ABSTRACT
PURPOSE: Despite the established role of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC, drug resistance inevitably ensues, with a paucity of treatment options especially in EGFR T790M-negative resistance. EXPERIMENTAL DESIGN: We performed whole-exome and transcriptome analysis of 59 patients with first- and second-generation EGFR TKI-resistant metastatic EGFR-mutated NSCLC to characterize and compare molecular alterations mediating resistance in T790M-positive (T790M+) and -negative (T790M-) disease. RESULTS: Transcriptomic analysis revealed ubiquitous loss of adenocarcinoma lineage gene expression in T790M- tumors, orthogonally validated using multiplex IHC. There was enrichment of genomic features such as TP53 alterations, 3q chromosomal amplifications, whole-genome doubling and nonaging mutational signatures in T790M- tumors. Almost half of resistant tumors were further classified as immunehot, with clinical outcomes conditional on immune cell-infiltration state and T790M status. Finally, using a Bayesian statistical approach, we explored how T790M- and T790M+ disease might be predicted using comprehensive genomic and transcriptomic profiles of treatment-naïve patients. CONCLUSIONS: Our results illustrate the interplay between genetic alterations, cell lineage plasticity, and immune microenvironment in shaping divergent TKI resistance and outcome trajectories in EGFR-mutated NSCLC. Genomic and transcriptomic profiling may facilitate the design of bespoke therapeutic approaches tailored to a tumor's adaptive potential.
Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , ErbB Receptors/genetics , Humans , Protein-Tyrosine Kinases/geneticsABSTRACT
Targeted therapies against the BCR-ABL1 kinase have revolutionized treatment of chronic phase (CP) chronic myeloid leukemia (CML). In contrast, management of blast crisis (BC) CML remains challenging because BC cells acquire complex molecular alterations that confer stemness features to progenitor populations and resistance to BCR-ABL1 tyrosine kinase inhibitors. Comprehensive models of BC transformation have proved elusive because of the rarity and genetic heterogeneity of BC, but are important for developing biomarkers predicting BC progression and effective therapies. To better understand BC, we performed an integrated multiomics analysis of 74 CP and BC samples using whole-genome and exome sequencing, transcriptome and methylome profiling, and chromatin immunoprecipitation followed by high-throughput sequencing. Employing pathway-based analysis, we found the BC genome was significantly enriched for mutations affecting components of the polycomb repressive complex (PRC) pathway. While transcriptomically, BC progenitors were enriched and depleted for PRC1- and PRC2-related gene sets respectively. By integrating our data sets, we determined that BC progenitors undergo PRC-driven epigenetic reprogramming toward a convergent transcriptomic state. Specifically, PRC2 directs BC DNA hypermethylation, which in turn silences key genes involved in myeloid differentiation and tumor suppressor function via so-called epigenetic switching, whereas PRC1 represses an overlapping and distinct set of genes, including novel BC tumor suppressors. On the basis of these observations, we developed an integrated model of BC that facilitated the identification of combinatorial therapies capable of reversing BC reprogramming (decitabine+PRC1 inhibitors), novel PRC-silenced tumor suppressor genes (NR4A2), and gene expression signatures predictive of disease progression and drug resistance in CP.
Subject(s)
Blast Crisis/genetics , Gene Expression Regulation, Leukemic/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Polycomb Repressive Complex 1/physiology , Polycomb Repressive Complex 2/physiology , Cell Differentiation , Chromatin Immunoprecipitation , DNA Methylation , Datasets as Topic , Enhancer of Zeste Homolog 2 Protein/physiology , Gene Dosage , Gene Ontology , High-Throughput Nucleotide Sequencing , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Mutation , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 2/genetics , Transcriptome , Exome Sequencing , Whole Genome SequencingABSTRACT
EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.
