Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 71
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
2.
J Mol Med (Berl) ; 98(1): 111-122, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31832701

RESUMO

Psoriasis is a very common chronic inflammatory skin disease characterized by epidermal thickening and scaling resulting from keratinocyte hyperproliferation and impaired differentiation. Pathomechanistic studies in psoriasis are often limited by using whole skin tissue biopsies, neglecting their stratification and cellular diversity. This study aimed at characterizing epidermal alterations in psoriasis at the level of keratinocyte populations. Epidermal cell populations were purified from skin biopsies of psoriasis patients and healthy donors using a novel cell type-specific approach. Molecular characterization of the transit-amplifying cells (TAC), the key players of epidermal renewal, was performed using immunocytofluorescence-technique and integrated multiscale-omics analyses. Already TAC from non-lesional psoriatic skin showed altered methylation and differential expression in 1.7% and 1.0% of all protein-coding genes, respectively. In psoriatic lesions, TAC were strongly expanded showing further increased differentially methylated (10-fold) and expressed (22-fold) genes numbers. Importantly, 17.2% of differentially expressed genes were associated with respective gene methylations. Compared with non-lesional TAC, pathway analyses revealed metabolic alterations as one feature predominantly changed in TAC derived from active psoriatic lesions. Overall, our study showed stage-specific molecular alterations, allows new insights into the pathogenesis, and implies the involvement of epigenetic mechanisms in lesion development in psoriasis. KEY MESSAGES: Transit amplifying cell (TAC) numbers are highly increased in psoriatic lesions Psoriatic TAC show profound molecular alterations & stage-specific identity TAC from unaffected areas already show first signs of molecular alterations Lesional TAC show a preference in metabolic-related alterations.

3.
Genes (Basel) ; 10(7)2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31323892

RESUMO

Hi-C, capture Hi-C (CHC) and Capture-C have contributed greatly to our present understanding of the three-dimensional organization of genomes in the context of transcriptional regulation by characterizing the roles of topological associated domains, enhancer promoter loops and other three-dimensional genomic interactions. The analysis is based on counts of chimeric read pairs that map to interacting regions of the genome. However, the processing and quality control presents a number of unique challenges. We review here the experimental and computational foundations and explain how the characteristics of restriction digests, sonication fragments and read pairs can be exploited to distinguish technical artefacts from valid read pairs originating from true chromatin interactions.


Assuntos
Cromatina/genética , Biologia Computacional , Genoma , Genômica , Mapeamento Cromossômico , Biologia Computacional/métodos , Bases de Dados Genéticas , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Controle de Qualidade
4.
Front Immunol ; 10: 767, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024575

RESUMO

Reactivation of the BK polyomavirus is known to lead to severe complications in kidney transplant patients. The current treatment strategy relies on decreasing the immunosuppression to allow the immune system to clear the virus. Recently, we demonstrated a clear association between the resolution of BKV reactivation and reconstitution of BKV-specific CD4+ T-cells. However, which factors determine the duration of viral infection clearance remains so far unclear. Here we apply a combination of in-depth multi-parametric flow cytometry and NGS-based CDR3 beta chain receptor repertoire analysis of BKV-specific T-cells to a cohort of 7 kidney transplant patients during the clinical course of BKV reactivation. This way we followed TCR repertoires at single clone levels and functional activity of BKV-specific T-cells during the resolution of BKV infection. The duration of BKV clearance did not depend on the number of peripheral blood BKV-specific T-cells nor on a few immunodominant BKV-specific T-cell clones. Rather, the T-cell receptor repertoire diversity and exhaustion status of BKV-specific T-cells affected the duration of viral clearance: high clonotype diversity and lack of PD1 and TIM3 exhaustion markers on BKV-specific T-cells was associated with short clearance time. Our data thus demonstrate how the diversity and the exhaustion state of the T-cells can determine the clinical course of BKV infection.

