RESUMO
Congenital plasminogen (Plg) deficiency leads to the development of ligneous membranes on mucosal surfaces. Here, we report our experience with local and intravenous fresh frozen plasma (FFP). We retrospectively reviewed medical files of 17 patients and their eight first-degree relatives. Conjunctivitis was the main complaint. Thirteen patients were treated both with intravenous and conjunctival FFP. Venous thrombosis did not develop in any. Genetic evaluation revealed heterogeneous mutations as well as polymorphisms. Diagnosis and treatment of Plg deficiency is challenging; topical and intravenous FFP may be an alternative treatment.
Assuntos
Transfusão de Componentes Sanguíneos , Conjuntivite/terapia , Doenças Genéticas Inatas/terapia , Plasma , Plasminogênio/deficiência , Pré-Escolar , Conjuntivite/diagnóstico , Conjuntivite/genética , Feminino , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Polimorfismo GenéticoRESUMO
BACKGROUND: Therapy discontinuations and toxicities occur because of significant interindividual variations in 6-mercaptopurine (6-MP) and methotrexate (MTX) response during maintenance therapy of childhood acute lymphoblastic leukemia (ALL). 6-MP/MTX intolerance in some of the patients cannot be explained by thiopurine S-methyl transferase (TPMT) gene variants. In this study, we aimed to investigate candidate pharmacogenetic determinants of 6-MP and MTX intolerance in Turkish ALL children. METHODS: In total, 48 children with ALL who had completed or were receiving maintenance therapy according to Children's Oncology Group (COG) protocols were enrolled. Fifteen single-nucleotide polymorphisms in 8 candidate genes that were related to drug toxicity or had a role in the 6-MP/MTX metabolism (TPMT, ITPA, MTHFR, IMPDH2, PACSIN2, SLCO1B1, ABCC4, and PYGL) were genotyped by competitive allele-specific PCR (KASP). Drug doses during maintenance therapy were modified according to the protocol. RESULTS: The median drug dose intensity was 50% (28% to 92%) for 6-MP and 58% (27% to 99%) for MTX in the first year of maintenance therapy, which were lower than that scheduled in all patients. Among the analyzed polymorphisms, variant alleles in SLCO1B1 rs4149056 and rs11045879 were found to be associated with lower 6-MP/MTX tolerance. CONCLUSIONS: SLCO1B1 rs4149056 and rs11045879 polymorphisms may be important genetic markers to individualize 6-MP/MTX doses.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Quimioterapia de Manutenção/efeitos adversos , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , TurquiaAssuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Proteínas com Domínio LIM/metabolismo , Linfoma Difuso de Grandes Células B , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Proteínas Proto-Oncogênicas/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Adulto , Idoso , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Humanos , Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Prednisona/administração & dosagem , Rituximab/administração & dosagem , Vincristina/administração & dosagemRESUMO
This research aims to assess the concentration of metals in human and canine placentas from the same geographic area and to investigate how these metal levels influence gene expression within the placenta. Placentas of 25 dogs and 60 women who had recently given birth residing in Ankara, Turkey were collected and subjected to metal analysis using ICP-OES. Placentas with detectable metal levels underwent further examination including Next Generation Sequencing, transcriptional analysis, single nucleotide polymorphism investigation, and extensive scrutiny across various groups. For women, placentas with concurrent detection of aluminum (Al), lead (Pb), and cadmium (Cd) underwent transcriptomic analysis based on metal analysis results. However, the metal load in dog placentas was insufficient for comparison. Paired-end sequencing with 100-base pair read lengths was conducted using the DNBseq platform. Sequencing quality control was evaluated using FastQC, fastq screen, and MultiQC. RNA-sequencing data is publicly available via PRJNA936158. Comparative analyses were performed between samples with detected metals and "golden" samples devoid of these metals, revealing significant gene lists and read counts. Normalization of read counts was based on estimated size factors. Principal Component Analysis (PCA) was applied to all genes using rlog-transformed count data. Results indicate that metal exposure significantly influences placental gene expression, impacting various biological processes and pathways, notably those related to protein synthesis, immune responses, and cellular structure. Upregulation of immune-related pathways and alterations in protein synthesis machinery suggest potential defense mechanisms against metal toxicity. Nonetheless, these changes may adversely affect placental function and fetal health, emphasizing the importance of monitoring and mitigating environmental exposure to metals during pregnancy.
