Alterations in colorectal cancer virome and its persistence after surgery.

Viruses are a key component of the colon microbiome, but the relationship between virome and colorectal cancer (CRC) remains poorly understood. We seek to identify alterations in the viral community that is characteristic of CRC and examine if they persist after surgery. Forty-nine fecal samples from 25 non-cancer (NC) individuals and 12 CRC patients, before and 6-months after surgery, were collected for metagenomic analysis. The fecal virome of CRC patients demonstrated an increased network connectivity as compared to NC individuals. Co-exclusion of influential viruses to bacterial species associated with healthy gut status was observed in CRC, suggesting an altered virome induced a change in the healthy gut bacteriome. Network analysis revealed lower connectivity within the virome and trans-kingdom interactions in NC. After surgery, the number of strong correlations decreased for trans-kingdom and within the bacteria and virome networks, indicating lower connectivity within the microbiome. Some co-occurrence patterns between dominant viruses and bacteria were also lost after surgery, suggesting a possible return to the healthy state of gut microbiome. Microbial signatures characteristic of CRC include an altered virome besides an altered bacterial composition. Elevated viral correlations and network connectivity were observed in CRC patients relative to healthy individuals, alongside distinct changes in the cross-kingdom correlation network unique to CRC patients. Some patterns of dysbiosis persist after surgery. Future studies should seek to verify if dysbiosis truly persists after surgery in a larger sample size with microbiome data collected at various time points after surgery to explore if there is field-change in the remaining colon, as well as to examine if persistent dysbiosis correlates with patient outcomes.

Integration and Reanalysis of Four RNA-Seq Datasets Including BALF, Nasopharyngeal Swabs, Lung Biopsy, and Mouse Models Reveals Common Immune Features of COVID-19.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), has spread over the world causing a pandemic which is still ongoing since its emergence in late 2019. A great amount of effort has been devoted to understanding the pathogenesis of COVID-19 with the hope of developing better therapeutic strategies. Transcriptome analysis using technologies such as RNA sequencing became a commonly used approach in study of host immune responses to SARS-CoV-2. Although substantial amount of information can be gathered from transcriptome analysis, different analysis tools used in these studies may lead to conclusions that differ dramatically from each other. Here, we re-analyzed four RNA-sequencing datasets of COVID-19 samples including human bronchoalveolar lavage fluid, nasopharyngeal swabs, lung biopsy and hACE2 transgenic mice using the same standardized method. The results showed that common features of COVID-19 include upregulation of chemokines including CCL2, CXCL1, and CXCL10, inflammatory cytokine IL-1ß and alarmin S100A8/S100A9, which are associated with dysregulated innate immunity marked by abundant neutrophil and mast cell accumulation. Downregulation of chemokine receptor genes that are associated with impaired adaptive immunity such as lymphopenia is another common feather of COVID-19 observed. In addition, a few interferon-stimulated genes but no type I IFN genes were identified to be enriched in COVID-19 samples compared to their respective control in these datasets. These features are in line with results from single-cell RNA sequencing studies in the field. Therefore, our re-analysis of the RNA-seq datasets revealed common features of dysregulated immune responses to SARS-CoV-2 and shed light to the pathogenesis of COVID-19.

Annexin-A1 deficiency attenuates stress-induced tumor growth via fatty acid metabolism in mice: an Integrated multiple omics analysis on the stress- microbiome-metabolite-epigenetic-oncology (SMMEO) axis.

