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1.
Gastroenterology ; 155(6): 1908-1922.e5, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30217742

RESUMO

BACKGROUND & AIMS: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants. METHODS: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls). We performed genome-wide association analyses and gene-based rare-variant burden tests to identify rare and common disease-associated variants and study their interactions. We obtained induced pluripotent stem cell (iPSC) lines from 4 patients with Hirschsprung disease and 2 control individuals, and we used these to generate enteric neural crest cells for transcriptomic analyses. We assessed the neuronal lineage differentiation capability of iPSC-derived enteric neural crest cells using an in vitro differentiation assay. RESULTS: We identified 4 susceptibility loci, including 1 in the phospholipase D1 gene (PLD1) (P = 7.4 × 10-7). The patients had a significant excess of rare protein-altering variants in genes previously associated with Hirschsprung disease and in the ß-secretase 2 gene (BACE2) (P = 2.9 × 10-6). The epistatic effects of common and rare variants across these loci provided a sensitized background that increased risk for the disease. In studies of the iPSCs, we observed common and distinct pathways associated with variants in RET that affect risk. In functional assays, we found variants in BACE2 to protect enteric neurons from apoptosis. We propose that alterations in BACE1 signaling via amyloid ß precursor protein and BACE2 contribute to pathogenesis of Hirschsprung disease. CONCLUSIONS: In whole-genome sequence analyses of patients with Hirschsprung disease, we identified rare and common variants associated with disease risk. Using iPSC cells, we discovered some functional effects of these variants.

2.
Eur J Hum Genet ; 26(6): 818-826, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29483666

RESUMO

Hirschsprung disease (HSCR) is a complex birth defect characterized by the lack of ganglion cells along a variable length of the distal intestine. A large proportion of HSCR patients remain genetically unexplained. We applied whole-genome sequencing (WGS) on 9 trios where the probands are sporadically affected with the most severe form of the disorder and harbor no coding sequence variants affecting the function of known HSCR genes. We found de novo protein-altering variants in three intolerant to change genes-CCT2, VASH1, and CYP26A1-for which a plausible link with the enteric nervous system (ENS) exists. De novo single-nucleotide and indel variants were present in introns and non-coding neighboring regions of ENS-related genes, including NRG1 and ERBB4. Joint analysis with those inherited rare variants found under recessive and/or digenic models revealed both patient-unique and shared genetic features where rare variants were found to be enriched in the extracellular matrix-receptor (ECM-receptor) pathway (p = 3.4 × 10-11). Delineation of the genetic profile of each patient might help finding common grounds that could lead to the discovery of shared molecules that could be used as drug targets for the currently ongoing cell therapy effort which aims at providing an alternative to the surgical treatment.

4.
Nat Commun ; 7: 12992, 2016 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-27703150

RESUMO

Hepatitis B virus (HBV) can integrate into the human genome, contributing to genomic instability and hepatocarcinogenesis. Here by conducting high-throughput viral integration detection and RNA sequencing, we identify 4,225 HBV integration events in tumour and adjacent non-tumour samples from 426 patients with HCC. We show that HBV is prone to integrate into rare fragile sites and functional genomic regions including CpG islands. We observe a distinct pattern in the preferential sites of HBV integration between tumour and non-tumour tissues. HBV insertional sites are significantly enriched in the proximity of telomeres in tumours. Recurrent HBV target genes are identified with few that overlap. The overall HBV integration frequency is much higher in tumour genomes of males than in females, with a significant enrichment of integration into chromosome 17. Furthermore, a cirrhosis-dependent HBV integration pattern is observed, affecting distinct targeted genes. Our data suggest that HBV integration has a high potential to drive oncogenic transformation.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/fisiologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Transformação Celular Neoplásica , Ilhas de CpG , DNA Viral/genética , Feminino , Genoma Humano , Genoma Viral , Hepatite B Crônica/genética , Humanos , Estimativa de Kaplan-Meier , Cirrose Hepática/genética , Cirrose Hepática/virologia , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise de Sequência de RNA , Integração Viral
5.
Medicine (Baltimore) ; 95(29): e4251, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27442652

RESUMO

BACKGROUND: Cases of multiple tumors are rarely reported in China. In our study, a 57-year-old female patient had concurrent squamous cell carcinoma, mucoepidermoid carcinoma, brain cancer, bone cancer, and thyroid cancer, which has rarely been reported to date. METHODS: To determine the relationship among these multiple cancers, available DNA samples from the thyroid, lung, and skin tumors and from normal thyroid tissue were sequenced using whole exome sequencing. RESULTS: The notable discrepancies of somatic mutations among the 3 tumor tissues indicated that they arose independently, rather than metastasizing from 1 tumor. A novel deleterious germline mutation (chr22:29091846, G->A, p.H371Y) was identified in CHEK2, a Li-Fraumeni syndrome causal gene. Examining the status of this novel mutation in the patient's healthy siblings revealed its de novo origin. CONCLUSION: Our study reports the first case of Li-Fraumeni syndrome-like in Chinese patients and demonstrates the important contribution of de novo mutations in this type of rare disease.