RESUMO
The placental cholinergic system; known as an important factor in intracellular metabolic activities, regulation of placental vascular tone, placental development, and neurotransmission; can be affected by persistent organic pesticides, particularly organochlorine pesticides(OCPs), which can influence various epigenetic regulations and molecular pathways. Although OCPs are legally prohibited, trace amounts of the persistent dichlorodiphenyltrichloroethane(DDT) are still found in the environment, making prenatal exposure inevitable. In this study, the effects of 2,4'-DDT and 4,4'-DDT; and its breakdown product 4,4'-DDE in the environment on placental cholinergic system were evaluated with regards to cholinergic genes. 40 human placentas were screened, where 42,50% (17 samples) were found to be positive for the tested compounds. Average concentrations were 10.44⯵g/kg; 15.07⯵g/kg and 189,42⯵g/kg for 4,4'-DDE; 2,4'-DDT and 4,4'-DDT respectively. RNA-Seq results revealed 2396 differentially expressed genes in positive samples; while an increase in CHRM1,CHRNA1,CHRNG and CHRNA2 genes at 1.28, 1.49, 1.59 and 0.4 fold change were found(p<0028). The increase for CHRM1 was also confirmed in tissue samples with immunohistochemistry. In vitro assays using HTR8/SVneo cells; revealed an increase in mRNA expression of CHRM1, CHRM3 and CHRN1 in DDT and DDE treated groups; which was also confirmed through western blot assays. An increase in the expression of CHRM1,CHRNA1, CHRNG(p<0001) and CHRNA2(p<0,05) were found from the OCPs exposed and non exposed groups.The present study reveals that intrauterine exposure to DDT affects the placental cholinergic system mainly through increased expression of muscarinic receptors. This increase in receptor expression is expected to enhance the sensitivity of the placental cholinergic system to acetylcholine.
Assuntos
DDT , Diclorodifenil Dicloroetileno , Placenta , Humanos , DDT/toxicidade , Feminino , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Diclorodifenil Dicloroetileno/toxicidade , Receptores Colinérgicos/metabolismo , Receptores Colinérgicos/genética , Adulto , Inseticidas/toxicidadeRESUMO
We present an autosomal-recessive frontonasal dysplasia (FND) characterized by bilateral extreme microphthalmia, bilateral oblique facial cleft, complete cleft palate, hypertelorism, wide nasal bridge with hypoplasia of the ala nasi, and low-set, posteriorly rotated ears in two distinct families. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this clinical entity to chromosome 12q21. In one of the families, three siblings were affected, and CNV analysis of the critical region showed a homozygous 3.7 Mb deletion containing the ALX1 (CART1) gene, which encodes the aristaless-like homeobox 1 transcription factor. In the second family we identified a homozygous donor-splice-site mutation (c.531+1G > A) in the ALX1 gene, providing evidence that complete loss of function of ALX1 protein causes severe disruption of early craniofacial development. Unlike loss of its murine ortholog, loss of human ALX1 does not result in neural-tube defects; however, it does severely affect the orchestrated fusion between frontonasal, nasomedial, nasolateral, and maxillary processes during early-stage embryogenesis. This study further expands the spectrum of the recently recognized autosomal-recessive ALX-related FND phenotype in humans.