Background: High emotional or psychophysical stress levels have been correlated with an increased risk and progression of various diseases. How stress impacts the gut microbiota to influence metabolism and subsequent cancer progression is unclear. Methods: Feces and serum samples from BALB/c ANXA1+/+ and ANXA1-/- mice with or without chronic restraint stress were used for 16S rRNA gene sequencing and GC-MS metabolomics analysis to investigate the effect of stress on microbiome and metabolomics during stress and breast tumorigenesis. Breast tumors samples from stressed and non-stressed mice were used to perform Whole-Genome Bisulfite Sequencing (WGBS) and RNAseq analysis to construct the potential network from candidate hub genes. Finally, machine learning and integrated analysis were used to map the axis from chronic restraint stress to breast cancer development. Results: We report that chronic stress promotes breast tumor growth via a stress-microbiome-metabolite-epigenetic-oncology (SMMEO) axis. Chronic restraint stress in mice alters the microbiome composition and fatty acids metabolism and induces an epigenetic signature in tumors xenografted after stress. Subsequent machine learning and systemic modeling analyses identified a significant correlation among microbiome composition, metabolites, and differentially methylated regions in stressed tumors. Moreover, silencing Annexin-A1 inhibits the changes in the gut microbiome and fatty acid metabolism after stress as well as basal and stress-induced tumor growth. Conclusions: These data support a physiological axis linking the microbiome and metabolites to cancer epigenetics and inflammation. The identification of this axis could propel the next phase of experimental discovery in further understanding the underlying molecular mechanism of tumorigenesis caused by physiological stress.

ZDOG: zooming in on dominating genes with mutations in cancer pathways.

BACKGROUND: Inference of cancer-causing genes and their biological functions are crucial but challenging due to the heterogeneity of somatic mutations. The heterogeneity of somatic mutations reveals that only a handful of oncogenes mutate frequently and a number of cancer-causing genes mutate rarely. RESULTS: We develop a Cytoscape app, named ZDOG, for visualization of the extent to which mutated genes may affect cancer pathways using the dominating tree model. The dominator tree model allows us to examine conveniently the positional importance of a gene in cancer signalling pathways. This tool facilitates the identification of mutated "master" regulators even with low mutation frequency in deregulated signalling pathways. CONCLUSIONS: We have presented a model for facilitating the examination of the extent to which mutation in a gene may affect downstream components in a signalling pathway through its positional information. The model is implemented in a user-friendly Cytoscape app which will be freely available upon publication. AVAILABILITY: Together with a user manual, the ZDOG app is freely available at GitHub (https://github.com/rudi2013/ZDOG). It is also available in the Cytoscape app store (http://apps.cytoscape.org/apps/ZDOG) and users can easily install it using the Cytoscape App Manager.

MOLGENIS research: advanced bioinformatics data software for non-bioinformaticians.

MOTIVATION: The volume and complexity of biological data increases rapidly. Many clinical professionals and biomedical researchers without a bioinformatics background are generating big '-omics' data, but do not always have the tools to manage, process or publicly share these data. RESULTS: Here we present MOLGENIS Research, an open-source web-application to collect, manage, analyze, visualize and share large and complex biomedical datasets, without the need for advanced bioinformatics skills. AVAILABILITY AND IMPLEMENTATION: MOLGENIS Research is freely available (open source software). It can be installed from source code (see http://github.com/molgenis), downloaded as a precompiled WAR file (for your own server), setup inside a Docker container (see http://molgenis.github.io), or requested as a Software-as-a-Service subscription. For a public demo instance and complete installation instructions see http://molgenis.org/research.

Gut microbiota composition and functional changes in inflammatory bowel disease and irritable bowel syndrome.

Changes in the gut microbiota have been associated with two of the most common gastrointestinal diseases, inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Here, we performed a case-control analysis using shotgun metagenomic sequencing of stool samples from 1792 individuals with IBD and IBS compared with control individuals in the general population. Despite substantial overlap between the gut microbiome of patients with IBD and IBS compared with control individuals, we were able to use gut microbiota composition differences to distinguish patients with IBD from those with IBS. By combining species-level profiles and strain-level profiles with bacterial growth rates, metabolic functions, antibiotic resistance, and virulence factor analyses, we identified key bacterial species that may be involved in two common gastrointestinal diseases.

Genomic and Expression Analyses Identify a Disease-Modifying Variant for Fibrostenotic Crohn's Disease.

