ABSTRACT
The immense progress in molecular biology observed in the last decades has led to a fundamental change in our understanding of the etiology of human diseases. Whole genome analyses, both DNA sequencing and microarray comparative genomic hybridization, allowed for identification of previously unknown diseases and syndromes. Therefore, in difficulttodiagnose cases, clinical diagnosis is being replaced by molecular diagnosis (molecular dysmorphology, genomic medicine). For both scientific development of human genetics and clinical characteristics of rare genetic diseases, the construction and sharing of internationally available large databases has become crucial. However, genetic data have to be considered on the individual level too; therefore, they have to be treated as sensitive personal information. The context of ethical and legal risks involved in genetic testing has been long analyzed, although recognition of personal data protection issues is a more recent topic. The respective legal acts and protective measures should take into account several different aspects. The present paper explores major benefits and risks associated with international sharing of vast databases of genetic material, and presents legal provisions applied in the European Union, the United States, and China. The latter part is based on the respective acts themselves, as well as on analyses and commentaries by other scholars.
Subject(s)
Genetic Testing , Genomic Medicine , Humans , United States , Comparative Genomic Hybridization , Computer SecurityABSTRACT
The remains of the heart tissue of Thaddeus Kosciuszko have been investigated as the possible cause of disease and death of the hero of Polish and American nations. Three specimens, DNA isolated from scrappings of wax surface, from the surface of a wooden plate, and from the linen cloth that have had contact with the object were subjected to nanosequencing. From the first two, among all reads identified, only one classified as Propionibacterium acnes (synonymous current name Cutibacterium acnes), had a purported clinical significance. The observed identity between the P. acnes sequences and reference was 89-90% consistent with the hypothesis that the identified reads represent the ancient P. acnes DNA (aDNA), which underwent fragmentation and sequence changes caused by its long-time presence in the environmental conditions conducive to degradation. We present a reasonable and entirely new hypothesis that the analyzed samples could reflect the presence of the bacteria in the original Kosciuszko's heart tissue and that the process of C. acnes infection was progressing inside the organ (endocarditis), not on its surface (pericarditis) leading to rapid deterioration of health and eventually death. We again point out that normal skin and mucosal membranes commensal, a causative agent of common skin acne, may be associated with various severe organ infections posing a threat to health and life.
Subject(s)
Endocarditis , Propionibacterium acnes , Humans , Cause of Death , PolandABSTRACT
We aimed to identify miRNAs and pathways specifically deregulated in adolescent and young adult (AYA) T-ALL patients. Small RNA-seq showed no major differences between AYA and pediatric T-ALL, but it revealed downregulation of miR-143-3p in T-ALL patients. Prediction algorithms identified several known and putative oncogenes targeted by this miRNA, including KRAS, FGF1, and FGF9. Pathway analysis indicated signaling pathways related to cell growth and proliferation, including FGFR signaling and PI3K-AKT signaling, with the majority of genes overrepresented in these pathways being predicted targets of hsa-miR-143-3p. By luciferase reporter assays, we validated direct interactions of this miRNA with KRAS, FGF1 and FGF9. In cell proliferation assays, we showed reduction of cell growth upon miR-143-3p overexpression in two T-ALL cell lines. Our study is the first description of the miRNA transcriptome in AYA T-ALL patients and the first report on tumor suppressor potential of miR-143-3p in T-ALL. Downregulation of this miRNA in T-ALL patients might contribute to enhanced growth and viability of leukemic cells. We also discuss the potential role of miR-143-3p in FGFR signaling. Although this requires more extensive validation, it might be an interesting direction, since FGFR inhibition proved promising in preclinical studies in various cancers.
Subject(s)
MicroRNAs , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Adolescent , Child , Fibroblast Growth Factor 1/metabolism , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins p21(ras)/genetics , RNA-Seq , Transcriptome , Young AdultABSTRACT
T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous and aggressive malignancy arising from T-cell precursors. MiRNAs are implicated in negative regulation of gene expression and when aberrantly expressed contribute to various cancer types, including T-ALL. Previously we demonstrated the oncogenic potential of miR-363-3p overexpression in a subgroup of T-ALL patients. Here, using combined proteomic and transcriptomic approaches, we show that miR-363-3p enhances cell growth of T-ALL in vitro via inhibition of PTPRC and SOCS2, which are implicated in repression of the JAK-STAT pathway. We propose that overexpression of miR-363-3p is a novel mechanism potentially contributing to overactivation of JAK-STAT pathway. Additionally, by combining the transcriptomic and methylation data of T-ALL patients, we show that promoter methylation may also contribute to downregulation of SOCS2 expression and thus potentially to JAK-STAT activation. In conclusion, we highlight aberrant miRNA expression and aberrant promoter methylation as mechanisms, alternative to mutations of JAK-STAT-related genes, which might lead to the upregulation of JAK-dependent signaling in T-ALL.
