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1.
Clin Chem Lab Med ; 60(2): 183-190, 2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-34761647

RESUMO

OBJECTIVES: Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF. METHODS: A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF. RESULTS: We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at -80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy. CONCLUSIONS: Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.


Assuntos
Ácidos Nucleicos Livres , Síndrome de Down , Síndrome de Down/diagnóstico , Feminino , Humanos , Masculino , Gravidez , Diagnóstico Pré-Natal , Trissomia , Síndrome da Trissomía do Cromossomo 18/diagnóstico
2.
BMC Genomics ; 14: 550, 2013 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-23941359

RESUMO

BACKGROUND: Chimeric transcripts, including partial and internal tandem duplications (PTDs, ITDs) and gene fusions, are important in the detection, prognosis, and treatment of human cancers. RESULTS: We describe Barnacle, a production-grade analysis tool that detects such chimeras in de novo assemblies of RNA-seq data, and supports prioritizing them for review and validation by reporting the relative coverage of co-occurring chimeric and wild-type transcripts. We demonstrate applications in large-scale disease studies, by identifying PTDs in MLL, ITDs in FLT3, and reciprocal fusions between PML and RARA, in two deeply sequenced acute myeloid leukemia (AML) RNA-seq datasets. CONCLUSIONS: Our analyses of real and simulated data sets show that, with appropriate filter settings, Barnacle makes highly specific predictions for three types of chimeric transcripts that are important in a range of cancers: PTDs, ITDs, and fusions. High specificity makes manual review and validation efficient, which is necessary in large-scale disease studies. Characterizing an extended range of chimera types will help generate insights into progression, treatment, and outcomes for complex diseases.


Assuntos
Duplicação Gênica/genética , Perfilação da Expressão Gênica/métodos , Fusão Gênica/genética , Genômica , Neoplasias da Mama/genética , Éxons/genética , Humanos , Leucemia Mieloide Aguda/genética , Anotação de Sequência Molecular , RNA Mensageiro/genética , Estatística como Assunto
3.
Stem Cell Res ; 71: 103153, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37385135

RESUMO

Variants in the sodium voltage-gated channel alpha subunit 5 gene (SCN5A) produce variable cardiac phenotypes including Brugada syndrome, conduction disease and cardiomyopathy. These phenotypes can lead to life-threatening arrhythmias, heart failure, and sudden cardiac death. Novel variants in splice-site regions of SCN5A require functional studies to characterise their pathogenicity as they are poorly understood. The generation of an induced pluripotent stem cell line provides a valuable resource to investigate the functional effects of potential splice-disrupting variants in SCN5A.


Assuntos
Células-Tronco Pluripotentes Induzidas , Fibrilação Ventricular , Humanos , Fibrilação Ventricular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença do Sistema de Condução Cardíaco , Arritmias Cardíacas , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Sódio/metabolismo , Mutação
4.
Nat Commun ; 14(1): 4537, 2023 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-37500618

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) originate from an endothelial-to-hematopoietic transition (EHT) during embryogenesis. Characterization of early hemogenic endothelial (HE) cells is required to understand what drives hemogenic specification and to accurately define cells capable of undergoing EHT. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), we define the early subpopulation of pre-HE cells based on both surface markers and transcriptomes. We identify the transcription factor Meis1 as an essential regulator of hemogenic cell specification in the embryo prior to Runx1 expression. Meis1 is expressed at the earliest stages of EHT and distinguishes pre-HE cells primed towards the hemogenic trajectory from the arterial endothelial cells that continue towards a vascular fate. Endothelial-specific deletion of Meis1 impairs the formation of functional Runx1-expressing HE which significantly impedes the emergence of pre-HSPC via EHT. Our findings implicate Meis1 in a critical fate-determining step for establishing EHT potential in endothelial cells.