5.
Front Immunol ; 10: 593, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019503

RESUMO

Influenza vaccination is a common approach to prevent seasonal and pandemic influenza. Pre-existing antibodies against close viral strains might impair antibody formation against previously unseen strains-a process called original antigenic sin. The role of this pre-existing cellular immunity in this process is, despite some hints from animal models, not clear. Here, we analyzed cellular and humoral immunity in healthy individuals before and after vaccination with seasonal influenza vaccine. Based on influenza-specific hemagglutination inhibiting (HI) titers, vaccinees were grouped into HI-negative and -positive cohorts followed by in-depth cytometric and TCR repertoire analysis. Both serological groups revealed cross-reactive T-cell memory to the vaccine strains at baseline that gave rise to the majority of vaccine-specific T-cells post vaccination. On the contrary, very limited number of vaccine-specific T-cell clones was recruited from the naive pool. Furthermore, baseline quantity of vaccine-specific central memory helper T-cells and clonotype richness of this population directly correlated with the vaccination efficacy. Our findings suggest that the deliberate recruitment of pre-existing cross-reactive cellular memory might help to improve vaccination outcome.

6.
Leukemia ; 33(8): 1895-1909, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30842609

RESUMO

Chromosomal rearrangements and specific aneuploidy patterns are initiating events and define subgroups in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here we analyzed 250 BCP-ALL cases and identified a novel subgroup ('PAX5-plus', n = 19) by distinct DNA methylation and gene expression profiles. All patients in this subgroup harbored mutations in the B-lineage transcription factor PAX5, with p.P80R as hotspot. Mutations either affected two independent codons, consistent with compound heterozygosity, or suffered LOH predominantly through chromosome 9p aberrations. These biallelic events resulted in disruption of PAX5 transcriptional programs regulating B-cell differentiation and tumor suppressor functions. Homozygous CDKN2A/B deletions and RAS-activating hotspot mutations were highly enriched as cooperating events in the genomic profile of PAX5-plus ALL. Together, this defined a specific pattern of triple alterations, exclusive to the novel subgroup. PAX5-plus ALL was observed in pediatric and adult patients. Although restricted by the limited sample size, a tendency for more favorable clinical outcome was observed, with 10 of 12 adult PAX5-plus patients achieving long-term survival. PAX5-plus represents the first BCP-ALL subgroup defined by sequence alterations in contrast to gross chromosomal events and exemplifies how deregulated differentiation (PAX5), impaired cell cycle control (CDKN2A/B) and sustained proliferative signaling (RAS) cooperatively drive leukemogenesis.


Assuntos
Mutação , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Cromossomos Humanos Par 9 , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Metabolismo Energético , Humanos , Perda de Heterozigosidade
7.
BMC Genomics ; 20(1): 40, 2019 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-30642251

RESUMO

BACKGROUND: Target enrichment combined with chromosome conformation capturing methodologies such as capture Hi-C (CHC) can be used to investigate spatial layouts of genomic regions with high resolution and at scalable costs. A common application of CHC is the investigation of regulatory elements that are in contact with promoters, but CHC can be used for a range of other applications. Therefore, probe design for CHC needs to be adapted to experimental needs, but no flexible tool is currently available for this purpose. RESULTS: We present a Java desktop application called GOPHER (Generator Of Probes for capture Hi-C Experiments at high Resolution) that implements three strategies for CHC probe design. GOPHER's simple approach is similar to the probe design of previous approaches that employ CHC to investigate all promoters, with one probe being placed at each margin of a single digest that overlaps the transcription start site (TSS) of each promoter. GOPHER's simple-patched approach extends this methodology with a heuristic that improves coverage of viewpoints in which the TSS is located near to one of the boundaries of the digest. GOPHER's extended approach is intended mainly for focused investigations of smaller gene sets. GOPHER can also be used to design probes for regions other than TSS such as GWAS hits or large blocks of genomic sequence. GOPHER additionally provides a number of features that allow users to visualize and edit viewpoints, and outputs a range of files useful for documentation, ordering probes, and downstream analysis. CONCLUSION: GOPHER is an easy-to-use and robust desktop application for CHC probe design. Source code and a precompiled executable can be downloaded from the GOPHER GitHub page at https://github.com/TheJacksonLaboratory/Gopher .


