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1.
Nat Biotechnol ; 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685959

RESUMO

The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells, but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk of genome instability, and by the inability to use the transposase protein directly. In this study, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (high-solubility SB, hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T cells, which exhibit potent antitumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.

2.
Nat Cell Biol ; 21(10): 1248-1260, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576060

RESUMO

While nuclear lamina abnormalities are hallmarks of human diseases, their interplay with epigenetic regulators and precise epigenetic landscape remain poorly understood. Here, we show that loss of the lysine acetyltransferase MOF or its associated NSL-complex members KANSL2 or KANSL3 leads to a stochastic accumulation of nuclear abnormalities with genomic instability patterns including chromothripsis. SILAC-based MOF and KANSL2 acetylomes identified lamin A/C as an acetylation target of MOF. HDAC inhibition or acetylation-mimicking lamin A derivatives rescue nuclear abnormalities observed in MOF-deficient cells. Mechanistically, loss of lamin A/C acetylation resulted in its increased solubility, defective phosphorylation dynamics and impaired nuclear mechanostability. We found that nuclear abnormalities include EZH2-dependent histone H3 Lys 27 trimethylation and loss of nascent transcription. We term this altered epigenetic landscape "heterochromatin enrichment in nuclear abnormalities" (HENA). Collectively, the NSL-complex-dependent lamin A/C acetylation provides a mechanism that maintains nuclear architecture and genome integrity.


Assuntos
Núcleo Celular/ultraestrutura , Histona Acetiltransferases/metabolismo , Lamina Tipo A/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Epigênese Genética , Fibroblastos , Heterocromatina , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Lamina Tipo A/química , Lamina Tipo A/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética
3.
Bioinformatics ; 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31589307

RESUMO

SUMMARY: VISOR is a tool for haplotype-specific simulations of simple and complex structural variants (SVs). The method is applicable to haploid, diploid or higher ploidy simulations for bulk or single-cell sequencing data. SVs are implanted into FASTA haplotypes at single-basepair resolution, optionally with nearby single-nucleotide variants. Short or long reads are drawn at random from these haplotypes using standard error profiles. Double- or single-stranded data can be simulated and VISOR supports the generation of haplotype-tagged BAM files. The tool further includes methods to interactively visualize simulated variants in single-stranded data. The versatility of VISOR is unmet by comparable tools and it lays the foundation to simulate haplotype-resolved cancer heterogeneity data in bulk or at single cell resolution. AVAILABILITY AND IMPLEMENTATION: VISOR is implemented in python 3.6, open-source and freely available at https://github.com/davidebolo1993/VISOR. Documentation is available at https://davidebolo1993.github.io/visordoc/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

4.
Nat Commun ; 10(1): 1784, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992455

RESUMO

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50 bp) and 27,622 SVs (≥50 bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.


Assuntos
Genoma Humano/genética , Variação Estrutural do Genoma , Genômica/métodos , Haplótipos/genética , Algoritmos , Mapeamento Cromossômico/métodos , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação INDEL , Sequenciamento Completo do Genoma/métodos
5.
Artigo em Inglês | MEDLINE | ID: mdl-30709874

RESUMO

Whole-genome and whole-exome sequencing of individual patients allow the study of rare and potentially causative genetic variation. In this study, we sequenced DNA of a trio comprising a boy with very-early-onset inflammatory bowel disease (veoIBD) and his unaffected parents. We identified a rare, X-linked missense variant in the NAPDH oxidase NOX1 gene (c.C721T, p.R241C) in heterozygous state in the mother and in hemizygous state in the patient. We discovered that, in addition, the patient was homozygous for a common missense variant in the CYBA gene (c.T214C, p.Y72H). CYBA encodes the p22phox protein, a cofactor for NOX1. Functional assays revealed reduced cellular ROS generation and antibacterial capacity of NOX1 and p22phox variants in intestinal epithelial cells. Moreover, the identified NADPH oxidase complex variants affected NOD2-mediated immune responses, and p22phox was identified as a novel NOD2 interactor. In conclusion, we detected missense variants in a veoIBD patient that disrupt the host response to bacterial challenges and reduce protective innate immune signaling via NOD2. We assume that the patient's individual genetic makeup favored disturbed intestinal mucosal barrier function.

6.
Bioinformatics ; 2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30520945

RESUMO

Summary/Motivation: Harmonizing quality control of large-scale second and third-generation sequencing datasets is key for enabling downstream computational and biological analyses. We present Alfred, an efficient and versatile command-line application that computes multi-sample quality control metrics in a read-group aware manner, across a wide variety of sequencing assays and technologies. In addition to standard quality control metrics such as GC bias, base composition, insert size and sequencing coverage distributions it supports haplotype-aware and allele-specific feature counting and feature annotation. The versatility of Alfred allows for easy pipeline integration in high-throughput settings, including DNA sequencing facilities and large-scale research initiatives, enabling continuous monitoring of sequence data quality and characteristics across samples. Alfred supports haplo-tagging of BAM/CRAM files to conduct haplotype-resolved analyses in conjunction with a variety of next-generation sequencing based assays. Alfred's companion web application enables interactive exploration of results and comparison to public data sets. Availability: Alfred is open-source and freely available at https://tobiasrausch.com/alfred/. Supplementary Information: Supplementary data are available at Bioinformatics online.

7.
EMBO Mol Med ; 2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389682

RESUMO

We compared 24 primary pediatric T-cell acute lymphoblastic leukemias (T-ALL) collected at the time of initial diagnosis and relapse from 12 patients and 24 matched patient-derived xenografts (PDXs). DNA methylation profile was preserved in PDX mice in 97.5% of the promoters (ρ = 0.99). Similarly, the genome-wide chromatin accessibility (ATAC-Seq) was preserved remarkably well (ρ = 0.96). Interestingly, both the ATAC regions, which showed a significant decrease in accessibility in PDXs and the regions hypermethylated in PDXs, were associated with immune response, which might reflect the immune deficiency of the mice and potentially the incomplete interaction between murine cytokines and human receptors. The longitudinal approach of this study allowed an observation that samples collected from patients who developed a type 1 relapse (clonal mutations maintained at relapse) preserved their genomic composition; whereas in patients who developed a type 2 relapse (subset of clonal mutations lost at relapse), the preservation of the leukemia's composition was more variable. In sum, this study underlines the remarkable genomic stability, and for the first time documents the preservation of the epigenomic landscape in T-ALL-derived PDX models.

8.
Haematologica ; 2018 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-30213827

RESUMO

T-cell/histiocyte-rich large B-cell lymphoma is a rare aggressive lymphoma showing histopathological overlap with nodular lymphocyte predominant Hodgkin lymphoma. Despite differences in tumor microenvironment and clinical behavior, the tumor cells of both entities show remarkable similarities, suggesting that both lymphomas might represent a spectrum of the same disease. To address this issue, we investigated if these entities share mutations. Ultra-deep targeted resequencing of 6 typical and 11 histopathological variants of nodular lymphocyte predominant Hodgkin lymphoma, and 9 T-cell/histiocyte-rich large B-cell lymphoma cases revealed that genes recurrently mutated in nodular lymphocyte predominant Hodgkin lymphoma are affected by mutations at similar frequencies in T-cell/histiocyte-rich large B-cell lymphoma. The most recurrently mutated genes were JUNB, DUSP2, SGK1, SOCS1 and CREBBP, which harbored mutations more frequently in T-cell/histiocyte-rich large B-cell lymphoma and the histopathological variants of nodular lymphocyte predominant Hodgkin lymphoma compared to its typical form. Mutations in JUNB, DUSP2, SGK1 and SOCS1 were highly enriched for somatic hypermutation hotspot sites, suggesting an important role of aberrant somatic hypermutation in the generation of these somatic mutations and thus in the pathogenesis of both lymphoma entities. Mutations in JUNB are generally rarely observed in malignant lymphomas and thus are relatively specific for nodular lymphocyte predominant Hodgkin lymphoma and T-cell/histiocyte-rich large B-cell lymphoma at such high frequencies (5/17 and 5/9 cases with JUNB mutations, respectively). Taken together, the present study further supports a close relationship of T-cell/histiocyte-rich large B-cell lymphoma and nodular lymphocyte predominant Hodgkin lymphoma by showing that they share highly recurrent genetic lesions.

9.
BMC Bioinformatics ; 19(1): 243, 2018 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-29940847

RESUMO

BACKGROUND: High-throughput bioinformatics analyses of next generation sequencing (NGS) data often require challenging pipeline optimization. The key problem is choosing appropriate tools and selecting the best parameters for optimal precision and recall. RESULTS: Here we introduce ToTem, a tool for automated pipeline optimization. ToTem is a stand-alone web application with a comprehensive graphical user interface (GUI). ToTem is written in Java and PHP with an underlying connection to a MySQL database. Its primary role is to automatically generate, execute and benchmark different variant calling pipeline settings. Our tool allows an analysis to be started from any level of the process and with the possibility of plugging almost any tool or code. To prevent an over-fitting of pipeline parameters, ToTem ensures the reproducibility of these by using cross validation techniques that penalize the final precision, recall and F-measure. The results are interpreted as interactive graphs and tables allowing an optimal pipeline to be selected, based on the user's priorities. Using ToTem, we were able to optimize somatic variant calling from ultra-deep targeted gene sequencing (TGS) data and germline variant detection in whole genome sequencing (WGS) data. CONCLUSIONS: ToTem is a tool for automated pipeline optimization which is freely available as a web application at https://totem.software .

10.
Lancet Oncol ; 19(6): 785-798, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29753700

RESUMO

BACKGROUND: Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines. METHODS: In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGroup3), and group 4 (MBGroup4). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma. FINDINGS: We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MBSHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MBSHH subgroup). Patients with germline APC mutations developed MBWNT and accounted for most (five [71%] of seven) cases of MBWNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MBSHH. Germline TP53 mutations presented only in childhood patients in the MBSHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MBSHH, MBGroup3, and MBGroup4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40-69) and 5-year overall survival was 65% (95% CI 52-81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes. INTERPRETATION: Genetic counselling and testing should be used as a standard-of-care procedure in patients with MBWNT and MBSHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and molecular tumour characteristics. FUNDING: German Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario.

11.
BMC Cancer ; 18(1): 23, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29301499

RESUMO

BACKGROUND: The Grainyhead-like (GRHL) transcription factors have been linked to many different types of cancer. However, no previous study has attempted to investigate potential correlations in expression of different GRHL genes in this context. Furthermore, there is very little information concerning damaging mutations and/or single nucleotide polymorphisms in GRHL genes that may be linked to cancer. METHODS: DNA and RNA were extracted from human non-melanoma skin cancers (NMSC) and adjacent normal tissues (n = 33 pairs of samples). The expression of GRHL genes was measured by quantitative real time PCR. Regulation of GRHL expression by miRNA was studied using cell transfection methods and dual-luciferase reporter system. Targeted deep sequencing of GRHL genes in tumor samples and control tissues were employed to search for mutations and single nucleotide polymorphisms. Single marker rs141193530 was genotyped with pyrosequencing in additional NMSC replication cohort (n = 176). Appropriate statistical and bioinformatic methods were used to analyze and interpret results. RESULTS: We discovered that the expression of two genes - GRHL1 and GRHL3 - is reduced in a coordinated manner in tumor samples, in comparison to the control healthy skin samples obtained from the same individuals. It is possible that both GRHL1 and GRHL3 are regulated, at least to some extent, by different strands of the same oncogenic microRNA - miR-21, what would at least partially explain observed correlation. No de novo mutations in the GRHL genes were detected in the examined tumor samples. However, some single nucleotide polymorphisms in the GRHL genes occur at significantly altered frequencies in the examined group of NMSC patients. CONCLUSIONS: Non-melanoma skin cancer growth is accompanied by coordinated reduced expression of epidermal differentiation genes: GRHL1 and GRHL3, which may be regulated by miR-21-3p and -5p, respectively. Some potentially damaging single nucleotide polymorphisms in GRHL genes occur with altered frequencies in NMSC patients, and they may in particular impair the expression of GRHL3 gene or functioning of encoded protein. The presence of these polymorphisms may indicate an increased risk of NMSC development in affected people.


Assuntos
Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Neoplasias Cutâneas/genética , Fatores de Transcrição/genética , Diferenciação Celular/genética , Epiderme/crescimento & desenvolvimento , Epiderme/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Mutação , Polimorfismo de Nucleotídeo Único/genética , Neoplasias Cutâneas/patologia
12.
EMBO Mol Med ; 10(1): 107-120, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29138229

RESUMO

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non-coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome-wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non-protein-coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non-coding RNAs and 1,756 protein-coding transcripts. 65.3% of the SV-eQTLs do not harbor a significant SNV-eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein-coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike.

13.
Viruses ; 9(8)2017 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-28749451

RESUMO

Although smallpox has been known for centuries, the oldest available variola virus strains were isolated in the early 1940s. At that time, large regions of the world were already smallpox-free. Therefore, genetic information of these strains can represent only the very last fraction of a long evolutionary process. Based on the genomes of 48 strains, two clades are differentiated: Clade 1 includes variants of variola major, and clade 2 includes West African and variola minor (Alastrim) strains. Recently, the genome of an almost 400-year-old Lithuanian mummy was determined, which fell basal to all currently sequenced strains of variola virus on phylogenetic trees. Here, we determined two complete variola virus genomes from human tissues kept in a museum in Prague dating back 60 and 160 years, respectively. Moreover, mass spectrometry-based proteomic, chemical, and microscopic examinations were performed. The 60-year-old specimen was most likely an importation from India, a country with endemic smallpox at that time. The genome of the 160-year-old specimen is related to clade 2 West African and variola minor strains. This sequence likely represents a new endemic European variant of variola virus circulating in the midst of the 19th century in Europe.


Assuntos
Genoma Viral , Museus , Varíola/virologia , Vírus da Varíola/genética , República Tcheca , DNA Viral/genética , Europa (Continente)/epidemiologia , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala , História do Século XIX , História do Século XX , Humanos , Índia/epidemiologia , Filogenia , Reação em Cadeia da Polimerase , Proteômica , Varíola/epidemiologia , Varíola/história , Vírus da Varíola/classificação
14.
Mol Carcinog ; 56(11): 2414-2423, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28543713

RESUMO

The involvement of Grainyhead-like (GRHL) transcription factors in various cancers is well documented. However, little is known about their role in clear cell renal cell carcinoma (ccRCC). We discovered that the expression of two of these factors-GRHL1 and GRHL2-are downregulated in ccRCC samples, and their expression is correlated with the expression of VHL gene. This suggests a functional link between the GRHL transcription factors and one of the best known tumor suppressors. Although the GRHL genes are not mutated in ccRCC, some of the single nucleotide polymorphisms in these genes may indicate an increased risk of ccRCC development and/or may allow to assess patients' prognoses and predict their responses to various forms of therapy. Silencing of GRHL2 expression in non-tumorigenic kidney cell line results in increased cell proliferation, increased resistance to apoptosis, as well as changes in the levels of selected proteins involved in the pathogenesis of ccRCC. These changes support the potential role for GRHL2 as a suppressor of ccRCC.


Assuntos
Carcinoma de Células Renais/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Rim/patologia , Fatores de Transcrição/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Rim/metabolismo , Neoplasias Renais/patologia , Masculino , Polimorfismo de Nucleotídeo Único , Proteínas Repressoras/genética
15.
Oncotarget ; 8(10): 16463-16472, 2017 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-28145886

RESUMO

Follicular dendritic cell (FDC)-sarcoma is a rare neoplasm with morphologic and phenotypic features of FDCs. It shows an extremely heterogeneous morphology, therefore, its diagnosis relys on the phenotype of tumor cells. Aim of the present study was the identification of new specific markers for FDC-sarcoma by whole transcriptome sequencing (WTS). Candidate markers were selected based on gene expression level and biological function. Immunohistochemistry was performed on reactive tonsils, on 22 cases of FDC-sarcomas and 214 control cases including 114 carcinomas, 87 soft tissue tumors, 5 melanomas, 5 thymomas and 3 interdigitating dendritic cell sarcomas. FDC secreted protein (FDCSP) and Serglycin (SRGN) proved to be specific markers of FDC and related tumor. They showed better specificity and sensitivity values than some well known markers used in FDC sarcoma diagnosis (specificity: 98.6%, and 100%, respectively; sensitivity: 72.73% and 68.18%, respectively). In our cohorts CXCL13, CD21, CD35, FDCSP and SRGN were the best markers for FDC-sarcoma diagnosis and could discriminate 21/22 FDC sarcomas from other mesenchymal tumors by linear discriminant analysis. In summary, by WTS we identified two novel FDC markers and by the analysis of a wide cohort of cases and controls we propose an efficient marker panel for the diagnosis of this rare and enigmatic tumor.


Assuntos
Biomarcadores Tumorais/genética , Sarcoma de Células Dendríticas Foliculares/genética , Proteínas/genética , Proteoglicanas/genética , Proteínas de Transporte Vesicular/genética , Idoso , Biomarcadores Tumorais/metabolismo , Sarcoma de Células Dendríticas Foliculares/metabolismo , Sarcoma de Células Dendríticas Foliculares/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Proteínas/metabolismo , Proteoglicanas/metabolismo , Análise Serial de Tecidos , Transcriptoma , Proteínas de Transporte Vesicular/metabolismo
16.
Hum Mutat ; 37(3): 257-68, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26615982

RESUMO

Immunodeficiency patients with DNA repair defects exhibit radiosensitivity and proneness to leukemia/lymphoma formation. Though progress has been made in identifying the underlying mutations, in most patients the genetic basis is unknown. Two de novo mutated candidate genes, MCM3AP encoding germinal center-associated nuclear protein (GANP) and POMP encoding proteasome maturation protein (POMP), were identified by whole-exome sequencing (WES) and confirmed by Sanger sequencing in a child with complex phenotype displaying immunodeficiency, genomic instability, skin changes, and myelodysplasia. GANP was previously described to promote B-cell maturation by nuclear targeting of activation-induced cytidine deaminase (AID) and to control AID-dependent hyperrecombination. POMP is required for 20S proteasome assembly and, thus, for efficient NF-κB signaling. Patient-derived cells were characterized by impaired homologous recombination, moderate radio- and cross-linker sensitivity associated with accumulation of damage, impaired DNA damage-induced NF-κB signaling, and reduced nuclear AID levels. Complementation by wild-type (WT)-GANP normalized DNA repair and WT-POMP rescued defective NF-κB signaling. In conclusion, we identified for the first time mutations in MCM3AP and POMP in an immunodeficiency patient. These mutations lead to cooperative effects on DNA recombination and damage signaling. Digenic/polygenic mutations may constitute a novel genetic basis in immunodeficiency patients with DNA repair defects.


Assuntos
Acetiltransferases/genética , Dano ao DNA/genética , Reparo do DNA/genética , Síndromes de Imunodeficiência/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Chaperonas Moleculares/genética , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Mutação/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
17.
Aging (Albany NY) ; 7(11): 911-27, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26546739

RESUMO

The TERT gene encodes for the reverse transcriptase activity of the telomerase complex and mutations in TERT can lead to dysfunctional telomerase activity resulting in diseases such as dyskeratosis congenita (DKC). Here, we describe a novel TERT mutation at position T1129P leading to DKC with progressive bone marrow (BM) failure in homozygous members of a consanguineous family. BM hematopoietic stem cells (HSCs) of an affected family member were 300-fold reduced associated with a significantly impaired colony forming capacity in vitro and impaired repopulation activity in mouse xenografts. Recent data in yeast suggested improved cellular checkpoint controls by mTOR inhibition preventing cells with short telomeres or DNA damage from dividing. To evaluate a potential therapeutic option for the patient, we treated her primary skin fibroblasts and BM HSCs with the mTOR inhibitor rapamycin. This led to prolonged survival and decreased levels of senescence in T1129P mutant fibroblasts. In contrast, the impaired HSC function could not be improved by mTOR inhibition, as colony forming capacity and multilineage engraftment potential in xenotransplanted mice remained severely impaired. Thus, rapamycin treatment did not rescue the compromised stem cell function of TERTT1129P mutant patient HSCs and outlines limitations of a potential DKC therapy based on rapamycin.


Assuntos
Antígenos CD34/análise , Senescência Celular , Disceratose Congênita/genética , Células-Tronco Hematopoéticas/fisiologia , Mutação , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Telomerase/genética , Animais , Feminino , Células HeLa , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Telômero
18.
Nature ; 526(7571): 75-81, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26432246

RESUMO

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.


Assuntos
Variação Genética/genética , Genoma Humano/genética , Mapeamento Físico do Cromossomo , Sequência de Aminoácidos , Predisposição Genética para Doença , Genética Médica , Genética Populacional , Estudo de Associação Genômica Ampla , Genômica , Genótipo , Haplótipos/genética , Homozigoto , Humanos , Dados de Sequência Molecular , Taxa de Mutação , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Análise de Sequência de DNA , Deleção de Sequência/genética
19.
Haematologica ; 100(11): 1442-50, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26294725

RESUMO

Relapsed precursor T-cell acute lymphoblastic leukemia is characterized by resistance against chemotherapy and is frequently fatal. We aimed at understanding the molecular mechanisms resulting in relapse of T-cell acute lymphoblastic leukemia and analyzed 13 patients at first diagnosis, remission and relapse by whole exome sequencing, targeted ultra-deep sequencing, multiplex ligation dependent probe amplification and DNA methylation array. Compared to primary T-cell acute lymphoblastic leukemia, in relapse the number of single nucleotide variants and small insertions and deletions approximately doubled from 11.5 to 26. Targeted ultra-deep sequencing sensitively detected subclones that were selected for in relapse. The mutational pattern defined two types of relapses. While both are characterized by selection of subclones and acquisition of novel mutations, 'type 1' relapse derives from the primary leukemia whereas 'type 2' relapse originates from a common pre-leukemic ancestor. Relapse-specific changes included activation of the nucleotidase NT5C2 resulting in resistance to chemotherapy and mutations of epigenetic modulators, exemplified by SUZ12, WHSC1 and SMARCA4. While mutations present in primary leukemia and in relapse were enriched for known drivers of leukemia, relapse-specific changes revealed an association with general cancer-promoting mechanisms. This study thus identifies mechanisms that drive progression of pediatric T-cell acute lymphoblastic leukemia to relapse and may explain the characteristic treatment resistance of this condition.


Assuntos
Metilação de DNA , DNA de Neoplasias , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Regiões Promotoras Genéticas , Adolescente , Criança , Pré-Escolar , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia
20.
Nat Methods ; 12(8): 780-6, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26121404

RESUMO

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.


Assuntos
Biologia Computacional/métodos , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo de Nucleotídeo Único , Algoritmos , Mapeamento Cromossômico , Diploide , Biblioteca Gênica , Variação Genética , Genoma , Haplótipos , Humanos , Nucleotídeos/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Sequências de Repetição em Tandem
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