RESUMEN
Hereditary sensory neuropathy type I (HSN I) is an axonal form of autosomal-dominant hereditary motor and sensory neuropathy distinguished by prominent sensory loss that leads to painless injuries. Unrecognized, these can result in delayed wound healing and osteomyelitis, necessitating distal amputations. To elucidate the genetic basis of an HSN I subtype in a family in which mutations in the few known HSN I genes had been excluded, we employed massive parallel exon sequencing of the 14.3 Mb disease interval on chromosome 14q. We detected a missense mutation (c.1065C>A, p.Asn355Lys) in atlastin-1 (ATL1), a gene that is known to be mutated in early-onset hereditary spastic paraplegia SPG3A and that encodes the large dynamin-related GTPase atlastin-1. The mutant protein exhibited reduced GTPase activity and prominently disrupted ER network morphology when expressed in COS7 cells, strongly supporting pathogenicity. An expanded screen in 115 additional HSN I patients identified two further dominant ATL1 mutations (c.196G>C [p.Glu66Gln] and c.976 delG [p.Val326TrpfsX8]). This study highlights an unexpected major role for atlastin-1 in the function of sensory neurons and identifies HSN I and SPG3A as allelic disorders.
Asunto(s)
GTP Fosfohidrolasas/genética , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Cromosomas Humanos Par 14/genética , Retículo Endoplásmico/enzimología , Exones , Femenino , Proteínas de Unión al GTP , Genes Dominantes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Proteínas de la Membrana , Datos de Secuencia Molecular , Mutación , Mutación Missense , Análisis de Secuencia de ADN , Paraplejía Espástica Hereditaria/genéticaRESUMEN
BACKGROUND: Hypoxia-induced genes are potential targets in cancer therapy. Responses to hypoxia have been extensively studied in vitro, however, they may differ in vivo due to the specific tumor microenvironment. In this study gene expression profiles were obtained from fresh human lung cancer tissue fragments cultured ex vivo under different oxygen concentrations in order to study responses to hypoxia in a model that mimics human lung cancer in vivo. METHODS: Non-small cell lung cancer (NSCLC) fragments from altogether 70 patients were maintained ex vivo in normoxia or hypoxia in short-term culture. Viability, apoptosis rates and tissue hypoxia were assessed. Gene expression profiles were studied using Affymetrix GeneChip 1.0 ST microarrays. RESULTS: Apoptosis rates were comparable in normoxia and hypoxia despite different oxygenation levels, suggesting adaptation of tumor cells to hypoxia. Gene expression profiles in hypoxic compared to normoxic fragments largely overlapped with published hypoxia-signatures. While most of these genes were up-regulated by hypoxia also in NSCLC cell lines, membrane metallo-endopeptidase (MME, neprilysin, CD10) expression was not increased in hypoxia in NSCLC cell lines, but in carcinoma-associated fibroblasts isolated from non-small cell lung cancers. High MME expression was significantly associated with poor overall survival in 342 NSCLC patients in a meta-analysis of published microarray datasets. CONCLUSIONS: The novel ex vivo model allowed for the first time to analyze hypoxia-regulated gene expression in preserved human lung cancer tissue. Gene expression profiles in human hypoxic lung cancer tissue overlapped with hypoxia-signatures from cancer cell lines, however, the elastase MME was identified as a novel hypoxia-induced gene in lung cancer. Due to the lack of hypoxia effects on MME expression in NSCLC cell lines in contrast to carcinoma-associated fibroblasts, a direct up-regulation of stroma fibroblast MME expression under hypoxia might contribute to enhanced aggressiveness of hypoxic cancers.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Fibroblastos/enzimología , Neoplasias Pulmonares/enzimología , Neprilisina/metabolismo , Células del Estroma/enzimología , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Hipoxia de la Célula , Línea Celular Tumoral , Supervivencia Celular , Fibroblastos/patología , Perfilación de la Expresión Génica/métodos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Neprilisina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Células del Estroma/patología , Técnicas de Cultivo de Tejidos , Regulación hacia ArribaRESUMEN
Eukaryote genomes contain multiple copies of nuclear ribosomal DNA (nrDNA) harboring both highly conserved and variable regions. This has made nrDNA the most popular genetic marker for phylogenetic studies and the region of choice for barcoding projects. Furthermore, many scientists believe that all copies of nrDNA within one nucleus are practically identical due to concerted evolution. Here, we investigate the model plant species Arabidopsis thaliana for intragenomic variation of the internal transcribed spacer (ITS) region of nrDNA. Based on a modified deep sequencing approach, we provide a comprehensive list of ITS polymorphisms present in the two most widely used accessions of A. thaliana-Col-0 and Ler. Interestingly, we found that some polymorphisms are shared between these genetically very distinct accessions. On the other hand, the high number of accession-specific polymorphisms shows that each accession can be clearly and easily characterized by its specific ITS polymorphism patterns and haplotypes. Network analysis based on the detected haplotypes demonstrates that the study of ITS polymorphism patterns and haplotypes is an extremely powerful tool for population genetics. Using the methods proposed here, it will now be possible to extend the traditionally species-bound barcoding concept to populations.