Subject(s)
Adenocarcinoma/genetics , ErbB Receptors/genetics , Exome Sequencing/methods , Genomics/methods , Lung Neoplasms/genetics , Mutation , Adenocarcinoma/ethnology , Adenocarcinoma/pathology , Asian People/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Genetic Predisposition to Disease/ethnology , Genetic Predisposition to Disease/genetics , Humans , Lung Neoplasms/ethnology , Lung Neoplasms/pathology , Protein Kinase Inhibitors/pharmacologyABSTRACT
Bosma arhinia microphthalmia syndrome (BAMS) is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects. We report here that missense mutations in the epigenetic regulator SMCHD1 mapping to the extended ATPase domain of the encoded protein cause BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus laevis embryos suggest that these mutations may behave as gain-of-function alleles. This finding is in contrast to the loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD) type 2. Our results establish SMCHD1 as a key player in nasal development and provide biochemical insight into its enzymatic function that may be exploited for development of therapeutics for FSHD.
Subject(s)
Choanal Atresia/genetics , Chromosomal Proteins, Non-Histone/genetics , Microphthalmos/genetics , Mutation, Missense/genetics , Nose/abnormalities , Animals , Cell Line , Child, Preschool , Epigenesis, Genetic/genetics , Female , Genetic Predisposition to Disease/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Muscular Dystrophy, Facioscapulohumeral/genetics , Xenopus laevis/geneticsABSTRACT
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.
Subject(s)
Claudins/genetics , GTPase-Activating Proteins/genetics , Oncogene Proteins, Fusion/metabolism , Stomach Neoplasms/pathology , Amino Acid Sequence , Animals , Cell Adhesion , Cell Line, Tumor , Cell Movement , Cell Proliferation , Clathrin/pharmacology , Claudins/metabolism , Dogs , Endocytosis/drug effects , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition , GTPase-Activating Proteins/metabolism , HeLa Cells , Humans , MCF-7 Cells , Madin Darby Canine Kidney Cells , Molecular Sequence Data , Oncogene Proteins, Fusion/genetics , Phenotype , Stomach Neoplasms/metabolism , rhoA GTP-Binding Protein/antagonists & inhibitors , rhoA GTP-Binding Protein/metabolismABSTRACT
Somatic mutations of TP53 are among the most common in cancer and germline mutations of TP53 (usually missense) can cause Li-Fraumeni syndrome (LFS). Recently, recurrent genomic rearrangements in intron 1 of TP53 have been described in osteosarcoma (OS), a highly malignant neoplasm of bone belonging to the spectrum of LFS tumors. Using whole-genome sequencing of OS, we found features of TP53 intron 1 rearrangements suggesting a unique mechanism correlated with transcription. Screening of 288 OS and 1,090 tumors of other types revealed evidence for TP53 rearrangements in 46 (16%) OS, while none were detected in other tumor types, indicating this rearrangement to be highly specific to OS. We revisited a four-generation LFS family where no TP53 mutation had been identified and found a 445 kb inversion spanning from the TP53 intron 1 towards the centromere. The inversion segregated with tumors in the LFS family. Cancers in this family had loss of heterozygosity, retaining the rearranged allele and resulting in TP53 expression loss. In conclusion, intron 1 rearrangements cause p53-driven malignancies by both germline and somatic mechanisms and provide an important mechanism of TP53 inactivation in LFS, which might in part explain the diagnostic gap of formerly classified "TP53 wild-type" LFS.
Subject(s)
Bone Neoplasms/genetics , Genes, p53 , Introns , Li-Fraumeni Syndrome/genetics , Osteosarcoma/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Base Sequence , Child , Child, Preschool , Gene Rearrangement , Genetic Predisposition to Disease , Germ-Line Mutation , Humans , Male , Middle Aged , Molecular Sequence Data , Pedigree , Young AdultABSTRACT
BACKGROUND: Next-generation sequencing (NGS) technologies have changed our understanding of the variability of the human genome. However, the identification of genome structural variations based on NGS approaches with read lengths of 35-300 bases remains a challenge. Single-molecule optical mapping technologies allow the analysis of DNA molecules of up to 2 Mb and as such are suitable for the identification of large-scale genome structural variations, and for de novo genome assemblies when combined with short-read NGS data. Here we present optical mapping data for two human genomes: the HapMap cell line GM12878 and the colorectal cancer cell line HCT116. FINDINGS: High molecular weight DNA was obtained by embedding GM12878 and HCT116 cells, respectively, in agarose plugs, followed by DNA extraction under mild conditions. Genomic DNA was digested with KpnI and 310,000 and 296,000 DNA molecules (≥ 150 kb and 10 restriction fragments), respectively, were analyzed per cell line using the Argus optical mapping system. Maps were aligned to the human reference by OPTIMA, a new glocal alignment method. Genome coverage of 6.8× and 5.7× was obtained, respectively; 2.9× and 1.7× more than the coverage obtained with previously available software. CONCLUSIONS: Optical mapping allows the resolution of large-scale structural variations of the genome, and the scaffold extension of NGS-based de novo assemblies. OPTIMA is an efficient new alignment method; our optical mapping data provide a resource for genome structure analyses of the human HapMap reference cell line GM12878, and the colorectal cancer cell line HCT116.