Assuntos
Hemangioblastos , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Proteína Meis1/genética , Proteína Meis1/metabolismo , Hematopoese/genética
5.
J Mol Diagn ; 23(9): 1145-1158, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34197922

RESUMO

Next-generation sequencing assays are capable of identifying cancer patients eligible for targeted therapies and can also detect germline variants associated with increased cancer susceptibility. However, these capabilities have yet to be routinely harmonized in a single assay because of challenges with accurately identifying germline variants from tumor-only data. We have developed the Oncology and Hereditary Cancer Program targeted capture panel, which uses tumor tissue to simultaneously screen for both clinically actionable solid tumor variants and germline variants across 45 genes. Validation using 14 tumor specimens, composed of patient samples and cell lines analyzed in triplicate, demonstrated high coverage with sensitive and specific identification of single-nucleotide variants and small insertions and deletions. Average coverage across all targets remained >2000× in 198 additional patient tumor samples. Analysis of 55 formalin-fixed, paraffin-embedded tumor samples for the detection of known germline variants within a subset of cancer-predisposition genes, including one multiexon deletion, yielded a 100% detection rate, demonstrating that germline variants can be reliably detected in tumor samples using a single panel. Combining targetable somatic and actionable germline variants into a single tumor tissue assay represents a streamlined approach that can inform treatment for patients with advanced cancers as well as identify those with potential germline variants who are eligible for confirmatory testing, but would not otherwise have been identified.


Assuntos
Predisposição Genética para Doença/genética , Células Germinativas , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Alelos , Estudos de Coortes , Variações do Número de Cópias de DNA , Confiabilidade dos Dados , Feminino , Testes Genéticos/métodos , Humanos , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Prognóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
6.
Nat Commun ; 12(1): 2474, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931648

RESUMO

As more clinically-relevant genomic features of myeloid malignancies are revealed, it has become clear that targeted clinical genetic testing is inadequate for risk stratification. Here, we develop and validate a clinical transcriptome-based assay for stratification of acute myeloid leukemia (AML). Comparison of ribonucleic acid sequencing (RNA-Seq) to whole genome and exome sequencing reveals that a standalone RNA-Seq assay offers the greatest diagnostic return, enabling identification of expressed gene fusions, single nucleotide and short insertion/deletion variants, and whole-transcriptome expression information. Expression data from 154 AML patients are used to develop a novel AML prognostic score, which is strongly associated with patient outcomes across 620 patients from three independent cohorts, and 42 patients from a prospective cohort. When combined with molecular risk guidelines, the risk score allows for the re-stratification of 22.1 to 25.3% of AML patients from three independent cohorts into correct risk groups. Within the adverse-risk subgroup, we identify a subset of patients characterized by dysregulated integrin signaling and RUNX1 or TP53 mutation. We show that these patients may benefit from therapy with inhibitors of focal adhesion kinase, encoded by PTK2, demonstrating additional utility of transcriptome-based testing for therapy selection in myeloid malignancy.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Estudos de Coortes , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Feminino , Fusão Gênica , Humanos , Mutação INDEL , Integrinas/genética , Integrinas/metabolismo , Leucemia Mieloide Aguda/genética , Masculino , Polimorfismo de Nucleotídeo Único , Prognóstico , Estudos Prospectivos , RNA-Seq , Fatores de Risco , Transdução de Sinais/genética , Análise de Sobrevida , Transcriptoma , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sequenciamento do Exoma , Sequenciamento Completo do Genoma
7.
J Cell Biol ; 169(5): 707-10, 2005 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-15928206

RESUMO

A recent convergence of data indicating a relationship between cilia and proliferative diseases, such as polycystic kidney disease, has revived the long-standing enigma of the reciprocal regulatory relationship between cilia and the cell cycle. Multiple signaling pathways are localized to cilia in mammalian cells, and some proteins have been shown to act both in the cilium and in cell cycle regulation. Work from the unicellular alga Chlamydomonas is providing novel insights as to how cilia and the cell cycle are coordinately regulated.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Cílios/fisiologia , Animais , Chlamydomonas/metabolismo , Humanos , Fosfotransferases/metabolismo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/fisiopatologia , Transdução de Sinais/fisiologia
8.
Cell Rep ; 27(6): 1769-1780.e4, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067462

RESUMO

The sterile alpha motif (SAM) and SRC homology 3 (SH3) domain containing protein 1 (Sash1) acts as a scaffold in TLR4 signaling. We generated Sash1-/- mice, which die in the perinatal period due to respiratory distress. Constitutive or endothelial-restricted Sash1 loss leads to a delay in maturation of alveolar epithelial cells causing reduced surfactant-associated protein synthesis. We show that Sash1 interacts with ß-arrestin 1 downstream of the TLR4 pathway to activate Akt and endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. Generation of nitric oxide downstream of Sash1 in endothelial cells affects alveolar epithelial cells in a cGMP-dependent manner, inducing maturation of alveolar type 1 and 2 cells. Thus, we identify a critical cell nonautonomous function for Sash1 in embryonic development in which endothelial Sash1 regulates alveolar epithelial cell maturation and promotes pulmonary surfactant production through nitric oxide signaling. Lung immaturity is a major cause of respiratory distress and mortality in preterm infants, and these findings identify the endothelium as a potential target for therapy.