Assuntos
Sondas de DNA/genética , Software , Redes Reguladoras de Genes , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Sítio de Iniciação de Transcrição
8.
Microbiome ; 6(1): 218, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30522523

RESUMO

BACKGROUND: The oral cavity comprises a rich and diverse microbiome, which plays important roles in health and disease. Previous studies have mostly focused on adult populations or in very young children, whereas the adolescent oral microbiome remains poorly studied. Here, we used a citizen science approach and 16S profiling to assess the oral microbiome of 1500 adolescents around Spain and its relationships with lifestyle, diet, hygiene, and socioeconomic and environmental parameters. RESULTS: Our results provide a detailed snapshot of the adolescent oral microbiome and how it varies with lifestyle and other factors. In addition to hygiene and dietary habits, we found that the composition of tap water was related to important changes in the abundance of several bacterial genera. This points to an important role of drinking water in shaping the oral microbiota, which has been so far poorly explored. Overall, the microbiome samples of our study can be clustered into two broad compositional patterns (stomatotypes), driven mostly by Neisseria and Prevotella, respectively. These patterns show striking similarities with those found in unrelated populations. CONCLUSIONS: We hypothesize that these stomatotypes represent two possible global optimal equilibria in the oral microbiome that reflect underlying constraints of the human oral niche. As such, they should be found across a variety of geographical regions, lifestyles, and ages.


Assuntos
Bactérias/classificação , Água Potável/microbiologia , Metagenômica/métodos , Boca/microbiologia , Adolescente , Adulto , Bactérias/genética , Bactérias/isolamento & purificação , DNA Bacteriano/genética , DNA Ribossômico/genética , Comportamento Alimentar , Humanos , Higiene , Estilo de Vida , Neisseria/classificação , Neisseria/genética , Neisseria/isolamento & purificação , Filogenia , Prevotella/classificação , Prevotella/genética , Prevotella/isolamento & purificação , RNA Ribossômico 16S/genética , Professores Escolares , Análise de Sequência de DNA , Espanha
9.
Development ; 145(7)2018 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-29511024

RESUMO

Connective tissues support organs and play crucial roles in development, homeostasis and fibrosis, yet our understanding of their formation is still limited. To gain insight into the molecular mechanisms of connective tissue specification, we selected five zinc-finger transcription factors - OSR1, OSR2, EGR1, KLF2 and KLF4 - based on their expression patterns and/or known involvement in connective tissue subtype differentiation. RNA-seq and ChIP-seq profiling of chick limb micromass cultures revealed a set of common genes regulated by all five transcription factors, which we describe as a connective tissue core expression set. This common core was enriched with genes associated with axon guidance and myofibroblast signature, including fibrosis-related genes. In addition, each transcription factor regulated a specific set of signalling molecules and extracellular matrix components. This suggests a concept whereby local molecular niches can be created by the expression of specific transcription factors impinging on the specification of local microenvironments. The regulatory network established here identifies common and distinct molecular signatures of limb connective tissue subtypes, provides novel insight into the signalling pathways governing connective tissue specification, and serves as a resource for connective tissue development.


Assuntos
Diferenciação Celular/genética , Galinhas/metabolismo , Tecido Conjuntivo/metabolismo , Fatores de Transcrição/metabolismo , Animais , Galinhas/genética , Clonagem Molecular , Extremidades , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Morfogênese/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Transdução de Sinais , Dedos de Zinco/genética
10.
Bone ; 110: 368-377, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29499418

RESUMO

Osteogenesis Imperfecta (OI) is a clinically and genetically heterogeneous disorder. Although differential diagnosis is greatly facilitated by next generation sequencing, its availability can vary considerably. In this study, we compared targeted gene panel or exome sequencing with clinical scoring and grouping in a cohort of 50 OI index patients recruited by a single Indian clinical center in an unselected fashion. In 48 patients we observed a total of 24 novel mutations and 24 known OI mutations, of which several were recurrent. In one patient neither gene panel nor exome sequencing revealed any significant mutation and another patient harbored a class III COL1A1 intronic variant. The percentage of autosomal recessive forms due to mutations in BMP1, FKBP10, LEPRE1, SERPINF1, and WNT1 was unusually high (48%). Grouping according to phenotypic and radiographic features revealed four individuals with Bruck syndrome due to FKBP10 mutations, three patients with hypertrophic callus caused by IFITM5 mutations, and twenty with pronounced bone bowing, of which eight carried WNT1 mutations. There was a clear correlation between genotype and phenotype severity: IFITM5=LEPRE1>WNT1>SERPINF1>COL1A1 (qualitative)>BMP1>FKBP10>COL1A2 (qualitative)>COL1A1 (quantitative)>COL1A2 (quantitative). In one patient we found heterozygous variants in COL1A1 and COL1A2 inherited from parents without an obvious bone phenotype indicating that both variants might contribute to the phenotype. Our findings demonstrate the clinical utility of gene panel testing for OI, but in cases with contractures, hypertrophic callus formation, or - to some extent - extensive bowing single gene analysis might still be more cost-effective.