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Arabidopsis/genética , Código de Barras del ADN Taxonómico , ADN Espaciador Ribosómico , Haplotipos , Arabidopsis/clasificación , Secuencia de Bases , Mutación INDEL , Datos de Secuencia Molecular , Filogenia , Polimorfismo GenéticoRESUMEN
BACKGROUND & AIMS: The liver controls central processes of lipid and bile acid homeostasis. We aimed to investigate whether alterations in lipid metabolism contribute to the pathogenesis of chronic cholestatic liver disease in mice. METHODS: We used microarray and metabolic profiling analyses to identify alterations in systemic and hepatic lipid metabolism in mice with disruption of the gene ATP-binding cassette sub-family B member 4 (Abcb4(-/-) mice), a model of inflammation-induced cholestatic liver injury, fibrosis, and cancer. RESULTS: Alterations in Abcb4(-/-) mice, compared with wild-type mice, included deregulation of genes that control lipid synthesis, storage, and oxidation; decreased serum levels of cholesterol and phospholipids; and reduced hepatic long-chain fatty acyl-CoAs (LCA-CoA). Feeding Abcb4(-/-) mice the side chain-modified bile acid 24-norursodeoxycholic acid (norUDCA) reversed their liver injury and fibrosis, increased serum levels of lipids, lowered phospholipase and triglyceride hydrolase activities, and restored hepatic LCA-CoA and triglyceride levels. Additional genetic and nutritional studies indicated that lipid metabolism contributed to chronic cholestatic liver injury; crossing peroxisome proliferator-activated receptor (PPAR)-α-deficient mice with Abcb4(-/-) mice (to create double knockouts) or placing Abcb4(-/-) mice on a high-fat diet protected against liver injury, with features similar to those involved in the response to norUDCA. Placing pregnant Abcb4(-/-) mice on high-fat diets prevented liver injury in their offspring. However, fenofibrate, an activator of PPARα, aggravated liver injury in Abcb4(-/-) mice. CONCLUSIONS: Alterations in lipid metabolism contribute to the pathogenesis and progression of cholestatic liver disease in mice.
