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Background: Familial dilated cardiomyopathy (DCM) causes heart failure and may lead to heart transplantation. DCM is typically a monogenic disorder with autosomal dominant inheritance. Currently disease-causing variants have been reported in over 60 genes that encode proteins in sarcomeres, nuclear lamina, desmosomes, cytoskeleton, and mitochondria. Over half of the patients undergoing comprehensive genetic testing are left without a molecular diagnosis even when patient selection follows strict DCM criteria. Methods and results: This study was a retrospective review of patients referred for genetic testing at Blueprint Genetics due to suspected inherited DCM. Next generation sequencing panels included 23-316 genes associated with cardiomyopathies and other monogenic cardiac diseases. Variants were considered diagnostic if classified as pathogenic (P) or likely pathogenic (LP). Of the 2,088 patients 514 (24.6%) obtained a molecular diagnosis; 534 LP/P variants were observed across 45 genes, 2.7% (14/514) had two diagnostic variants in dominant genes. Nine copy number variants were identified: two multigene and seven intragenic. Diagnostic variants were observed most often in TTN (45.3%), DSP (6.7%), LMNA (6.7%), and MYH7 (5.2%). Clinical characteristics independently associated with molecular diagnosis were: a lower age at diagnosis, family history of DCM, paroxysmal atrial fibrillation, absence of left bundle branch block, and the presence of an implantable cardioverter-defibrillator. Conclusions: Panel testing provides good diagnostic yield in patients with clinically suspected DCM. Causative variants were identified in 45 genes. In minority, two diagnostic variants were observed in dominant genes. Our results support the use of genetic panels in clinical settings in DCM patients with suspected genetic etiology.
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Background: Familial dilated cardiomyopathy (DCM) is a monogenic disorder typically inherited in an autosomal dominant pattern. We have identified two Finnish families with familial cardiomyopathy that is not explained by a variant in any previously known cardiomyopathy gene. We describe the cardiac phenotype related to homozygous truncating GCOM1 variants. Methods and Results: This study included two probands and their relatives. All the participants are of Finnish ethnicity. Whole-exome sequencing was used to test the probands; bi-directional Sanger sequencing was used to identify the GCOM1 variants in probands' family members. Clinical evaluation was performed, medical records and death certificates were obtained. Immunohistochemical analysis of myocardial samples was conducted. A homozygous GCOM1 variant was identified altogether in six individuals, all considered to be affected. None of the nine heterozygous family members fulfilled any cardiomyopathy criteria. Heart failure was the leading clinical feature, and the patients may have had a tendency for atrial arrhythmias. Conclusions: This study demonstrates the significance of GCOM1 variants as a cause of human cardiomyopathy and highlights the importance of searching for new candidate genes when targeted gene panels do not yield a positive outcome.
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BACKGROUND: Methylation of DNA at CpG sites is an epigenetic modification and a potential modifier of disease risk, possibly mediating environmental effects. Currently, DNA methylation is commonly assessed using specific microarrays that sample methylation at a few % of all methylated sites. METHODS: To understand if significant information on methylation can be added by a more comprehensive analysis of methylation, we set up a quantitative method, bisulfite oligonucleotide-selective sequencing (Bs-OS-seq), and compared the data with microarray-derived methylation data. We assessed methylation at two asthma-associated genes, IL13 and ORMDL3, in blood samples collected from children with and without asthma and fractionated white blood cell types from healthy adult controls. RESULTS: Our results show that Bs-OS-seq can uncover vast amounts of methylation variation not detected by commonly used array methods. We found that high-density methylation information from even one gene can delineate the main white blood cell lineages. CONCLUSIONS: We conclude that high-resolution methylation studies can yield clinically important information at selected specific loci missed by array-based methods, with potential implications for future studies of methylation-disease associations.
