RESUMO
Genetic variation in the human leukocyte antigen (HLA) loci is associated with risk of immune-mediated diseases, but the molecular effects of HLA polymorphism are unclear. Here we examined the effects of HLA genetic variation on the expression of 2940 plasma proteins across 45,330 Europeans in the UK Biobank, with replication analyses across multiple ancestry groups. We detected 504 proteins affected by HLA variants (HLA-pQTL), including widespread trans effects by autoimmune disease risk alleles. More than 80% of the HLA-pQTL fine-mapped to amino acid positions in the peptide binding groove. HLA-I and II affected proteins expressed in similar cell types but in different pathways of both adaptive and innate immunity. Finally, we investigated potential HLA-pQTL effects on disease by integrating HLA-pQTL with fine-mapped HLA-disease signals in the UK Biobank. Our data reveal the diverse effects of HLA genetic variation and aid the interpretation of associations between HLA alleles and immune-mediated diseases.
Assuntos
Alelos , Proteínas Sanguíneas , Variação Genética , Antígenos HLA , Humanos , Antígenos HLA/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Reino Unido , Predisposição Genética para Doença , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , População Branca/genética , Imunidade Inata/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Chinese hamster ovary (CHO) cells are the most extensively used mammalian production system for biologics intended for use in humans. A critical step in the establishment of production cell lines is single cell cloning, with the objective of achieving high productivity and product quality. Despite general use, knowledge of the effects of this process is limited. Importantly, single cell cloned cells display a wide array of observed phenotypes, which so far was attributed to the instability and variability of the CHO genome. In this study we present data indicating that the emergence of diverse phenotypes during single cell cloning is associated with changes in DNA methylation patterns and transcriptomes that occur during the subcloning process. The DNA methylation pattern of each analyzed subclone, randomly picked from all outgrowing clones of the experiment, had unique changes preferentially found in regulatory regions of the genome such as enhancers, and de-enriched in actively transcribed sequences (not including the respective promoters), indicating that these changes resulted in adaptations of the relative gene expression pattern. The transcriptome of each subclone also had a significant number of individual changes. These results indicate that epigenetic regulation is a hidden, but important player in cell line development with a major role in the establishment of high performing clones with improved characteristics for bioprocessing.
Assuntos
Metilação de DNA , Epigênese Genética , Animais , Células CHO , Cricetulus , DNA , Metilação de DNA/genética , HumanosRESUMO
Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of nucleosome occupancy and methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7 and MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding, and determination of the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate a fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and noncancerous cells.
Assuntos
Neoplasias da Mama/genética , Cromatina/genética , Metilação de DNA/genética , Sequenciamento por Nanoporos/métodos , Linhagem Celular Tumoral , Ilhas de CpG/genética , DNA/metabolismo , Epigenoma/genética , Feminino , Genoma Humano/genética , Humanos , Células MCF-7 , Metiltransferases/metabolismo , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNARESUMO
Improved identification of structural variants (SVs) in cancer can lead to more targeted and effective treatment options as well as advance our basic understanding of the disease and its progression. We performed whole-genome sequencing of the SKBR3 breast cancer cell line and patient-derived tumor and normal organoids from two breast cancer patients using Illumina/10x Genomics, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT) sequencing. We then inferred SVs and large-scale allele-specific copy number variants (CNVs) using an ensemble of methods. Our findings show that long-read sequencing allows for substantially more accurate and sensitive SV detection, with between 90% and 95% of variants supported by each long-read technology also supported by the other. We also report high accuracy for long reads even at relatively low coverage (25×-30×). Furthermore, we integrated SV and CNV data into a unifying karyotype-graph structure to present a more accurate representation of the mutated cancer genomes. We find hundreds of variants within known cancer-related genes detectable only through long-read sequencing. These findings highlight the need for long-read sequencing of cancer genomes for the precise analysis of their genetic instability.
