Cytosolic nucleic acid sensing and mitochondrial transcriptomic changes as early triggers of metabolic disease in db/db mice.

Animal models of diabetes, such as db/db mice, are a useful tool for deciphering the genetic background of molecular changes at the initial stages of disease development. Our goal was to find early transcriptomic changes in three tissues involved in metabolism regulation in db/db mice: adipose tissue, muscle tissue and liver tissue. Nine animals (three per time point) were studied. Tissues were collected at 8, 12 and 16 weeks of age. Transcriptome-wide analysis was performed using mRNA-seq. Libraries were sequenced on NextSeq (Illumina). Differential expression (DE) analysis was performed with edgeR. The analysis of the gene expression profile shared by all three tissues revealed eight upregulated genes (Irf7, Sp100, Neb, Stat2, Oas2, Rtp4, H2-T24 and Oasl2) as early as between 8 and 12 weeks of age. The most pronounced differences were found in liver tissue: nine DE genes between 8 and 12 weeks of age (Irf7, Ly6a, Ly6g6d, H2-Dma, Pld4, Ly86, Fcer1g, Ly6e and Idi1) and five between 12 and 16 weeks of age (Irf7, Plac8, Ifi44, Xaf1 and Ly6a) (adj. p-value < 0.05). The mitochondrial transcriptomic profile also changed with time: we found two downregulated genes in mice between 8 and 12 weeks old (Ckmt2 and Cox6a2) and five DE genes between 12 and 16 weeks of age (Mavs, Tomm40L, Mtfp1, Ckmt2 and Cox6a2). The KEGG pathway analysis showed significant enrichment in pathways related to the autoimmune response and cytosolic DNA sensing. Our results suggest an important involvement of the immunological response, mainly cytosolic nucleic acid sensing, and mitochondrial signalling in the early stages of diabetes and obesity.

Intratumor heterogeneity in colorectal cancer: Distribution of tumor suppressor gene variants with regard to patient lymph node status.

Intratumor heterogeneity (ITH) results from accumulation of somatic mutations in the fractions of successive cancer cell generations. We aimed to use deep sequencing to investigate ITH in colorectal tumors with particular emphasis on variants in oncogenes (ONC) and tumor suppressor genes (TSG). Samples were collected from 16 patients with colorectal cancer and negative or positive lymph node status (n = 8 each). We deep-sequenced a panel of 56 cancer-related genes in the central and peripheral locations of T3 size primary tumors and healthy mucosa. The central region of T3 tumors has a different frequency profile and composition of genetic variants. This mutation profile is capable of independently discriminating patients with different lymph node status (p = 0.028) in the central region. We noted an increasing number of mutations outside of the central region of the tumor and a higher number of mutations in tumors from node-positive patients. Unexpectedly, in the healthy mucosa, we identified somatic mutations with variant allele frequencies, characteristic not only of heterozygotes and homozygotes but also of other discrete peaks (e.g., around 10%, 20%), suggestive of clonal expansion of certain mutant alleles. We found differences in the distribution of variant allele frequencies in TSGs when comparing node-negative and node-positive tumors (p = 0.029), as well as central and peripheral regions (p = 0.00399). TSGs may play an important role in the escape of the tumor toward metastatic colonization.

Genome-Wide DNA Methylation and Gene Expression in Patients with Indolent Systemic Mastocytosis.

