RESUMO
Mosaic loss of the X chromosome (mLOX) is the most common clonal somatic alteration in leukocytes of female individuals1,2, but little is known about its genetic determinants or phenotypic consequences. Here, to address this, we used data from 883,574 female participants across 8 biobanks; 12% of participants exhibited detectable mLOX in approximately 2% of leukocytes. Female participants with mLOX had an increased risk of myeloid and lymphoid leukaemias. Genetic analyses identified 56 common variants associated with mLOX, implicating genes with roles in chromosomal missegregation, cancer predisposition and autoimmune diseases. Exome-sequence analyses identified rare missense variants in FBXO10 that confer a twofold increased risk of mLOX. Only a small fraction of associations was shared with mosaic Y chromosome loss, suggesting that distinct biological processes drive formation and clonal expansion of sex chromosome missegregation. Allelic shift analyses identified X chromosome alleles that are preferentially retained in mLOX, demonstrating variation at many loci under cellular selection. A polygenic score including 44 allelic shift loci correctly inferred the retained X chromosomes in 80.7% of mLOX cases in the top decile. Our results support a model in which germline variants predispose female individuals to acquiring mLOX, with the allelic content of the X chromosome possibly shaping the magnitude of clonal expansion.
Assuntos
Aneuploidia , Cromossomos Humanos X , Células Clonais , Leucócitos , Mosaicismo , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Alelos , Doenças Autoimunes/genética , Bancos de Espécimes Biológicos , Segregação de Cromossomos/genética , Cromossomos Humanos X/genética , Cromossomos Humanos Y/genética , Células Clonais/metabolismo , Células Clonais/patologia , Exoma/genética , Proteínas F-Box/genética , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa , Leucemia/genética , Leucócitos/metabolismo , Modelos Genéticos , Herança Multifatorial/genética , Mutação de Sentido Incorreto/genéticaRESUMO
Hispanic/Latino children have the highest risk of acute lymphoblastic leukemia (ALL) in the US compared to other racial/ethnic groups, yet the basis of this remains incompletely understood. Through genetic fine-mapping analyses, we identified a new independent childhood ALL risk signal near IKZF1 in self-reported Hispanic/Latino individuals, but not in non-Hispanic White individuals, with an effect size of â¼1.44 (95% confidence interval = 1.33-1.55) and a risk allele frequency of â¼18% in Hispanic/Latino populations and <0.5% in European populations. This risk allele was positively associated with Indigenous American ancestry, showed evidence of selection in human history, and was associated with reduced IKZF1 expression. We identified a putative causal variant in a downstream enhancer that is most active in pro-B cells and interacts with the IKZF1 promoter. This variant disrupts IKZF1 autoregulation at this enhancer and results in reduced enhancer activity in B cell progenitors. Our study reveals a genetic basis for the increased ALL risk in Hispanic/Latino children.
Assuntos
Predisposição Genética para Doença , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Criança , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Hispânico ou Latino/genética , Fator de Transcrição Ikaros/genéticaRESUMO
Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a â¼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.
Assuntos
Neoplasias Hematológicas , Fosfoproteínas , Elongação da Transcrição Genética , Fatores de Transcrição , Humanos , Neoplasias Hematológicas/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fosfoproteínas/genéticaRESUMO
The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived haematopoietic stem cells (HSCs)1. Perturbations to this process underlie diverse diseases, but the clonal contributions to human haematopoiesis and how this changes with age remain incompletely understood. Although recent insights have emerged from barcoding studies in model systems2-5, simultaneous detection of cell states and phylogenies from natural barcodes in humans remains challenging. Here we introduce an improved, single-cell lineage-tracing system based on deep detection of naturally occurring mitochondrial DNA mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs and map the physiological state and output of clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as both differences in total HSC output and biases towards the production of different mature cell types. We also find that the diversity of HSC clones decreases markedly with age, leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides a clonally resolved and cell-state-aware atlas of human haematopoiesis at single-cell resolution, showing an unappreciated functional diversity of human HSC clones and, more broadly, paving the way for refined studies of clonal dynamics across a range of tissues in human health and disease.