Background and Aims: Crohn's disease [CD] is a chronic inflammatory disease with unpredictable behaviour. More than half of CD patients eventually develop complications such as stenosis, for which they then require endoscopic dilatation or surgery, as no anti-fibrotic drugs are currently available. We aim to identify disease-modifying genes associated with fibrostenotic CD. Methods: We performed a within-case analysis comparing 'extreme phenotypes' using the Immunochip and replication of the top single nucleotide polymorphisms [SNPs] with Agena Bioscience in two independent case-control cohorts totalling 322 cases with fibrostenotis [recurrent after surgery] and 619 cases with purely inflammatory CD. Results: Combined meta-analysis resulted in a genome-wide significant signal for SNP rs11861007 [p = 6.0910-11], located on chromosome 16, in lncRNA RP11-679B19.1, an lncRNA of unknown function, and close to exon 9 of the WWOX gene, which codes for WW domain-containing oxidoreductase. We analysed mRNA expression of TGF-ß and downstream genes in ileocecal resection material from ten patients with and without the WWOX risk allele. Patients carrying the risk allele [A] showed enhanced colonic expression of TGF-ß compared to patients homozygous for the wild-type [G] allele [p = 0.0079]. Conclusion: We have identified a variant in WWOX and in lncRNA RP11-679B19.1 as a disease-modifying genetic variant associated with recurrent fibrostenotic CD and replicated this association in an independent cohort. WWOX can potentially play a crucial role in fibrostenosis in CD, being positioned at the crossroads of inflammation and fibrosis.

Genetic association analysis identifies variants associated with disease progression in primary sclerosing cholangitis.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is a genetically complex, inflammatory bile duct disease of largely unknown aetiology often leading to liver transplantation or death. Little is known about the genetic contribution to the severity and progression of PSC. The aim of this study is to identify genetic variants associated with PSC disease progression and development of complications. DESIGN: We collected standardised PSC subphenotypes in a large cohort of 3402 patients with PSC. After quality control, we combined 130 422 single nucleotide polymorphisms of all patients-obtained using the Illumina immunochip-with their disease subphenotypes. Using logistic regression and Cox proportional hazards models, we identified genetic variants associated with binary and time-to-event PSC subphenotypes. RESULTS: We identified genetic variant rs853974 to be associated with liver transplant-free survival (p=6.07×10-9). Kaplan-Meier survival analysis showed a 50.9% (95% CI 41.5% to 59.5%) transplant-free survival for homozygous AA allele carriers of rs853974 compared with 72.8% (95% CI 69.6% to 75.7%) for GG carriers at 10 years after PSC diagnosis. For the candidate gene in the region, RSPO3, we demonstrated expression in key liver-resident effector cells, such as human and murine cholangiocytes and human hepatic stellate cells. CONCLUSION: We present a large international PSC cohort, and report genetic loci associated with PSC disease progression. For liver transplant-free survival, we identified a genome-wide significant signal and demonstrated expression of the candidate gene RSPO3 in key liver-resident effector cells. This warrants further assessments of the role of this potential key PSC modifier gene.

Interplay of host genetics and gut microbiota underlying the onset and clinical presentation of inflammatory bowel disease.

OBJECTIVE: Patients with IBD display substantial heterogeneity in clinical characteristics. We hypothesise that individual differences in the complex interaction of the host genome and the gut microbiota can explain the onset and the heterogeneous presentation of IBD. Therefore, we performed a case-control analysis of the gut microbiota, the host genome and the clinical phenotypes of IBD. DESIGN: Stool samples, peripheral blood and extensive phenotype data were collected from 313 patients with IBD and 582 truly healthy controls, selected from a population cohort. The gut microbiota composition was assessed by tag-sequencing the 16S rRNA gene. All participants were genotyped. We composed genetic risk scores from 11 functional genetic variants proven to be associated with IBD in genes that are directly involved in the bacterial handling in the gut: NOD2, CARD9, ATG16L1, IRGM and FUT2. RESULTS: Strikingly, we observed significant alterations of the gut microbiota of healthy individuals with a high genetic risk for IBD: the IBD genetic risk score was significantly associated with a decrease in the genus Roseburia in healthy controls (false discovery rate 0.017). Moreover, disease location was a major determinant of the gut microbiota: the gut microbiota of patients with colonic Crohn's disease (CD) is different from that of patients with ileal CD, with a decrease in alpha diversity associated to ileal disease (p=3.28×10-13). CONCLUSIONS: We show for the first time that genetic risk variants associated with IBD influence the gut microbiota in healthy individuals. Roseburia spp are acetate-to-butyrate converters, and a decrease has already been observed in patients with IBD.

