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.
Assuntos
Doenças Inflamatórias Intestinais/genética , Mutação de Sentido Incorreto , NADPH Oxidase 1/genética , NADPH Oxidases/genética , Linhagem Celular Tumoral , Cromossomos Humanos X , Homozigoto , Humanos , Doenças Inflamatórias Intestinais/enzimologia , Masculino , Proteína Adaptadora de Sinalização NOD2/genética , Polimorfismo de Nucleotídeo Único , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
OBJECTIVE: Breath testing and duodenal culture studies suggest that a significant proportion of irritable bowel syndrome (IBS) patients have small intestinal bacterial overgrowth. In this study, we extended these data through 16S rDNA amplicon sequencing and quantitative PCR (qPCR) analyses of duodenal aspirates from a large cohort of IBS, non-IBS and control subjects. MATERIALS AND METHODS: Consecutive subjects presenting for esophagogastroduodenoscopy only and healthy controls were recruited. Exclusion criteria included recent antibiotic or probiotic use. Following extensive medical work-up, patients were evaluated for symptoms of IBS. DNAs were isolated from duodenal aspirates obtained during endoscopy. Microbial populations in a subset of IBS subjects and controls were compared by 16S profiling. Duodenal microbes were then quantitated in the entire cohort by qPCR and the results compared with quantitative live culture data. RESULTS: A total of 258 subjects were recruited (21 healthy, 163 non-healthy non-IBS, and 74 IBS). 16S profiling in five IBS and five control subjects revealed significantly lower microbial diversity in the duodenum in IBS, with significant alterations in 12 genera (false discovery rate < 0.15), including overrepresentation of Escherichia/Shigella (p = 0.005) and Aeromonas (p = 0.051) and underrepresentation of Acinetobacter (p = 0.024), Citrobacter (p = 0.031) and Microvirgula (p = 0.036). qPCR in all 258 subjects confirmed greater levels of Escherichia coli in IBS and also revealed increases in Klebsiella spp, which correlated strongly with quantitative culture data. CONCLUSIONS: 16S rDNA sequencing confirms microbial overgrowth in the small bowel in IBS, with a concomitant reduction in diversity. qPCR supports alterations in specific microbial populations in IBS.
Assuntos
DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Duodeno/microbiologia , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Síndrome do Intestino Irritável/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Endoscopia Gastrointestinal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Spatial and temporal dissection of the genomic changes occurring during the evolution of human non-small cell lung cancer (NSCLC) may help elucidate the basis for its dismal prognosis. We sequenced 25 spatially distinct regions from seven operable NSCLCs and found evidence of branched evolution, with driver mutations arising before and after subclonal diversification. There was pronounced intratumor heterogeneity in copy number alterations, translocations, and mutations associated with APOBEC cytidine deaminase activity. Despite maintained carcinogen exposure, tumors from smokers showed a relative decrease in smoking-related mutations over time, accompanied by an increase in APOBEC-associated mutations. In tumors from former smokers, genome-doubling occurred within a smoking-signature context before subclonal diversification, which suggested that a long period of tumor latency had preceded clinical detection. The regionally separated driver mutations, coupled with the relentless and heterogeneous nature of the genome instability processes, are likely to confound treatment success in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Heterogeneidade Genética , Instabilidade Genômica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Desaminase APOBEC-1 , Carcinógenos/toxicidade , Carcinoma Pulmonar de Células não Pequenas/induzido quimicamente , Citidina Desaminase/genética , Evolução Molecular , Dosagem de Genes , Humanos , Neoplasias Pulmonares/induzido quimicamente , Mutação , Recidiva Local de Neoplasia/genética , Prognóstico , Fumar/efeitos adversos , Translocação Genética , Células Tumorais CultivadasRESUMO
The somatic mutation burden in healthy white blood cells (WBCs) is not well known. Based on deep whole-genome sequencing, we estimate that approximately 450 somatic mutations accumulated in the nonrepetitive genome within the healthy blood compartment of a 115-yr-old woman. The detected mutations appear to have been harmless passenger mutations: They were enriched in noncoding, AT-rich regions that are not evolutionarily conserved, and they were depleted for genomic elements where mutations might have favorable or adverse effects on cellular fitness, such as regions with actively transcribed genes. The distribution of variant allele frequencies of these mutations suggests that the majority of the peripheral white blood cells were offspring of two related hematopoietic stem cell (HSC) clones. Moreover, telomere lengths of the WBCs were significantly shorter than telomere lengths from other tissues. Together, this suggests that the finite lifespan of HSCs, rather than somatic mutation effects, may lead to hematopoietic clonal evolution at extreme ages.