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Metilación de ADN/genética , Epigénesis Genética/genética , Epigenómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Interleucina-13/genética , Proteínas de la Membrana/genética , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Femenino , Humanos , Interleucina-13/sangre , Masculino , Proteínas de la Membrana/sangre , SulfitosRESUMEN
BACKGROUND: Genetic testing in hypertrophic cardiomyopathy (HCM) is a published guideline-based recommendation. The diagnostic yield of genetic testing and corresponding HCM-associated genes have been largely documented by single center studies and carefully selected patient cohorts. Our goal was to evaluate the diagnostic yield of genetic testing in a heterogeneous cohort of patients with a clinical suspicion of HCM, referred for genetic testing from multiple centers around the world. METHODS: A retrospective review of patients with a suspected clinical diagnosis of HCM referred for genetic testing at Blueprint Genetics was undertaken. The analysis included syndromic, myopathic and metabolic etiologies. Genetic test results and variant classifications were extracted from the database. Variants classified as pathogenic (P) or likely pathogenic (LP) were considered diagnostic. RESULTS: A total of 1376 samples were analyzed. Three hundred and sixty-nine tests were diagnostic (26.8%); 373 P or LP variants were identified. Only one copy number variant was identified. The majority of diagnostic variants involved genes encoding the sarcomere (85.0%) followed by 4.3% of diagnostic variants identified in the RASopathy genes. Two percent of diagnostic variants were in genes associated with a cardiomyopathy other than HCM or an inherited arrhythmia. Clinical variables that increased the likelihood of identifying a diagnostic variant included: an earlier age at diagnosis (p < 0.0001), a higher maximum wall thickness (MWT) (p < 0.0001), a positive family history (p < 0.0001), the absence of hypertension (p = 0.0002), and the presence of an implantable cardioverter-defibrillator (ICD) (p = 0.0004). CONCLUSION: The diagnostic yield of genetic testing in this heterogeneous cohort of patients with a clinical suspicion of HCM is lower than what has been reported in well-characterized patient cohorts. We report the highest yield of diagnostic variants in the RASopathy genes identified in a laboratory cohort of HCM patients to date. The spectrum of genes implicated in this unselected cohort highlights the importance of pre-and post-test counseling when offering genetic testing to the broad HCM population.
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Cardiomiopatía Hipertrófica/diagnóstico , Pruebas Genéticas , Variación Genética , Adolescente , Adulto , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/fisiopatología , Niño , Preescolar , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Lactante , Masculino , Fenotipo , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Adulto JovenRESUMEN
BACKGROUND: Familial dilated cardiomyopathy (DCM) is typically a monogenic disorder with dominant inheritance. Although over 40 genes have been linked to DCM, more than half of the patients undergoing comprehensive genetic testing are left without molecular diagnosis. Recently, biallelic protein-truncating variants (PTVs) in the nebulin-related anchoring protein gene (NRAP) were identified in a few patients with sporadic DCM. METHODS AND RESULTS: We determined the frequency of rare NRAP variants in a cohort of DCM patients and control patients to further evaluate role of this gene in cardiomyopathies. A retrospective analysis of our internal variant database consisting of 31,639 individuals who underwent genetic testing (either panel or direct exome sequencing) was performed. The DCM group included 577 patients with either a confirmed or suspected DCM diagnosis. A control cohort of 31,062 individuals, including 25,912 individuals with non-cardiac (control group) and 5,150 with non-DCM cardiac indications (Non-DCM cardiac group). Biallelic (n = 6) or two (n = 5) NRAP variants (two PTVs or PTV+missense) were identified in 11 unrelated probands with DCM (1.9%) but none of the controls. None of the 11 probands had an alternative molecular diagnosis. Family member testing supports co-segregation. Biallelic or potentially biallelic NRAP variants were enriched in DCM vs. controls (OR 1052, p<0.0001). Based on the frequency of NRAP PTVs in the gnomAD reference population, and predicting full penetrance, biallelic NRAP variants could explain 0.25%-2.46% of all DCM cases. CONCLUSION: Loss-of-function in NRAP is a cause for autosomal recessive dilated cardiomyopathy, supporting its inclusion in comprehensive genetic testing.