Assuntos
Células Endoteliais/metabolismo , Pulmão/crescimento & desenvolvimento , Óxido Nítrico/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Linhagem Celular , GMP Cíclico/metabolismo , Perda do Embrião/metabolismo , Perda do Embrião/patologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Endotélio/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Pulmão/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Alvéolos Pulmonares/patologia , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , beta-Arrestinas/metabolismo
9.
J Mol Diagn ; 21(4): 705-717, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31055024

RESUMO

Formalin fixation is the standard method for the preservation of tissue for diagnostic purposes, including pathologic review and molecular assays. However, this method is known to cause artifacts that can affect the accuracy of molecular genetic test results. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade, laboratory-developed targeted resequencing assay. Several parameters relating to sequencing quality and variant calling were examined and quantified in tumor and normal colon epithelial tissues. We identified an alternative fixative that suppresses many formalin-related artifacts while retaining adequate morphology for pathologic review.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Fixação de Tecidos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Imuno-Histoquímica , Inclusão em Parafina , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas
10.
Cold Spring Harb Mol Case Stud ; 2(2): a000679, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27148583

RESUMO

In an attempt to assess potential treatment options, whole-genome and transcriptome sequencing were performed on a patient with an unclassifiable small lymphoproliferative disorder. Variants from genome sequencing were prioritized using a combination of comparative variant distributions in a spectrum of lymphomas, and meta-analyses of gene expression profiling. In this patient, the molecular variants that we believe to be most relevant to the disease presentation most strongly resemble a diffuse large B-cell lymphoma (DLBCL), whereas the gene expression data are most consistent with a low-grade chronic lymphocytic leukemia (CLL). The variant of greatest interest was a predicted NOTCH2-truncating mutation, which has been recently reported in various lymphomas.

11.
Cytoskeleton (Hoboken) ; 67(7): 425-30, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20506243

RESUMO

Cilia are necessary for normal tissue development and homeostasis and are generally present during interphase, but not in mitosis. The precise mechanism of premitotic ciliary loss has been controversial, with data supporting either sequential disassembly through the transition zone or, alternatively, a severing event at the base of the cilia. Here we show by live cell imaging and immunofluorescence microscopy that resorbing flagella of Chlamydomonas leave remnants associated with the mother cell wall. We postulated that the remnants are the product of severing of doublet microtubules between the basal bodies and the flagellar transition zone, thereby freeing the centrioles to participate in spindle organization. We show via TEM that flagellar remnants are indeed flagellar transition zones encased in vesicles derived from the flagellar membrane. This transition zone vesicle can be lodged within the cell wall or it can be expelled into the environment. This process is observable in Chlamydomonas, first because the released flagellar remnants can remain associated with the cell by virtue of attachments to the cell wall, and second because the Chlamydomonas transition zone is particularly rich with electron-dense structure. However, release of basal bodies for spindle-associated function is likely to be conserved among the eukaryotes.


Assuntos
Centríolos/metabolismo , Chlamydomonas/citologia , Chlamydomonas/metabolismo , Cílios/metabolismo , Mitose , Sobrevivência Celular , Centríolos/ultraestrutura , Chlamydomonas/ultraestrutura , Cílios/ultraestrutura , Flagelos/ultraestrutura , Imunofluorescência
12.
Mol Biol Cell ; 20(1): 379-88, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19005222

RESUMO

Katanin is a microtubule-severing protein that participates in the regulation of cell cycle progression and in ciliary disassembly, but its precise role is not known for either activity. Our data suggest that in Chlamydomonas, katanin severs doublet microtubules at the proximal end of the flagellar transition zone, allowing disengagement of the basal body from the flagellum before mitosis. Using an RNA interference approach we have discovered that severe knockdown of the p60 subunit of katanin, KAT1, is achieved only in cells that also carry secondary mutations that disrupt ciliogenesis. Importantly, we observed that cells in the process of cell cycle-induced flagellar resorption sever the flagella from the basal bodies before resorption is complete, and we find that this process is defective in KAT1 knockdown cells.