Drug Repositioning in Inflammatory Bowel Disease Based on Genetic Information.

BACKGROUND: Currently, 200 genetic risk loci have been identified for inflammatory bowel disease (IBD). Although these findings have significantly advanced our insight into IBD biology, there has been little progress in translating this knowledge toward clinical practice, like more cost-efficient drug development. Our aim was to use genetic knowledge to identify drugs that warrant further investigation in IBD treatment. METHODS: We hypothesized that proteins encoded by IBD candidate genes are potential IBD drug targets because genetic information can increase successful drug identification. We identified drugs that target the proteins encoded by IBD candidate genes using the DrugBank. We included proteins that are in direct protein-protein interaction with proteins encoded by IBD risk genes. Promising potential IBD drugs were selected based on a manual literature search of all identified drugs (PubMed, ClinicalTrials.gov). RESULTS: We have identified 113 drugs that could potentially be used in IBD treatment. Fourteen are known IBD drugs, 48 drugs have been, or are being investigated in IBD, 19 are being used or being investigated in other inflammatory disorders treatment, and 32 are investigational new drugs that have not yet been registered for clinical use. CONCLUSIONS: We confirm that proteins encoded by IBD candidate genes are targeted by approved IBD therapies. Furthermore, we show that Food and Drug Administration-approved drugs could possibly be repositioned for IBD treatment. We also identify investigational new drugs that warrant further investigation for IBD treatment. Incorporating this process in IBD drug development will improve the utilization of genetic data and could lead to the improvement of IBD treatment.

Pooled Resequencing of 122 Ulcerative Colitis Genes in a Large Dutch Cohort Suggests Population-Specific Associations of Rare Variants in MUC2.

Genome-wide association studies have revealed several common genetic risk variants for ulcerative colitis (UC). However, little is known about the contribution of rare, large effect genetic variants to UC susceptibility. In this study, we performed a deep targeted re-sequencing of 122 genes in Dutch UC patients in order to investigate the contribution of rare variants to the genetic susceptibility to UC. The selection of genes consists of 111 established human UC susceptibility genes and 11 genes that lead to spontaneous colitis when knocked-out in mice. In addition, we sequenced the promoter regions of 45 genes where known variants exert cis-eQTL-effects. Targeted pooled re-sequencing was performed on DNA of 790 Dutch UC cases. The Genome of the Netherlands project provided sequence data of 500 healthy controls. After quality control and prioritization based on allele frequency and pathogenicity probability, follow-up genotyping of 171 rare variants was performed on 1021 Dutch UC cases and 1166 Dutch controls. Single-variant association and gene-based analyses identified an association of rare variants in the MUC2 gene with UC. The associated variants in the Dutch population could not be replicated in a German replication cohort (1026 UC cases, 3532 controls). In conclusion, this study has identified a putative role for MUC2 on UC susceptibility in the Dutch population and suggests a population-specific contribution of rare variants to UC.

Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight shared genetic risk across populations.

Ulcerative colitis and Crohn's disease are the two main forms of inflammatory bowel disease (IBD). Here we report the first trans-ancestry association study of IBD, with genome-wide or Immunochip genotype data from an extended cohort of 86,640 European individuals and Immunochip data from 9,846 individuals of East Asian, Indian or Iranian descent. We implicate 38 loci in IBD risk for the first time. For the majority of the IBD risk loci, the direction and magnitude of effect are consistent in European and non-European cohorts. Nevertheless, we observe genetic heterogeneity between divergent populations at several established risk loci driven by differences in allele frequency (NOD2) or effect size (TNFSF15 and ATG16L1) or a combination of these factors (IL23R and IRGM). Our results provide biological insights into the pathogenesis of IBD and demonstrate the usefulness of trans-ancestry association studies for mapping loci associated with complex diseases and understanding genetic architecture across diverse populations.

Performance of the Montreal classification for inflammatory bowel diseases.