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Cardiomiopatía Dilatada , Proteínas Musculares/genética , Adulto , Cardiomiopatía Dilatada/diagnóstico , Cardiomiopatía Dilatada/genética , Preescolar , Femenino , Pruebas Genéticas , Humanos , Mutación con Pérdida de Función , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
The genetic basis of pulmonary arterial hypertension (PAH) among Finnish PAH patients is poorly understood. We adopted a novel-targeted next-generation sequencing (NGS) approach called Oligonucleotide-Selective Sequencing (OS-Seq) and developed a custom data analysis and interpretation pipeline to identify pathogenic base substitutions, insertions, and deletions in seven genes associated with PAH (BMPR2, BMPR1B, ACVRL1, ENG, SMAD9, CAV1, and KCNK3) from Finnish PAH patients. This study represents the first clinical study with OS-Seq technology on patients suffering from a rare genetic disorder. We analyzed DNA samples from 21 Finnish PAH patients, whose BMPR2 and ACVRL1 mutation status had been previously studied using Sanger sequencing. Our sequencing panel covered 100% of the targeted base pairs with >15× sequencing depth. Pathogenic base substitutions were identified in the BMPR2 gene in 29% of the Finnish PAH cases. Two of the pathogenic variant-positive patients had been previously tested negative using Sanger sequencing. No clinically significant variants were identified in the six other PAH genes. Our study validates the use of targeted OS-Seq for genetic diagnostics of PAH and revealed pathogenic variants that had been previously missed using Sanger sequencing.
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AIMS: Despite our increased understanding of the genetic basis of dilated cardiomyopathy (DCM), the clinical utility and yield of clinically meaningful findings of comprehensive next-generation sequencing (NGS)-based genetic diagnostics in DCM has been poorly described. We utilized a high-quality oligonucleotide-selective sequencing (OS-Seq)-based targeted sequencing panel to investigate the genetic landscape of DCM in Finnish population and to evaluate the utility of OS-Seq technology as a novel comprehensive diagnostic tool. METHODS AND RESULTS: Using OS-Seq, we targeted and sequenced the coding regions and splice junctions of 101 genes associated with cardiomyopathies in 145 unrelated Finnish patients with DCM. We developed effective bioinformatic variant filtering strategy and implemented strict variant classification scheme to reveal diagnostic yield and genotype-phenotype correlations. Implemented OS-Seq technology provided high coverage of the target region (median coverage 410× and 99.42% of the nucleotides were sequenced at least 15× read depth). Diagnostic yield was 35.2% (familial 47.6% and sporadic 25.6%, P = 0.004) when both pathogenic and likely pathogenic variants are considered as disease causing. Of these, 20 (53%) were titin (TTN) truncations (non-sense and frameshift) affecting all TTN transcripts. TTN truncations accounted for 20.6% and 14.6% of the familial and sporadic DCM cases, respectively. CONCLUSION: Panel-based, high-quality NGS enables high diagnostic yield especially in the familial form of DCM, and bioinformatic variant filtering is a reliable step in the process of interpretation of genomic data in a clinical setting.
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Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/epidemiología , Femenino , Finlandia/epidemiología , Estudios de Seguimiento , Mutación del Sistema de Lectura/genética , Genotipo , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Mutación Missense/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Linaje , Fenotipo , RecurrenciaRESUMEN
Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p<0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The results rather suggest that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study suggests novel non-enzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation.
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Perfilación de la Expresión Génica , Gliadina/genética , Mucosa Intestinal/metabolismo , Pepsina A/metabolismo , Tripsina/metabolismo , Células CACO-2 , Humanos , Mucosa Intestinal/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Uterine leiomyomas are benign but affect the health of millions of women. A better understanding of the molecular mechanisms involved may provide clues to the prevention and treatment of these lesions. METHODS: We performed whole-genome sequencing and gene-expression profiling of 38 uterine leiomyomas and the corresponding myometrium from 30 women. RESULTS: Identical variants observed in some separate tumor nodules suggested that these nodules have a common origin. Complex chromosomal rearrangements resembling chromothripsis were a common feature of leiomyomas. These rearrangements are best explained by a single event of multiple chromosomal breaks and random reassembly. The rearrangements created tissue-specific changes consistent with a role in the initiation of leiomyoma, such as translocations of the HMGA2 and RAD51B loci and aberrations at the COL4A5-COL4A6 locus, and occurred in the presence of normal TP53 alleles. In some cases, separate events had occurred more than once in single tumor-cell lineages. CONCLUSIONS: Chromosome shattering and reassembly resembling chromothripsis (a single genomic event that results in focal losses and rearrangements in multiple genomic regions) is a major cause of chromosomal abnormalities in uterine leiomyomas; we propose that tumorigenesis occurs when tissue-specific tumor-promoting changes are formed through these events. Chromothripsis has previously been associated with aggressive cancer; its common occurrence in leiomyomas suggests that it also has a role in the genesis and progression of benign tumors. We observed that multiple separate tumors could be seeded from a single lineage of uterine leiomyoma cells. (Funded by the Academy of Finland Center of Excellence program and others.).