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Proliferación Celular , Colestasis Intrahepática/metabolismo , Hepatitis/metabolismo , Metabolismo de los Lípidos , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Ácidos y Sales Biliares/metabolismo , Ácidos y Sales Biliares/farmacología , Colestasis Intrahepática/tratamiento farmacológico , Colestasis Intrahepática/genética , Colestasis Intrahepática/patología , Enfermedad Crónica , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ácidos Grasos/metabolismo , Femenino , Fenofibrato/farmacología , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Hepatitis/tratamiento farmacológico , Hepatitis/genética , Hepatitis/patología , Hipolipemiantes/farmacología , Metabolismo de los Lípidos/genética , Hígado/efectos de los fármacos , Hígado/patología , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Metabolómica , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , PPAR gamma/deficiencia , PPAR gamma/genética , Embarazo , Efectos Tardíos de la Exposición Prenatal , Triglicéridos/metabolismo , Ácido Ursodesoxicólico/análogos & derivados , Ácido Ursodesoxicólico/farmacología , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND: Reports on common mutations in neuroendocrine tumors (NET) are rare and clonality of NET metastases has not been investigated in this tumor entity yet. We selected one NET and the corresponding lymph node and liver metastases as well as the derivative cell lines to screen for somatic mutations in the primary NET and to track the fate of genetic changes during metastasis and in vitro progression. RESULTS: Applying microarray based sequence capture resequencing including 4,935 Exons from of 203 cancer-associated genes and high-resolution copy number and genotype analysis identified multiple somatic mutations in the primary NET, affecting BRCA2, CTNNB1, ERCC5, HNF1A, KIT, MLL, RB1, ROS1, SMAD4, and TP53. All mutations were confirmed in the patients' lymph node and liver metastasis tissue as well as early cell line passages. In contrast to the tumor derived cell line, higher passages of the metastases derived cell lines lacked somatic mutations and chromosomal alterations, while expression of the classical NET marker serotonin was maintained. CONCLUSION: Our study reveals that both metastases have evolved from the same pair of genetically differing NET cell clones. In both metastases, the in vivo dominating "mutant" tumor cell clone has undergone negative selection in vitro being replaced by the "non-mutant" tumor cell population. This is the first report of a bi-clonal origin of NET derived metastases, indicating selective advantage of interclonal cooperation during metastasis. In addition, this study underscores the importance to monitor cell line integrity using high-resolution genome analysis tools.
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Neoplasias Hepáticas/genética , Metástasis Linfática/genética , Tumores Neuroendocrinos/genética , Línea Celular Tumoral , Cromosomas/genética , Cromosomas/metabolismo , Variaciones en el Número de Copia de ADN , Exones , Genotipo , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Mutación , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Serotonina/genética , Serotonina/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Inherited disorders characterized by motor neuron loss and muscle weakness are genetically heterogeneous. The recent identification of mutations in the gene encoding transient receptor potential vanilloid 4 (TRPV4) in distal spinal muscular atrophy (dSMA) prompted us to screen for TRPV4 mutations in a small group of children with compatible phenotype. In a girl with dSMA and vocal cord paralysis, we detected a new variant (p.P97R) localized in the cytosolic N-terminus of the TRPV4 protein, upstream of the ankyrin-repeat domain, where the great majority of disease-associated mutations reside. In another child with congenital dSMA, in this case associated with bone abnormalities, we detected a previously reported mutation (p.R232C). Functional analysis of the novel p.P97R mutation in a heterologous system demonstrated a loss-of-function mechanism. Protein localization studies in muscle, skin, and cultured skin fibroblasts from both patients showed normal protein expression. No TRPV4 mutations were detected in four children with dSMA without bone or vocal cord involvement. Adding to the clinical and molecular heterogeneity of TRPV4-associated diseases, our results suggest that molecular testing of the TRPV4 gene is warranted in cases of congenital dSMA with bone abnormalities and vocal cord paralysis.
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Atrofia Muscular Espinal/genética , Mutación , Canales Catiónicos TRPV/genética , Adulto , Niño , Preescolar , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Variación Genética , Genotipo , Humanos , Intrones , Masculino , Atrofia Muscular Espinal/congénito , Linaje , Fenotipo , Parálisis de los Pliegues Vocales/patologíaRESUMEN
To identify the disease-causing gene responsible for an autosomal dominantly inherited Charcot-Marie-Tooth neuropathy subtype in a family excluded for mutations in the common Charcot-Marie-Tooth genes, we used array-based sequence capture to simultaneously analyse the disease-linked protein coding exome at chromosome 14q32. A missense mutation in fibulin-5, encoding a widely expressed constituent of the extracellular matrix that has an essential role in elastic fibre assembly and has been shown to cause cutis laxa, was detected as the only novel non-synonymous sequence variant within the disease interval. Screening of 112 index probands with unclassified Charcot-Marie-Tooth neuropathies detected two further fibulin-5 missense mutations in two families with Charcot-Marie-Tooth disease and hyperextensible skin. Since fibulin-5 mutations have been described in patients with age-related macular degeneration, an additional 300 probands with exudative age-related macular degeneration were included in this study. Two further fibulin-5 missense mutations were identified in six patients. A mild to severe peripheral neuropathy was detected in the majority of patients with age-related macular degeneration carrying mutations in fibulin-5. This study identifies fibulin-5 as a gene involved in Charcot-Marie-Tooth neuropathies and reveals heterozygous fibulin-5 mutations in 2% of our patients with age-related macular degeneration. Furthermore, it adumbrates a new syndrome by linking concurrent pathologic alterations affecting peripheral nerves, eyes and skin to mutations in the fibulin-5 gene.