Subject(s)
MicroRNAs , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Cell Line, Tumor , Child , Humans , Janus Kinases/genetics , Leukocyte Common Antigens/metabolism , Methylation , MicroRNAs/genetics , MicroRNAs/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proteomics , STAT Transcription Factors/genetics , STAT Transcription Factors/metabolism , Signal Transduction , Suppressor of Cytokine Signaling Proteins/genetics , Suppressor of Cytokine Signaling Proteins/metabolismABSTRACT
miRNAs form a class of noncoding RNAs, involved in post-transcriptional regulation of gene expression, broadly studied for their involvement in physiological and pathological context. Inhibition of mature miRNA transcripts, commonly used in miRNA loss-of-function experiments, may not be specific in case of miRNAs with high sequence homology, e.g. miRNAs from the same seed family. Phenotypic effects of miRNA repression might be biased by the repression of highly similar miRNAs. Another challenge is simultaneous inhibition of multiple miRNAs encoded within policistronic clusters, potentially co-regulating common biological processes. To elucidate roles of miRNA clusters and miRNAs with high sequence homology, it is of key importance to selectively repress only the miRNAs of interest. Targeting miRNAs on genomic level with CRISPR/dCas9-based methods is an attractive alternative to blocking mature miRNAs. Yet, so far no clear guidelines on the design of CRISPR inhibition (CRISPRi) experiments, specifically for miRNA repression, have been proposed. To address this need, here we propose a strategy for effective inhibition of miRNAs and miRNA clusters using CRISPRi. We provide clues on how to approach the challenges in using CRISPR/dCas in miRNA studies, which include prediction of miRNA transcription start sites (TSSs) and the design of single guide RNAs (sgRNAs). The strategy implements three TSS prediction online tools, dedicated specifically for miRNAs: miRStart, FANTOM 5 miRNA atlas, DIANA-miRGen, and CRISPOR tool for sgRNAs design; it includes testing and selection of optimal sgRNAs. We demonstrate that compared to siRNA/shRNA-based miRNA silencing, CRISPRi improves the repression specificity for miRNAs with highly similar sequence and contribute to higher uniformity of the effects of silencing the whole miRNA clusters. This strategy may be adapted for CRISPR-mediated activation (CRISPRa) of miRNA expression.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , MicroRNAs , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Sequence Homology , Transcription Initiation SiteABSTRACT
Primary ciliary dyskinesia (PCD) is a ciliopathy caused by genetically determined impairment of motile cilia-organelles present on the surface of many types of cells [...].
Subject(s)
Ciliary Motility Disorders , Cilia , Ciliary Motility Disorders/genetics , Humans , MutationABSTRACT
Primary cultures of the human airway epithelium (AE) cells are an indispensable tool in studies of pathophysiology of genetic and environmental pulmonary diseases, including cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and chronic obstructive pulmonary disease (COPD). Air-liquid interface (ALI) culture is the best method to follow the differentiation of ciliated cells, whose dysfunction forms the basis of PCD. Here, we used custom-designed Taqman Low Density Array (TLDA), qRT-PCR-based assay, to analyze expression of 14 AE genes in cells from healthy donors, cultured in ALI settings using Pneumacult medium, with the focus on genes involved in cilia differentiation and in PCD pathogenesis. The results of TLDA assay were compared with the bulk RNAseq analysis, and placed in the cellular context using immunofluorescent staining (IF) of ALI cultured cells. Expression analysis revealed culture time-related upregulation of the majority of cilia-related genes, followed by the appearance of respective protein signals visualized by IF. Strong correlation of TLDA with RNAseq results indicated that TLDA assay is a reliable and scalable approach to analyze expression of selected genes specific for different AE cell types. Characterization of temporal and inter-donor changes in the expression of these genes, performed in healthy donors and in well-defined ALI/Pnemacult culture conditions, provides a useful reference relevant for a broad spectrum of functional studies where the in vitro AE differentiation is in focus.