Assuntos
Linhagem da Célula , Hematopoese , Células-Tronco Hematopoéticas , Humanos , Cromatina/genética , Cromatina/metabolismo , Células Clonais/classificação , Células Clonais/citologia , Células Clonais/metabolismo , DNA Mitocondrial/genética , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Mutação , Análise de Célula Única , Transcrição Gênica , EnvelhecimentoRESUMO
Mitochondria (MT) participate in most metabolic activities of mammalian cells. A near-unidirectional mitochondrial transfer from T cells to cancer cells was recently observed to "metabolically empower" cancer cells while "depleting immune cells," providing new insights into tumor-T cell interaction and immune evasion. Here, we leverage single-cell RNA-seq technology and introduce MERCI, a statistical deconvolution method for tracing and quantifying mitochondrial trafficking between cancer and T cells. Through rigorous benchmarking and validation, MERCI accurately predicts the recipient cells and their relative mitochondrial compositions. Application of MERCI to human cancer samples identifies a reproducible MT transfer phenotype, with its signature genes involved in cytoskeleton remodeling, energy production, and TNF-α signaling pathways. Moreover, MT transfer is associated with increased cell cycle activity and poor clinical outcome across different cancer types. In summary, MERCI enables systematic investigation of an understudied aspect of tumor-T cell interactions that may lead to the development of therapeutic opportunities.
Assuntos
DNA Mitocondrial , Neoplasias , Animais , Humanos , DNA Mitocondrial/genética , Linfócitos T/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Mamíferos/genética , Mamíferos/metabolismoRESUMO
Clonal hematopoiesis (CH)-age-related expansion of mutated hematopoietic clones-can differ in frequency and cellular fitness by CH type (e.g., mutations in driver genes (CHIP), gains/losses and copy-neutral loss of chromosomal segments (mCAs), and loss of sex chromosomes). Co-occurring CH raises questions as to their origin, selection, and impact. We integrate sequence and genotype array data in up to 482,378 UK Biobank participants to demonstrate shared genetic architecture across CH types. Our analysis suggests a cellular evolutionary trade-off between different types of CH, with LOY occurring at lower rates in individuals carrying mutations in established CHIP genes. We observed co-occurrence of CHIP and mCAs with overlap at TET2, DNMT3A, and JAK2, in which CHIP precedes mCA acquisition. Furthermore, individuals carrying overlapping CH had high risk of future lymphoid and myeloid malignancies. Finally, we leverage shared genetic architecture of CH traits to identify 15 novel loci associated with leukemia risk.
Assuntos
Evolução Biológica , Hematopoiese Clonal , Humanos , Hematopoiese Clonal/genética , Genótipo , Células Clonais , Metilases de Modificação do DNARESUMO
Hematopoiesis requires balance between self-renewal of stem cells and differentiation into mature blood cells, orchestrated by pathways such as thrombopoietin signaling. In this issue of Cell, Tsutsumi et al. report the structure of the thrombopoietin ligand-receptor complex and demonstrate the potential to decouple its roles in self-renewal and hematopoietic differentiation.
Assuntos
Hematopoese , Trombopoetina , Diferenciação Celular , Membrana Celular , Transdução de SinaisRESUMO
MECOM deficiency is a recently identified inborn error of immunity and inherited bone marrow failure syndrome caused by haploinsufficiency of the hematopoietic transcription factor MECOM. It is unique among inherited bone marrow failure syndromes, many of which present during later childhood or adolescence, because of the early age of onset and severity of the pancytopenia, emphasizing the importance and gene dose dependency of MECOM during hematopoiesis. B-cell lymphopenia and hypogammaglobulinemia have been described in a subset of patients with MECOM deficiency. While the mechanisms underlying the B-cell deficiency are currently unknown, recent work has provided mechanistic insights into the function of MECOM in hematopoietic stem cell (HSC) maintenance. MECOM binds to regulatory enhancers that control the expression of a network of genes essential for HSC maintenance and self-renewal. Heterozygous mutations, as seen in MECOM-deficient bone marrow failure, lead to dysregulated MECOM network expression. Extra-hematopoietic manifestations of MECOM deficiency, including renal and cardiac anomalies, radioulnar synostosis, clinodactyly, and hearing loss, have been reported. Individuals with specific genotypes have some of the systemic manifestations with isolated mild thrombocytopenia or without hematologic abnormalities, highlighting the tissue specificity of mutations in some MECOM domains. Those infants with MECOM-associated bone marrow failure require HSC transplantation for survival. Here, we review the expanding cohort of patient phenotypes and accompanying genotypes resulting in MECOM deficiency, and the proposed mechanisms underlying MECOM regulation of human HSC maintenance and B-cell development.