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Aberraciones Cromosómicas , Fumarato Hidratasa/deficiencia , Leiomioma/genética , Complejo Mediador/genética , Neoplasias Uterinas/genética , Rotura Cromosómica , Deleción Cromosómica , Colágeno Tipo IV/genética , Femenino , Fumarato Hidratasa/genética , Perfilación de la Expresión Génica , Reordenamiento Génico , Estudio de Asociación del Genoma Completo , Humanos , Mutación , Miometrio/química , Regulación hacia ArribaRESUMEN
BACKGROUND: The growth of high-throughput technologies such as microarrays and next generation sequencing has been accompanied by active research in data analysis methodology, producing new analysis methods at a rapid pace. While most of the newly developed methods are freely available, their use requires substantial computational skills. In order to enable non-programming biologists to benefit from the method development in a timely manner, we have created the Chipster software. RESULTS: Chipster (http://chipster.csc.fi/) brings a powerful collection of data analysis methods within the reach of bioscientists via its intuitive graphical user interface. Users can analyze and integrate different data types such as gene expression, miRNA and aCGH. The analysis functionality is complemented with rich interactive visualizations, allowing users to select datapoints and create new gene lists based on these selections. Importantly, users can save the performed analysis steps as reusable, automatic workflows, which can also be shared with other users. Being a versatile and easily extendable platform, Chipster can be used for microarray, proteomics and sequencing data. In this article we describe its comprehensive collection of analysis and visualization tools for microarray data using three case studies. CONCLUSIONS: Chipster is a user-friendly analysis software for high-throughput data. Its intuitive graphical user interface enables biologists to access a powerful collection of data analysis and integration tools, and to visualize data interactively. Users can collaborate by sharing analysis sessions and workflows. Chipster is open source, and the server installation package is freely available.
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Análisis por Micromatrices/métodos , Programas Informáticos , Algoritmos , Bases de Datos Genéticas , Regulación de la Expresión Génica , MicroARNs/análisis , Interfaz Usuario-ComputadorRESUMEN
Uterine leiomyomas, or fibroids, are benign tumors that affect millions of women worldwide and that can cause considerable morbidity. To study the genetic basis of this tumor type, we examined 18 uterine leiomyomas derived from 17 different patients by exome sequencing and identified tumor-specific mutations in the mediator complex subunit 12 (MED12) gene in 10. Through analysis of 207 additional tumors, we determined that MED12 is altered in 70% (159 of 225) of tumors from a total of 80 patients. The Mediator complex is a 26-subunit transcriptional regulator that bridges DNA regulatory sequences to the RNA polymerase II initiation complex. All mutations resided in exon 2, suggesting that aberrant function of this region of MED12 contributes to tumorigenesis.
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Leiomioma/genética , Complejo Mediador/genética , Neoplasias Uterinas/genética , Codón , Exones , Femenino , Perfilación de la Expresión Génica , Humanos , Mutación INDEL , Intrones , Leiomioma/metabolismo , Mutación , Mutación Missense , Transducción de Señal , Neoplasias Uterinas/metabolismoRESUMEN
We report an autosomal recessive lethal syndrome characterized by multiple fetal malformations, the most obvious anomalies being the defective face and seemingly absent limbs, which are bound to the trunk and encased under the skin. We identified the molecular defect that causes this syndrome, using a combined strategy of gene-expression arrays, candidate-gene analysis, clinical studies, and genealogic investigations. A point mutation in two affected fetuses led to the loss of the conserved helixloophelix ubiquitous kinase (CHUK), also known as IκB kinase α. CHUK has an essential role in the development of skin epidermis and its derivatives, along with various other morphogenetic events. (Funded by the Academy of Finland and others.).