Assuntos
Fissura Palatina/genética , Proteínas de Homeodomínio/genética , Microftalmia/genética , Anormalidades Musculoesqueléticas/genética , Mutação , Orelha/anormalidades , Face/anormalidades , Homozigoto , Humanos , Fenótipo , Sítios de Splice de RNA/genética , Deleção de Sequência/genéticaRESUMO
More than 270 million people worldwide have hearing loss that affects normal communication. Although astonishing progress has been made in the identification of more than 50 genes for deafness during the past decade, the majority of deafness genes are yet to be identified. In this study, we mapped a previously unknown autosomal-recessive nonsyndromic sensorineural hearing loss locus (DFNB91) to chromosome 6p25 in a consanguineous Turkish family. The degree of hearing loss was moderate to severe in affected individuals. We subsequently identified a nonsense mutation (p.E245X) in SERPINB6, which is located within the linkage interval for DFNB91 and encodes for an intracellular protease inhibitor. The p.E245X mutation cosegregated in the family as a completely penetrant autosomal-recessive trait and was absent in 300 Turkish controls. The mRNA expression of SERPINB6 was reduced and production of protein was absent in the peripheral leukocytes of homozygotes, suggesting that the hearing loss is due to loss of function of SERPINB6. We also demonstrated that SERPINB6 was expressed primarily in the inner ear hair cells. We propose that SERPINB6 plays an important role in the inner ear in the protection against leakage of lysosomal content during stress and that loss of this protection results in cell death and sensorineural hearing loss.
Assuntos
Códon sem Sentido , Perda Auditiva Neurossensorial/genética , Perda Auditiva/genética , Mutação , Serpinas/genética , Consanguinidade , Família , Hereditariedade , Homozigoto , HumanosRESUMO
Introduction: Pluripotent stem cells have been used by various researchers to differentiate and characterize endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) for the clinical treatment of vascular injuries. Studies continue to differentiate and characterize the cells with higher vascularization potential and low risk of malignant transformation to the recipient. Unlike previous studies, this research aimed to differentiate induced pluripotent stem (iPS) cells into endothelial progenitor cells (EPCs) and VSMCs using a step-wise technique. This was achieved by elucidating the spatio-temporal expressions of the stage-specific genes and proteins during the differentiation process. The presence of highly expressed oncogenes in iPS cells was also investigated during the differentiation period. Methods: Induced PS cells were differentiated into lateral mesoderm cells (Flk1+). The Flk1+ populations were isolated on day 5.5 of the mesodermal differentiation period. Flk1+ cells were further differentiated into EPCs and VSMCs using VEGF165 and platelet-derived growth factor-BB (PDGF-BB), respectively, and then characterized using gene expression levels, immunocytochemistry (ICC), and western blot (WB) methods. During the differentiation steps, the expression levels of the marker genes and proto-oncogenic Myc and Klf4 genes were simultaneously studied. Results: The optimal time for the isolation of Flk1+ cells was on day 5.5. EPCs and VSMCs were differentiated from Flk1+ cells and characterized with EPC-specific markers, including Kdr, Pecam1, CD133, Cdh5, Efnb2, Vcam1; and VSMC-specific markers, including Acta2, Cnn1, Des, and Myh11. Differentiated cells were validated based on their temporal gene expressions, protein synthesis, and localization at certain time points. Significant decreases in Myc and Klf4 gene expression levels were observed during the EPCs and VSMC differentiation period. Conclusion: EPCs and VSMCs were successfully differentiated from iPS cells and characterized by gene expression levels, ICC, and WB. We observed significant decreases in oncogene expression levels in the differentiated EPCs and VSMCs. In terms of safety, the described methodology provided a better safety margin. EPCs and VSMC obtained using this method may be good candidates for transplantation and vascular regeneration.
