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In this study we used the Illumina Infinium Methylation array to investigate in a cohort of matched archival human tissue samples (n = 32) from 14 individuals with soft tissue sarcomas if genome-wide methylation changes occur during metastatic and recurrent (Met/Rec) disease. A range of sarcoma types were selected for this study: leiomyosarcoma (LMS), myxofibrosarcoma (MFS), rhabdomyosarcoma (RMS) and synovial sarcoma (SS). We identified differential methylation in all Met/Rec matched samples, demonstrating that epigenomic differences develop during the clonal evolution of sarcomas. Differentially methylated regions and genes were detected, not been previously implicated in sarcoma progression, including at PTPRN2 and DAXX in LMS, WT1-AS and TNXB in SS, VENTX and NTRK3 in pleomorphic RMS and MEST and the C14MC / miR-379/miR-656 in MFS. Our overall findings indicate the presence of objective epigenetic differences across primary and Met/Rec human tissue samples not previously reported.

Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design., Author summary Poxvirus vector systems have been widely developed for vaccine applications. Despite considerable progress, so far only one recombinant poxvirus vectored vaccine has to date been licensed for human use, with ongoing efforts seeking to enhance immunogenicity whilst minimizing reactogenicity. The latter two characteristics are often determined by early post-vaccination events at the injection site. We therefore undertook an injection site vaccinology approach to analyzing gene expression at the vaccination site after intramuscular inoculation with a recombinant, multiplication defective, vaccinia-based vaccine. This provided detailed insights into inter alia expression of vector-encoded immunoregulatory genes, as well as host innate and adaptive immune responses. We propose that such injection site vaccinology can inform rational vaccine vector design, and we discuss how the information and approach elucidated herein might be used to improve immunogenicity and limit reactogenicity of poxvirus-based vaccine vector systems.

Whole exome sequencing (WES) is a popular and successful technology which is widely used in both research and clinical settings. However, there is a paucity of reference data for Aboriginal Australians to underpin the translation of health-based genomic research. Here we provide a catalogue of variants called after sequencing the exomes of 50 Aboriginal individuals from the Northern Territory (NT) of Australia and compare these to 72 previously published exomes from a Western Australian (WA) population of Martu origin. Sequence data for both NT and WA samples were processed using an ‘intersect-then-combine’ (ITC) approach, using GATK and SAMtools to call variants. A total of 289,829 variants were identified in at least one individual in the NT cohort and 248,374 variants in at least one individual in the WA cohort. Of these, 166,719 variants were present in both cohorts, whilst 123,110 variants were private to the NT cohort and 81,655 were private to the WA cohort. Our data set provides a useful reference point for genomic studies on Aboriginal Australians., Measurement(s)Aboriginal Australian • DNA • sequence feature annotationTechnology Type(s)Whole Exome Sequencing • DNA sequencing • sequence annotationFactor Type(s)ancestry • sex • ageSample Characteristic - OrganismHomo sapiensSample Characteristic - LocationNorthern Territory Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12040638

Poxvirus systems have been extensively used as vaccine vectors. Herein a systems vaccinology analysis of intramuscular injection sites provides detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. Adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such innate systems vaccinology approaches should inform refinements in poxvirus-based vector design.

Background Progesterone receptor membrane component 1 (PGRMC1) is often elevated in cancers, and exists in alternative states of phosphorylation. A motif centered on PGRMC1 Y180 was evolutionarily acquired concurrently with the embryological gastrulation organizer that orchestrates vertebrate tissue differentiation. Results Here, we show that mutagenic manipulation of PGRMC1 phosphorylation alters cell metabolism, genomic stability, and CpG methylation. Each of several mutants elicited distinct patterns of genomic CpG methylation. Mutation of S57A/Y180/S181A led to increased net hypermethylation, reminiscent of embryonic stem cells. Pathways enrichment analysis suggested modulation of processes related to animal cell differentiation status and tissue identity, as well as cell cycle control and ATM/ATR DNA damage repair regulation. We detected different genomic mutation rates in culture. Conclusions A companion manuscript shows that these cell states dramatically affect protein abundances, cell and mitochondrial morphology, and glycolytic metabolism. We propose that PGRMC1 phosphorylation status modulates cellular plasticity mechanisms relevant to early embryological tissue differentiation.

Additional file 6 Fig. S6. Methylation status of CpG associated with annotated enhancers. Related to Fig. 7. Significant differentially methylated probes were annotated with the 450k_Enhancer feature and the proportion of probes attributed to each feature counted. Arrows indicate increased hypomethylation and reduced hypermethylation of enhancers in the TM/DM comparison.