Subject(s)
Cilia , Ciliopathies , Cell Differentiation , Cells, Cultured , Epithelial Cells , Epithelium , HumansABSTRACT
Based on a macroscopic analysis of the heart of Frederic Chopin performed in 2014, it can be stated with high probability that the composer suffered from a long lasting tuberculosis as a primary disease, which was the cause of progressive deterioration of his physical condition and numerous symptoms mainly from the respiratory tract. Tuberculous pericarditis rapidly progressing within a rather short period of time, a relatively rare complication of diffuse tuberculosis, might have been an immediate cause of death. This would aptly coincide with a startling opinion that in an autopsy picture the composer's heart had been more affected by the disease than the lungs.
Subject(s)
Famous Persons , Music , Tuberculosis , HumansABSTRACT
Distinct DNA methylation signatures, related to different prognosis, have been observed across many cancers, including T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological neoplasm. By global methylation analysis, two major phenotypes might be observed in T-ALL: hypermethylation related to better outcome and hypomethylation, which is a candidate marker of poor prognosis. Moreover, DNA methylation holds more than a clinical meaning. It reflects the replicative history of leukemic cells and most likely different mechanisms underlying leukemia development in these T-ALL subtypes. The elucidation of the mechanisms and aberrations specific to (epi-)genomic subtypes might pave the way towards predictive diagnostics and precision medicine in T-ALL. We present the current state of knowledge on the role of DNA methylation in T-ALL. We describe the involvement of DNA methylation in normal hematopoiesis and T-cell development, focusing on epigenetic aberrations contributing to this leukemia. We further review the research investigating distinct methylation phenotypes in T-ALL, related to different outcomes, pointing to the most recent research aimed to unravel the biological mechanisms behind differential methylation. We highlight how technological advancements facilitated broadening the perspective of the investigation into DNA methylation and how this has changed our understanding of the roles of this epigenetic modification in T-ALL.
Subject(s)
DNA Methylation , Epigenesis, Genetic , Gene Expression Regulation, Leukemic , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , T-Lymphocytes/pathology , Cell Differentiation/genetics , Cell Proliferation/genetics , Hematopoiesis/genetics , Humans , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/blood , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , PrognosisABSTRACT
To provide a comprehensive analysis of the SARS-CoV-2 sequence diversity in Poland in the European context. All publicly available (n = 115; GISAID database) whole-genome SARS-Cov-2 sequences from Polish samples, including those obtained during coronavirus testing performed in our COVID-19 Lab, were examined. Multiple sequence alignment of Polish isolates, phylogenetic analysis (ML tree), and multidimensional scaling (based on the pairwise DNA distances) were complemented by the comparison of the coronavirus clades frequency and diversity in the subset of over 5000 European GISAID sequences. Approximately seventy-seven percent of isolates in the European dataset carried frequent and ubiquitously found haplotypes; the remaining haplotype diversity was population-specific and resulted from population-specific mutations, homoplasies, and recombinations. Coronavirus strains circulating in Poland represented the variability found in other European countries. The prevalence of clades circulating in Poland was shifted in favor of GR, both in terms of the diversity (number of distinct haplotypes) and the frequency (number of isolates) of the clade. Polish-specific haplotypes were rare and could be explained by changes affecting common European strains. The analysis of the whole viral genomes allowed detection of several tight clusters of isolates, presumably reflecting local outbreaks. New mutations, homoplasies, and, to a smaller extent, recombinations increase SARS-CoV-2 haplotype diversity, but the majority of these variants do not increase in frequency and remains rare and population-specific. The spectrum of SARS-CoV-2 haplotypes in the Polish dataset reflects many independent transfers from a variety of sources, followed by many local outbreaks. The prevalence of the sequences belonging to the GR clade among Polish isolates is consistent with the European trend of the GR clade frequency increase.