RESUMO
INTRODUCTION: Behçet syndrome (BS) is a chronic, multisystemic inflammatory condition with unanswered questions regarding its pathogenesis and rational therapeutics. A microarray-based comparative transcriptomic analysis was performed to elucidate the molecular mechanisms of BS and identify any potential therapeutic targets. METHODS: Twenty-nine BS patients (B) and 15 age and sex-matched control subjects (C) were recruited. Patients were grouped as mucocutaneous (M), ocular (O), and vascular (V) according to their clinical phenotypes. GeneChip Human Genome U133 Plus 2.0 arrays were used for expression profiling on peripheral blood samples of the patients and the control subjects. Following documentation of the differentially expressed gene (DEG) sets, the data were further evaluated with bioinformatics analysis, visualization, and enrichment tools. Validation of the microarray data was performed using quantitative reverse transcriptase polymerase chain reaction. RESULTS: When p ≤ 0.05 and fold change ≥2.0 were chosen, the following numbers of DEGs were obtained; B versus C: 28, M versus C: 20, O versus C: 8, V versus C: 555, M versus O: 6, M versus V: 324, O versus V: 142. Venn diagram analysis indicated only two genes, CLEC12A and IFI27, in the intersection of M versus C â© O versus C â© V versus C. Another noteworthy gene appeared as CLC in the DEG sets. Cluster analyses successfully clustered distinct clinical phenotypes of BS. While innate immunity-related processes were enriched in the M group, adaptive immunity-specific processes were significantly enriched in the O and V groups. CONCLUSIONS: Distinct clinical phenotypes of BS patients displayed distinct expression profiles. In Turkish BS patients, expression differences regarding the genes CLEC12A, IFI27, and CLC seemed to be operative in the disease pathogenesis. Based on these findings, future research should consider the immunogenetic heterogeneity of BS clinical phenotypes. Two anti-inflammatory genes, namely CLEC12A and CLC, may be valuable as therapeutic targets and may also help design an experimental model in BS.
Assuntos
Síndrome de Behçet , Humanos , Síndrome de Behçet/genética , Biologia Computacional , Expressão Gênica , Perfilação da Expressão Gênica , Lectinas Tipo C/genética , Proteínas de Membrana/genética , Fenótipo , Receptores Mitogênicos/genéticaRESUMO
PURPOSE: The aim is to describe the behavior of pilocytic astrocytoma (PAs) and its effects on patient prognosis by using flow cytometric, immunohistochemical and cytogenetic methods. We also aim to find out whether there is any difference between differently localized tumors by the above mentioned analyses. METHODS: We studied DNA index, expression of p53, p16, pRb, MMAC/PTEN1, VEGF, MIB-1 index and chromosomal anomalies which can be detected by array comparative genomic hybridization (CGH) technique. We analyzed the association of the results of these studies with clinical prognosis and tumor localization. We included 53 patients (18 cerebellar, 20 chiasmatic/hypothalamic and 15 hemispheric). Samples were studied from paraffin embedded tumors. RESULTS: We found that PAs are mostly diploid and ploidy pattern does not affect the prognosis. The expression of p53, p16, pRb, MMAC/PTEN1 and VEGF was not significantly different between different localizations and could not predict the prognosis. Frequently seen copy number aberrations (CNAs) are: amplification in 1p36.33, 2p11.2, 9p11.2, 9q12, 16p11.2, 19q13.12-q13.2, Xp22.2-p21.3, Xp11.3-p11.22, Xq11.1-q12, Xq13.1, Xq21.1-q21.31, Xq22.3, Xq26.3 and homozygous deletion in 2p11.2, 8p23.1, 16p12.3. Among them, 2p11.2 amp, 9p11.2 amp and 1p36.21 hom del were correlated with prognosis. Moreover, we found a significant correlation between 16p11.2 amp and tumor localization. CONCLUSIONS: Differently localized PAs have different properties which make them behave with different biological aggressiveness. PAs demonstrate a significant amount of CNAs that can be detected by a high-resolution study. However, tumor suppressor genes p53, p16, pRb, MMAC/PTEN1 and expression patterns do not play a significant role in PAs.