Assuntos
Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/genética , Adolescente , Adulto , Grupo com Ancestrais do Continente Asiático , Criança , Pré-Escolar , Exoma/genética , Feminino , Estudos de Associação Genética , Genótipo , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/genética , Mutação/genética , Osteogênese Imperfeita/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Proteína Wnt1/genética , Adulto Jovem
11.
Nat Genet ; 50(2): 238-249, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29335546

RESUMO

Chromosomal architecture is known to influence gene expression, yet its role in controlling cell fate remains poorly understood. Reprogramming of somatic cells into pluripotent stem cells (PSCs) by the transcription factors (TFs) OCT4, SOX2, KLF4 and MYC offers an opportunity to address this question but is severely limited by the low proportion of responding cells. We have recently developed a highly efficient reprogramming protocol that synchronously converts somatic into pluripotent stem cells. Here, we used this system to integrate time-resolved changes in genome topology with gene expression, TF binding and chromatin-state dynamics. The results showed that TFs drive topological genome reorganization at multiple architectural levels, often before changes in gene expression. Removal of locus-specific topological barriers can explain why pluripotency genes are activated sequentially, instead of simultaneously, during reprogramming. Together, our results implicate genome topology as an instructive force for implementing transcriptional programs and cell fate in mammals.


Assuntos
Reprogramação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Estruturas Cromossômicas/genética , Genoma , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação/genética , Células Cultivadas , Estruturas Cromossômicas/metabolismo , Compensação de Dosagem (Genética)/genética , Feminino , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/fisiologia , Camundongos , Camundongos Transgênicos , Ligação Proteica , Inativação do Cromossomo X/genética
12.
Am J Hum Genet ; 101(5): 833-843, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100093

RESUMO

Gorlin-Chaudhry-Moss syndrome (GCMS) is a dysmorphic syndrome characterized by coronal craniosynostosis and severe midface hypoplasia, body and facial hypertrichosis, microphthalmia, short stature, and short distal phalanges. Variable lipoatrophy and cutis laxa are the basis for a progeroid appearance. Using exome and genome sequencing, we identified the recurrent de novo mutations c.650G>A (p.Arg217His) and c.649C>T (p.Arg217Cys) in SLC25A24 in five unrelated girls diagnosed with GCMS. Two of the girls had pronounced neonatal progeroid features and were initially diagnosed with Wiedemann-Rautenstrauch syndrome. SLC25A24 encodes a mitochondrial inner membrane ATP-Mg/Pi carrier. In fibroblasts from affected individuals, the mutated SLC25A24 showed normal stability. In contrast to control cells, the probands' cells showed mitochondrial swelling, which was exacerbated upon treatment with hydrogen peroxide (H2O2). The same effect was observed after overexpression of the mutant cDNA. Under normal culture conditions, the mitochondrial membrane potential of the probands' fibroblasts was intact, whereas ATP content in the mitochondrial matrix was lower than that in control cells. However, upon H2O2 exposure, the membrane potential was significantly elevated in cells harboring the mutated SLC25A24. No reduction of mitochondrial DNA copy number was observed. These findings demonstrate that mitochondrial dysfunction with increased sensitivity to oxidative stress is due to the SLC25A24 mutations. Our results suggest that the SLC25A24 mutations induce a gain of pathological function and link mitochondrial ATP-Mg/Pi transport to the development of skeletal and connective tissue.


Assuntos
Anormalidades Múltiplas/genética , Antiporters/genética , Proteínas de Ligação ao Cálcio/genética , Anormalidades Craniofaciais/genética , Craniossinostoses/genética , Permeabilidade do Canal Arterial/genética , Hipertricose/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mutação/genética , Trifosfato de Adenosina/genética , Adolescente , Criança , Pré-Escolar , Cútis Laxa/genética , DNA Mitocondrial/genética , Exoma/genética , Feminino , Retardo do Crescimento Fetal/genética , Fibroblastos/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Lactente , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/genética , Progéria/genética
13.
BMC Cancer ; 17(1): 239, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28376765