Mastocytosis is a clinically heterogenous, usually acquired disease of the mast cells with a survival time that depends on the time of onset. It ranges from skin-limited to systemic disease, including indolent and more aggressive variants. The presence of the oncogenic KIT p. D816V gene somatic mutation is a crucial element in the pathogenesis. However, further epigenetic regulation may also affect the expression of genes that are relevant to the pathology. Epigenetic alterations are responsible for regulating the expression of genes that do not modify the DNA sequence. In general, it is accepted that DNA methylation inhibits the binding of transcription factors, thereby down-regulating gene expression. However, so far, little is known about the epigenetic factors leading to the clinical onset of mastocytosis. Therefore, it is essential to identify possible epigenetic predictors, indicators of disease progression, and their link to the clinical picture to establish appropriate management and a therapeutic strategy. The aim of this study was to analyze genome-wide methylation profiles to identify differentially methylated regions (DMRs) in patients with mastocytosis compared to healthy individuals, as well as the genes located in those regulatory regions. Genome-wide DNA methylation profiling was performed in peripheral blood collected from 80 adult patients with indolent systemic mastocytosis (ISM), the most prevalent subvariant of mastocytosis, and 40 healthy adult volunteers. A total of 117 DNA samples met the criteria for the bisulfide conversion step and microarray analysis. Genome-wide DNA methylation analysis was performed using a MethylationEPIC BeadChip kit. Further analysis was focused on the genomic regions rather than individual CpG sites. Co-methylated regions (CMRs) were assigned via the CoMeBack method. To identify DMRs between the groups, a linear regression model with age as the covariate on CMRs was performed using Limma. Using the available data for cases only, an association analysis was performed between methylation status and tryptase levels, as well as the context of allergy, and anaphylaxis. KEGG pathway mapping was used to identify genes differentially expressed in anaphylaxis. Based on the DNA methylation results, the expression of 18 genes was then analyzed via real-time PCR in 20 patients with mastocytosis and 20 healthy adults. A comparison of the genome-wide DNA methylation profile between the mastocytosis patients and healthy controls revealed significant differences in the methylation levels of 85 selected CMRs. Among those, the most intriguing CMRs are 31 genes located within the regulatory regions. In addition, among the 10 CMRs located in the promoter regions, 4 and 6 regions were found to be either hypo- or hypermethylated, respectively. Importantly, three oncogenes-FOXQ1, TWIST1, and ERG-were identified as differentially methylated in mastocytosis patients, for the first time. Functional annotation revealed the most important biological processes in which the differentially methylated genes were involved as transcription, multicellular development, and signal transduction. The biological process related to histone H2A monoubiquitination (GO:0035518) was found to be enriched in association with higher tryptase levels, which may be associated with more aberrant mast cells and, therefore, more atypical mast cell disease. The signal in the BAIAP2 gene was detected in the context of anaphylaxis, but no significant differential methylation was found in the context of allergy. Furthermore, increased expression of genes encoding integral membrane components (GRM2 and KRTCAP3) was found in mastocytosis patients. This study confirms that patients with mastocytosis differ significantly in terms of methylation levels in selected CMRs of genes involved in specific molecular processes. The results of gene expression profiling indicate the increased expression of genes belonging to the integral component of the membrane in mastocytosis patients (GRM2 and KRTCAP3). Further work is warranted, especially in relation to the disease subvariants, to identify links between the methylation status and the symptoms and novel therapeutic targets.

MicroRNA composition of plasma extracellular vesicles: a harbinger of late cardiotoxicity of doxorubicin.

BACKGROUND: The use of doxorubicin is associated with an increased risk of acute and long-term cardiomyopathy. Despite the constantly growing number of cancer survivors, little is known about the transcriptional mechanisms which progress in the time leading to a severe cardiac outcome. It is also unclear whether long-term transcriptomic alterations related to doxorubicin use are similar to transcriptomic patterns present in patients suffering from other cardiomyopathies. METHODS: We have sequenced miRNA from total plasma and extracellular vesicles (EVs) from 66 acute lymphoblastic leukemia (ALL) survivors and 61 healthy controls (254 samples in total). We then analyzed processes regulated by differentially expressed circulating miRNAs and cross-validated results with the data of patients with clinically manifested cardiomyopathies. RESULTS: We found that especially miRNAs contained within EVs may be informative in terms of cardiomyopathy development and may regulate pathways related to neurotrophin signaling, transforming growth factor beta (TGFß) or epidermal growth factor receptors (ErbB). We identified vesicular miR-144-3p and miR-423-3p as the most variable between groups and significantly correlated with echocardiographic parameters and, respectively, for plasma: let-7g-5p and miR-16-2-3p. Moreover, vesicular miR-144-3p correlates with the highest number of echocardiographic parameters and is differentially expressed in the circulation of patients with dilated cardiomyopathy. We also found that distribution of particular miRNAs between of plasma and EVs (proportion between compartments) e.g., miR-184 in ALL, is altered, suggesting changes within secretory and miRNA sorting mechanisms. CONCLUSIONS: Our results show that transcriptomic changes resulting from doxorubicin induced myocardial injury are reflected in circulating miRNA levels and precede development of the late onset cardiomyopathy phenotype. Among miRNAs related to cardiac function, we found vesicular miR-144-3p and miR-423-3p, as well as let-7g-5p and miR-16-2-3p contained in the total plasma. Selection of source for such studies (plasma or EVs) is of critical importance, as distribution of some miRNA between plasma and EVs is altered in ALL survivors, in comparison to healthy people, which suggests that doxorubicin-induced changes include miRNA sorting and export to extracellular space.

