RESUMO
BACKGROUND: Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed. RESULTS: We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate. CONCLUSIONS: This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.
Assuntos
Elementos de DNA Transponíveis/genética , Polimorfismo Genético/genética , Alelos , Genótipo , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodosRESUMO
Insertions of the human-specific subfamily of LINE-1 (L1) retrotransposon are highly polymorphic across individuals and can critically influence the human transcriptome. We hypothesized that L1 insertions could represent genetic variants determining important human phenotypic traits, and performed an integrated analysis of L1 elements and single nucleotide polymorphisms (SNPs) in several human populations. We found that a large fraction of L1s were in high linkage disequilibrium with their surrounding genomic regions and that they were well tagged by SNPs. However, L1 variants were only partially captured by SNPs on standard SNP arrays, so that their potential phenotypic impact would be frequently missed by SNP array-based genome-wide association studies. We next identified potential phenotypic effects of L1s by looking for signatures of natural selection linked to L1 insertions; significant extended haplotype homozygosity was detected around several L1 insertions. This finding suggests that some of these L1 insertions may have been the target of recent positive selection.
Assuntos
Genoma Humano/genética , Desequilíbrio de Ligação/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos/genética , Evolução Molecular , Regulação da Expressão Gênica/genética , Genética Populacional/métodos , Estudo de Associação Genômica Ampla , Haplótipos , Homozigoto , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Seleção Genética/genéticaRESUMO
CTL clear virus-infected cells and tumorigenic cells by releasing potent cytotoxic enzymes stored in preformed lytic granules. The exocytosis process includes polarization of lytic granules toward the immunological synapse, tethering of lytic granules to the plasma membrane and finally fusion of lytic granules with the plasma membrane to release cytotoxic enzymes. Although much is known about the molecular machineries necessary for the earlier steps in lytic granule exocytosis, the molecular machinery governing the final step in the fusion process has not been identified. Here, we show using control and VAMP8 KO mice that VAMP8 is localized to the CTL lytic granules. While the immunological synapse and granule polarization appears normal in both VAMP8 KO and control CTL, CTL-mediated killing was reduced for the Vamp8(-/-) CTL. Analysis of lytic enzyme secretion demonstrated that granzyme A and granzyme B secretion is significantly compromised in VAMP8(-/-) CTL, while the levels of the lytic enzymes in the cells are unaffected. Our results clearly show that VAMP8 is one of the v-SNARE that regulate the lytic ability of CTL by influencing the ability of the lytic granules to fuse with the plasma membrane and release its contents.