AIM: To validate the Montreal classification system for Crohn's disease (CD) and ulcerative colitis (UC) within the Netherlands. METHODS: A selection of 20 de-identified medical records with an appropriate representation of the inflammatory bowel disease (IBD) sub phenotypes were scored by 30 observers with different professions (gastroenterologist specialist in IBD, gastroenterologist in training and IBD-nurses) and experience level with IBD patient care. Patients were classified according to the Montreal classification. In addition, participants were asked to score extra-intestinal manifestations (EIM) and disease severity in CD based on their clinical judgment. The inter-observer agreement was calculated by percentages of correct answers (answers identical to the "expert evaluation") and Fleiss-kappa (κ). Kappa cut-offs: < 0.4-poor; 0.41-0.6-moderate; 0.61-0.8-good; > 0.8 excellent. RESULTS: The inter-observer agreement was excellent for diagnosis (κ = 0.96), perianal disease (κ = 0.92) and disease location in CD (κ = 0.82) and good for age of onset (κ = 0.67), upper gastrointestinal disease (κ = 0.62), disease behaviour in CD (κ = 0.79) and disease extent in UC (κ = 0.65). Disease severity in UC was scored poor (κ = 0.23). The additional items resulted in a good inter-observer agreement for EIM (κ = 0.68) and a moderate agreement for disease severity in CD (κ = 0.44). Percentages of correct answers over all Montreal items give a good reflection of the inter-observer agreement (> 80%), except for disease severity (48%-74%). IBD-nurses were significantly worse in scoring upper gastrointestinal disease in CD compared to gastroenterologists (P = 0.008) and gastroenterologists in training (P = 0.040). Observers with less than 10 years of experience were significantly better at scoring UC severity than observers with 10-20 years (P = 0.003) and more than 20 years (P = 0.003) of experience with IBD patient care. Observers with 10-20 years of experience with IBD patient care were significantly better at scoring upper gastrointestinal disease in CD than observers with less than 10 years (P = 0.007) and more than 20 years (P = 0.007) of experience with IBD patient care. CONCLUSION: We found a good to excellent inter-observer agreement for all Montreal items except for disease severity in UC (poor).

Systems genetics of liver fibrosis: identification of fibrogenic and expression quantitative trait loci in the BXD murine reference population.

The progression of liver fibrosis in response to chronic injury varies considerably among individual patients. The underlying genetics is highly complex due to large numbers of potential genes, environmental factors and cell types involved. Here, we provide the first toxicogenomic analysis of liver fibrosis induced by carbon tetrachloride in the murine 'genetic reference panel' of recombinant inbred BXD lines. Our aim was to define the core of risk genes and gene interaction networks that control fibrosis progression. Liver fibrosis phenotypes and gene expression profiles were determined in 35 BXD lines. Quantitative trait locus (QTL) analysis identified seven genomic loci influencing fibrosis phenotypes (pQTLs) with genome-wide significance on chromosomes 4, 5, 7, 12, and 17. Stepwise refinement was based on expression QTL mapping with stringent selection criteria, reducing the number of 1,351 candidate genes located in the pQTLs to a final list of 11 cis-regulated genes. Our findings demonstrate that the BXD reference population represents a powerful experimental resource for shortlisting the genes within a regulatory network that determine the liver's vulnerability to chronic injury.

ATR-FTIR spectroscopy reveals genomic loci regulating the tissue response in high fat diet fed BXD recombinant inbred mouse strains.