Assuntos
Quinase do Ponto de Checagem 2/genética , Análise Mutacional de DNA , Mutação em Linhagem Germinativa/genética , Síndrome de Li-Fraumeni/genética , Fenótipo , Doenças Raras , China , Comparação Transcultural , Exoma/genética , Feminino , Humanos , Síndrome de Li-Fraumeni/diagnóstico , Síndrome de Li-Fraumeni/etnologia , Pessoa de Meia-Idade
6.
Nat Genet ; 48(7): 740-6, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27213287

RESUMO

The human major histocompatibility complex (MHC) region has been shown to be associated with numerous diseases. However, it remains a challenge to pinpoint the causal variants for these associations because of the extreme complexity of the region. We thus sequenced the entire 5-Mb MHC region in 20,635 individuals of Han Chinese ancestry (10,689 controls and 9,946 patients with psoriasis) and constructed a Han-MHC database that includes both variants and HLA gene typing results of high accuracy. We further identified multiple independent new susceptibility loci in HLA-C, HLA-B, HLA-DPB1 and BTNL2 and an intergenic variant, rs118179173, associated with psoriasis and confirmed the well-established risk allele HLA-C*06:02. We anticipate that our Han-MHC reference panel built by deep sequencing of a large number of samples will serve as a useful tool for investigating the role of the MHC region in a variety of diseases and thus advance understanding of the pathogenesis of these disorders.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Complexo Principal de Histocompatibilidade/genética , Polimorfismo de Nucleotídeo Único/genética , Psoríase/genética , Butirofilinas/genética , Estudos de Casos e Controles , China/epidemiologia , Predisposição Genética para Doença , Antígenos HLA-B/genética , Antígenos HLA-C/genética , Cadeias beta de HLA-DP/genética , Humanos , Psoríase/epidemiologia
7.
Oncotarget ; 7(3): 2629-45, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26625313

RESUMO

Bladder cancer (BC) is distinguished by high rate of recurrence after surgery, but the underlying mechanisms remain poorly understood. Here we performed the whole-exome sequencing of 37 BC individuals including 20 primary and 17 recurrent samples in which the primary and recurrent samples were not from the same patient. We uncovered that MLL, EP400, PRDM2, ANK3 and CHD5 exclusively altered in recurrent BCs. Specifically, the recurrent BCs and bladder cancer cells with MLL mutation displayed increased histone H3 tri-methyl K4 (H3K4me3) modification in tissue and cell levels and showed enhanced expression of GATA4 and ETS1 downstream. What's more, MLL mutated bladder cancer cells obtained with CRISPR/Cas9 showed increased ability of drug-resistance to epirubicin (a chemotherapy drug for bladder cancer) than wild type cells. Additionally, the BC patients with high expression of GATA4 and ETS1 significantly displayed shorter lifespan than patients with low expression. Our study provided an overview of the genetic basis of recrudescent bladder cancer and discovered that genetic alterations of MLL were involved in BC relapse. The increased modification of H3K4me3 and expression of GATA4 and ETS1 would be the promising targets for the diagnosis and therapy of relapsed bladder cancer.


Assuntos
Biomarcadores Tumorais/genética , Exoma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histona-Lisina N-Metiltransferase/genética , Mutação/genética , Proteína de Leucina Linfoide-Mieloide/genética , Recidiva Local de Neoplasia/genética , Neoplasias da Bexiga Urinária/genética , Animais , Apoptose , Western Blotting , Estudos de Casos e Controles , Proliferação de Células , Imunoprecipitação da Cromatina , Feminino , Fator de Transcrição GATA4/genética , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Recidiva Local de Neoplasia/patologia , Prognóstico , Proteína Proto-Oncogênica c-ets-1/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Am J Hum Genet ; 96(4): 597-611, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-25839328

RESUMO

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and the fourth most lethal cancer in China. However, although genomic studies have identified some mutations associated with ESCC, we know little of the mutational processes responsible. To identify genome-wide mutational signatures, we performed either whole-genome sequencing (WGS) or whole-exome sequencing (WES) on 104 ESCC individuals and combined our data with those of 88 previously reported samples. An APOBEC-mediated mutational signature in 47% of 192 tumors suggests that APOBEC-catalyzed deamination provides a source of DNA damage in ESCC. Moreover, PIK3CA hotspot mutations (c.1624G>A [p.Glu542Lys] and c.1633G>A [p.Glu545Lys]) were enriched in APOBEC-signature tumors, and no smoking-associated signature was observed in ESCC. In the samples analyzed by WGS, we identified focal (<100 kb) amplifications of CBX4 and CBX8. In our combined cohort, we identified frequent inactivating mutations in AJUBA, ZNF750, and PTCH1 and the chromatin-remodeling genes CREBBP and BAP1, in addition to known mutations. Functional analyses suggest roles for several genes (CBX4, CBX8, AJUBA, and ZNF750) in ESCC. Notably, high activity of hedgehog signaling and the PI3K pathway in approximately 60% of 104 ESCC tumors indicates that therapies targeting these pathways might be particularly promising strategies for ESCC. Collectively, our data provide comprehensive insights into the mutational signatures of ESCC and identify markers for early diagnosis and potential therapeutic targets.