RESUMO

BACKGROUND: The defects in DNA repair genes are potentially linked to development and response to therapy in medulloblastoma. Therefore the purpose of this study was to establish the spectrum and frequency of germline variants in selected DNA repair genes and their impact on response to chemotherapy in medulloblastoma patients. METHODS: The following genes were investigated in 102 paediatric patients: MSH2 and RAD50 using targeted gene panel sequencing and NBN variants (p.I171V and p.K219fs*19) by Sanger sequencing. In three patients with presence of rare life-threatening adverse events (AE) and no detected variants in the analyzed genes, whole exome sequencing was performed. Based on combination of molecular and immunohistochemical evaluations tumors were divided into molecular subgroups. Presence of variants was tested for potential association with the occurrence of rare life-threatening AE and other clinical features. RESULTS: We have identified altogether six new potentially pathogenic variants in MSH2 (p.A733T and p.V606I), RAD50 (p.R1093*), FANCM (p.L694*), ERCC2 (p.R695C) and EXO1 (p.V738L), in addition to two known NBN variants. Five out of twelve patients with defects in either of MSH2, RAD50 and NBN genes suffered from rare life-threatening AE, more frequently than in control group (p = 0.0005). When all detected variants were taken into account, the majority of patients (8 out of 15) suffered from life-threatening toxicity during chemotherapy. CONCLUSION: Our results, based on the largest systematic study performed in a clinical setting, provide preliminary evidence for a link between defects in DNA repair genes and treatment related toxicity in children with medulloblastoma. The data suggest that patients with DNA repair gene variants could need special vigilance during and after courses of chemotherapy.


Assuntos
Proteínas de Ciclo Celular/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Meduloblastoma/genética , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Adolescente , Antineoplásicos/efeitos adversos , Criança , Pré-Escolar , DNA Helicases/genética , Reparo do DNA/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Exodesoxirribonucleases/genética , Mutação em Linhagem Germinativa , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Sequenciamento Completo do Exoma , Proteína Grupo D do Xeroderma Pigmentoso/genética
14.
Sci Rep ; 7: 44138, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28281571

RESUMO

Opitz trigonocephaly C syndrome (OTCS) is a rare genetic disorder characterized by craniofacial anomalies, variable intellectual and psychomotor disability, and variable cardiac defects with a high mortality rate. Different patterns of inheritance and genetic heterogeneity are known in this syndrome. Whole exome and genome sequencing of a 19-year-old girl (P7), initially diagnosed with OTCS, revealed a de novo nonsense mutation, p.Q638*, in the MAGEL2 gene. MAGEL2 is an imprinted, maternally silenced, gene located at 15q11-13, within the Prader-Willi region. Patient P7 carried the mutation in the paternal chromosome. Recently, mutations in MAGEL2 have been described in Schaaf-Yang syndrome (SHFYNG) and in severe arthrogryposis. Patient P7 bears resemblances with SHFYNG cases but has other findings not described in this syndrome and common in OTCS. We sequenced MAGEL2 in nine additional OTCS patients and no mutations were found. This study provides the first clear molecular genetic basis for an OTCS case, indicates that there is overlap between OTCS and SHFYNG syndromes, and confirms that OTCS is genetically heterogeneous. Genes encoding MAGEL2 partners, either in the retrograde transport or in the ubiquitination-deubiquitination complexes, are promising candidates as OTCS disease-causing genes.


Assuntos
Craniossinostoses , Deficiência Intelectual , Mutação de Sentido Incorreto , Proteínas , Adulto , Craniossinostoses/genética , Craniossinostoses/metabolismo , Feminino , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Proteínas/genética , Proteínas/metabolismo
15.
PLoS Genet ; 13(1): e1006567, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28103242