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Enfermedad de Charcot-Marie-Tooth/genética , Proteínas de la Matriz Extracelular/genética , Predisposición Genética a la Enfermedad , Degeneración Macular/genética , Mutación Missense/genética , Enfermedades Cutáneas Genéticas/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Enfermedad de Charcot-Marie-Tooth/complicaciones , Enfermedad de Charcot-Marie-Tooth/patología , Biología Computacional , Análisis Mutacional de ADN/métodos , Evolución Molecular , Salud de la Familia , Femenino , Humanos , Desequilibrio de Ligamiento , Degeneración Macular/complicaciones , Degeneración Macular/patología , Masculino , Persona de Mediana Edad , Músculos/patología , Conducción Nerviosa/genética , Piel/patología , Enfermedades Cutáneas Genéticas/complicaciones , Enfermedades Cutáneas Genéticas/patología , Adulto JovenRESUMEN
In lung transplant patients undergoing immunosuppression, more than one human cytomegalovirus (HCMV) genotype may emerge during follow-up, and this could be critical for the outcome of HCMV infection. Up to now, many cases of infection with multiple HCMV genotypes were probably overlooked due to the limitations of the current genotyping approaches. We have now analyzed mixed-genotype infections in 17 clinical samples from 9 lung transplant patients using the highly sensitive ultradeep-pyrosequencing (UDPS) technology. UDPS genotyping was performed at three variable HCMV genes, coding for glycoprotein N (gN), glycoprotein O (gO), and UL139. Simultaneous analysis of a mean of 10,430 sequence reads per amplicon allowed the relative amounts of distinct genotypes in the samples to be determined down to 0.1% to 1% abundance. Complex mixtures of up to six different HCMV genotypes per sample were observed. In all samples, no more than two major genotypes accounted for at least 88% of the HCMV DNA load, and these were often accompanied by up to four low-abundance genotypes at frequencies of 0.1% to 8.6%. No evidence for the emergence of new genotypes or sequence changes over time was observed. However, analysis of different samples withdrawn from the same patients at different time points revealed that the relative levels of replication of the individual HCMV genotypes changed within a mixed-genotype population upon reemergence of the virus. Our data show for the first time that, similar to what has been hypothesized for the murine model, HCMV reactivation in humans seems to occur stochastically.
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Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Trasplante de Pulmón , Análisis de Secuencia de ADN , Animales , Secuencia de Bases , Citomegalovirus/clasificación , Infecciones por Citomegalovirus/etiología , ADN Viral/genética , Genotipo , Humanos , Trasplante de Pulmón/efectos adversos , Ratones , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN/métodos , Factores de Tiempo , Carga ViralRESUMEN
BACKGROUND: Ventricular tachycardia (VT) is a major cause of sudden cardiac death (SCD). Clinical investigations can sometimes fail to identify the underlying cause of VT and the event is classified as idiopathic (iVT). VT contributes significantly to the morbidity and mortality in patients with coronary artery disease (CAD) and dilated cardiomyopathy (DCM). Since mutations in arrhythmia-associated genes frequently determine arrhythmia susceptibility screening for disease-predisposing variants could improve VT diagnostics and prevent SCD in patients. METHODS: Ninety-two patients diagnosed with coronary heart disease (CHD), DCM, or iVT were included in our study. We evaluated genetic profiles and variants in known cardiac risk genes by targeted next generation sequencing (NGS) using a newly designed custom panel of 96 genes. We hypothesized that shared morphological and phenotypical features among these subgroups