Additional file 2 Fig. S2. PGRMC1 phosphorylation mutants do not affect AG-205-induced death. Related to Fig. 2. (A) AG-205-induced cell death is unaffected by PGRMC1 phosphorylation status. Cells were incubated in the presence of the indicated AG-205 concentrations (n = 8: 3x line 1, 3x line 2, 2 x line 3) or DMSO vehicle control (n = 3: 1x each cell line), and percentage viable cells was calculated relative to untreated cell controls (n = 9: 3 replicates per cell line) using MTT assay. (B) AUC results for values from A greater than 25 μM reveal no significant differences in response to AG-205 treatment between cell lines (p > 0.85, post-hoc Bonferroni after 1 way ANOVA for AG-205 treatment). The apparently greater survival of DM cells at 25 μM AG-205 observed in this panel was never observed in multiple other repeat experiments.

Additional file 5 Fig. S5. Methylation status of CpG associated with annotated coding genes. Related to Fig. 7. Significant differentially methylated probes were annotated with the UCSC gene feature and the proportion of probes attributed to each feature counted. The y-axis shows the frequency of hypermethylated-hypomethylated probes per feature, and the x-axis shows the difference in those frequencies between WT/MP, DM/WT and TM/DM. 3’UTR: 3′ untranslated region; 5’UTR: 5′ untranslated region; Body: Between the ATG and stop codon; irrespective of the presence of introns, exons, TSS, or promoters; ExonBnd: exon boundaries: TSS1500/TSS200: within indicated number of residues of transcription start site (TSS).

Additional file 1 Fig. S1. Representative hyperspectral autofluorescence cell images from the measurements. Related to Fig. 1. (A) Mean cellular intensity of hyperspectral autofluorescence channel 18 [495 nm(Ex), 700 nm(Em)], which may reflect porphyrin or protein-bound red-shifted flavin emission [46], is significantly affected by PGRMC1-HA phosphorylation status. The table provides Kolmogorov-Smirnov test p values from pair wise comparisons. (B) The ratio of hyperspectral autofluorescence channels 3 [375 nm(Ex), 450 nm(Em)] to channel 12 [435 nm(Ex), 587 nm(Em)] differs significantly between cells. The table follows C. (C) Individual channels #3 and #4 from (B) as listed in Table S1 (left) and the same two channels superimposed (right).

Background Rheumatic heart disease (RHD) after group A streptococcus (GAS) infections is heritable and prevalent in Indigenous populations. Molecular mimicry between human and GAS proteins triggers proinflammatory cardiac valve-reactive T cells. Methods Genome-wide genetic analysis was undertaken in 1263 Aboriginal Australians (398 RHD cases; 865 controls). Single-nucleotide polymorphisms were genotyped using Illumina HumanCoreExome BeadChips. Direct typing and imputation was used to fine-map the human leukocyte antigen (HLA) region. Epitope binding affinities were mapped for human cross-reactive GAS proteins, including M5 and M6. Results The strongest genetic association was intronic to HLA-DQA1 (rs9272622; P = 1.86 × 10-7). Conditional analyses showed rs9272622 and/or DQA1*AA16 account for the HLA signal. HLA-DQA1*0101_DQB1*0503 (odds ratio [OR], 1.44; 95% confidence interval [CI], 1.09-1.90; P = 9.56 × 10-3) and HLA-DQA1*0103_DQB1*0601 (OR, 1.27; 95% CI, 1.07-1.52; P = 7.15 × 10-3) were risk haplotypes; HLA_DQA1*0301-DQB1*0402 (OR 0.30, 95%CI 0.14-0.65, P = 2.36 × 10-3) was protective. Human myosin cross-reactive N-terminal and B repeat epitopes of GAS M5/M6 bind with higher affinity to DQA1/DQB1 alpha/beta dimers for the 2-risk haplotypes than the protective haplotype. Conclusions Variation at HLA_DQA1-DQB1 is the major genetic risk factor for RHD in Aboriginal Australians studied here. Cross-reactive epitopes bind with higher affinity to alpha/beta dimers formed by risk haplotypes, supporting molecular mimicry as the key mechanism of RHD pathogenesis.

BackgroundRheumatic heart disease (RHD) following Group A Streptococcus (GAS) infections is heritable and prevalent in Indigenous populations. Molecular mimicry between human and GAS proteins triggers pro-inflammatory cardiac valve-reactive T-cells.MethodsGenome-wide genetic analysis was undertaken in 1263 Aboriginal Australians (398 RHD cases; 865 controls). Single nucleotide polymorphisms (SNPs) were genotyped using Illumina HumanCoreExome BeadChips. Direct typing and imputation was used to fine-map the human leukocyte antigen (HLA) region. Epitope binding affinities were mapped for human cross-reactive GAS proteins, including M5 and M6.ResultsThe strongest genetic association was intronic to HLA-DQA1 (rs9272622; P=1.86x10−7). Conditional analyses showed rs9272622 and/or DQA1*AA16 account for the HLA signal. HLA-DQA1*0101_DQB1*0503 (OR 1.44, 95%CI 1.09-1.90, P=9.56x10−3) and HLA-DQA1*0103_DQB1*0601 (OR 1.27, 95%CI 1.07-1.52, P=7.15x10−3) were risk haplotypes; HLA_DQA1*0301-DQB1*0402 (OR 0.30, 95%CI 0.14-0.65, P=2.36x10−3) was protective. Human myosin cross-reactive N-terminal and B repeat epitopes of GAS M5/M6 bind with higher affinity to DQA1/DQB1 alpha/beta dimers for the two risk haplotypes than the protective haplotype.ConclusionsVariation at HLA_DQA1-DQB1 is the major genetic risk factor for RHD in Aboriginal Australians studied here. Cross-reactive epitopes bind with higher affinity to alpha/beta dimers formed by risk haplotypes, supporting molecular mimicry as the key mechanism of RHD pathogenesis.