Variants influencing age at diagnosis of HNF1A-MODY.

BACKGROUND: HNF1A-MODY is a monogenic form of diabetes caused by variants in the HNF1A gene. Different HNF1A variants are associated with differences in age of disease onset, but other factors are postulated to influence this trait. Here, we searched for genetic variants influencing age of HNF1A-MODY onset. METHODS: Blood samples from 843 HNF1A-MODY patients from Czech Republic, France, Poland, Slovakia, the UK and the US were collected. A validation set consisted of 121 patients from the US. We conducted a genome-wide association study in 843 HNF1A-MODY patients. Samples were genotyped using Illumina Human Core arrays. The core analysis was performed using the GENESIS package in R statistical software. Kinship coefficients were estimated with the KING and PC-Relate algorithms. In the linear mixed model, we accounted for year of birth, sex, and location of the HNF1A causative variant. RESULTS: A suggestive association with age of disease onset was observed for rs2305198 (p = 2.09E-07) and rs7079157 (p = 3.96E-06) in the HK1 gene, rs2637248 in the LRMDA gene (p = 2.44E-05), and intergenic variant rs2825115 (p = 2.04E-05). Variant rs2637248 reached nominal significance (p = 0.019), while rs7079157 (p = 0.058) and rs2825115 (p = 0.068) showed suggestive association with age at diabetes onset in the validation set. CONCLUSIONS: rs2637248 in the LRMDA gene is associated with age at diabetes onset in HNF1A-MODY patients.

The Analysis of a Genome-Wide Association Study (GWAS) of Overweight and Obesity in Psoriasis.

There is evidence that the concomitance of psoriasis and obesity may originate from the interplay between multiple genetic pathways and involve gene−gene interactions. The aim of this study was to compare the genetic background related to obesity among psoriatic patients versus healthy controls by means of a Genome-Wide Association Study (GWAS). A total of 972 psoriatic patients and a total of 5878 healthy donors were enrolled in this study. DNA samples were genotyped for over 500,000 single nucleotide polymorphisms (SNPs) using Infinium CoreExome BeadChips (Illumina, San Diego, CA, USA). Statistical analysis identified eleven signals (p < 1 × 10−5) associated with BMI across the study groups and revealed a varying effect size in each sub-cohort. Seven of the alternative alleles (rs1558902 in the FTO gene, rs696574 in the CALCRL gene, as well as rs10968110, rs4551082, rs4609724, rs9320269, and rs2338833,) are associated with increased BMI among all psoriatic patients and four (rs1556519 in the ITLN2 gene, rs12972098 in the AC003006.7 gene, rs12676670 in the PAG1 gene, and rs1321529) are associated with lower BMI. The results of our study may lead to further insights into the understanding of the pathogenesis of obesity among psoriatic patients.

The transcriptome-wide association search for genes and genetic variants which associate with BMI and gestational weight gain in women with type 1 diabetes.

BACKGROUND: Clinical data suggest that BMI and gestational weight gain (GWG) are strongly interconnected phenotypes; however, the genetic basis of the latter is rather unclear. Here we aim to find genes and genetic variants which influence BMI and/or GWG. METHODS: We have genotyped 316 type 1 diabetics using Illumina Infinium Omni Express Exome-8 v1.4 arrays. The GIANT, ARIC and T2D-GENES summary statistics were used for TWAS (performed with PrediXcan) in adipose tissue. Next, the analysis of association of imputed expression with BMI in the general and diabetic cohorts (Analysis 1 and 2) or GWG (Analysis 3 and 4) was performed, followed by variant association analysis (1 Mb around identified loci) with the mentioned phenotypes. RESULTS: In Analysis 1 we have found 175 BMI associated genes and 19 variants (p < 10-4) which influenced GWG, with the strongest association for rs11465293 in CCL24 (p = 3.18E-06). Analysis 2, with diabetes included in the model, led to discovery of 1812 BMI associated loci and 207 variants (p < 10-4) influencing GWG, with the strongest association for rs9690213 in PODXL (p = 9.86E-07). In Analysis 3, among 648 GWG associated loci, 2091 variants were associated with BMI (FDR < 0.05). In Analysis 4, 7 variants in GWG associated loci influenced BMI in the ARIC cohort. CONCLUSIONS: Here, we have shown that loci influencing BMI might have an impact on GWG and GWG associated loci might influence BMI, both in the general and T1DM cohorts. The results suggest that both phenotypes are related to insulin signaling, glucose homeostasis, mitochondrial metabolism, ubiquitinoylation and inflammatory responses.

