorithm uses a sliding-window framework and employs a logarithm of the odds score measure of autozygosity coupled with permutation-based methods to identify disease-related regions. We illustrate the advantage of the algorithm with its application to a genome-wide association study on Parkinson's disease.
Assuntos
Mapeamento Cromossômico/estatística & dados numéricos , Genoma Humano , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Algoritmos , Alelos , Cromossomos Humanos Par 20/genética , Consanguinidade , Feminino , Frequência do Gene , Genética Médica/estatística & dados numéricos , Homozigoto , Humanos , Escore Lod , Masculino , Epidemiologia Molecular/estatística & dados numéricos , Doença de Parkinson/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) is a common event in many cancers and typically activates the maternally silenced allele. The resulting biallelic IGF2 expression correlates strongly with the hypomethylation of a differentially methylated region (DMR) near its promoter. It has also been shown that IGF2 undergoes overexpression in human malignancies; nevertheless, this phenomenon and its link to aberrant DMR methylation have not been reported in colorectal cancer (CRC). The aim of this study was to determine the relationship between IGF2 LOI, overexpression and DMR hypomethylation in CRC. By analyzing IGF2 and H19 methylation in 97 primary CRC and 64 matched normal colorectal tissues, we have shown a significant correlation between IGF2 LOI and DMR hypomethylation of IGF2 and H19. Additionally, when analyzing Affymetrix expression data of 167 primary CRC tumors and 32 normal tissues, 15% of tumors showed marked IGF2 elevation. We further investigated if substantially elevated IGF2 levels were linked to IGF2 or H19 hypomethylation, but found no significant correlation. However, we demonstrated that noticeable IGF2 overexpression, rather than LOI, negatively correlated with CRC microsatellite instability. These observations indicate that IGF2 expression, particularly when transcribed at significantly high levels, is a result of mechanisms unrelated to LOI. Our results suggest that IGF2 participates in CRC tumorigenesis through 2 different forms of aberrant gene expression.
Assuntos
Neoplasias Colorretais/genética , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , Metilação de DNA , Humanos , Polimorfismo Genético , Regiões Promotoras GenéticasRESUMO
Detection of pathogenic bacteria and viruses require strategies that can signal the presence of these targets in near real-time due to the potential threats created by rapid dissemination into water and/or food supplies. In this paper, we report an innovative strategy that can rapidly detect bacterial pathogens using reporter sequences found in their genome without requiring polymerase chain reaction (PCR). A pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) or acceptor (Cy5.5) dye. In the presence of the target bacterium, the primers were joined using a ligase detection reaction (LDR) only when the primers were completely complementary to the target sequence to form a reverse molecular beacon (rMB), thus bringing Cy5 (donor) and Cy5.5 (acceptor) into close proximity to allow fluorescence resonance energy transfer (FRET) to occur. These rMBs were subsequently analyzed using single-molecule detection of the FRET pairs (single-pair FRET; spFRET). The LDR was performed using a continuous flow thermal cycling process configured in a cyclic olefin copolymer (COC) microfluidic device using either 2 or 20 thermal cycles. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a simple laser-induced fluorescence (LIF) instrument. The spFRET signatures from the target pathogens were reported in as little as 2.6 min using spFRET.
Assuntos
Alcenos/química , Bactérias/isolamento & purificação , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Técnicas Analíticas Microfluídicas/métodos , Sondas de Oligonucleotídeos/química , Carbocianinas/química , Ciclização , Contaminação de Alimentos , Ligases/metabolismo , Polímeros/química , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genéticaRESUMO
BACKGROUND: In colorectal cancer (CRC), microsatellite instability (MSI) is a valuable marker of defective DNA mismatch repair that identifies cancers with distinct phenotypic properties, including favorable survival. However, the optimal assay for MSI status is unknown. We have evaluated a simplified 3-marker assay for MSI and compared it with the 5-marker (NCI) assay to see if technical variations in MSI testing are important. MATERIALS AND METHODS: DNA samples from 357 CRCs were evaluated for MSI using the 5 microsatellite markers recommended for the NCI assay (BAT 25, BAT26, D2S123, D5S346, and D17S250). Results were compared with a simplified 3-marker assay (BAT25, BAT26, and D2S123). CRCs identified as MSI were evaluated for their clinical, pathological, and genetic characteristics. RESULTS: The 5-marker assay identified 96 cancers as MSI. Only 56 of these were MSI by the 3-marker assay (3-marker+ group), leaving 40 cases identified as MSI only by NCI criteria (3-marker- group). The remaining 261 cancers were microsatellite stable (MSS). The 3-marker+ MSI tumors had features characteristic of MSI tumors: more proximal, poorly differentiated, associated with hereditary nonpolyposis colorectal cancer (HNPCC), more BRAF mutations, fewer KRAS mutations, better 5-year disease-specific survival, more frequent mismatch repair (MMR) protein loss, and less likely to be metastatic on presentation (P < .05). Chromosomal arm loss was observed only in 3-marker- MSI and MSS cancers (P < .05). CONCLUSION: The 3-marker MSI assay outperforms the traditional 5-marker assay for identifying patients with favorable prognosis and homogeneous clinical and genetic features. More accurate MSI testing should improve prognostic and predictive scoring systems for colorectal cancer.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Enzimas Reparadoras do DNA/genética , Mutação em Linhagem Germinativa/genética , Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Adenocarcinoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bioensaio , Neoplasias Colorretais/patologia , Hibridização Genômica Comparativa , Reparo do DNA , Feminino , Seguimentos , Testes Genéticos , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Estudos Prospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