Subject(s)
Genetic Variation , Genome, Viral , Phylogeny , SARS-CoV-2/genetics , Haplotypes , Humans , Mutation , Poland , RNA, Viral/geneticsABSTRACT
BACKGROUND: Epigenetics is one of the factors shaping natural variability observed among human populations. A small proportion of heritable inter-population differences are observed in the context of both the genome-wide methylation level and the methylation status of individual CpG sites. It has been demonstrated that a limited number of carefully selected differentially methylated sites may allow discrimination between main human populations. However, most of the few published results have been performed exclusively on B-lymphocyte cell lines. RESULTS: The goal of our study was to identify a set of CpG sites sufficient to discriminate between populations of European and Chinese ancestry based on the difference in the DNA methylation profile not only in cell lines but also in primary cell samples. The preliminary selection of CpG sites differentially methylated in these two populations (pop-CpGs) was based on the analysis of two groups of commercially available ethnically-specific B-lymphocyte cell lines, performed using Illumina Infinium Human Methylation 450 BeadChip Array. A subset of 10 pop-CpGs characterized by the best differentiating criteria (|Mdiff| > 1, q < 0.05; lack of the confounding genomic features), and 10 additional CpGs in their immediate vicinity, were further tested using pyrosequencing technology in both B-lymphocyte cell lines and in the primary samples of the peripheral blood representing two analyzed populations. To assess the population-discriminating potential of the selected set of CpGs (further referred to as "composite pop (CEU-CHB)-CpG marker"), three classification methods were applied. The predictive ability of the composite 8-site pop (CEU-CHB)-CpG marker was assessed using 10-fold cross-validation method on two independent sets of samples. CONCLUSIONS: Our results showed that less than 10 pop-CpG sites may distinguish populations of European and Chinese ancestry; importantly, this small composite pop-CpG marker performs well in both lymphoblastoid cell lines and in non-homogenous blood samples regardless of a gender.
Subject(s)
CpG Islands , DNA Methylation , Genetics, Population , Adult , Cell Line , China , Europe , Female , Genetics, Population/methods , Humans , Leukocytes, Mononuclear , MaleABSTRACT
Primary ciliary dyskinesia, a rare disease causing bronchiectasis, lacks a sound evidence base for treatment. @beatpcd proposes 1) forming a PCD European clinical trial network to address this situation and 2) conducting n-of-1 trials to access medication. https://bit.ly/3j5blfM.
ABSTRACT
There is an agreement about joint genetic and environmental background of musical reception and performance. Musical abilities tend to cluster in families. The studies done on a random population, twins and families of gifted musicians provided a strong support for genetic contribution. Modern biomolecular techniques exploring linkage analysis, variation of gene copy number, scanning for whole-genome expression helped to identify genes, or chromosome regions associated with musical aptitude. Some studies were focused on rare ability to recognize tone without reference that is known as a perfect pitch where a far ethnic differentiation was established. On the other hand, gene deletion leading to dysfunction in amusical individuals also indicated appropriate loci "by negation." The strongest support for an association of genes with musicality was provided for genes: AVPR1 (12q14.2), SLC6A4 (17q11.2), GALM (2p22), PCDH7 (4p15.1), GATA2 (3q21.3), and few others as well for 4q22, 4q23, and 8q13-21 chromosome bands.
Subject(s)
Music , Pitch Discrimination , Cadherins/genetics , Carbohydrate Epimerases/genetics , GATA2 Transcription Factor/genetics , Gene Dosage , Humans , Inheritance Patterns , Protocadherins , Receptors, Vasopressin/genetics , Serotonin Plasma Membrane Transport Proteins/genetics , Twin Studies as TopicABSTRACT
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy arising from T lymphocyte precursors. We have previously shown by miRNA-seq, that miRNAs from the mir-106a-363 cluster are overexpressed in pediatric T-ALL. In silico analysis indicated their potential involvement in the regulation of apoptosis. Here, we aimed to test the hypothesis on the pro-tumorigenic roles of these miRNAs in T-ALL cells in vitro. We demonstrate, for the first time, that hsa-miR-20b-5p and hsa-miR-363-3p from the mir-106a-363 cluster, when upregulated in T-ALL cells in vitro, protect leukemic cells from apoptosis, enhance proliferation, and contribute to growth advantage. We show, using dual luciferase reporter assays, Ago2-RNA immunoprecipitation, RT-qPCR, and Western blots, that the oncogenic effects of these upregulated miRNAs might, at least in part, be mediated by the downregulation of two important tumor suppressor genes, PTEN and BIM, targeted by both miRNAs. Additionally, we demonstrate the cooperative effects of these two miRNAs by simultaneous inhibition of both miRNAs as compared to the inhibition of single miRNAs. We postulate that hsa-miR-20b-5p and hsa-miR-363-3p from the mir-106a-363 cluster might serve as oncomiRs in T-ALL, by contributing to post-transcriptional repression of key tumor suppressors, PTEN and BIM.