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Anomalías Múltiples/genética , Cara/anomalías , Quinasa I-kappa B/genética , Deformidades Congénitas de las Extremidades/genética , Mutación Puntual , Animales , Expresión Génica , Genes Recesivos , Humanos , Ratones , Ratones Noqueados/genética , LinajeRESUMEN
The lymphatic vasculature is important for the regulation of tissue fluid homeostasis, immune response, and lipid absorption, and the development of in vitro models should allow for a better understanding of the mechanisms regulating lymphatic vascular growth, repair, and function. Here we report isolation and characterization of lymphatic endothelial cells from human intestine and show that intestinal lymphatic endothelial cells have a related but distinct gene expression profile from human dermal lymphatic endothelial cells. Furthermore, we identify liprin beta1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, as highly expressed in intestinal lymphatic endothelial cells in vitro and lymphatic vasculature in vivo, and show that it plays an important role in the maintenance of lymphatic vessel integrity in Xenopus tadpoles.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Células Endoteliales/citología , Mucosa Intestinal/citología , Vasos Linfáticos/citología , Proteínas de Xenopus/metabolismo , Xenopus laevis/fisiología , Animales , Proteínas Portadoras/genética , Células Cultivadas , Dermis/citología , Células Endoteliales/fisiología , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Larva/fisiología , Linfangiogénesis/fisiología , Vasos Linfáticos/fisiología , Modelos Animales , Organismos Modificados Genéticamente , Proteínas de Xenopus/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
BACKGROUND: Hydrolethalus syndrome (HLS) is a severe fetal malformation syndrome characterized by multiple developmental anomalies, including central nervous system (CNS) malformation such as hydrocephaly and absent midline structures of the brain, micrognathia, defective lobation of the lungs and polydactyly. Microscopically, immature cerebral cortex, abnormalities in radial glial cells and hypothalamic hamartoma are among key findings in the CNS of HLS fetuses. HLS is caused by a substitution of aspartic acid by glycine in the HYLS1 protein, whose function was previously unknown. RESULTS: To provide insight into the disease mechanism(s) of this lethal disorder we have studied different aspects of HLS and HYLS1. A genome-wide gene expression analysis indicated several upregulated genes in cell cycle regulatory cascades and in specific signal transduction pathways while many downregulated genes were associated with lipid metabolism. These changes were supported by findings in functional cell biology studies, which revealed an increased cell cycle rate and a decreased amount of apoptosis in HLS neuronal progenitor cells. Also, changes in lipid metabolism gene expression were reflected by a significant increase in the cholesterol levels of HLS liver tissues. In addition, based on our functional studies of HYLS1, we propose that HYLS1 is a transcriptional regulator that shuffles between the cytoplasm and the nucleus, and that when HYLS1 is mutated its function is significantly altered. CONCLUSION: In this study, we have shown that the HYLS1 mutation has significant consequences in the cellular and tissue levels in HLS fetuses. Based on these results, it can be suggested that HYLS1 is part of the cellular transcriptional regulatory machinery and that the genetic defect has a widespread effect during embryonic and fetal development. These findings add a significant amount of new information to the pathogenesis of HLS and strongly suggest an essential role for HYLS1 in normal fetal development.
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Leiomyosarcoma (LMS) is the third most common type of soft tissue sarcoma after malignant fibrous histiocytoma (MFH) and liposarcoma. Comparative genomic hybridization (CGH) has shown similar DNA copy number imbalances in LMS and MFH. It has been suggested that both tumors may correspond to different differentiation states of a single tumor entity and that a large proportion of MFHs could correspond to undifferentiated LMS. We report CGH results from 102 MFH and 82 LMS cases, as well as a subsequent clustering analysis. The distribution pattern of DNA copy number changes could not differentiate LMS from MFH, suggesting that most MFHs could represent an ultimate state of tumor progression of LMS. Even if an oncogenic pattern common to LMS and MFH is valid, the genes relevant to smooth muscle cell differentiation may reside in one or more chromosomal imbalances that are not shared by both tumor types. Further explorative analysis identified a small cluster of tumors (9% of the samples: 2 LMS and 10 MFH) characterized by the presence of high-level amplifications at 1p33 approximately p34.3, 17q22 approximately q23, 17q25 approximately qter, 19p, 22p, and 22q, and associated with a higher proportion of tumors located in the thigh (P=0.003) and with male sex (P=0.079).