BACKGROUND: Obesity-associated organ-specific pathological states can be ensued from the dysregulation of the functions of the adipose tissues, liver and muscle. However, the influence of genetic differences underlying gross-compositional differences in these tissues is largely unknown. In the present study, the analytical method of ATR-FTIR spectroscopy has been combined with a genetic approach to identify genetic differences responsible for phenotypic alterations in adipose, liver and muscle tissues. RESULTS: Mice from 29 BXD recombinant inbred mouse strains were put on high fat diet and gross-compositional changes in adipose, liver and muscle tissues were measured by ATR-FTIR spectroscopy. The analysis of genotype-phenotype correlations revealed significant quantitative trait loci (QTL) on chromosome 12 for the content of fat and collagen, collagen integrity, and the lipid to protein ratio in adipose tissue and on chromosome 17 for lipid to protein ratio in liver. Using gene expression and sequence information, we suggest Rsad2 (viperin) and Colec11 (collectin-11) on chromosome 12 as potential quantitative trait candidate genes. Rsad2 may act as a modulator of lipid droplet contents and lipid biosynthesis; Colec11 might play a role in apoptopic cell clearance and maintenance of adipose tissue. An increased level of Rsad2 transcripts in adipose tissue of DBA/2J compared to C57BL/6J mice suggests a cis-acting genetic variant leading to differential gene activation. CONCLUSION: The results demonstrate that the analytical method of ATR-FTIR spectroscopy effectively contributed to decompose the macromolecular composition of tissues that accumulate fat and to link this information with genetic determinants. The candidate genes in the QTL regions may contribute to obesity-related diseases in humans, in particular if the results can be verified in a bigger BXD cohort.

Analysis of the lung transcriptome in Mycobacterium tuberculosis-infected mice reveals major differences in immune response pathways between TB-susceptible and resistant hosts.

Using whole genome microarrays, we compared changes in gene expression patterns in the lungs of TB-resistant A/Sn and TB-susceptible I/St mice at day 14 following infection with Mycobacterium tuberculosis H37Rv. Analyses of differentially expressed genes for representation of gene ontology terms and activation of regulatory pathways revealed interstrain differences in antigen presentation, NK, T and B cell activation pathways. In general, resistant A/Sn mice exhibited a more complex pattern and stronger activation of host defense pathways compared to the TB-susceptible I/St mouse strain. In addition, in I/St mice elevated activation of genes involved in neutrophil response was observed and confirmed by quantitative RT-PCR and histopathology. Furthermore, a specific post infection upregulation of cysteine protease inhibitors was found in susceptible I/St mice.

Distinct gene loci control the host response to influenza H1N1 virus infection in a time-dependent manner.

BACKGROUND: There is strong but mostly circumstantial evidence that genetic factors modulate the severity of influenza infection in humans. Using genetically diverse but fully inbred strains of mice it has been shown that host sequence variants have a strong influence on the severity of influenza A disease progression. In particular, C57BL/6J, the most widely used mouse strain in biomedical research, is comparatively resistant. In contrast, DBA/2J is highly susceptible. RESULTS: To map regions of the genome responsible for differences in influenza susceptibility, we infected a family of 53 BXD-type lines derived from a cross between C57BL/6J and DBA/2J strains with influenza A virus (PR8, H1N1). We monitored body weight, survival, and mean time to death for 13 days after infection. Qivr5 (quantitative trait for influenza virus resistance on chromosome 5) was the largest and most significant QTL for weight loss. The effect of Qivr5 was detectable on day 2 post infection, but was most pronounced on days 5 and 6. Survival rate mapped to Qivr5, but additionally revealed a second significant locus on chromosome 19 (Qivr19). Analysis of mean time to death affirmed both Qivr5 and Qivr19. In addition, we observed several regions of the genome with suggestive linkage. There are potentially complex combinatorial interactions of the parental alleles among loci. Analysis of multiple gene expression data sets and sequence variants in these strains highlights about 30 strong candidate genes across all loci that may control influenza A susceptibility and resistance. CONCLUSIONS: We have mapped influenza susceptibility loci to chromosomes 2, 5, 16, 17, and 19. Body weight and survival loci have a time-dependent profile that presumably reflects the temporal dynamic of the response to infection. We highlight candidate genes in the respective intervals and review their possible biological function during infection.

Bioinformatics tools and database resources for systems genetics analysis in mice--a short review and an evaluation of future needs.

During a meeting of the SYSGENET working group 'Bioinformatics', currently available software tools and databases for systems genetics in mice were reviewed and the needs for future developments discussed. The group evaluated interoperability and performed initial feasibility studies. To aid future compatibility of software and exchange of already developed software modules, a strong recommendation was made by the group to integrate HAPPY and R/qtl analysis toolboxes, GeneNetwork and XGAP database platforms, and TIQS and xQTL processing platforms. R should be used as the principal computer language for QTL data analysis in all platforms and a 'cloud' should be used for software dissemination to the community. Furthermore, the working group recommended that all data models and software source code should be made visible in public repositories to allow a coordinated effort on the use of common data structures and file formats.