Subject(s)
Bcl-2-Like Protein 11/genetics , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , MicroRNAs/metabolism , PTEN Phosphohydrolase/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Apoptosis/genetics , Bcl-2-Like Protein 11/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Down-Regulation/genetics , HEK293 Cells , Humans , MicroRNAs/genetics , PTEN Phosphohydrolase/metabolismABSTRACT
BACKGROUND: Primary ciliary dyskinesia (PCD) is a motile ciliopathy, whose symptoms include airway infections, male infertility and situs inversus. Apart from the typical forms of PCD, rare syndromic PCD forms exist. Mutations of the X-linked OFD1 gene cause several syndromic ciliopathies, including oral-facial-digital syndrome type 1, Joubert syndrome type 10 (JBTS10), and Simpson-Golabi-Behmel syndrome type 2, the latter causing the X-linked syndromic form of PCD. Neurological and skeletal symptoms are characteristic for these syndromes, with their severity depending on the location of the mutation within the gene. OBJECTIVES: To elucidate the role of motile cilia defects in the respiratory phenotype of PCD patients with C-terminal OFD1 mutations. METHODS: Whole-exome sequencing in a group of 120 Polish PCD patients, mutation screening of the OFD1 coding sequence, analysis of motile cilia, and magnetic resonance brain imaging. RESULTS: Four novel hemizygous OFD1 mutations, in exons 20 and 21, were found in men with a typical PCD presentation but without severe neurological, skeletal or renal symptoms characteristic for other OFD1-related syndromes. Magnetic resonance brain imaging in two patients did not show a molar tooth sign typical for JBTS10. Cilia in the respiratory epithelium were sparse, unusually long and displayed a defective motility pattern. CONCLUSION: Consistent with the literature, truncations of the C-terminal part of OFD1 (exons 16-22) almost invariably cause a respiratory phenotype (due to motile cilia defects) while their impact on the primary cilia function is limited. We suggest that exons 20-21 should be included in the panel for regular mutation screening in PCD.
Subject(s)
Ciliary Motility Disorders/genetics , Exons/genetics , Mutation/genetics , Proteins/genetics , Cerebellar Diseases/genetics , Cilia/genetics , Exome/genetics , Female , Genetic Diseases, X-Linked/genetics , Humans , Male , Muscle Hypotonia/genetics , Pedigree , PhenotypeABSTRACT
Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous hereditary disease from a class of ciliopathies. In spite of the recent progress, the genetic basis of PCD in one-third of patients remains unknown. In search for new genes and/or mutations, whole-exome sequencing was performed in 120 unrelated Polish patients with PCD, in whom no genetic cause of PCD was earlier identified. Among a number of pathogenic variants in PCD genes, mutations in CFAP300 (alias C11orf70) were detected. Extended screening in the whole Polish PCD cohort revealed the relatively high frequency (3.6%) of otherwise rare c.[198_200 del_insCC] variant, indicating that it should be included in population-specific genetic tests for PCD in Slavic populations. Immunofluorescence analysis of the respiratory epithelial cells from patients with CFAP300 mutations revealed the absence or aberrant localization of outer and inner dynein arm markers, consistent with transmission electron microscope images indicating the lack of both dynein arms. Interestingly, the disparate localization of DNAH5 and DNALI1 proteins in patients with CFAP300 mutations suggested differential mechanisms for the trafficking of preassembled outer and inner dynein arms to the axoneme. The profile of CFAP300 expression during ciliogenesis in suspension culture was consistent with its role in cilia assembly. Gene silencing experiments, performed in a model organism, Schmidtea mediterranea (flatworm), pointed to the conserved role of CFAP300 in ciliary function.
Subject(s)
Cilia/physiology , Ciliary Motility Disorders/genetics , Cytoskeletal Proteins/genetics , Dyneins/metabolism , Ethnicity/genetics , INDEL Mutation , Adolescent , Adult , Animals , Axoneme/metabolism , Axoneme/ultrastructure , Cell Movement , Child , Child, Preschool , Cilia/ultrastructure , Ciliary Motility Disorders/ethnology , Conserved Sequence , Cytoskeletal Proteins/physiology , Female , Helminth Proteins/genetics , Humans , Infant , Locomotion , Male , Poland , Protein Transport , RNA Interference , Exome Sequencing , Young Adult , Zebrafish Proteins/geneticsABSTRACT
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy originating from T-cell precursors. The genetic landscape of T-ALL has been largely characterized by next-generation sequencing. Yet, the transcriptome of miRNAs (miRNome) of T-ALL has been less extensively studied. Using small RNA sequencing, we characterized the miRNome of 34 pediatric T-ALL samples, including the expression of isomiRs and the identification of candidate novel miRNAs (not previously annotated in miRBase). For the first time, we show that immunophenotypic subtypes of T-ALL present different miRNA expression profiles. To extend miRNome characteristics in T-ALL (to 82â¯T-ALL cases), we combined our small RNA-seq results with data available in Gene Expression Omnibus. We report on miRNAs most abundantly expressed in pediatric T-ALL and miRNAs differentially expressed in T-ALL versus normal mature T-lymphocytes and thymocytes, representing candidate oncogenic and tumor suppressor miRNAs. Using eight target prediction algorithms and pathway enrichment analysis, we identified differentially expressed miRNAs and their predicted targets implicated in processes (defined in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) of potential importance in pathogenesis of T-ALL, including interleukin-6-mediated signaling, mTOR signaling, and regulation of apoptosis. We finally focused on hsa-mir-106a-363 cluster and functionally validated direct interactions of hsa-miR-20b-5p and hsa-miR-363-3p with 3' untranslated regions of their predicted targets (PTEN, SOS1, LATS2), overrepresented in regulation of apoptosis. hsa-mir-106a-363 is a paralogue of prototypic oncogenic hsa-mir-17-92 cluster with yet unestablished role in the pathogenesis of T-ALL. Our study provides a firm basis and data resource for functional analyses on the role of miRNA-mRNA interactions in T-ALL.
Subject(s)
Gene Expression Regulation, Leukemic , MicroRNAs/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , RNA Interference , RNA, Messenger/genetics , Transcriptome , Apoptosis/genetics , Computational Biology/methods , Gene Expression Profiling , Genes, Reporter , Immunophenotyping , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/diagnosisSubject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , PTEN Phosphohydrolase/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Adolescent , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Asparaginase/administration & dosage , Asparaginase/adverse effects , Child , Child, Preschool , Daunorubicin/administration & dosage , Daunorubicin/adverse effects , Disease-Free Survival , Female , Humans , Infant , Male , PTEN Phosphohydrolase/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Predictive Value of Tests , Prednisone/administration & dosage , Prednisone/adverse effects , Survival Rate , Vincristine/administration & dosage , Vincristine/adverse effectsABSTRACT
Optimal endogenous controls enable reliable normalization of microRNA (miRNA) expression in reverse-transcription quantitative PCR (RT-qPCR). This is particularly important when miRNAs are considered as candidate diagnostic or prognostic biomarkers. Universal endogenous controls are lacking, thus candidate normalizers must be evaluated individually for each experiment. Here we present a strategy that we applied to the identification of optimal control miRNAs for RT-qPCR profiling of miRNA expression in T-cell acute lymphoblastic leukemia (T-ALL) and in normal cells of T-lineage. First, using NormFinder for an iterative analysis of miRNA stability in our miRNA-seq data, we established the number of control miRNAs to be used in RT-qPCR. Then, we identified optimal control miRNAs by a comprehensive analysis of miRNA stability in miRNA-seq data and in RT-qPCR by analysis of RT-qPCR amplification efficiency and expression across a variety of T-lineage samples and T-ALL cell line culture conditions. We then showed the utility of the combination of three miRNAs as endogenous normalizers (hsa-miR-16-5p, hsa-miR-25-3p, and hsa-let-7a-5p). These miRNAs might serve as first-line candidate endogenous controls for RT-qPCR analysis of miRNAs in different types of T-lineage samples: T-ALL patient samples, T-ALL cell lines, normal immature thymocytes, and mature T-lymphocytes. The strategy we present is universal and can be transferred to other RT-qPCR experiments.
Subject(s)
Gene Expression Regulation, Neoplastic , MicroRNAs/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Algorithms , Cell Line, Tumor , Humans , Jurkat Cells , RNA Stability , Real-Time Polymerase Chain ReactionABSTRACT
Chopin's heart, generally enlarged, presented morphologic features pathognomonic for fibrinous pericarditis presumably of tubercular origin: multiple nodular hyalinization foci-tuberculomas and fibrillary coating covering the whole surface of pericardium ("frosted heart"). We show that these features differ significantly from post mortem-formed inorganic crystalline deposits, mold colonies, or fat deposits known from various preserved anatomical objects stored for a long period of time. In our opinion, these pathologies fully justify the claim that chronic cavernous pulmonary, laryngeal, and intestinal tuberculosis presents itself as the most plausible diagnosis of Frederic Chopin and that rapidly progressing tubercular pericarditis became the immediate cause of his death.