Assuntos
Astrocitoma/genética , Astrocitoma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Adolescente , Astrocitoma/patologia , Astrocitoma/cirurgia , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Criança , Pré-Escolar , Mapeamento Cromossômico , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Estimativa de Kaplan-Meier , Estudos Longitudinais , Masculino , Estudos Retrospectivos , Estatística como AssuntoRESUMO
OBJECTIVES: Transcriptomics of atrial fibrillation (AFib) in the setting of chronic primary mitral regurgitation (MR) remains to be characterized. We aimed to compare the gene expression profiles of patients with degenerative MR in AFib and sinus rhythm (SR) for a clearer picture of AFib pathophysiology. METHODS: After transcriptomic analysis and bioinformatics (n = 59), differentially expressed genes were defined using 1.5-fold change as the threshold. Additionally, independent datasets from GEO were included as meta-analyses. RESULTS: QRT-PCR analysis confirmed that AFib persistence was associated with increased expression molecular changes underlying a transition to heart failure (NPPB, P = 0.002; ANGPTL2, P = 0.002; IGFBP2, P = 0.010), structural remodeling including changes in the extracellular matrix and cellular stress response (COLQ, P = 0.003; COMP, P = 0.028; DHRS9, P = 0.038; CHGB, P = 0.038), and cellular stress response (DNAJA4, P = 0.038). Furthermore, AFib persistence was associated with decreased expression of the targets of structural remodeling (BMP7, P = 0.021) and electrical remodeling (CACNB2, P = 0.035; MCOLN3, P = 0.035) in both left and right atrial samples. The transmission electron microscopic analysis confirmed ultrastructural atrial remodeling and autophagy in human AFib atrial samples. CONCLUSIONS: Atrial cardiomyocyte remodeling in persistent AFib is closely linked to alterations in gene expression profiles compared to SR in patients with primary MR. Study findings may lead to novel therapeutic targets. This trial is registered with ClinicalTrials.gov identifier: NCT00970034.
Assuntos
Apêndice Atrial , Fibrilação Atrial , Remodelamento Atrial , Insuficiência da Valva Mitral , Canais de Potencial de Receptor Transitório , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/genética , Átrios do Coração , Humanos , Insuficiência da Valva Mitral/genéticaRESUMO
The first observation of homozygote Hb Q-Iran (α1 75(EF4) Asp-His) is presented in this report. The clinical and hematological data of the index case, his father and mother showed that homozygous or heterozygous Hb Q-Iran has no clinical importance.
RESUMO
Addressing the global burden of cancer, understanding its diverse biology, and promoting appropriate prevention and treatment strategies around the world has become a priority for the United Nations and International Atomic Energy Agency (IAEA), the WHO, and International Agency for Research on Cancer (IARC). The IAEA sponsored an international prospective cohort study to better understand biology, treatment response, and outcomes of diffuse large B-cell lymphoma (DLBCL) in low and middle-income countries across five UN-defined geographical regions. We report an analysis of biological variation in DLBCL across seven ethnic and environmentally diverse populations. In this cohort of 136 patients treated to a common protocol, we demonstrate significant biological differences between countries, characterized by a validated prognostic gene expression score (p < .0001), but International Prognostic Index (IPI)-adjusted survivals in all participating countries were similar. We conclude that DLBCL treatment outcomes in these populations can be benchmarked to international standards, despite biological heterogeneity.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/epidemiologia , Adulto , Idoso , Anticorpos Monoclonais Murinos/uso terapêutico , Biomarcadores Tumorais , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Feminino , Saúde Global , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/genética , Masculino , Pessoa de Meia-Idade , Vigilância da População , Prednisona/uso terapêutico , Prognóstico , Rituximab , Resultado do Tratamento , Vincristina/uso terapêuticoRESUMO