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Aberraciones Cromosómicas , ADN de Neoplasias/genética , Histiocitoma Fibroso Maligno/genética , Leiomiosarcoma/genética , Hibridación de Ácido Nucleico/genética , Actinas/genética , Algoritmos , Secuencia de Bases , Cromosomas Humanos/genética , Análisis por Conglomerados , Amplificación de Genes , HumanosRESUMEN
The Drosophila transcription factor Prospero functions as a tumor suppressor, and it has been suggested that the human counterpart of Prospero, PROX1, acts similarly in human cancers. However, we show here that PROX1 promotes dysplasia in colonic adenomas and colorectal cancer progression. PROX1 expression marks the transition from benign colon adenoma to carcinoma in situ, and its loss inhibits growth of human colorectal tumor xenografts and intestinal adenomas in Apc(min/+) mice, while its transgenic overexpression promotes colorectal tumorigenesis. Furthermore, in intestinal tumors PROX1 is a direct and dose-dependent target of the beta-catenin/TCF signaling pathway, responsible for the neoplastic transformation. Our data underscore the complexity of cancer pathogenesis and implicate PROX1 in malignant tumor progression through the regulation of cell polarity and adhesion.
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Adenoma/genética , Neoplasias del Colon/genética , Proteínas de Homeodominio/genética , Proteínas Supresoras de Tumor/genética , Adenoma/patología , Carcinoma in Situ/genética , Línea Celular Tumoral , Neoplasias del Colon/patología , Neoplasias Colorrectales/genética , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Fenotipo , beta Catenina/fisiologíaRESUMEN
BACKGROUND: The neuronal ceroid lipofuscinoses (NCL) are a group of children's inherited neurodegenerative disorders, characterized by blindness, early dementia and pronounced cortical atrophy. The similar pathological and clinical profiles of the different forms of NCL suggest that common disease mechanisms may be involved. To explore the NCL-associated disease pathology and molecular pathways, we have previously produced targeted knock-out mice for Cln1 and Cln5. Both mouse-models replicate the NCL phenotype and neuropathology; the Cln1-/- model presents with early onset, severe neurodegenerative disease, whereas the Cln5-/- model produces a milder disease with a later onset. RESULTS: Here we have performed quantitative gene expression profiling of the cortex from 1 and 4 month old Cln1-/- and Cln5-/- mice. Combined microarray datasets from both mouse models exposed a common affected pathway: genes regulating neuronal growth cone stabilization display similar aberrations in both models. We analyzed locus specific gene expression and showed regional clustering of Cln1 and three major genes of this pathway, further supporting a close functional relationship between the corresponding gene products; adenylate cyclase-associated protein 1 (Cap1), protein tyrosine phosphatase receptor type F (Ptprf) and protein tyrosine phosphatase 4a2 (Ptp4a2). The evidence from the gene expression data, indicating changes in the growth cone assembly, was substantiated by the immunofluorescence staining patterns of Cln1-/- and Cln5-/- cortical neurons. These primary neurons displayed abnormalities in cytoskeleton-associated proteins actin and beta-tubulin as well as abnormal intracellular distribution of growth cone associated proteins GAP-43, synapsin and Rab3. CONCLUSION: Our data provide the first evidence for a common molecular pathogenesis behind neuronal degeneration in INCL and vLINCL. Since CLN1 and CLN5 code for proteins with distinct functional roles these data may have implications for other forms of NCLs as well.