Assuntos
Carcinoma de Células Escamosas/genética , Citidina Desaminase/genética , Neoplasias Esofágicas/genética , Predisposição Genética para Doença/genética , Genoma Humano/genética , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/genética , Desaminase APOBEC-1 , Análise de Variância , Sequência de Bases , Proteína de Ligação a CREB/genética , Linhagem Celular Tumoral , China , Classe I de Fosfatidilinositol 3-Quinases , Variações do Número de Cópias de DNA/genética , Carcinoma de Células Escamosas do Esôfago , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Proteínas com Domínio LIM/genética , Ligases , Dados de Sequência Molecular , Receptores Patched , Receptor Patched-1 , Complexo Repressor Polycomb 1/genética , Proteínas do Grupo Polycomb/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/genética , Análise de Sequência de DNA , Sais de Tetrazólio , Tiazóis , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genética
9.
Nature ; 509(7498): 91-5, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24670651

RESUMO

Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide. Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.


Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Genoma Humano/genética , Mutação/genética , Consumo de Bebidas Alcoólicas/efeitos adversos , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/patologia , Ciclo Celular/genética , Cromossomos Humanos Par 11/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Exoma/genética , Feminino , Genômica , Histonas/metabolismo , Humanos , Masculino , MicroRNAs/genética , Oncogenes/genética , Fenótipo , Receptores Notch/genética , Fatores de Risco , Via de Sinalização Wnt/genética
10.
PLoS One ; 9(3): e90343, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24614680

RESUMO

Despite an increase in the number of molecular epidemiological studies conducted in recent years to evaluate the association between human papillomavirus (HPV) and the risk of breast carcinoma, these studies remain inconclusive. Here we aim to detect HPV DNA in various tissues from patients with breast carcinoma using the method of HPV capture combined with massive paralleled sequencing (MPS). To validate the confidence of our methods, 15 cervical cancer samples were tested by PCR and the new method. Results showed that there was 100% consistence between the two methods.DNA from peripheral blood, tumor tissue, adjacent lymph nodes and adjacent normal tissue were collected from seven malignant breast cancer patients, and HPV type 16 (HPV16) was detected in 1/7, 1/7, 1/7 and 1/7 of patients respectively. Peripheral blood, tumor tissue and adjacent normal tissue were also collected from two patients with benign breast tumor, and 1/2, 2/2 and 2/2 was detected to have HPV16 DNA respectively. MPS metrics including mapping ratio, coverage, depth and SNVs were provided to characterize HPV in samples. The average coverage was 69% and 61.2% for malignant and benign samples respectively. 126 SNVs were identified in all 9 samples. The maximum number of SNVs was located in the gene of E2 and E4 among all samples. Our study not only provided an efficient method to capture HPV DNA, but detected the SNVS, coverage, SNV type and depth. The finding has provided further clue of association between HPV16 and breast cancer.


Assuntos
Neoplasias da Mama/virologia , DNA Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Neoplasias do Colo do Útero/virologia
11.
Genomics ; 102(4): 338-44, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23867110

RESUMO

We reported HIVID (high-throughput Viral Integration Detection), a novel experimental and computational method to detect the location of Hepatitis B Virus (HBV) integration breakpoints in Hepatocellular Carcinoma (HCC) genome. In this method, the fragments with HBV sequence were enriched by a set of HBV probes and then processed to high-throughput sequencing. In order to evaluate the performance of HIVID, we compared the results of HIVID with that of whole genome sequencing method (WGS) in 28 HCC tumors. We detected a total of 246 HBV integration breakpoints in HCC genome, 113 out of which were within 400bp upstream or downstream of 125 breakpoints identified by WGS method, covering 89.3% (125/140) of total breakpoints. The integration was located in the gene TERT, MLL4, and CCNE1. In addition, we discovered 133 novel breakpoints missed by WGS method, with 66.7% (10/15) of validation rate. Our study shows HIVID is a cost-effective methodology with high specificity and sensitivity to identify viral integration in human genome.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Integração Viral , China , Ciclina E/genética , Quebras de DNA , Proteínas de Ligação a DNA/genética , Genoma Humano , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Proteínas Oncogênicas/genética , Telomerase/genética
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