Amplification of 8p11-12 is a well-known alteration in human breast cancers but the driving oncogene has not been identified. We have developed a high-resolution comparative genomic hybridization array covering 8p11-12 and analysed 33 primary breast tumors, 20 primary ovarian tumors and 27 breast cancer cell lines. Expression analysis of the genes in the region was carried out by using real-time quantitative PCR and/or oligo-microarray profiling. In all, 24% (8/33) of the breast tumors, 5% (1/20) of the ovary tumors and 15% (4/27) of the cell lines showed 8p11-12 amplification. We identified a 1 Mb segment of common amplification that excludes previously proposed candidate genes. Some of the amplified genes did not show overexpression, whereas for others, overexpression was not specifically attributable to amplification. The genes FLJ14299, C8orf2, BRF2 and RAB11FIP, map within the 8p11-12 minimal amplicon, two have a putative function consistent with an oncogenic role, these four genes showed a strong correlation between amplification and overexpression and are therefore the best candidate driver oncogenes at 8p12.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 8/genética , Amplificação de Genes , Perfilação da Expressão Gênica , Oncogenes/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Post-translational modification of histones resulting in chromatin remodelling plays a key role in the regulation of gene expression. Here we report characteristic patterns of expression of 12 members of 3 classes of chromatin modifier genes in 6 different cancer types: histone acetyltransferases (HATs)- EP300, CREBBP, and PCAF; histone deacetylases (HDACs)- HDAC1, HDAC2, HDAC4, HDAC5, HDAC7A, and SIRT1; and histone methyltransferases (HMTs)- SUV39H1and SUV39H2. Expression of each gene in 225 samples (135 primary tumours, 47 cancer cell lines, and 43 normal tissues) was analysedby QRT-PCR, normalized with 8 housekeeping genes, and given as a ratio by comparison with a universal reference RNA. RESULTS: This involved a total of 13,000 PCR assays allowing for rigorous analysis by fitting a linear regression model to the data. Mutation analysis of HDAC1, HDAC2, SUV39H1, and SUV39H2 revealed only two out of 181 cancer samples (both cell lines) with significant coding-sequence alterations. Supervised analysis and Independent Component Analysis showed that expression of many of these genes was able to discriminate tumour samples from their normal counterparts. Clustering based on the normalized expression ratios of the 12 genes also showed that most samples were grouped according to tissue type. Using a linear discriminant classifier and internal cross-validation revealed that with as few as 5 of the 12 genes, SIRT1, CREBBP, HDAC7A, HDAC5 and PCAF, most samples were correctly assigned. CONCLUSION: The expression patterns of HATs, HDACs, and HMTs suggest these genes are important in neoplastic transformation and have characteristic patterns of expression depending on tissue of origin, with implications for potential clinical application.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Acetiltransferases/genética , Código das Histonas/genética , Histona Desacetilases/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Processamento de Proteína Pós-Traducional/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Transformação Celular Neoplásica/genética , Análise por Conglomerados , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , DNA Complementar/genética , DNA de Neoplasias/genética , Feminino , Histona Acetiltransferases/biossíntese , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/biossíntese , Humanos , Modelos Lineares , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proteínas MetiltransferasesRESUMO
X-linked agammaglobulinemia (XLA) is a primary B cell immunodeficiency disorder, caused by a defect in the Bruton tyrosine kinase (BTK) gene. Here, we describe a novel four base pair mutation (838delGAGT) in intron 9 of the BTK gene leading to the skipping of exon 9 in a 2.5-year-old boy with this disorder.
Assuntos
Agamaglobulinemia/genética , Cromossomos Humanos X , Proteínas Tirosina Quinases/genética , Domínios de Homologia de src , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/enzimologia , Pré-Escolar , Éxons/genética , Ligação Genética , Humanos , Íntrons/genética , Masculino , Mutação , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples.