may have an overlapping molecular base. To our knowledge, this was the first study of the deep sequencing of 96 targeted cardiac genes in Kazakhstan. The clinical significance of the sequence variants was interpreted according to the guidelines developed by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015. The ClinVar and Varsome databases were used to determine the variant classifications. RESULTS: Targeted sequencing and stepwise filtering of the annotated variants identified a total of 307 unique variants in 74 genes, totally 456 variants in the overall study group. We found 168 mutations listed in the Human Genome Mutation Database (HGMD) and another 256 rare/unique variants with elevated pathogenic potential. There was a predominance of high- to intermediate pathogenicity variants in LAMA2, MYBPC3, MYH6, KCNQ1, GAA, and DSG2 in CHD VT patients. Similar frequencies were observed in DCM VT, and iVT patients, pointing to a common molecular disease association. TTN, GAA, LAMA2, and MYBPC3 contained the most variants in the three subgroups which confirm the impact of these genes in the complex pathogenesis of cardiomyopathies and VT. The classification of 307 variants according to ACMG guidelines showed that nine (2.9%) variants could be classified as pathogenic, nine (2.9%) were likely pathogenic, 98 (31.9%) were of uncertain significance, 73 (23.8%) were likely benign, and 118 (38.4%) were benign. CHD VT patients carry rare genetic variants with increased pathogenic potential at a comparable frequency to DCM VT and iVT patients in genes related to sarcomere function, nuclear function, ion flux, and metabolism. CONCLUSIONS: In this study we showed that in patients with VT secondary to coronary artery disease, DCM, or idiopathic etiology multiple rare mutations and clinically significant sequence variants in classic cardiac risk genes associated with cardiac channelopathies and cardiomyopathies were found in a similar pattern and at a comparable frequency.
RESUMEN
BACKGROUND: Octamer-binding factor 6 (Oct-6, Pou3f1, SCIP, Tst-1) is a transcription factor of the Pit-Oct-Unc (POU) family. POU proteins regulate key developmental processes and have been identified from a diverse range of species. Oct-6 expression is described to be confined to the developing brain, Schwann cells, oligodendrocyte precursors, testes, and skin. Its function is primarily characterised in Schwann cells, where it is required for correctly timed transition to the myelinating state. In the present study, we report that Oct-6 is an interferon (IFN)-inducible protein and show for the first time expression in murine fibroblasts and macrophages. RESULTS: Oct-6 was induced by type I and type II IFN, but not by interleukin-6. Induction of Oct-6 after IFNbeta treatment was mainly dependent on signal transducer and activator of transcription 1 (Stat1) and partially on tyrosine kinase 2 (Tyk2). Chromatin immunopreciptitation experiments revealed binding of Stat1 to the Oct-6 promoter in a region around 500 bp upstream of the transcription start site, a region different from the downstream regulatory element involved in Schwann cell-specific Oct-6 expression. Oct-6 was also induced by dsRNA treatment and during viral infections, in both cases via autocrine/paracrine actions of IFNalpha/beta. Using microarray and RT-qPCR, we furthermore show that Oct-6 is involved in the regulation of transcriptional responses to dsRNA, in particular in the gene regulation of serine/threonine protein kinase 40 (Stk40) and U7 snRNA-associated Sm-like protein Lsm10 (Lsm10). CONCLUSION: Our data show that Oct-6 expression is not as restricted as previously assumed. Induction of Oct-6 by IFNs and viruses in at least two different cell types, and involvement of Oct-6 in gene regulation after dsRNA treatment, suggest novel functions of Oct-6 in innate immune responses.