Assuntos
Adenosina Trifosfatases/metabolismo , Chlamydomonas reinhardtii , Cílios/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas de Protozoários/metabolismo , Adenosina Trifosfatases/genética , Animais , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/metabolismo , Cílios/ultraestrutura , Técnicas de Silenciamento de Genes , Katanina , Microtúbulos/ultraestrutura , Proteínas de Protozoários/genética , Interferência de RNA
13.
J Cell Sci ; 122(Pt 5): 611-24, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19208769

RESUMO

Meckel syndrome (MKS) is a ciliopathy characterized by encephalocele, cystic renal disease, liver fibrosis and polydactyly. An identifying feature of MKS1, one of six MKS-associated proteins, is the presence of a B9 domain of unknown function. Using phylogenetic analyses, we show that this domain occurs exclusively within a family of three proteins distributed widely in ciliated organisms. Consistent with a ciliary role, all Caenorhabditis elegans B9-domain-containing proteins, MKS-1 and MKS-1-related proteins 1 and 2 (MKSR-1, MKSR-2), localize to transition zones/basal bodies of sensory cilia. Their subcellular localization is largely co-dependent, pointing to a functional relationship between the proteins. This localization is evolutionarily conserved, because the human orthologues also localize to basal bodies, as well as cilia. As reported for MKS1, disrupting human MKSR1 or MKSR2 causes ciliogenesis defects. By contrast, single, double and triple C. elegans mks/mksr mutants do not display overt defects in ciliary structure, intraflagellar transport or chemosensation. However, we find genetic interactions between all double mks/mksr mutant combinations, manifesting as an increased lifespan phenotype, which is due to abnormal insulin-IGF-I signaling. Our findings therefore demonstrate functional interactions between a novel family of proteins associated with basal bodies or cilia, providing new insights into the molecular etiology of a pleiotropic human disorder.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Cílios/patologia , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/classificação , Proteínas de Caenorhabditis elegans/genética , Cílios/metabolismo , Evolução Molecular , Humanos , Dados de Sequência Molecular , Fenótipo , Filogenia , Proteínas/classificação , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia
14.
PLoS One ; 2(10): e1076, 2007 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-17957258

RESUMO

BACKGROUND: NIMA-related kinases (Neks) have been studied in diverse eukaryotes, including the fungus Aspergillus and the ciliate Tetrahymena. In the former, a single Nek plays an essential role in cell cycle regulation; in the latter, which has more than 30 Neks in its genome, multiple Neks regulate ciliary length. Mammalian genomes encode an intermediate number of Neks, several of which are reported to play roles in cell cycle regulation and/or localize to centrosomes. Previously, we reported that organisms with cilia typically have more Neks than organisms without cilia, but were unable to establish the evolutionary history of the gene family. METHODOLOGY/PRINCIPLE FINDINGS: We have performed a large-scale analysis of the Nek family using Bayesian techniques, including tests of alternate topologies. We find that the Nek family had already expanded in the last common ancestor of eukaryotes, a ciliated cell which likely expressed at least five Neks. We suggest that Neks played an important role in the common ancestor in regulating cilia, centrioles, and centrosomes with respect to mitotic entry, and that this role continues today in organisms with cilia. Organisms that lack cilia generally show a reduction in the number of Nek clades represented, sometimes associated with lineage specific expansion of a single clade, as has occurred in the plants. CONCLUSION/SIGNIFICANCE: This is the first rigorous phylogenetic analysis of a kinase family across a broad array of phyla. Our findings provide a coherent framework for the study of Neks and their roles in coordinating cilia and cell cycle progression.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Cílios/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Teorema de Bayes , Ciclo Celular , Evolução Molecular , Genoma , Humanos , Quinase 1 Relacionada a NIMA , Fosforilação , Fosfotransferases/química , Filogenia , Transdução de Sinais , Software , Tetrahymena
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