Subject(s)
Colorectal Neoplasms/genetics , DNA, Neoplasm , Genome, Human , Sequence Analysis, DNA/methods , Cell Line, Tumor , Chromosome Mapping/methods , HapMap Project , High-Throughput Nucleotide Sequencing/methods , HumansABSTRACT
Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is a rare autosomal recessive renal disease caused by mutations in genes for the tight junction transmembrane proteins Claudin-16 (CLDN16) and Claudin-19 (CLDN19). We present the first case report of a Mexican family with three affected sisters carrying a p.Gly20Asp mutation in CLDN19 whose heterozygous mother showed evident hypercalciuria and normal low magnesemia without any other clinical, laboratory, and radiological symptoms of renal disease making of her an unsuitable donor. The affected sisters showed variable phenotypic expression including age of first symptoms, renal urinary tract infections, nephrolithiasis, nephrocalcinosis, and eye symptoms consisting in retinochoroiditis, strabismus, macular scars, bilateral anisocoria, and severe myopia and astigmatism. End stage renal disease due to renal failure needed kidney transplantation in the three of them. Interesting findings were a heterozygous mother with asymptomatic hypercalciuria warning on the need of carefully explore clinical, laboratory, kidney ultrasonograpy, and mutation status in first degree asymptomatic relatives to avoid inappropriate kidney donors; an evident variable phenotypic expression among patients; the identification of a mutation almost confined to Spanish cases and a 3.5 Mb block of genomic homozygosis strongly suggesting a common remote parental ancestor for the gene mutation reported.
Subject(s)
Claudins/genetics , Hypercalciuria , Kidney Failure, Chronic , Nephrocalcinosis , Renal Tubular Transport, Inborn Errors , Adult , Female , Genetic Carrier Screening , Humans , Hypercalciuria/complications , Hypercalciuria/diagnosis , Hypercalciuria/ethnology , Hypercalciuria/genetics , Hypercalciuria/physiopathology , Kidney Failure, Chronic/etiology , Kidney Failure, Chronic/surgery , Kidney Transplantation , Mexico , Middle Aged , Mutation , Nephrocalcinosis/complications , Nephrocalcinosis/diagnosis , Nephrocalcinosis/ethnology , Nephrocalcinosis/genetics , Nephrocalcinosis/physiopathology , Pedigree , Renal Tubular Transport, Inborn Errors/complications , Renal Tubular Transport, Inborn Errors/diagnosis , Renal Tubular Transport, Inborn Errors/ethnology , Renal Tubular Transport, Inborn Errors/genetics , Renal Tubular Transport, Inborn Errors/physiopathologyABSTRACT
Chromosomal structural variations play an important role in determining the transcriptional landscape of human breast cancers. To assess the nature of these structural variations, we analyzed eight breast tumor samples with a focus on regions of gene amplification using mate-pair sequencing of long-insert genomic DNA with matched transcriptome profiling. We found that tandem duplications appear to be early events in tumor evolution, especially in the genesis of amplicons. In a detailed reconstruction of events on chromosome 17, we found large unpaired inversions and deletions connect a tandemly duplicated ERBB2 with neighboring 17q21.3 amplicons while simultaneously deleting the intervening BRCA1 tumor suppressor locus. This series of events appeared to be unusually common when examined in larger genomic data sets of breast cancers albeit using approaches with lesser resolution. Using siRNAs in breast cancer cell lines, we showed that the 17q21.3 amplicon harbored a significant number of weak oncogenes that appeared consistently coamplified in primary tumors. Down-regulation of BRCA1 expression augmented the cell proliferation in ERBB2-transfected human normal mammary epithelial cells. Coamplification of other functionally tested oncogenic elements in other breast tumors examined, such as RIPK2 and MYC on chromosome 8, also parallel these findings. Our analyses suggest that structural variations efficiently orchestrate the gain and loss of cancer gene cassettes that engage many oncogenic pathways simultaneously and that such oncogenic cassettes are favored during the evolution of a cancer.