INTRODUCTION: We examined two genetic markers established early in colorectal tumor development, microsatellite instability (MSI) and mutation of the KRAS proto-oncogene, to see if these genetic changes influence metastatic disease progression and survival. PATIENTS AND METHODS: MSI and KRAS mutation status were assessed in 532 primary adenocarcinomas (stage I-IV) from patients treated by colon resection. Median follow-up was 4.1 years (range 0-13.3 years) overall, 5.4 years for survivors. RESULTS: MSI and KRAS mutation were detected in 12 and 36% of cases, respectively. MSI was more common in early-stage disease (I, 15%; II, 21%; III, 10%; IV, 2%; P = 0.0001). Prevalence of KRAS mutation did not vary with stage (I, 36%; II, 34%; III, 35%; IV, 40%; P = ns). Disease-specific survival was far superior for MSI tumors than for microsatellite stability (MSS) tumors (5-year survival 92 vs. 59%, P < 0.0001). KRAS mutation was a marker of poor survival (5-year survival 55 vs. 68%, P = 0.0002). Using Cox regression analysis MSI, KRAS mutation, and stage were strong independent predictors of survival in the entire patient population. A high-mortality group with MSS/KRAS-mutant tumors was identified within the stage I and II cohort. CONCLUSIONS: MSI and KRAS mutation provide fundamental genetic signatures influencing tumor behavior across patient subsets and stages of tumor development.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Progressão da Doença , Feminino , Seguimentos , Marcadores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas p21(ras) , Reto/metabolismo , Reto/patologia , Taxa de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
The analysis of CpG methylation in circulating tumor DNA fragments has emerged as a promising approach for the noninvasive early detection of solid tumors, including colorectal cancer (CRC). The most commonly employed assay involves bisulfite conversion of circulating tumor DNA, followed by targeted PCR, then real-time quantitative PCR (alias methylation-specific PCR). This report demonstrates the ability of a multiplex bisulfite PCR-ligase detection reaction-real-time quantitative PCR assay to detect seven methylated CpG markers (CRC or colon specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with approximately 3000 copies of fragmented peripheral blood DNA) and CRC patient-derived cell-free DNAs. This scalable assay is designed for multiplexing and incorporates steps for improved sensitivity and specificity, including the enrichment of methylated CpG fragments, ligase detection reaction, the incorporation of ribose bases in primers, and use of uracil DNA glycosylase. Six of the seven CpG markers (located in promoter regions of PPP1R16B, KCNA3, CLIP4, GDF6, SEPT9, and GSG1L) were identified through integrated analyses of genome-wide methylation data sets for 31 different types of cancer. These markers were mapped to CpG sites at the promoter region of VIM; VIM and SEPT9 are established epigenetic markers of CRC. Additional bioinformatics analyses show that the methylation at these CpG sites negatively correlates with the transcription of their corresponding genes.
Assuntos
Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Metilação de DNA/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Bases/genética , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Estudos de Coortes , Neoplasias Colorretais/diagnóstico , Biologia Computacional/métodos , Ilhas de CpG/genética , Células HT29 , Humanos , Ligases/genética , Regiões Promotoras Genéticas/genética , Septinas/sangue , Septinas/genética , Vimentina/sangue , Vimentina/genéticaRESUMO
ß-Tryptase, a homotetrameric serine protease, has four identical active sites facing a central pore, presenting an optimized setting for the rational design of bivalent inhibitors that bridge two adjacent sites. Using diol, hydroxymethyl phenols or benzoyl methyl hydroxamates, and boronic acid chemistries to reversibly join two [3-(1-acylpiperidin-4-yl)phenyl]methanamine core ligands, we have successfully produced a series of self-assembling heterodimeric inhibitors. These heterodimeric tryptase inhibitors demonstrate superior activity compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and compounds demonstrated high selectivity against related proteases, good target engagement, and tryptase inhibition in HMC1 xenograft models. Screening 3872 possible combinations from 44 boronic acid and 88 diol derivatives revealed several combinations that produced nanomolar inhibition, and seven unique pairs produced greater than 100-fold improvement in potency over monomeric inhibition. These heterodimeric tryptase inhibitors demonstrate the power of target-driven combinatorial chemistry to deliver bivalent drugs in a small molecule form.