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Fibroblastos/metabolismo , Macrófagos/metabolismo , Factor 6 de Transcripción de Unión a Octámeros/metabolismo , Virosis/metabolismo , Animales , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibroblastos/virología , Inmunidad Innata/genética , Interferón beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/patología , Macrófagos/virología , Ratones , Ratones Noqueados , Análisis por Micromatrices , Morfogénesis/genética , Factor 6 de Transcripción de Unión a Octámeros/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , ARN Bicatenario/farmacología , ARN Viral/farmacología , Ribonucleoproteína Nuclear Pequeña U7/biosíntesis , Ribonucleoproteína Nuclear Pequeña U7/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Activación Transcripcional/efectos de los fármacos , Virosis/genética , Virosis/inmunologíaRESUMEN
BACKGROUND: Research on mesenchymal stromal cells has created high expectations for a variety of therapeutic applications. Extensive propagation to yield enough mesenchymal stromal cells for therapy may result in replicative senescence and thus hamper long-term functionality in vivo. Highly variable proliferation rates of mesenchymal stromal cells in the course of long-term expansions under varying culture conditions may already indicate different propensity for cellular senescence. We hypothesized that senescence-associated regulated genes differ in mesenchymal stromal cells propagated under different culture conditions. DESIGN AND METHODS: Human bone marrow-derived mesenchymal stromal cells were cultured either by serial passaging or by a two-step protocol in three different growth conditions. Culture media were supplemented with either fetal bovine serum in varying concentrations or pooled human platelet lysate. RESULTS: All mesenchymal stromal cell preparations revealed significant gene expression changes upon long-term culture. Especially genes involved in cell differentiation, apoptosis and cell death were up-regulated, whereas genes involved in mitosis and proliferation were down-regulated. Furthermore, overlapping senescence-associated gene expression changes were found in all mesenchymal stromal cell preparations. CONCLUSIONS: Long-term cell growth induced similar gene expression changes in mesenchymal stromal cells independently of isolation and expansion conditions. In advance of therapeutic application, this panel of genes might offer a feasible approach to assessing mesenchymal stromal cell quality with regard to the state of replicative senescence.
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Células de la Médula Ósea/metabolismo , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Senescencia Celular/fisiología , Regulación de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
BRAF V600E is the predominantly occurring mutation of the cytoplasmic kinase BRAF, and, in colorectal cancer, its determination provides a diagnostic exclusion criterion for hereditary nonpolyposis colorectal cancer. The aim of our study was to develop a sensitive BRAF V600E high resolution melting (HRM) assay. We first established and optimized the BRAF HRM assay using a cell line dilution model, enabling us to detect 1% mutant DNA in a background of wild-type DNA. In a comparison, DNA sequencing and real-time allele-specific PCR in the cell line dilution model HRM assay proved to be more sensitive than DNA sequencing and denaturing high performance liquid chromatography, retaining the same sensitivity as real-time allele-specific PCR. In a learning set of 13 patients with known BRAF V600 status, the mutation was detected with high concordance by all four methods. Finally, we validated the HRM assay on 60 formalin-fixed, paraffin-embedded colorectal cancer samples. Although all mutated samples were correctly identified by HRM, the detection limit of the HRM assay decreased when using low-quality DNA derived from formalin-fixed, paraffin-embedded samples. In conclusion, HRM analysis is a powerful diagnostic tool for detection of BRAF V600E mutation with a high sensitivity and high-throughput capability. Despite the expected decrease in sensitivity, HRM can reliably be applied in archival formalin-fixed, paraffin-embedded samples tissues.
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Neoplasias Colorrectales/diagnóstico , Análisis Mutacional de ADN/métodos , Técnicas de Diagnóstico Molecular/métodos , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Formaldehído/química , Humanos , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Fijación del Tejido , Temperatura de TransiciónRESUMEN
In previous experiments, we observed signs of cardiac failure in mice overexpressing lipoprotein lipase (LPL) under the control of a muscle specific promotor and in peroxisome proliferators activated receptor alpha (PPARalpha) knockout mice overexpressing LPL under the control of the same promotor. In our current investigations, we focussed on morphological consequences and changes in mRNA and protein expression in hearts from these animals. mRNA expression was analysed by differential display analysis and Northern blot as well as by cDNA microarray analysis followed by pathway analysis. Protein expression was examined using immunoblot and immunohistochemistry. Fibrosis was determined by chromotrope aniline blue staining for collagen. A distinct increase in the expression of alpha-tubulin mRNA was noted in hearts of all mutant mouse strains compared with the control. This result was paralleled by increased alpha-tubulin protein expression. Using cDNA microarray analysis, we detected an activation of apoptosis, in particular an increase of caspase-3 expression in hearts of mice overexpressing LPL but not in PPARalpha knockout mice overexpressing LPL. This finding was confirmed immunohistochemically. In addition, we identified a distinct interstitial increase in collagen and an increase around blood vessels. In our mouse model, we detect mRNA and protein changes typical for cardiomyopathy even before overt clinical signs of heart failure. In addition, a small but distinct increase in the rate of apoptosis of cardiomyocytes and fibrotic changes contributes to cardiac failure in mice overexpressing LPL, whereas additional deficiency in PPARalpha seems to protect hearts from these effects.