RESUMO

Homeotic genes code for key transcription factors (HOX-TFs) that pattern the animal body plan. During embryonic development, Hox genes are expressed in overlapping patterns and function in a partially redundant manner. In vitro biochemical screens probing the HOX-TF sequence specificity revealed largely overlapping sequence preferences, indicating that co-factors might modulate the biological function of HOX-TFs. However, due to their overlapping expression pattern, high protein homology, and insufficiently specific antibodies, little is known about their genome-wide binding preferences. In order to overcome this problem, we virally expressed tagged versions of limb-expressed posterior HOX genes (HOXA9-13, and HOXD9-13) in primary chicken mesenchymal limb progenitor cells (micromass). We determined the effect of each HOX-TF on cellular differentiation (chondrogenesis) and gene expression and found that groups of HOX-TFs induce distinct regulatory programs. We used ChIP-seq to determine their individual genome-wide binding profiles and identified between 12,721 and 28,572 binding sites for each of the nine HOX-TFs. Principal Component Analysis (PCA) of binding profiles revealed that the HOX-TFs are clustered in two subgroups (Group 1: HOXA/D9, HOXA/D10, HOXD12, and HOXA13 and Group 2: HOXA/D11 and HOXD13), which are characterized by differences in their sequence specificity and by the presence of cofactor motifs. Specifically, we identified CTCF binding sites in Group 1, indicating that this subgroup of HOX-proteins cooperates with CTCF. We confirmed this interaction by an independent biological assay (Proximity Ligation Assay) and demonstrated that CTCF is a novel HOX cofactor that specifically associates with Group 1 HOX-TFs, pointing towards a possible interplay between HOX-TFs and chromatin architecture.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genoma , Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/metabolismo , Ativação Transcricional , Animais , Fator de Ligação a CCCTC , Galinhas , Condrogênese , Cromatina/metabolismo , Mesoderma/metabolismo , Ligação Proteica
16.
BMC Genomics ; 17(1): 873, 2016 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-27814676

RESUMO

BACKGROUND: ChIP-nexus, an extension of the ChIP-exo protocol, can be used to map the borders of protein-bound DNA sequences at nucleotide resolution, requires less input DNA and enables selective PCR duplicate removal using random barcodes. However, the use of random barcodes requires additional preprocessing of the mapping data, which complicates the computational analysis. To date, only a very limited number of software packages are available for the analysis of ChIP-exo data, which have not yet been systematically tested and compared on ChIP-nexus data. RESULTS: Here, we present a comprehensive software package for ChIP-nexus data that exploits the random barcodes for selective removal of PCR duplicates and for quality control. Furthermore, we developed bespoke methods to estimate the width of the protected region resulting from protein-DNA binding and to infer binding positions from ChIP-nexus data. Finally, we applied our peak calling method as well as the two other methods MACE and MACS2 to the available ChIP-nexus data. CONCLUSIONS: The Q-nexus software is efficient and easy to use. Novel statistics about duplication rates in consideration of random barcodes are calculated. Our method for the estimation of the width of the protected region yields unbiased signatures that are highly reproducible for biological replicates and at the same time very specific for the respective factors analyzed. As judged by the irreproducible discovery rate (IDR), our peak calling algorithm shows a substantially better reproducibility. An implementation of Q-nexus is available at http://charite.github.io/Q/ .


Assuntos
Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Software , Algoritmos , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
17.
Macromol Biosci ; 16(12): 1776-1791, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27689917

RESUMO

Common strategies for biofunctionalization of surfaces comprise the immobilization of bioactive molecules used as cell-binding ligands for cell recruitment. Besides covalent binding, multivalent noncovalent physical forces between substrate and ligand are an alternative way to equip surfaces with biomacromolecules. In this study, polymer binding ligands are screened by means of a DNA-based in vitro selection process. As candidate biomaterials poly(ether imide) (PEI), polystyrene, and poly[ethylene-co-(vinyl acetate)] are selected, due to their different chemical structure, but similar macroscopic interface properties, allowing physical interaction with nucleotide bases by varying valences. Multivalent interacting aptamers are successfully enriched by SELEX method and an area-wide surface functionalization is achieved, which can be used for further binding of bioactive molecules. In vitro selection against the polymers result in thymine-dominated aptamer binding motifs. The preferential interaction with thymine is attributed to its chemical structure, connected with a decreased electrostatic repulsion of the π-system and the hydrophobic character maximizing entropy. The aptamer binding stability correlates with available valences for interaction, resulting in a more stable functionalization of PEI.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Polímeros/química , Polímeros/metabolismo , Técnica de Seleção de Aptâmeros/métodos , Propriedades de Superfície
18.
Am J Med Genet A ; 170A(4): 1080-5, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26799614

RESUMO

Osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS) are variable genetic disorders that overlap in different ways [Cole 1993; Grahame 1999]. Here, we describe a boy presenting with severe muscular hypotonia, multiple fractures, and joint hyperflexibility, features that are compatible with mild OI and hypermobility type EDS, respectively. By whole exome sequencing, we identified both a COL1A1 mutation (c.4006-1G > A) inherited from the patient's mildly affected mother and biallelic missense variants in TNXB (p.Val1213Ile, p.Gly2592Ser). Analysis of cDNA showed that the COL1A1 splice site mutation led to intron retention causing a frameshift (p.Phe1336Valfs*72). Type 1 collagen secretion by the patient's skin fibroblasts was reduced. Immunostaining of a muscle biopsy obtained from the patient revealed a clear reduction of tenascin-X in the extracellular matrix compared to a healthy control. These findings imply that the combination of the COL1A1 mutation with the TNXB variants might cause the patient's unique phenotype.