Traditional genome-wide scans for positive selection have mainly uncovered selective sweeps associated with monogenic traits. While selection on quantitative traits is much more common, very few signals have been detected because of their polygenic nature. We searched for positive selection signals underlying coronary artery disease (CAD) in worldwide populations, using novel approaches to quantify relationships between polygenic selection signals and CAD genetic risk. We identified new candidate adaptive loci that appear to have been directly modified by disease pressures given their significant associations with CAD genetic risk. These candidates were all uniquely and consistently associated with many different male and female reproductive traits suggesting selection may have also targeted these because of their direct effects on fitness. This suggests the presence of widespread antagonistic-pleiotropic tradeoffs on CAD loci, which provides a novel explanation for the maintenance and high prevalence of CAD in modern humans. Lastly, we found that positive selection more often targeted CAD gene regulatory variants using HapMap3 lymphoblastoid cell lines, which further highlights the unique biological significance of candidate adaptive loci underlying CAD. Our study provides a novel approach for detecting selection on polygenic traits and evidence that modern human genomes have evolved in response to CAD-induced selection pressures and other early-life traits sharing pleiotropic links with CAD.Author SummaryHow genetic variation contributes to disease is complex, especially for those such as coronary artery disease (CAD) that develop over the lifetime of individuals. One of the fundamental questions about CAD — whose progression begins in young adults with arterial plaque accumulation leading to life-threatening outcomes later in life — is why natural selection has not removed or reduced this costly disease. It is the leading cause of death worldwide and has been present in human populations for thousands of years, implying considerable pressures that natural selection should have operated on. Our study provides new evidence that genes underlying CAD have recently been modified by natural selection and that these same genes uniquely and extensively contribute to human reproduction, which suggests that natural selection may have maintained genetic variation contributing to CAD because of its beneficial effects on fitness. This study provides novel evidence that CAD has been maintained in modern humans as a byproduct of the fitness advantages those genes provide early in human lifecycles.

Additional file 8 Table S1. Spectral channel filters employed for hyperspectral autofluorescence imaging. Related to Fig. 1. For further details of this approach see Gosnell et al. [44].

Additional file 8 Table S1. Spectral channel filters employed for hyperspectral autofluorescence imaging. Related to Fig. 1. For further details of this approach see Gosnell et al. [44].

Additional file 3 Fig. S3. SWATH-MS proteomic quantification of Nicotinamide phosphoribosyltransferase. Related to Fig. 6. The figure shows the abundance profile of P43490 nicotinamide phosphoribosyltransferase (NAMPT) from the SWATH-MS proteomics quantification of the accompanying manuscript [10].

Additional file 10 Table S3. Unique KEGG pathways detected for TM/DM hyper and hypo-methylated gene sets. Related to Fig. 7. The identities of the 11 hypermethylated and 7 hypomethylated pathways unique to the TM/DM comparison from Fig. S7B are given. Full results are available in File S1.

Additional file 9 Table S2. GO pathways enrichment results. Related to Fig. 7. The top ten GO enrichments for each cell comparison from Fig. 7. Full results are available in File S1.

Additional file 10 Table S3. Unique KEGG pathways detected for TM/DM hyper and hypo-methylated gene sets. Related to Fig. 7. The identities of the 11 hypermethylated and 7 hypomethylated pathways unique to the TM/DM comparison from Fig. S7B are given. Full results are available in File S1.

Additional file 9 Table S2. GO pathways enrichment results. Related to Fig. 7. The top ten GO enrichments for each cell comparison from Fig. 7. Full results are available in File S1.

Additional file 4 Fig. S4. PGRMC1 phosphorylation status affects the NNMT pathway. Related to Fig. 6. (A) Metabolomics quantification of S-adenosyl-Methionine (SAM) levels for the indicated cell lines, representing n = 12 (four technical replicates for each of three independent cell lines per PGRMC1 condition). (B) RT-PCR quantification of NNMT mRNA levels after treatment by an NNMT-specific shRNA (NN) or a random scramble shRNA control (scr) in TM cells stably expressing lentiplasmid-driven shRNAs. RT-PCR Methods follow A. p

Additional file 3 Fig. S3. SWATH-MS proteomic quantification of Nicotinamide phosphoribosyltransferase. Related to Fig. 6. The figure shows the abundance profile of P43490 nicotinamide phosphoribosyltransferase (NAMPT) from the SWATH-MS proteomics quantification of the accompanying manuscript [10].