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1.
Mol Oncol ; 2024 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-38770541

RESUMEN

Early identification of resistant cancer cells is currently a major challenge, as their expansion leads to refractoriness. To capture the dynamics of these cells, we made a comprehensive analysis of disease progression and treatment response in a chronic lymphocytic leukemia (CLL) patient using a combination of single-cell and bulk genomic methods. At diagnosis, the patient presented with unfavorable genetic markers, including notch receptor 1 (NOTCH1) mutation and loss(11q). The initial and subsequent treatment lines did not lead to a durable response and the patient developed refractory disease. Refractory CLL cells featured substantial dysregulation in B-cell phenotypic markers such as human leukocyte antigen (HLA) genes, immunoglobulin (IG) genes, CD19 molecule (CD19), membrane spanning 4-domains A1 (MS4A1; previously known as CD20), CD79a molecule (CD79A) and paired box 5 (PAX5), indicating B-cell de-differentiation and disease transformation. We described the clonal evolution and characterized in detail two cell populations that emerged during the refractory disease phase, differing in the presence of high genomic complexity. In addition, we successfully tracked the cells with high genomic complexity back to the time before treatment, where they formed a rare subpopulation. We have confirmed that single-cell RNA sequencing enables the characterization of refractory cells and the monitoring of their development over time.

2.
Geroscience ; 46(3): 3005-3019, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38172489

RESUMEN

Biological age is typically estimated using biomarkers whose states have been observed to correlate with chronological age. A persistent limitation of such aging clocks is that it is difficult to establish how the biomarker states are related to the mechanisms of aging. Somatic mutations could potentially form the basis for a more fundamental aging clock since the mutations are both markers and drivers of aging and have a natural timescale. Cell lineage trees inferred from these mutations reflect the somatic evolutionary process, and thus, it has been conjectured, the aging status of the body. Such a timer has been impractical thus far, however, because detection of somatic variants in single cells presents a significant technological challenge. Here, we show that somatic mutations detected using single-cell RNA sequencing (scRNA-seq) from thousands of cells can be used to construct a cell lineage tree whose structure correlates with chronological age. De novo single-nucleotide variants (SNVs) are detected in human peripheral blood mononuclear cells using a modified protocol. A default model based on penalized multiple regression of chronological age on 31 metrics characterizing the phylogenetic tree gives a Pearson correlation of 0.81 and a median absolute error of ~4 years between predicted and chronological ages. Testing of the model on a public scRNA-seq dataset yields a Pearson correlation of 0.85. In addition, cell tree age predictions are found to be better predictors of certain clinical biomarkers than chronological age alone, for instance glucose, albumin levels, and leukocyte count. The geometry of the cell lineage tree records the structure of somatic evolution in the individual and represents a new modality of aging timer. In addition to providing a numerical estimate of "cell tree age," it unveils a temporal history of the aging process, revealing how clonal structure evolves over life span. Cell Tree Rings complements existing aging clocks and may help reduce the current uncertainty in the assessment of geroprotective trials.


Asunto(s)
Envejecimiento , Leucocitos Mononucleares , Humanos , Filogenia , Envejecimiento/genética , Longevidad , Biomarcadores
3.
Biochim Biophys Acta Mol Cell Res ; 1869(10): 119321, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35779629

RESUMEN

Single-cell transcriptomics has emerged as a powerful tool to investigate cells' biological landscape and focus on the expression profile of individual cells. Major advantage of this approach is an analysis of highly complex and heterogeneous cell populations, such as a specific subpopulation of T helper cells that are known to differentiate into distinct subpopulations. The need for distinguishing the specific expression profile is even more important considering the T cell plasticity. However, importantly, the universal pipelines for single-cell analysis are usually not sufficient for every cell type. Here, the aims are to analyze the diversity of T cell phenotypes employing classical in vitro cytokine-mediated differentiation of human T cells isolated from human peripheral blood by single-cell transcriptomic approach with support of labelled antibodies and a comprehensive bioinformatics analysis using combination of Seurat, Nebulosa, GGplot and others. The results showed high expression similarities between Th1 and Th17 phenotype and very distinct Th2 expression profile. In a case of Th2 highly specific marker genes SPINT2, TRIB3 and CST7 were expressed. Overall, our results demonstrate how donor difference, Th plasticity and cell cycle influence the expression profiles of distinct T cell populations. The results could help to better understand the importance of each step of the analysis when working with T cell single-cell data and observe the results in a more practical way by using our analyzed datasets.


Asunto(s)
Activación de Linfocitos , Células Th2 , Diferenciación Celular/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Análisis de Secuencia de ARN , Células Th17 , Células Th2/metabolismo
4.
Int J Mol Sci ; 23(2)2022 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-35055070

RESUMEN

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.


Asunto(s)
Alelos , Síndrome de Bernard-Soulier/diagnóstico , Síndrome de Bernard-Soulier/genética , Predisposición Genética a la Enfermedad , Variación Genética , Fenotipo , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Síndrome de Bernard-Soulier/sangre , Plaquetas/metabolismo , Plaquetas/ultraestructura , República Checa , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Humanos , Inmunofenotipificación , Masculino , Linaje , Recuento de Plaquetas , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Trombocitopenia/sangre , Trombocitopenia/diagnóstico
5.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-33348918

RESUMEN

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCß4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.


Asunto(s)
Biomarcadores de Tumor/genética , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Células Neoplásicas Circulantes/patología , Neoplasias de la Úvea/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Deleción Cromosómica , Cromosomas Humanos Par 3/genética , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Femenino , Estudios de Seguimiento , Humanos , Biopsia Líquida , Masculino , Melanoma/secundario , Melanoma/terapia , Persona de Mediana Edad , Células Neoplásicas Circulantes/metabolismo , Pronóstico , Neoplasias de la Úvea/secundario , Neoplasias de la Úvea/terapia
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