Assuntos
Evolução Clonal , Hematopoese , Leucócitos/metabolismo , Longevidade/genética , Mutação , Sequência Rica em At , Idoso de 80 Anos ou mais , Linhagem da Célula , Sequência Conservada , Feminino , Frequência do Gene , Genoma , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Leucócitos/citologia , Leucócitos/fisiologia , Telômero/genética , Encurtamento do TelômeroRESUMO
The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction-based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.
Assuntos
DNA de Neoplasias/sangue , Neoplasias/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias/genética , Neoplasias/patologia , Adulto JovemRESUMO
Intra-tumor heterogeneity is a hallmark of many cancers and may lead to therapy resistance or interfere with personalized treatment strategies. Here, we combined topographic mapping of somatic breakpoints and transcriptional profiling to probe intra-tumor heterogeneity of treatment-naïve stage IIIC/IV epithelial ovarian cancer. We observed that most substantial differences in genomic rearrangement landscapes occurred between metastases in the omentum and peritoneum versus tumor sites in the ovaries. Several cancer genes such as NF1, CDKN2A, and FANCD2 were affected by lesion-specific breakpoints. Furthermore, the intra-tumor variability involved different mutational hallmarks including lesion-specific kataegis (local mutation shower coinciding with genomic breakpoints), rearrangement classes, and coding mutations. In one extreme case, we identified two independent TP53 mutations in ovary tumors and omentum/peritoneum metastases, respectively. Examination of gene expression dynamics revealed up-regulation of key cancer pathways including WNT, integrin, chemokine, and Hedgehog signaling in only subsets of tumor samples from the same patient. Finally, we took advantage of the multilevel tumor analysis to understand the effects of genomic breakpoints on qualitative and quantitative gene expression changes. We show that intra-tumor gene expression differences are caused by site-specific genomic alterations, including formation of in-frame fusion genes. These data highlight the plasticity of ovarian cancer genomes, which may contribute to their strong capacity to adapt to changing environmental conditions and give rise to the high rate of recurrent disease following standard treatment regimes.
Assuntos
Aberrações Cromossômicas , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Neoplasias Ovarianas/genética , Idoso , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Neurofibromatose 1/genética , Omento/metabolismo , Omento/patologia , Proteínas de Fusão Oncogênica/genética , Neoplasias Ovarianas/patologia , Peritônio/metabolismo , Peritônio/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory. METHODS: We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine™ (PGM™) platform. RESULTS: The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500× to 3500×, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region. CONCLUSIONS: With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/sangue , Dosagem de Genes , Humanos , Mutação , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (â¼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use.
Assuntos
Bactérias/genética , Infecções Bacterianas/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenoma/genética , RNA Ribossômico 16S/genética , Bactérias/classificação , Técnicas de Tipagem Bacteriana/métodos , Fibrose Cística/genética , Fibrose Cística/microbiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Microbiota/genética , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/classificação , Reprodutibilidade dos Testes , Especificidade da Espécie , Escarro/microbiologiaRESUMO
Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160x average target coverage, 93% of nucleotides had at least 20x coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.