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Apoptosis , Cardiomiopatías/complicaciones , Insuficiencia Cardíaca/etiología , Animales , Northern Blotting/métodos , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Fibrosis , Expresión Génica , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Lipoproteína Lipasa/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Miocardio/patología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , PPAR alfa/deficiencia , ARN Mensajero/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoAsunto(s)
COVID-19 , SARS-CoV-2 , Estudios de Cohortes , Humanos , Inmunidad , Reacción en Cadena de la PolimerasaRESUMEN
Recurrent disease following high-dose chemotherapy is a major problem in patients with acute myeloid leukemia (AML). To identify its characteristics, we performed expression profiling in blasts from untreated AML and relapse, using a specific cDNA microarray comprising 4128 genes generated by cDNA subtraction supplemented with cancer-associated genes. Expression analysis of 18 AML bone marrow specimens showed that recurrent AML is commonly associated with the mRNA expression changes in a set of 58 genes. Increased cellular proliferation was indicated by the overexpression of the transferrin receptor, proliferating cell nuclear antigen, and G1 cyclins. An immunohistochemical study for Ki-67-positive blasts in 18 paired bone marrow biopsy samples confirmed a highly significant (P<0.0001) increase in the proliferation fraction at relapse. In addition, we found enhanced activation of the RAF/MEK/ERK cascade as mRNAs of MKP-1, c-jun, c-fos, and egr-1 were significantly increased at relapse. Immunohistochemistry and immunoblotting analyses for biphosphorylated ERK1/2 protein provide additional evidence for enhanced activation of the RAF/MEK/ERK pathway. The degree of increase is significantly correlated with the increased proliferation. Furthermore, the genes identified provide a rationale for further studies on predictive diagnosis and therapeutic intervention.
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División Celular , Expresión Génica , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Enfermedad Aguda , Adulto , Anciano , Western Blotting , ADN Complementario , Humanos , Inmunohistoquímica , Leucemia Mieloide/enzimología , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Channelopathies, caused by disturbed potassium or calcium ion management in cardiac myocytes are a major cause of heart failure and sudden cardiac death worldwide. The human ryanodine receptor 2 (RYR2) is one of the key players tightly regulating calcium efflux from the sarcoplasmic reticulum to the cytosol and found frequently mutated (<60%) in context of catecholaminergic polymorphic ventricular tachycardia (CPVT1). We tested 35 Kazakhstani patients with episodes of ventricular arrhythmia, two of those with classical CPVT characteristics and 33 patients with monomorphic idiopathic ventricular arrhythmia, for variants in the hot-spot regions of the RYR2 gene. This approach revealed two novel variants; one de-novo RYR2 mutation (c13892A>T; p.D4631V) in a CPVT patient and a novel rare variant (c5428G>C; p.V1810L) of uncertain significance in a patient with VT of idiopathic origin which we suggest represents a low-penetrance or susceptibility variant. In addition we identified a known variant previously associated with arrhythmogenic right ventricular dysplasia type2 (ARVD2). Combining sets of prediction scores and reference databases appeared fundamental to predict the pathogenic potential of novel and rare missense variants in populations where genotype data are rare.