RESUMO
Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase important in cell proliferation, growth and protein translation. Rapamycin, a wellknown anticancer agent and immunosuppressant drug, inhibits mTOR activity in different taxa including zebrafish. In the present study, the effect of rapamycin exposure on the transcriptome of a zebrafish fibroblast cell line, ZF4, was investigated. Microarray analysis demonstrated that rapamycin treatment modulated a large set of genes with varying functions including protein synthesis, assembly of mitochondrial and proteasomal machinery, cell cycle, metabolism and oxidative phosphorylation in ZF4 cells. A mild however, coordinated reduction in the expression of proteasomal and mitochondrial ribosomal subunits was detected, while the expression of numerous ribosomal subunits increased. Metaanalysis of heterogeneous mouse rapamycin microarray datasets enabled the comparison of zebrafish and mouse pathways modulated by rapamycin, using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway analysis. The analyses demonstrated a high degree of functional conservation between zebrafish and mice in response to rapamycin. In addition, rapamycin treatment resulted in a marked dosedependent reduction in body size and pigmentation in zebrafish embryos. The present study is the first, to the best of our knowledge, to evaluate the conservation of rapamycinmodulated functional pathways between zebrafish and mice, in addition to the dosedependent growth curves of zebrafish embryos upon rapamycin exposure.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Camundongos , Especificidade da EspécieRESUMO
BACKGROUND: Genome-wide gene expression profiling analysis of FFPE tissue samples is indispensable for cancer research and provides the opportunity to evaluate links between molecular and clinical information, however, working with FFPE samples is challenging due to extensive cross-linking, fragmentation and limited quantities of nucleic acid. Thus, processing of FFPE tissue samples from RNA extraction to microarray analysis still needs optimization. MATERIALS AND METHODS: In this study, a modified deparaffinization protocol was conducted prior to RNA isolation. Trizol, Qiagen RNeasy FFPE and Arcturus PicoPure RNA Isolation kits were used in parallel to compare their impact on RNA isolation. We also evaluated the effect of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) on the percentage of present calls. RESULTS: Our optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol gave a significantly higher percentage of present calls than the 3' IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms. CONCLUSION: Our study paves the way for future high-throughput transcriptional analysis by optimizing FFPE tissue sample processing from RNA isolation to microarray analysis.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/isolamento & purificação , Análise Serial de Tecidos/métodos , Formaldeído , Humanos , Inclusão em Parafina , Fixação de Tecidos/métodosRESUMO
Behçet's disease (BD) is a chronic, relapsing, multisystemic inflammatory disorder with unanswered questions regarding its etiology/pathogenesis and classification. Distinct manifestation based subsets, pronounced geographical variations in expression, and discrepant immunological abnormalities raised the question whether Behçet's is "a disease or a syndrome". To answer the preceding question we aimed to display and compare the molecular mechanisms underlying distinct subsets of BD. For this purpose, the expression data of the gene expression profiling and association study on BD by Xavier et al (2013) was retrieved from GEO database and reanalysed by gene expression data analysis/visualization and bioinformatics enrichment tools. There were 15 BD patients (B) and 14 controls (C). Three subsets of BD patients were generated: MB (isolated mucocutaneous manifestations, n = 7), OB (ocular involvement, n = 4), and VB (large vein thrombosis, n = 4). Class comparison analyses yielded the following numbers of differentially expressed genes (DEGs); B vs C: 4, MB vs C: 5, OB vs C: 151, VB vs C: 274, MB vs OB: 215, MB vs VB: 760, OB vs VB: 984. Venn diagram analysis showed that there were no common DEGs in the intersection "MB vs C" â© "OB vs C" â© "VB vs C". Cluster analyses successfully clustered distinct expressions of BD. During gene ontology term enrichment analyses, categories with relevance to IL-8 production (MB vs C) and immune response to microorganisms (OB vs C) were differentially enriched. Distinct subsets of BD display distinct expression profiles and different disease associated pathways. Based on these clear discrepancies, the designation as "Behçet's syndrome" (BS) should be encouraged and future research should take into consideration the immunogenetic heterogeneity of BS subsets. Four gene groups, namely, negative regulators of inflammation (CD69, CLEC12A, CLEC12B, TNFAIP3), neutrophil granule proteins (LTF, OLFM4, AZU1, MMP8, DEFA4, CAMP), antigen processing and presentation proteins (CTSS, ERAP1), and regulators of immune response (LGALS2, BCL10, ITCH, CEACAM8, CD36, IL8, CCL4, EREG, NFKBIZ, CCR2, CD180, KLRC4, NFAT5) appear to be instrumental in BS immunopathogenesis.