Assuntos
Genes Recessivos/genética , Triagem de Portadores Genéticos/métodos , Testes Genéticos/métodos , Análise de Sequência de DNA/métodos , Sequência de Bases , Criança , Bases de Dados Genéticas , Feminino , Testes Genéticos/economia , Genoma Humano , Heterozigoto , Humanos , Dados de Sequência Molecular , Mutação , Gravidez , Diagnóstico Pré-Natal , Alinhamento de Sequência , Análise de Sequência de DNA/economiaRESUMO
Cancer is driven by mutation. Worldwide, tobacco smoking is the principal lifestyle exposure that causes cancer, exerting carcinogenicity through >60 chemicals that bind and mutate DNA. Using massively parallel sequencing technology, we sequenced a small-cell lung cancer cell line, NCI-H209, to explore the mutational burden associated with tobacco smoking. A total of 22,910 somatic substitutions were identified, including 134 in coding exons. Multiple mutation signatures testify to the cocktail of carcinogens in tobacco smoke and their proclivities for particular bases and surrounding sequence context. Effects of transcription-coupled repair and a second, more general, expression-linked repair pathway were evident. We identified a tandem duplication that duplicates exons 3-8 of CHD7 in frame, and another two lines carrying PVT1-CHD7 fusion genes, indicating that CHD7 may be recurrently rearranged in this disease. These findings illustrate the potential for next-generation sequencing to provide unprecedented insights into mutational processes, cellular repair pathways and gene networks associated with cancer.
Assuntos
Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Mutação/genética , Nicotiana/efeitos adversos , Carcinoma de Pequenas Células do Pulmão/etiologia , Carcinoma de Pequenas Células do Pulmão/genética , Fumar/efeitos adversos , Carcinógenos/toxicidade , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA/efeitos dos fármacos , Variações do Número de Cópias de DNA/genética , Dano ao DNA/genética , DNA Helicases/genética , Análise Mutacional de DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Éxons/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Humanos , Mutagênese Insercional/efeitos dos fármacos , Mutagênese Insercional/genética , Mutação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Deleção de Sequência/genéticaRESUMO
We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.
Assuntos
Pareamento de Bases , Biologia Computacional/métodos , Variação Genética , Genoma Humano , Ligases , Análise de Sequência de DNA/métodos , África , Sequência de Bases , Genômica , Genótipo , Heterozigoto , Homozigoto , Humanos , Polimorfismo de Nucleotídeo Único , Padrões de ReferênciaRESUMO
Massively parallel, tag-based sequencing systems, such as the SOLiD system, hold the promise of revolutionizing the study of whole genome gene expression due to the number of data points that can be generated in a simple and cost-effective manner. We describe the development of a 5'-end transcriptome workflow for the SOLiD system and demonstrate the advantages in sensitivity and dynamic range offered by this tag-based application over traditional approaches for the study of whole genome gene expression. 5'-end transcriptome analysis was used to study whole genome gene expression within a colon cancer cell line, HT-29, treated with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5Aza). More than 20 million 25-base 5'-end tags were obtained from untreated and 5Aza-treated cells and matched to sequences within the human genome. Seventy three percent of the mapped unique tags were associated with RefSeq cDNA sequences, corresponding to approximately 14,000 different protein-coding genes in this single cell type. The level of expression of these genes ranged from 0.02 to 4,704 transcripts per cell. The sensitivity of a single sequence run of the SOLiD platform was 100-1,000 fold greater than that observed from 5'end SAGE data generated from the analysis of 70,000 tags obtained by Sanger sequencing. The high-resolution 5'end gene expression profiling presented in this study will not only provide novel insight into the transcriptional machinery but should also serve as a basis for a better understanding of cell biology.
Assuntos
Regiões 5' não Traduzidas , Perfilação da Expressão Gênica/instrumentação , Expressão Gênica , Análise de Sequência de DNA/instrumentação , Regiões 5' não Traduzidas/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Éxons , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Humanos , Íntrons , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodosRESUMO
We have systematically investigated the effect of aggregation of a transmembrane peptide on its diffusion in dimyristoylphosphatidylcholine and in palmitoyloleoylphosphatidylcholine model membranes. The hydrophobic segment of the b subunit from E. coli F(1)F(0)-ATP synthase was modified with a histidine tag at the carbonyl terminus and was aggregated selectively by using a series of multivalent, dendritic chelating agents with nitrilotriacetic acid functional groups. Peptide complexes ranging from monomers to hexamers were formed and studied in giant unilamellar vesicles. The rate of diffusion for the transmembrane peptide complexes were found to depend on the size of the complex. The results agree with predictions from the free area model for monomers and dimers, and the hydrodynamic continuum model for tetramers, pentamers, and hexamers. Comparisons with diffusion of lipids confirm that the diffusion of a transmembrane peptide is enhanced by coupling of density fluctuations between the two monolayers.