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Mutación Missense , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Adulto , Animales , Secuencia de Bases , Estudios de Cohortes , Electrocardiografía , Femenino , Expresión Génica , Humanos , Kazajstán , Masculino , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Taquicardia Ventricular/fisiopatologíaRESUMEN
Reference genes (RGs) with uniform expression are used for normalization of reverse transcription quantitative PCR (RT-qPCR) data. Their optimization for a specific biological context, e.g. a specific tissue, has been increasingly considered. In this article, we compare RGs identified by expression data meta-analysis restricted to the context tissue, the jejunum of Mus musculus domesticus, i) to traditional RGs, ii) to expressed interspersed repeated DNA elements, and iii) to RGs identified by meta-analysis of expression data from diverse tissues and conditions. To select the set of candidate RGs, we developed a novel protocol for the cross-platform meta-analysis of microarray data. The expression stability of twenty-four putative RGs was analysed by RT-qPCR in at least 14 jejunum samples of the mouse strains C57Bl/6N, CD1, and OF1. Across strains, the levels of expression of the novel RGs Plekha7, Zfx, and Ube2v1 as well as of Oaz1 varied less than two-fold irrespective of genotype, sex or their combination. The gene set consisting of Plekha7 and Oaz1 showed superior expression stability analysed with the tool RefFinder. The novel RGs are functionally diverse. This facilitates expression studies over a wide range of conditions. The highly uniform expression of the optimized RGs in the jejunum points towards their involvement in tightly regulated pathways in this tissue. We also applied our novel protocol of cross-microarray platform meta-analysis to the identification of RGs in the duodenum, the ileum and the entire small intestine. The selection of RGs with improved expression stability in a specific biological context can reduce the number of RGs for the normalization step of RT-qPCR expression analysis, thus reducing the number of samples and experimental costs.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Yeyuno/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Algoritmos , Animales , Femenino , Perfilación de la Expresión Génica/normas , Perfilación de la Expresión Génica/estadística & datos numéricos , Redes Reguladoras de Genes , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Metaanálisis como Asunto , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Proteínas/genética , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Análisis de Secuencia de ADN , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Chordomas are rare mesenchymal tumors occurring exclusively in the midline from clivus to sacrum. Early tumor detection is extremely important as these tumors are resistant to chemotherapy and irradiation. Despite continuous research efforts surgical excision remains the main treatment option. Because of the often challenging anatomic location early detection is important to enable complete tumor resection and to reduce the high incidence of local recurrences. The aim of this study was to explore whether DNA methylation, a well known epigenetic marker, may play a role in chordoma development and if hypermethylation of specific CpG islands may serve as potential biomarkers correlated with SNP analyses in chordoma. The study was performed on tumor samples from ten chordoma patients. We found significant genomic instability by Affymetrix 6.0. It was interesting to see that all chordomas showed a loss of 3q26.32 (PIK 3CA) and 3q27.3 (BCL6) thus underlining the potential importance of the PI3K pathway in chordoma development. By using the AITCpG360 methylation assay we elucidated 20 genes which were hyper/hypomethylated compared to normal blood. The most promising candidates were nine hyper/hypomethylated genes C3, XIST, TACSTD2, FMR1, HIC1, RARB, DLEC1, KL, and RASSF1. In summary, we have shown that chordomas are characterized by a significant genomic instability and furthermore we demonstrated a characteristic DNA methylation pattern. These findings add new insights into chordoma development, diagnosis and potential new treatment options.
Asunto(s)
Cordoma/genética , Metilación de ADN/genética , Adulto , Anciano , Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Largo no Codificante/genética , Receptores de Ácido Retinoico/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Chordomas are rare, low to intermediate grade malignant bone tumors of the axial skeleton. Current treatment options are limited to surgical procedures, as chordomas are largely resistant to conventional radiation and chemotherapy. Cell lines are valuable tools for exploring molecular mechan-isms involved in tumorigenesis and they have a fundamental impact on the development of new anticancer agents. To date, only two chordoma cell lines exist world-wide. In the present study we report a third chordoma cell line, MUG-Chor1, as well as corresponding cultured fibroblasts established from a recur-rent morphologically 'classic' sacrococcygeal chordoma of a 58-year-old Caucasian female. The cells are brachyury-positive and have the characteristics of chordoma. The genetic profile of the primary chordoma and the established chordoma cell line was investigated during the culturing period (early and late passage). MUG-Chor1 is karyotypically, <2n>43-47,XX,del(3)(q1?)[11], +7,del(9)(p1?),der(9;15)(q10;q10),-10,+der(12)t(9;12)(p2?;q1?),der (12)t(12;19)(p;p)t(17;19)(q;q),-15,der(17;21)(q10;q10),der(20)t(10;20) (q25?26?;q11?12?),-21,-22[20]/idemx2[5] and displays known, chordoma-typical genetic changes, such as chromosomal gains at T/brachyury locus (6q27), losses at 9p24.3-p13.1 (includes the CDKN2a/CDKN2b locus), 10p15.3-q23.32 (includes the PTEN locus) and losses of 10q25.2 (includes the PDCD4 locus). MUG-Chor1 bears a marked resemblance to chordomas in vivo and is, therefore, an optimal in vitro chordoma model.