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Encéfalo/metabolismo , Regulación de la Expresión Génica/genética , Conos de Crecimiento/patología , Glicoproteínas de Membrana/genética , Lipofuscinosis Ceroideas Neuronales/genética , Tioléster Hidrolasas/genética , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Western Blotting , Células Cultivadas , Proteína GAP-43/metabolismo , Perfilación de la Expresión Génica , Genotipo , Conos de Crecimiento/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Inmunohistoquímica , Proteínas de Membrana de los Lisosomas , Ratones , Ratones Noqueados , Lipofuscinosis Ceroideas Neuronales/patología , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Sinapsinas/metabolismo , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of children, characterized by selective death of neocortical neurons. To understand early disease mechanisms in INCL, we have studied Ppt1(Deltaex4) knock-out mouse neurons in culture and acute brain slices. Global transcript profiling showed deregulation of key neuronal functions in knock-out mice including cholesterol metabolism, neuronal maturation, and calcium homeostasis. Cholesterol metabolism showed major changes; sterol biosynthesis was enhanced and steady-state amounts of sterols were altered at the cellular level. Changes were also present in early maturation of Ppt1(Deltaex4) neurons indicated by increased proliferative capacity of neuronal stem cells. Knock-out neurons presented unaltered electrophysiological properties suggesting uncompromised synaptic function in young animals. However, knock-out neurons exhibited more efficient recovery from glutamate-induced calcium transients, possibly indicating neuroprotective activation. This study established that the neuronal deregulation in INCL is linked to neuronal maturation, lipid metabolism and calcium homeostasis.
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Calcio/metabolismo , Colesterol/metabolismo , Homeostasis , Neuronas/metabolismo , Tioléster Hidrolasas/deficiencia , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Hipocampo/metabolismo , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Lipofuscinosis Ceroideas Neuronales/fisiopatología , Neuronas/citología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células Madre/citología , Sinapsis/metabolismo , Sinapsis/patología , Tioléster Hidrolasas/genéticaRESUMEN
Rare monogenic dementias have repeatedly exposed novel pathways guiding to details of the molecular pathogenesis behind this complex clinical phenotype. In this paper, we have studied polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), an early onset dementia with bone fractures caused by mutations in TYROBP (DAP12) and TREM2 genes, which encode important signaling molecules in human dendritic cells (DCs). To identify the pathways and biological processes associated with DAP12/TREM2-mediated signaling, we performed genome wide transcript analysis of in vitro differentiated DCs of PLOSL patients representing functional knockouts of either DAP12 or TREM2. Both DAP12- and TREM2-deficient cells differentiated into DCs and responded to pathogenic stimuli. However, the DCs showed morphological differences compared to control cells due to defects in the actin filaments. Not unexpectedly, transcript profiles of the patient DCs showed differential expression of genes involved in immune response. Importantly, significantly diverging transcript levels were also evident for genes earlier associated with other disorders of the central nervous system (CNS) and genes involved in the remodeling of bone, linking these two immunological genes with critical tissue phenotypes of patients. The data underline the functional diversity of the molecules of the innate immune system and implies their significant contribution also in demyelinating CNS disorders, including those resulting in dementia.
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Enfermedades del Sistema Nervioso Central/genética , Enfermedades Desmielinizantes/genética , Células Dendríticas/metabolismo , Perfilación de la Expresión Génica , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Northern Blotting , Diferenciación Celular/genética , Enfermedades del Sistema Nervioso Central/sangre , Citoesqueleto/metabolismo , Enfermedades Desmielinizantes/sangre , Células Dendríticas/citología , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Regulación de la Expresión Génica , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Microscopía Confocal , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteocondrodisplasias/sangre , Osteocondrodisplasias/genética , Receptores Inmunológicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Precursor cells of the human central nervous system can be cultured in vitro to reveal pathogenesis of diseases or developmental disorders. Here, we have studied the biology of neural precursor cells (NPCs) from patients of lethal congenital contracture syndrome (LCCS), a severe motoneuron disease leading to prenatal death before the 32nd gestational week. LCCS fetuses are immobile because of a motoneuron defect, seen as degeneration of the anterior horn and descending tracts of the developing spinal cord. The genetic defect for the syndrome is unknown. We show that NPCs isolated postmortem from LCCS fetuses grow and are maintained in culture, but display increased cell cycle activity. Global transcript analysis of undifferentiated LCCS precursor cells present with changes in EGF-related signaling when compared with healthy age-matched human controls. Further, we show that LCCS-derived NPCs differentiate into cells of neuronal and glial lineage and that the initial differentiation is not accompanied by overt apoptosis. Cells expressing markers Islet-1 and Hb9 are also generated from the LCCS NPCs, suggesting that the pathogenic mechanism of LCCS does not directly affect the differentiation capacity or survival of the cells, but the absence of motoneurons in LCCS may be caused by a noncell autonomous mechanism.