The 1000 Genomes Project (1kGP) is the largest fully open resource of whole-genome sequencing (WGS) data consented for public distribution without access or use restrictions. The final, phase 3 ...release of the 1kGP included 2,504 unrelated samples from 26 populations and was based primarily on low-coverage WGS. Here, we present a high-coverage 3,202-sample WGS 1kGP resource, which now includes 602 complete trios, sequenced to a depth of 30X using Illumina. We performed single-nucleotide variant (SNV) and short insertion and deletion (INDEL) discovery and generated a comprehensive set of structural variants (SVs) by integrating multiple analytic methods through a machine learning model. We show gains in sensitivity and precision of variant calls compared to phase 3, especially among rare SNVs as well as INDELs and SVs spanning frequency spectrum. We also generated an improved reference imputation panel, making variants discovered here accessible for association studies.
We have examined the roles of enzyme activity and the nontoxic AB complex of heat-labile toxin (LT) from Escherichia coli on its adjuvant and immunomodulatory properties. LTK63, an LT mutant that is ...completely devoid of enzyme activity, enhanced Th1 responses to coinjected Ags at low adjuvant dose. In contrast, LTR72, a partially detoxified mutant, enhanced Th2 responses and when administered intranasally to mice before infection with Bordetella pertussis suppressed Th1 responses and delayed bacterial clearance from the lungs. LTR72 or wild-type LT inhibited Ag-induced IFN-gamma production by Th1 cells, and LT enhanced IL-5 production by Th2 cells in vitro. Each of the toxins enhanced B7-1 expression on macrophages, but enhancement of B7-2 expression was dependent on enzyme activity. We also observed distinct effects of the nontoxic AB complex and enzyme activity on inflammatory cytokine production. LT and LTR72 suppressed LPS and IFN-gamma induced TNF-alpha and IL-12 production, but enhanced IL-10 secretion by macrophages in vitro and suppressed IL-12 production in vivo in a murine model of LPS-induced shock. In contrast, LTK63 augmented the production of IL-12 and TNF-alpha. Furthermore, LTK63 enhanced NF-kappaB translocation, whereas low doses of LTR72 or LT failed to activate NF-kappaB, but stimulated cAMP production. Thus, E. coli LT appears to be capable of suppressing Th1 responses and enhancing Th2 responses through the modulatory effects of enzyme activity on NF-kappaB activation and IL-12 production. In contrast, the nontoxic AB complex can stimulate acquired immune responses by activating components of the innate immune system.
Acellular pertussis vaccines (Pa) protect against severe pertussis in children. However, serum antibody responses decline quickly after immunization. Studies in animal models suggest that ...cell‐mediated immunity also contributes to protection against Bordetella pertussis, and it has already been demonstrated that Pa induce T cells that secrete type‐1 and type‐2 cytokines in children. In this study we examined the persistence of the T cell response and the effect of booster immunization in 4–6‐year‐old children. Cell‐mediated immunity to B. pertussis antigens was detected in a high proportion of children more than 42 months after their last immunization. Peripheral blood mononuclear cells (PBMC) from the majority of children secreted interferon‐gamma (IFN‐γ) and a smaller proportion IL‐5, in response to specific antigen stimulation in vitro. However, following booster immunization, significantly higher concentrations of IL‐5, but not IFN‐γ, were produced by PBMC in response to B. pertussis antigens. Furthermore, plasma IL‐4 and IL‐5 concentrations were increased, whereas IFN‐γ concentrations were reduced following booster immunization. It has been suggested that childhood immunization with Th2‐inducing vaccines may predispose some children to atopic disease. Although we found that pertussis toxin (PT)‐specific IgE was significantly increased after booster immunization in both atopic and non‐atopic children, the levels of IgE to common allergens and the prevalence of positive skin prick test were unaffected by the booster vaccination. Thus, despite the enhancement of type‐2 responses to B. pertussis antigens, booster vaccination with Pa does not appear to be a risk factor for allergy.
By characterizing the geographic and functional spectrum of human genetic variation, the 1000 Genomes Project aims to build a resource to help to understand the genetic contribution to disease. Here ...we describe the genomes of 1,092 individuals from 14 populations, constructed using a combination of low-coverage whole-genome and exome sequencing. By developing methods to integrate information across several algorithms and diverse data sources, we provide a validated haplotype map of 38 million single nucleotide polymorphisms, 1.4 million short insertions and deletions, and more than 14,000 larger deletions. We show that individuals from different populations carry different profiles of rare and common variants, and that low-frequency variants show substantial geographic differentiation, which is further increased by the action of purifying selection. We show that evolutionary conservation and coding consequence are key determinants of the strength of purifying selection, that rare-variant load varies substantially across biological pathways, and that each individual contains hundreds of rare non-coding variants at conserved sites, such as motif-disrupting changes in transcription-factor-binding sites. This resource, which captures up to 98% of accessible single nucleotide polymorphisms at a frequency of 1% in related populations, enables analysis of common and low-frequency variants in individuals from diverse, including admixed, populations.
Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent-child trio data. We present 64 assembled ...haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average minimum contig length needed to cover 50% of the genome: 26 million base pairs) integrate all forms of genetic variation, even across complex loci. We identified 107,590 structural variants (SVs), of which 68% were not discovered with short-read sequencing, and 278 SV hotspots (spanning megabases of gene-rich sequence). We characterized 130 of the most active mobile element source elements and found that 63% of all SVs arise through homology-mediated mechanisms. This resource enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1526 expression quantitative trait loci as well as SV candidates for adaptive selection within the human population.
Current paediatric immunization programmes include too many injections in the first months of life. Oral or nasal vaccine delivery eliminates the requirement for needles and can induce immunity at ...the site of infection. However, protein antigens are poorly immunogenic when so delivered and can induce tolerance. Novel ways to enhance immune responses to protein or polysaccharide antigens have opened up new possibilities for the design of effective mucosal vaccines. Here, we discuss the immunological principles underlying mucosal vaccine development and review the application of immunomodulatory molecules and delivery systems to the selective enhancement of protective immune responses at mucosal surfaces.
Abstract The skeletal muscle ryanodine receptor plays a crucial role in excitation–contraction (EC) coupling and is implicated in various congenital myopathies. The periodic paralyses are a ...heterogeneous, dominantly inherited group of conditions mainly associated with mutations in the SCN4A and the CACNA1S genes. The interaction between RyR1 and DHPR proteins underlies depolarization-induced Ca2+ release during EC coupling in skeletal muscle. We report a 35-year-old woman presenting with signs and symptoms of a congenital myopathy at birth and repeated episodes of generalized, atypical normokalaemic paralysis in her late teens. Genetic studies of this patient revealed three heterozygous RYR1 substitutions (p.Arg2241X, p.Asp708Asn and p.Arg2939Lys) associated with marked reduction of the RyR1 protein and abnormal DHPR distribution. We conclude that RYR1 mutations may give rise to both myopathies and atypical periodic paralysis, and RYR1 mutations may underlie other unresolved cases of periodic paralysis with unusual features.
The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present ...results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data set. On average, each person is found to carry approximately 250 to 300 loss-of-function variants in annotated genes and 50 to 100 variants previously implicated in inherited disorders. We demonstrate how these results can be used to inform association and functional studies. From the two trios, we directly estimate the rate of de novo germline base substitution mutations to be approximately 10(-8) per base pair per generation. We explore the data with regard to signatures of natural selection, and identify a marked reduction of genetic variation in the neighbourhood of genes, due to selection at linked sites. These methods and public data will support the next phase of human genetic research.
Because synonymous mutations do not change the amino acid sequence of a protein, they are generally considered to be selectively neutral. Empiric data suggest, however, that a significant fraction of ...viral mutational fitness effects may be attributable to synonymous mutation. Bias in synonymous codon usage in viruses may result from selection for translational efficiency, mutational bias, base pairing requirements in RNA structures, or even selection against specific dinucleotides by innate immune effectors. Experimental analyses of codon usage and genome evolution have been facilitated by advances in synthetic biology, which now make it feasible to generate viral genomes that contain large numbers of synonymous mutations. The generally pleiotropic effects of synonymous mutation on viral fitness have, at times, made it difficult to define the mechanistic basis for the observed attenuation of these heavily mutated viruses. We have addressed this problem by developing a bioinformatic tool for the generation and analysis of viral sequences with large-scale synonymous mutation. A variety of permutation strategies are applied to shuffle codons within an open reading frame. After measuring the dinucleotide frequency, codon usage, codon pair bias, and free energy of RNA folding for each permuted genome, we used z-score normalization and a least squares regression model to quantify their overall distance from the starting sequence. Using this approach, the user can easily identify a large number of synonymously mutated sequences with varying similarity to a wild-type genome across a range of nucleic-acid-based determinants of viral fitness. We believe that this tool will be useful in designing genomes for subsequent experimental studies of the fitness impacts of synonymous mutation.
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