ABSTRACT
The lack of accessible noninvasive tools to examine the molecular alterations occurring in the brain limits our understanding of the causes and progression of Alzheimer's disease (AD), as well as the identification of effective therapeutic strategies. Here, we conducted a comprehensive profiling of circulating, cell-free messenger RNA (cf-mRNA) in plasma of 126 patients with AD and 116 healthy controls of similar age. We identified 2591 dysregulated genes in the cf-mRNA of patients with AD, which are enriched in biological processes well known to be associated with AD. Dysregulated genes included brain-specific genes and resembled those identified to be dysregulated in postmortem AD brain tissue. Furthermore, we identified disease-relevant circulating gene transcripts that correlated with the severity of cognitive impairment. These data highlight the potential of high-throughput cf-mRNA sequencing to evaluate AD-related pathophysiological alterations in the brain, leading to precision healthcare solutions that could improve AD patient management.
Subject(s)
Alzheimer Disease , Cell-Free Nucleic Acids , Alzheimer Disease/genetics , Brain , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Humans , RNA, Messenger/geneticsABSTRACT
Circulating cell-free mRNA (cf-mRNA) holds great promise as a non-invasive diagnostic biomarker. However, cf-mRNA composition and its potential clinical applications remain largely unexplored. Here we show, using Next Generation Sequencing-based profiling, that cf-mRNA is enriched in transcripts derived from the bone marrow compared to circulating cells. Further, longitudinal studies involving bone marrow ablation followed by hematopoietic stem cell transplantation in multiple myeloma and acute myeloid leukemia patients indicate that cf-mRNA levels reflect the transcriptional activity of bone marrow-resident hematopoietic lineages during bone marrow reconstitution. Mechanistically, stimulation of specific bone marrow cell populations in vivo using growth factor pharmacotherapy show that cf-mRNA reflects dynamic functional changes over time associated with cellular activity. Our results shed light on the biology of the circulating transcriptome and highlight the potential utility of cf-mRNA to non-invasively monitor bone marrow involved pathologies.
Subject(s)
Biomarkers, Tumor/isolation & purification , Bone Marrow/pathology , Cell-Free Nucleic Acids/isolation & purification , Leukemia, Myeloid, Acute/diagnosis , Multiple Myeloma/diagnosis , RNA, Messenger/isolation & purification , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Bone Marrow/drug effects , Cell-Free Nucleic Acids/blood , Cell-Free Nucleic Acids/genetics , Feasibility Studies , Gene Expression Profiling/methods , Granulocyte Colony-Stimulating Factor/administration & dosage , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/drug effects , High-Throughput Nucleotide Sequencing/methods , Humans , Leukemia, Myeloid, Acute/blood , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/therapy , Longitudinal Studies , Middle Aged , Multiple Myeloma/blood , Multiple Myeloma/pathology , Multiple Myeloma/therapy , RNA, Messenger/blood , RNA, Messenger/genetics , Sequence Analysis, RNA/methods , Treatment Outcome , Young AdultABSTRACT
Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy.Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care.Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03).Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR.
Subject(s)
Biomarkers, Tumor , Circulating Tumor DNA , High-Throughput Nucleotide Sequencing , Neoplasms/diagnosis , Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Female , Genetic Testing , High-Throughput Nucleotide Sequencing/methods , High-Throughput Nucleotide Sequencing/standards , Humans , Male , Middle Aged , Mutation , Neoplasms/mortality , Prognosis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain.
Subject(s)
Transcriptome , Cell Nucleus , Cerebral Cortex , Gene Expression Profiling , Humans , Neurons , Sequence Analysis, RNAABSTRACT
Skin wounds comprise a serious medical issue for which few pharmacological interventions are available. Moreover, the inflammatory, angiogenic, and proliferative facets of a typical response to a wound each have broader relevance in other pathological conditions. Here we describe a genomics-driven approach to identify secreted proteins that modulate wound healing in a mouse ear punch model. We show that adiponectin, when injected into the wound edge, accelerates wound healing. Notably, adiponectin injection causes upregulation of keratin gene transcripts within hours of treatment, and subsequently promotes collagen organization, formation of pilosebaceous units, and proliferation of cells in the basal epithelial cell layer and pilosebaceous units of healing tissue. The globular domain of adiponectin is sufficient to mediate accelerated dorsal skin wound closure, and the effects are lost in mice that are homozygous null for the adiponectin receptor 1 gene. These findings extend recent observations of a protective role of adiponectin in other tissue injury settings, suggest modulation of AdipoR1 for the clinical management of wounds, and demonstrate a new approach to the identification of regulators of a wound healing response.
Subject(s)
Adiponectin/physiology , Skin/injuries , Skin/physiopathology , Wound Healing/physiology , Adiponectin/pharmacology , Animals , Collagen/metabolism , Keratins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Receptors, Adiponectin/deficiency , Receptors, Adiponectin/genetics , Receptors, Adiponectin/physiology , Skin/metabolism , Up-Regulation/drug effects , Wound Healing/drug effectsABSTRACT
Temperature compensation contributes to the accuracy of biological timing by preventing circadian rhythms from running more quickly at high than at low temperatures. We previously identified quantitative trait loci (QTL) with temperature-specific effects on the circadian rhythm of leaf movement, including a QTL linked to the transcription factor FLOWERING LOCUS C (FLC). We have now analyzed FLC alleles in near-isogenic lines and induced mutants to eliminate other candidate genes. We showed that FLC lengthened the circadian period specifically at 27 degrees C, contributing to temperature compensation of the circadian clock. Known upstream regulators of FLC expression in flowering time pathways similarly controlled its circadian effect. We sought to identify downstream targets of FLC regulation in the molecular mechanism of the circadian clock using genome-wide analysis to identify FLC-responsive genes and 3503 transcripts controlled by the circadian clock. A Bayesian clustering method based on Fourier coefficients allowed us to discriminate putative regulatory genes. Among rhythmic FLC-responsive genes, transcripts of the transcription factor LUX ARRHYTHMO (LUX) correlated in peak abundance with the circadian period in flc mutants. Mathematical modeling indicated that the modest change in peak LUX RNA abundance was sufficient to cause the period change due to FLC, providing a molecular target for the crosstalk between flowering time pathways and circadian regulation.