Subject(s)
BRCA1 Protein/genetics , Breast Neoplasms/genetics , Chromosome Aberrations , Chromosomes, Human, Pair 17/genetics , Receptor, ErbB-2/genetics , Base Sequence , Cell Line, Tumor , Female , Gene Amplification , Gene Duplication , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
Asian nonsmoking populations have a higher incidence of lung cancer compared with their European counterparts. There is a long-standing hypothesis that the increase of lung cancer in Asian never-smokers is due to environmental factors such as second-hand smoke. We analyzed whole-genome sequencing of 30 Asian lung cancers. Unsupervised clustering of mutational signatures separated the patients into two categories of either all the never-smokers or all the smokers or ex-smokers. In addition, nearly one third of the ex-smokers and smokers classified with the never-smoker-like cluster. The somatic variant profiles of Asian lung cancers were similar to that of European origin with G.C>T.A being predominant in smokers. We found EGFR and TP53 to be the most frequently mutated genes with mutations in 50% and 27% of individuals, respectively. Among the 16 never-smokers, 69% had an EGFR mutation compared with 29% of 14 smokers/ex-smokers. Asian never-smokers had lung cancer signatures distinct from the smoker signature and their mutation profiles were similar to European never-smokers. The profiles of Asian and European smokers are also similar. Taken together, these results suggested that the same mutational mechanisms underlie the etiology for both ethnic groups. Thus, the high incidence of lung cancer in Asian never-smokers seems unlikely to be due to second-hand smoke or other carcinogens that cause oxidative DNA damage, implying that routine EGFR testing is warranted in the Asian population regardless of smoking status.
Subject(s)
DNA Damage/genetics , Lung Neoplasms/epidemiology , Lung Neoplasms/genetics , Tobacco Smoke Pollution/adverse effects , Asian People/genetics , ErbB Receptors/genetics , Female , Genome, Human , High-Throughput Nucleotide Sequencing , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Mutation , Risk Factors , Tumor Suppressor Protein p53/geneticsABSTRACT
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).
Subject(s)
Chromosome Breakpoints , Developmental Disabilities/genetics , Language Development Disorders/genetics , Base Sequence , Chromosome Inversion , DNA Copy Number Variations , Female , Genetic Association Studies , High-Throughput Nucleotide Sequencing , Humans , Male , Molecular Sequence Data , Pedigree , Sequence Analysis, DNA , Translocation, GeneticABSTRACT
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.
Subject(s)
DNA Mutational Analysis/methods , High-Throughput Nucleotide Sequencing/methods , Stomach Neoplasms/genetics , Adenocarcinoma/genetics , Chromosomal Instability , Deamination , Exome , Genomics , Microsatellite Instability , Mutation , Reactive Oxygen Species/metabolismABSTRACT
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.
