RESUMO
As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to â¼ 100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Linfoide/genética , Meduloblastoma/genética , Mutação , Genoma Humano , HumanosRESUMO
Natural killer (NK) cells are lymphocytes involved in antimicrobial and antitumoral immune responses. Using N-ethyl-N-nitrosourea mutagenesis in mice, we identified a mutant with increased resistance to viral infections because of the presence of hyperresponsive NK cells. Whole-genome sequencing and functional analysis revealed a loss-of-function mutation in the Ncr1 gene encoding the activating receptor NKp46. The down-regulation of NK cell activity by NKp46 was associated with the silencing of the Helios transcription factor in NK cells. NKp46 was critical for the subsequent development of antiviral and antibacterial T cell responses, which suggests that the regulation of NK cell function by NKp46 allows for the optimal development of adaptive immune responses. NKp46 blockade enhanced NK cell reactivity in vivo, which could enable the design of immunostimulation strategies in humans.
Assuntos
Antígenos Ly/fisiologia , Proteínas de Ligação a DNA/genética , Infecções por Herpesviridae/imunologia , Células Matadoras Naturais/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/fisiologia , Linfócitos T/imunologia , Fatores de Transcrição/genética , Imunidade Adaptativa , Substituição de Aminoácidos , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Antígenos Ly/genética , Antígenos Ly/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Teste de Complementação Genética , Infecções por Herpesviridae/virologia , Memória Imunológica , Listeriose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muromegalovirus/fisiologia , Mutagênese , Receptor 1 Desencadeador da Citotoxicidade Natural/antagonistas & inibidores , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Carga ViralRESUMO
BACKGROUND/AIMS: In primary biliary cirrhosis (PBC), pathogenesis is influenced by genetic factors that remain poorly elucidated up to now. We investigated the impact of sequence diversity in candidate genes involved in immunity (CTLA-4 and TNFalpha), in bile formation (10 hepatobiliary transporter genes) and in the adaptative response to cholestasis (three nuclear receptor genes) on the susceptibility and severity of PBC. METHODS: A total of 42 Ht SNPs were identified and compared in 258 PBC patients and two independent groups of 286 and 269 healthy controls. All participants were white continental individuals with French ancestry. RESULTS: Ht SNPs of CTLA-4 and TNFalpha genes were significantly associated with susceptibility to PBC. The progression rate of liver disease under ursodeoxycholic acid (UDCA) therapy was significantly linked to SNPs of TNFalpha and SLC4A2/anion exchanger 2 (AE2) genes. A multivariate Cox regression analysis including clinical and biochemical parameters showed that SLC4A2/AE2 variant was an independent prognostic factor. CONCLUSIONS: These data point to a primary role of genes encoding regulators of the immune system in the susceptibility to PBC. They also demonstrate that allelic variations in TNFalpha and SLC4A2/AE2 have a significant impact on the evolutive profile of PBC under UDCA therapy.
Assuntos
Testes Genéticos , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/imunologia , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de Doença , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Proteínas de Transporte de Ânions/genética , Antígenos CD/genética , Antiporters/genética , Antígeno CTLA-4 , Estudos de Casos e Controles , Antiportadores de Cloreto-Bicarbonato , Proteínas de Ligação a DNA/genética , Feminino , França , Predisposição Genética para Doença , Humanos , Estimativa de Kaplan-Meier , Receptores X do Fígado , Masculino , Pessoa de Meia-Idade , Receptores Nucleares Órfãos , Prognóstico , Modelos de Riscos Proporcionais , Receptores Citoplasmáticos e Nucleares/genética , Proteínas SLC4A , Fator de Necrose Tumoral alfa/genéticaRESUMO
Pluripotential human embryonal carcinoma (EC) cell linesundergo differentiation programs resembling those occurring in embryonal stem cells during development. Expression profiling was performed during the terminal differentiation of the EC cell line, NTera2/Clone D1 by all-trans-retinoic acid. Time-response analysis via clustering of >12,000 human transcripts revealed distinct stages in the transition from an EC cell to neuronal progenitor cells expressing patterning markers compatible with posterior hindbrain fates followed by the appearance of immature postmitotic neurons with an evolving synaptic apparatus. Global analysis of gene expression allows monitoring cell fate and differentiation of EC cells in vitro and may provide insight into human embryonal stem cell development.