Expression QTL mapping in regulatory and helper T cells from the BXD family of strains reveals novel cell-specific genes, gene-gene interactions and candidate genes for auto-immune disease.

BACKGROUND: Regulatory T cells (Tregs) play an essential role in the control of the immune response. Treg cells represent important targets for therapeutic interventions of the immune system. Therefore, it will be very important to understand in more detail which genes are specifically activated in Treg cells versus T helper (Th) cells, and which gene regulatory circuits may be involved in specifying and maintaining Treg cell homeostasis. RESULTS: We isolated Treg and Th cells from a genetically diverse family of 31 BXD type recombinant inbred strains and the fully inbred parental strains of this family--C57BL/6J and DBA/2J. Subsequently genome-wide gene expression studies were performed from the isolated Treg and Th cells. A comparative analysis of the transcriptomes of these cell populations allowed us to identify many novel differentially expressed genes. Analysis of cis- and trans-expression Quantitative Trait Loci (eQTLs) highlighted common and unique regulatory mechanisms that are active in the two cell types. Trans-eQTL regions were found for the Treg functional genes Nrp1, Stat3 and Ikzf4. Analyses of the respective QTL intervals suggested several candidate genes that may be involved in regulating these genes in Treg cells. Similarly, possible candidate genes were found which may regulate the expression of F2rl1, Ctla4, Klrb1f. In addition, we identified a focused group of candidate genes that may be important for the maintenance of self-tolerance and the prevention of allergy. CONCLUSIONS: Variation of expression across the strains allowed us to find many novel gene-interaction networks in both T cell subsets. In addition, these two data sets enabled us to identify many differentially expressed genes and to nominate candidate genes that may have important functions for the maintenance of self-tolerance and the prevention of allergy.

Genome-wide analysis of the mouse lung transcriptome reveals novel molecular gene interaction networks and cell-specific expression signatures.

BACKGROUND: The lung is critical in surveillance and initial defense against pathogens. In humans, as in mice, individual genetic differences strongly modulate pulmonary responses to infectious agents, severity of lung disease, and potential allergic reactions. In a first step towards understanding genetic predisposition and pulmonary molecular networks that underlie individual differences in disease vulnerability, we performed a global analysis of normative lung gene expression levels in inbred mouse strains and a large family of BXD strains that are widely used for systems genetics. Our goal is to provide a key community resource on the genetics of the normative lung transcriptome that can serve as a foundation for experimental analysis and allow predicting genetic predisposition and response to pathogens, allergens, and xenobiotics. METHODS: Steady-state polyA+ mRNA levels were assayed across a diverse and fully genotyped panel of 57 isogenic strains using the Affymetrix M430 2.0 array. Correlations of expression levels between genes were determined. Global expression QTL (eQTL) analysis and network covariance analysis was performed using tools and resources in GeneNetwork http://www.genenetwork.org. RESULTS: Expression values were highly variable across strains and in many cases exhibited a high heritability factor. Several genes which showed a restricted expression to lung tissue were identified. Using correlations between gene expression values across all strains, we defined and extended memberships of several important molecular networks in the lung. Furthermore, we were able to extract signatures of immune cell subpopulations and characterize co-variation and shared genetic modulation. Known QTL regions for respiratory infection susceptibility were investigated and several cis-eQTL genes were identified. Numerous cis- and trans-regulated transcripts and chromosomal intervals with strong regulatory activity were mapped. The Cyp1a1 P450 transcript had a strong trans-acting eQTL (LOD 11.8) on Chr 12 at 36 ± 1 Mb. This interval contains the transcription factor Ahr that has a critical mis-sense allele in the DBA/2J haplotype and evidently modulates transcriptional activation by AhR. CONCLUSIONS: Large-scale gene expression analyses in genetic reference populations revealed lung-specific and immune-cell gene expression profiles and suggested specific gene regulatory interactions.