Subject(s)
Genome, Human , Genomic Structural Variation , Mutation , Neoplasms/genetics , Open Reading Frames , Sequence Analysis, DNA/methods , Algorithms , Cell Line, Tumor , Chromosome Mapping , DNA Copy Number Variations , Genomic Library , Humans , Mutagenesis, InsertionalABSTRACT
Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/genetics , Drug Resistance, Neoplasm/drug effects , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Lung Neoplasms/genetics , Membrane Proteins/genetics , Polymorphism, Genetic/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/genetics , Sequence Deletion/genetics , Adult , Aged , Aged, 80 and over , Annexins/metabolism , BH3 Interacting Domain Death Agonist Protein/genetics , Bcl-2-Like Protein 11 , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Cohort Studies , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Enzyme-Linked Immunosorbent Assay/methods , ErbB Receptors/genetics , Exons/genetics , Female , Follow-Up Studies , Gene Expression Regulation, Neoplastic/drug effects , Gene Frequency , Genotype , Humans , International Cooperation , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Lung Neoplasms/drug therapy , Male , Middle Aged , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/metabolism , Statistics, Nonparametric , TransfectionABSTRACT
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.
Subject(s)
Base Pairing/genetics , Breast Neoplasms/genetics , Chromosome Mapping/methods , Genome, Human/genetics , Genomic Structural Variation/genetics , Stomach Neoplasms/genetics , Cell Line, Tumor , Computational Biology , DNA/genetics , Female , Gene Rearrangement , Humans , Sequence Analysis, DNAABSTRACT
Sensitization to house dust mites (HDM) is highly prevalent among the young atopic population in Singapore. Previously published data suggest that individuals with skin allergies show preferred sensitization to Dermatophagoides pteronyssinus while individuals with pure respiratory allergies show preferred sensitization to Blomia tropicalis. The aim of our study was to compare the sensitization profiles between children with asthma and those with eczema to D. pteronyssinus and B. tropicalis and their specific allergens. A total of 60 children, 30 with asthma and 30 with eczema were recruited. IgE levels specific for a panel of HDM allergens from the two mite species were measured using enzyme-linked immunosorbent assay. The asthma group showed highest sensitization to Blo t5 while the eczema group showed highest sensitization to Der p5. Comparison between the two disease groups showed that the eczema group had significantly higher IgE levels for Der p (p = 0.042) and its allergens Der p1 (p = 0.019) and Der p5 (p = 0.001). Generally, the eczema group was more sensitized to the panel of allergens compared to the asthma group. Individuals with asthma and those with eczema showed different sensitization profiles to HDM. These findings highlighted possible mechanisms for different manifestation of allergy.
Subject(s)
Antigens, Dermatophagoides/immunology , Antigens, Plant/immunology , Asthma/immunology , Eczema/immunology , Adolescent , Allergens , Animals , Arthropod Proteins , Asthma/blood , Asthma/diagnosis , Asthma/physiopathology , Child , Cysteine Endopeptidases , Eczema/blood , Eczema/diagnosis , Eczema/physiopathology , Exanthema , Female , Humans , Immunization , Immunoglobulin E/blood , Immunoglobulin E/immunology , Male , Pyroglyphidae , Respiratory SoundsABSTRACT
Blo t 11, a dust-mite (Blomia tropicalis) paramyosin, is an allergen with significant IgE reactivity that has potential as a diagnostic/therapeutic reagent for house-dust-mite allergy. The present study describes the successful expression of Blo t 11 and its immunodominant peptide fD in insect cells using a baculovirus expression system. The Blo t 11 and fD genes were cloned into the pMelBacA vector and the resulting vectors were co-transfected into Sf9 insect cells with Bac-N-Blue DNA. Plaque assay was used to select for recombinant virus that was then used to infect High Five insect cells for protein expression. Secreted proteins were harvested by immuno-affinity purification using monoclonal antibodies to Blo t 11. Purified proteins were analysed by immunoblotting, N-terminal sequencing and ELISA. Immunoblot analyses revealed the full-length Blo t 11 cDNA expressed as a minor protein band of approx. 200 kDa and two major protein bands of approx. 60 and 70 kDa. Clones expressing fD cDNA fragment produced a protein of approx. 30 kDa that was confirmed to be fD by N-terminal sequencing. Approx. 4-7.5 mg/l of fD and 1 mg/l of Blo t 11 were obtained by affinity purification. ELISA results showed that human IgE reactivity to these recombinant allergens was lower as compared with that of the native Blo t 11, suggesting that these baculovirus-expressed allergens exhibiting reduced allergenicity could be useful for the development of immunotherapeutic reagents for mite allergy.