Assuntos
Colágeno Tipo I/genética , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/genética , Heterozigoto , Mutação , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/genética , Fenótipo , Tenascina/genética , Alelos , Colágeno Tipo I/metabolismo , Análise Mutacional de DNA , Exoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Lactente , Masculino , Linhagem
19.
Am J Med Genet A ; 170(3): 615-21, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26581570

RESUMO

Synpolydactyly (SPD) is a rare congenital limb disorder characterized by syndactyly between the third and fourth fingers and an additional digit in the syndactylous web. In most cases SPD is caused by heterozygous mutations in HOXD13 resulting in the expansion of a N-terminal polyalanine tract. If homozygous, the mutation results in severe shortening of all metacarpals and phalanges with a morphological transformation of metacarpals to carpals. Here, we describe a novel homozygous missense mutation in a family with unaffected consanguineous parents and severe brachydactyly and metacarpal-to-carpal transformation in the affected child. We performed whole exome sequencing on the index patient, followed by Sanger sequencing of parents and patient to investigate cosegregation. The DNA-binding ability of the mutant protein was tested with electrophoretic mobility shift assays. We demonstrate that the c.938C>G (p.313T>R) mutation in the DNA-binding domain of HOXD13 prevents binding to DNA in vitro. Our results show to our knowledge for the first time that a missense mutation in HOXD13 underlies severe brachydactyly with metacarpal-to-carpal transformation. The mutation is non-penetrant in heterozygous carriers. In conjunction with the literature we propose the possibility that the metacarpal-to-carpal transformation results from a homozygous loss of functional HOXD13 protein in humans in combination with an accumulation of non-functional HOXD13 that might be able to interact with other transcription factors in the developing limb.


Assuntos
Braquidactilia/genética , Proteínas de Homeodomínio/genética , Homozigoto , Mutação de Sentido Incorreto , Sindactilia/genética , Fatores de Transcrição/genética , Adulto , Sequência de Bases , Braquidactilia/diagnóstico , Braquidactilia/patologia , Ossos do Carpo/anormalidades , Ossos do Carpo/metabolismo , Pré-Escolar , Consanguinidade , Ensaio de Desvio de Mobilidade Eletroforética , Exoma , Feminino , Expressão Gênica , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Ossos Metacarpais/anormalidades , Ossos Metacarpais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Sindactilia/diagnóstico , Sindactilia/patologia
20.
Bioinformatics ; 31(22): 3577-83, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26249812

RESUMO

MOTIVATION: When analyzing a case group of patients with ultra-rare disorders the ethnicities are often diverse and the data quality might vary. The population substructure in the case group as well as the heterogeneous data quality can cause substantial inflation of test statistics and result in spurious associations in case-control studies if not properly adjusted for. Existing techniques to correct for confounding effects were especially developed for common variants and are not applicable to rare variants. RESULTS: We analyzed strategies to select suitable controls for cases that are based on similarity metrics that vary in their weighting schemes. We simulated different disease entities on real exome data and show that a similarity-based selection scheme can help to reduce false positive associations and to optimize the performance of the statistical tests. Especially when data quality as well as ethnicities vary a lot in the case group, a matching approach that puts more weight on rare variants shows the best performance. We reanalyzed collections of unrelated patients with Kabuki make-up syndrome, Hyperphosphatasia with Mental Retardation syndrome and Catel-Manzke syndrome for which the disease genes were recently described. We show that rare variant association tests are more sensitive and specific in identifying the disease gene than intersection filters and should thus be considered as a favorable approach in analyzing even small patient cohorts. AVAILABILITY AND IMPLEMENTATION: Datasets used in our analysis are available at ftp://ftp.1000genomes.ebi.ac.uk./vol1/ftp/ CONTACT: : peter.krawitz@charite.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Estudos de Associação Genética , Variação Genética , Estudos de Casos e Controles , Confiabilidade dos Dados , Doença/genética , Grupos Étnicos/genética , Humanos , Curva ROC , Análise de Sequência de DNA
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA