RESUMO
Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long-span paired-end-tag (PET) sequencing approach using 10-Kb genomic DNA inserts to study human genome structural variations (SVs). The use of a 10-Kb insert size allows the identification of breakpoints within repetitive or homology-containing regions of a few kilobases in size and results in a higher physical coverage compared with small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and two noncancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germ-line SVs, whereas tandem duplications, unpaired inversions, interchromosomal translocations, and complex rearrangements are over-represented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA-PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures that are compatible with breakage-fusion-bridge cycles, and we discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers.
Assuntos
Pareamento de Bases/genética , Neoplasias da Mama/genética , Mapeamento Cromossômico/métodos , Genoma Humano/genética , Variação Estrutural do Genoma/genética , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Biologia Computacional , DNA/genética , Feminino , Rearranjo Gênico , Humanos , Análise de Sequência de DNARESUMO
Genome rearrangements, a hallmark of cancer, can result in gene fusions with oncogenic properties. Using DNA paired-end-tag (DNA-PET) whole-genome sequencing, we analyzed 15 gastric cancers (GCs) from Southeast Asians. Rearrangements were enriched in open chromatin and shaped by chromatin structure. We identified seven rearrangement hot spots and 136 gene fusions. In three out of 100 GC cases, we found recurrent fusions between CLDN18, a tight junction gene, and ARHGAP26, a gene encoding a RHOA inhibitor. Epithelial cell lines expressing CLDN18-ARHGAP26 displayed a dramatic loss of epithelial phenotype and long protrusions indicative of epithelial-mesenchymal transition (EMT). Fusion-positive cell lines showed impaired barrier properties, reduced cell-cell and cell-extracellular matrix adhesion, retarded wound healing, and inhibition of RHOA. Gain of invasion was seen in cancer cell lines expressing the fusion. Thus, CLDN18-ARHGAP26 mediates epithelial disintegration, possibly leading to stomach H(+) leakage, and the fusion might contribute to invasiveness once a cell is transformed.
Assuntos
Claudinas/genética , Proteínas Ativadoras de GTPase/genética , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias Gástricas/patologia , Sequência de Aminoácidos , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Clatrina/farmacologia , Claudinas/metabolismo , Cães , Endocitose/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Humanos , Células MCF-7 , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Fenótipo , Neoplasias Gástricas/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7-11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide (GNAQ), RNA-binding protein, fox-1 homolog (RBFOX3), unc-5 homolog D (C.elegans) (UNC5D), transmembrane protein 47 (TMEM47), and X-linked inhibitor of apoptosis (XIAP). Among them, XIAP is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).
Assuntos
Pontos de Quebra do Cromossomo , Deficiências do Desenvolvimento/genética , Transtornos do Desenvolvimento da Linguagem/genética , Sequência de Bases , Inversão Cromossômica , Variações do Número de Cópias de DNA , Feminino , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Análise de Sequência de DNA , Translocação GenéticaRESUMO
Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. While genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs, the current application of PET sequencing with short insert size DNA can be insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We employed a recently developed procedure to generate PET sequencing data using large DNA inserts of 10-20 kb and compared their characteristics with short insert (1 kb) libraries for their ability to identify SVs. Our results suggest that although short insert libraries bear an advantage in identifying small deletions, they do not provide significantly better breakpoint resolution. In contrast, large inserts are superior to short inserts in providing higher physical genome coverage for the same sequencing cost and achieve greater sensitivity, in practice, for the identification of several classes of SVs, such as copy number neutral and complex events. Furthermore, our results confirm that large insert libraries allow for the identification of SVs within repetitive sequences, which cannot be spanned by short inserts. This provides a key advantage in studying rearrangements in cancer, and we show how it can be used in a fusion-point-guided-concatenation algorithm to study focally amplified regions in cancer.
Assuntos
Genoma Humano , Variação Estrutural do Genoma , Mutação , Neoplasias/genética , Fases de Leitura Aberta , Análise de Sequência de DNA/métodos , Algoritmos , Linhagem Celular Tumoral , Mapeamento Cromossômico , Variações do Número de Cópias de DNA , Biblioteca Genômica , Humanos , Mutagênese InsercionalRESUMO
BACKGROUND: Gastric cancer is the second highest cause of global cancer mortality. To explore the complete repertoire of somatic alterations in gastric cancer, we combined massively parallel short read and DNA paired-end tag sequencing to present the first whole-genome analysis of two gastric adenocarcinomas, one with chromosomal instability and the other with microsatellite instability. RESULTS: Integrative analysis and de novo assemblies revealed the architecture of a wild-type KRAS amplification, a common driver event in gastric cancer. We discovered three distinct mutational signatures in gastric cancer--against a genome-wide backdrop of oxidative and microsatellite instability-related mutational signatures, we identified the first exome-specific mutational signature. Further characterization of the impact of these signatures by combining sequencing data from 40 complete gastric cancer exomes and targeted screening of an additional 94 independent gastric tumors uncovered ACVR2A, RPL22 and LMAN1 as recurrently mutated genes in microsatellite instability-positive gastric cancer and PAPPA as a recurrently mutated gene in TP53 wild-type gastric cancer. CONCLUSIONS: These results highlight how whole-genome cancer sequencing can uncover information relevant to tissue-specific carcinogenesis that would otherwise be missed from exome-sequencing data.
Assuntos
Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Gástricas/genética , Adenocarcinoma/genética , Instabilidade Cromossômica , Desaminação , Exoma , Genômica , Instabilidade de Microssatélites , Mutação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Loss of parkin function is a predominant cause of familial Parkinsonism. Emerging evidence also suggests that parkin expression variability may confer a risk for sporadic Parkinson disease. We have recently demonstrated that a wide variety of Parkinson disease-linked stressors, including dopamine (DA), induce parkin solubility alterations and promote its aggregation within the cell, a phenomenon that may underlie the progressive susceptibility of the brain to degeneration. The vulnerability of parkin to stress-induced modification is likely due to its abundance of cysteine residues. Here, we performed a comprehensive mutational analysis and demonstrate that Cys residues residing both within and outside of the RING-IBR (in between RING fingers)-RING domain of parkin are important in maintaining its solubility. The majority of these Cys residues are highly conserved in parkin across different species and potentially fulfil important structural roles. Further, we found that both parkin and HHARI (human homologue of Drosophila ariadne), another RING-IBR-RING-type ubiquitin ligase, are comparably more susceptible to solubility alterations induced by oxidative and nitrosative stress when compared with other non-RING-IBR-RING Cys-containing enzymes. However, parkin appears to be uniquely sensitive to DA-mediated stress, the specificity of which is likely due to DA modification of 2 Cys residues on parkin (Cys-268 and Cys-323) that are distinct from other RING-IBR-RING members.
Assuntos
Cisteína/química , Ubiquitina-Proteína Ligases/fisiologia , Animais , Linhagem Celular Tumoral , Análise Mutacional de DNA , Dopamina/metabolismo , Drosophila , Humanos , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Solubilidade , Especificidade da Espécie , Ubiquitina-Proteína Ligases/químicaRESUMO
Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease indistinguishable from idiopathic disease. The mechanisms whereby missense alterations in the LRRK2 gene initiate neurodegeneration remain unknown. Here, we demonstrate that seven of 10 suspected familial-linked mutations result in increased kinase activity. Functional and disease-associated mutations in conserved residues reveal the critical link between intrinsic guanosine triphosphatase (GTPase) activity and downstream kinase activity. LRRK2 kinase activity requires GTPase activity, whereas GTPase activity functions independently of kinase activity. Both LRRK2 kinase and GTPase activity are required for neurotoxicity and potentiate peroxide-induced cell death, although LRRK2 does not function as a canonical MAP-kinase-kinase-kinase. These results suggest a link between LRRK2 kinase activity and pathogenic mechanisms relating to neurodegeneration, further supporting a gain-of-function role for LRRK2 mutations.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Mutação/genética , Degeneração Neural/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Degeneração Neural/genética , Neurônios/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/toxicidadeRESUMO
rRNA genes are thought unlikely to be laterally transferred, because rRNA must coevolve with a large number of cellular components to form the highly sophisticated translation apparatus and perform protein synthesis. In this paper, the authors first hypothesized that lateral gene transfer (LGT) might occur to rRNA genes via replacement of gene segments encoding individual domains of rRNA: the 'simplified complexity hypothesis'. Comparative sequence analyses of the 16S and 23S rRNA genes from a large number of actinomycete species frequently identified rRNA genes containing short segments with an abnormally high number of non-random base variations. These variations were nearly always characterized by complementing covariations of several paired bases within the stem of a hairpin. The nature of these base variations is not consistent with random mutations but satisfies well the predictions of the 'simplified complexity hypothesis'. The most parsimonious explanation for this phenomenon is the lateral transfer of rRNA gene segments between different bacterial species. This mode of LGT may create mosaic rRNA genes and occur repeatedly in different regions of a gene, gradually destroying the evolutionary history recorded in the nucleotide sequence.