Assuntos
Pancitopenia , Trombocitopenia , Humanos , Criança , Pancitopenia/genética , Fatores de Transcrição/genética , Transtornos da Insuficiência da Medula Óssea , Células-Tronco Hematopoéticas , Regulação da Expressão Gênica , Síndrome Congênita de Insuficiência da Medula Óssea , Hematopoese/genética , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proteína do Locus do Complexo MDS1 e EVI1/metabolismoRESUMO
Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.
Assuntos
DNA Mitocondrial , Doenças Mitocondriais , Humanos , DNA Mitocondrial/genética , Multiômica , Doenças Mitocondriais/genética , Mitocôndrias/genética , MutaçãoRESUMO
Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.
Assuntos
Edição de Genes , Células-Tronco Hematopoéticas , Humanos , Diferenciação Celular , Sistemas CRISPR-Cas , Genoma , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Engenharia Genética , Análise de Célula ÚnicaRESUMO
The MECOM gene encodes multiple protein isoforms that are essential for hematopoietic stem cell self-renewal and maintenance. Germline MECOM variants have been associated with congenital thrombocytopenia, radioulnar synostosis and bone marrow failure; however, the phenotypic spectrum of MECOM-associated syndromes continues to expand and novel pathogenic variants continue to be identified. We describe eight unrelated patients who add to the previously known phenotypes and genetic defects of MECOM-associated syndromes. As each subject presented with unique MECOM variants, the series failed to demonstrate clear genotype-to-phenotype correlation but may suggest a role for additional modifiers that affect gene expression and subsequent phenotype. Recognition of the expanded hematologic and non-hematologic clinical features allows for rapid molecular diagnosis, early identification of life-threatening complications, and improved genetic counseling for families. A centralized international publicly accessible database to share annotated MECOM variants would advance their clinical interpretation and provide a foundation to perform functional MECOM studies.
Assuntos
Doenças da Medula Óssea , Doenças Hematológicas , Pancitopenia , Humanos , Doenças da Medula Óssea/diagnóstico , Doenças da Medula Óssea/genética , Síndrome , Transtornos da Insuficiência da Medula Óssea , Fatores de Transcrição/genética , Fenótipo , Proteína do Locus do Complexo MDS1 e EVI1/genéticaRESUMO
ß-thalassemia is caused by mutations that reduce ß-globin production, causing globin chain imbalance, ineffective erythropoiesis, and consequent anemia. Increased fetal hemoglobin (HbF) levels can ameliorate the severity of ß-thalassemia by compensating for the globin chain imbalance. Careful clinical observations paired with population studies and advances in human genetics have enabled the discovery of major regulators of HbF switching (i.e. BCL11A, ZBTB7A) and led to pharmacological and genetic therapies for treating ß-thalassemia patients. Recent functional screens using genome editing and other emerging tools have identified many new HbF regulators, which may improve therapeutic HbF induction in the future.
Assuntos
Hemoglobina Fetal , Talassemia beta , Humanos , Hemoglobina Fetal/genética , Talassemia beta/genética , Proteínas de Ligação a DNA , Linhagem Celular Tumoral , Fatores de TranscriçãoRESUMO
Mosaic loss of the X chromosome (mLOX) is the most commonly occurring clonal somatic alteration detected in the leukocytes of women, yet little is known about its genetic determinants or phenotypic consequences. To address this, we estimated mLOX in >900,000 women across eight biobanks, identifying 10% of women with detectable X loss in approximately 2% of their leukocytes. Out of 1,253 diseases examined, women with mLOX had an elevated risk of myeloid and lymphoid leukemias and pneumonia. Genetic analyses identified 49 common variants influencing mLOX, implicating genes with established roles in chromosomal missegregation, cancer predisposition, and autoimmune diseases. Complementary exome-sequence analyses identified rare missense variants in FBXO10 which confer a two-fold increased risk of mLOX. A small fraction of these associations were shared with mosaic Y chromosome loss in men, suggesting different biological processes drive the formation and clonal expansion of sex chromosome missegregation events. Allelic shift analyses identified alleles on the X chromosome which are preferentially retained, demonstrating that variation at many loci across the X chromosome is under cellular selection. A novel polygenic score including 44 independent X chromosome allelic shift loci correctly inferred the retained X chromosomes in 80.7% of mLOX cases in the top decile. Collectively our results support a model where germline variants predispose women to acquiring mLOX, with the allelic content of the X chromosome possibly shaping the magnitude of subsequent clonal expansion.
RESUMO
Hematopoiesis is a continuous process of blood and immune cell production. It is orchestrated by thousands of gene products that respond to extracellular signals by guiding cell fate decisions to meet the needs of the organism. Although much of our knowledge of this process comes from work in model systems, we have learned a great deal from studies on human genetic variation. Considerable insight has emerged from studies on presumed monogenic blood disorders, which continue to provide key insights into the mechanisms critical for hematopoiesis. Furthermore, the emergence of large-scale biobanks and cohorts has uncovered thousands of genomic loci associated with blood cell traits and diseases. Some of these blood cell trait-associated loci act as modifiers of what were once thought to be monogenic blood diseases. However, most of these loci await functional validation. Here, we discuss the validation bottleneck and emerging methods to more effectively connect variant to function. In particular, we highlight recent innovations in genome editing, which have paved the path forward for high-throughput functional assessment of loci. Finally, we discuss existing barriers to progress, including challenges in manipulating the genomes of primary hematopoietic cells.
Assuntos
Edição de Genes , Hematopoese , Humanos , Diferenciação Celular/genética , GenômicaRESUMO
Hematopoietic stem cells (HSCs) have a number of unique physiologic adaptations that enable lifelong maintenance of blood cell production, including a highly regulated rate of protein synthesis. Yet, the precise vulnerabilities that arise from such adaptations have not been fully characterized. Here, inspired by a bone marrow failure disorder due to the loss of the histone deubiquitinase MYSM1, characterized by selectively disadvantaged HSCs, we show how reduced protein synthesis in HSCs results in increased ferroptosis. HSC maintenance can be fully rescued by blocking ferroptosis, despite no alteration in protein synthesis rates. Importantly, this selective vulnerability to ferroptosis not only underlies HSC loss in MYSM1 deficiency but also characterizes a broader liability of human HSCs. Increasing protein synthesis rates via MYSM1 overexpression makes HSCs less susceptible to ferroptosis, more broadly illustrating the selective vulnerabilities that arise in somatic stem cell populations as a result of physiologic adaptations.
Assuntos
Ferroptose , Células-Tronco Hematopoéticas , Humanos , Endopeptidases/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Transativadores/metabolismo , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Natural sequence variation within mitochondrial DNA (mtDNA) contributes to human phenotypes and may serve as natural genetic markers in human cells for clonal and lineage tracing. We recently developed a single-cell multi-omic approach, called 'mitochondrial single-cell assay for transposase-accessible chromatin with sequencing' (mtscATAC-seq), enabling concomitant high-throughput mtDNA genotyping and accessible chromatin profiling. Specifically, our technique allows the mitochondrial genome-wide inference of mtDNA variant heteroplasmy along with information on cell state and accessible chromatin variation in individual cells. Leveraging somatic mtDNA mutations, our method further enables inference of clonal relationships among native ex vivo-derived human cells not amenable to genetic engineering-based clonal tracing approaches. Here, we provide a step-by-step protocol for the use of mtscATAC-seq, including various cell-processing and flow cytometry workflows, by using primary hematopoietic cells, subsequent single-cell genomic library preparation and sequencing that collectively take ~3-4 days to complete. We discuss experimental and computational data quality control metrics and considerations for the extension to other mammalian tissues. Overall, mtscATAC-seq provides a broadly applicable platform to map clonal relationships between cells in human tissues, investigate fundamental aspects of mitochondrial genetics and enable additional modes of multi-omic discovery.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Cromatina , Animais , Humanos , Cromatina/genética , Multiômica , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA Mitocondrial/genética , Genótipo , Mamíferos/genéticaRESUMO
Lineage tracing using fluorescent proteins, genetic barcodes, and various other strategies has provided critical insights into the dynamics of both fetal and adult hematopoiesis in model organisms. However, these technologies cannot be readily used to study hematopoiesis in human beings. Therefore, there is a critical need to develop strategies to assess cellular dynamics within human hematopoietic tissues in vivo. Recently, researchers have used naturally acquired somatic mutations, coupled with other single-cell technologies, to retrospectively analyze clonal cellular dynamics. In summer 2022, the International Society for Experimental Hematology's New Investigator Committee hosted a webinar focused on novel approaches to dissect fetal and adult hematopoiesis, with presentations from Drs. Ana Cvejic and Vijay Sankaran. Here, we provide an overview of these exciting technological advances and some of the novel insights they have already provided in studying human hematopoiesis.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Adulto , Humanos , Mutação , Estudos Retrospectivos , Células-Tronco Hematopoéticas/metabolismo , Hematopoese/genéticaRESUMO
The molecular regulation of human hematopoietic stem cell (HSC) maintenance is therapeutically important, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. Here, we have studied a rare genetic disorder due to MECOM haploinsufficiency, characterized by an early-onset absence of HSCs in vivo. By generating a faithful model of this disorder in primary human HSCs and coupling functional studies with integrative single-cell genomic analyses, we uncover a key transcriptional network involving hundreds of genes that is required for HSC maintenance. Through our analyses, we nominate cooperating transcriptional regulators and identify how MECOM prevents the CTCF-dependent genome reorganization that occurs as HSCs differentiate. We show that this transcriptional network is co-opted in high-risk leukemias, thereby enabling these cancers to acquire stem cell properties. Collectively, we illuminate a regulatory network necessary for HSC self-renewal through the study of a rare experiment of nature.
Assuntos
Leucemia , Neoplasias , Humanos , Células-Tronco Hematopoéticas , Leucemia/genética , Fatores de Transcrição/genética , Diferenciação Celular/genéticaRESUMO
Sepsis continues to be associated with high morbidity and mortality. Currently, sepsis is managed only conservatively. In sepsis, a substantial number of neutrophils is required, leading to accelerated neutrophil production. Immature neutrophils are released into the circulation to meet a demand, despite their less effective functioning in microbial eradication. Although an intervention to provide more mature neutrophils may serve as a potential sepsis treatment, the mechanism of neutrophil differentiation and maturation remains poorly understood. We discovered that CD11c, traditionally known as a dendritic cell marker, was expressed in neutrophils and regulated neutrophil maturation and effector functions. In the absence of CD11c, neutrophil maturation was impaired in the bone marrow, concomitant with a significant increase in the proliferation and apoptosis of preneutrophils, associated with less effector functions. Under lipopolysaccharide challenge, inducing an emergent neutrophil production in the bone marrow, CD11c deficiency exaggerated the release of immature neutrophils into the circulation, associated with a significant proliferation and apoptosis of preneutrophils. In contrast, constitutively active CD11c knock-in mice showed accelerated neutrophil maturation associated with enhanced effector functions, which further supports the notion that CD11c regulates neutrophil maturation. Furthermore, the constitutively active CD11c knock-in mice offered enhanced bacterial eradication. Taken together, we discovered that CD11c was critical for the regulation of neutrophil maturation, and CD11c activation could serve as a potential target for sepsis treatment.