Assuntos
Neoplasias da Mama/genética , Variação Estrutural do Genoma , Sequenciamento Completo do Genoma/métodos , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Metilação de DNA , DNA de Neoplasias , Feminino , Humanos , Nanoporos , Organoides , RNA-SeqRESUMO
Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1-4 are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3 µg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sequenciamento por Nanoporos/métodos , RNA Guia de Cinetoplastídeos/metabolismo , Animais , Células Cultivadas , Cromossomos Humanos/genética , Loci Gênicos/genética , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNARESUMO
Telomerase reverse transcriptase (TERT) activation plays an important role in cancer development by enabling the immortalization of cells. TERT regulation is multifaceted, and its promoter methylation has been implicated in controlling expression through alteration in transcription factor binding. We have characterized TERT promoter methylation, transcription factor binding, and TERT expression levels in five differentiated thyroid cancer (DTC) cell lines and six normal thyroid tissue samples by targeted bisulfite sequencing, ChIP-qPCR, and qRT-PCR. DTC cell lines express varying levels of TERT and exhibit TERT promoter methylation patterns similar to patterns seen in other telomerase positive cancer cell lines. The minimal promoter immediately surrounding the transcription start site is hypomethylated, while further upstream portions show dense methylation. In contrast, the TERT promoter in normal thyroid tissue is largely unmethylated throughout and expresses TERT minimally. Transcription factor binding is also affected by TERT mutation status. The E-twenty-six (ETS) factor GABPA exhibits TERT binding in the TERT mutant DTC cells only, and allele-specific methylation patterns at the minimal promoter were observed as well, which may indicate allele-specific factor recruitment at the minimal promoter. Furthermore, we identified binding sites for activators MYC and GSC in the hypermethylated upstream region, pointing to its possible importance in TERT regulation. Overall, TERT expression and telomerase activity depend on the interplay of multiple regulatory mechanisms including TERT promoter methylation, mutation status, and recruitment of transcription factors. This work explores of the interplay between these regulatory mechanisms and offers insight into cellular control of active telomerase in human cancer.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Telomerase/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Sítios de Ligação , Linhagem Celular Tumoral , Ilhas de CpG , Humanos , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Neoplasias da Glândula Tireoide/patologia , Sítio de Iniciação de TranscriçãoRESUMO
Whole genome bisulfite sequencing (WGBS) analysis of DNA methylation uses massively parallel next generation sequencing technology to characterize global epigenetic patterns and fluctuations throughout a range of tissue samples. Development of the vertebrate retina is thought to involve extensive epigenetic reprogramming during embryogenesis. The chicken embryo (Gallus gallus) is a classic model system for studying developmental biology and retinogenesis, however, there are currently no publicly available data sets describing the developing chicken retinal methylome. Here we used Illumina WGBS analysis to characterize genome-wide patterns of DNA methylation in the developing chicken retina as well as cornea and brain in an effort to further our understanding of retina-specific epigenetic regulation. These data will be valuable to the vision research community for correlating global changes in DNA methylation to differential gene expression between ocular and neural tissues during critical developmental time points of retinogenesis in the chicken retina.
Assuntos
Encéfalo , Galinhas , Córnea , Metilação de DNA , Retina , Animais , Genoma , Sequenciamento Completo do GenomaRESUMO
Advanced hardware components embedded in modern smartphones have the potential to serve as widely available medical diagnostic devices, particularly when used in conjunction with custom software and tested algorithms. The goal of the present pilot study was to develop a smartphone application that could quantify the severity of Parkinson's disease (PD) motor symptoms, and in particular, bradykinesia. We developed an iPhone application that collected kinematic data from a small cohort of PD patients during guided movement tasks and extracted quantitative features using signal processing techniques. These features were used in a classification model trained to differentiate between overall motor impairment of greater and lesser severity using standard clinical scores provided by a trained neurologist. Using a support vector machine classifier, a classification accuracy of 0.945 was achieved under 6-fold cross validation, and several features were shown to be highly discriminatory between more severe and less severe motor impairment by area under the receiver operating characteristic curve (AUC > 0.85). Accurate classification for discriminating between more severe and less severe bradykinesia was not achieved with these methods. We discuss future directions of this work and suggest that this platform is a first step toward development of a smartphone application that has the potential to provide clinicians with a method for monitoring patients between clinical appointments.
Assuntos
Telefone Celular , Monitorização Fisiológica/instrumentação , Doença de Parkinson/diagnóstico , Doença de Parkinson/fisiopatologia , Processamento de Sinais Assistido por Computador/instrumentação , Software , Idoso , Idoso de 80 Anos ou mais , Feminino , Dedos/fisiopatologia , Humanos , Masculino , Projetos Piloto , Máquina de Vetores de Suporte , Análise e Desempenho de TarefasRESUMO
Barium titanate nanoparticles (BT NP) belong to a class of second harmonic generating (SHG) nanoprobes that have recently demonstrated promise in biological imaging. Unfortunately, BT NPs display low cellular uptake efficiencies, which may be a problem if cellular internalization is desired or required for a particular application. To overcome this issue, while concomitantly developing a particle platform that can also deliver nucleic acids into cells, we coated the BT NPs with the cationic polymer polyethylenimine (PEI)-one of the most effective nonviral gene delivery agents. Coating of BT with PEI yielded complexes with positive zeta potentials and resulted in an 8-fold increase in cellular uptake of the BT NPs. Importantly, we were able to achieve high levels of gene delivery with the BT-PEI/DNA complexes, supporting further efforts to generate BT platforms for coupled imaging and gene therapy.