Dysregulation of Transcription Factor Activity During Formation of Cancer-Associated Fibroblasts.

The reciprocal interactions between cancer cells and the quiescent fibroblasts leading to the activation of cancer-associated fibroblasts (CAFs) serve an important role in cancer progression. Here, we investigated the activation of transcription factors (TFs) in prostate fibroblasts (WPMY cell line) co-cultured with normal prostate or tumorous cells (RWPE1 and RWPE2 cell lines, respectively). After indirect co-cultures, we performed mRNA-seq and predicted TF activity using mRNA expression profiles with the Systems EPigenomics Inference of Regulatory Activity (SEPIRA) package and the GTEx and mRNA-seq data of 483 cultured fibroblasts. The initial differential expression analysis between time points and experimental conditions showed that co-culture with normal epithelial cells mainly promotes an inflammatory response in fibroblasts, whereas with the cancerous epithelial, it stimulates transformation by changing the expression of the genes associated with microfilaments. TF activity analysis revealed only one positively regulated TF in the RWPE1 co-culture alone, while we observed dysregulation of 45 TFs (7 decreased activity and 38 increased activity) uniquely in co-culture with RWPE2. Pathway analysis showed that these 45 dysregulated TFs in fibroblasts co-cultured with RWPE2 cells may be associated with the RUNX1 and PTEN pathways. Moreover, we showed that observed dysregulation could be associated with FER1L4 expression. We conclude that phenotypic changes in fibroblast responses to co-culturing with cancer epithelium result from orchestrated dysregulation of signaling pathways that favor their transformation and motility rather than proinflammatory status. This dysregulation can be observed both at the TF and transcriptome levels.

Thymus-derived regulatory T cells contribute to tolerance to commensal microbiota.

Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4(+) Foxp3(+) regulatory T (Treg) cells generated in the thymus or extrathymically by induction of naive CD4(+) Foxp3(-) T cells. Previous studies suggested that the T-cell receptor repertoires of thymic Treg cells and induced Treg cells are biased towards self and non-self antigens, respectively, but their relative contribution in controlling immunopathology, such as colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved. The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which Treg cells and other T-cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage and favour tolerogenic presentation of antigens to naive CD4(+) T cells, suggesting that intestinal homeostasis depends on microbiota-specific induced Treg cells. Here, to identify the origin and antigen-specificity of intestinal Treg cells, we performed single-cell and high-throughput sequencing of the T-cell receptor repertoires of CD4(+) Foxp3(+) and CD4(+) Foxp3(-) T cells, and analysed their reactivity against specific commensal species. We show that thymus-derived Treg cells constitute most Treg cells in all lymphoid and intestinal organs, including the colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that thymic Treg cells, and not induced Treg cells, dominantly mediate tolerance to antigens produced by intestinal commensals.

Combined genetic influence of the nicotinic receptor gene cluster CHRNA5/A3/B4 on nicotine dependence.

BACKGROUND: The CHRNA5/A3/B4 gene locus is associated with nicotine dependence and other smoking related disorders. While the non-synonymous CHRNA5 variant rs16969968 appears to be the main risk factor, linkage disequilibrium (LD) bins in the gene cluster carry frequent variants that regulate expression. Pairwise LD and haplotype analyses had identified at least three haplotype tagging SNPs including rs16969968 as main genetic risk factors. Searching for variants with evidence of regulatory functions, we have reported interactions between CHRNA5 and CHRNA3 enhancer variants (tagged by rs880395 and rs1948, respectively) and rs16969968, forming 3-SNP haplotypes and diplotypes that may more accurately reflect the cluster's combined effects on nicotine dependence (Barrie et al., Hum Mutat 38:112-9, 2017). Here we address further contributions by variants affecting CHRNB4, a possibly limiting component of nicotinic receptors. RESULTS: We identify an LD bin (tagged by rs4887074) associated with expression of CHRNB4. Additive logistic regression models indicate that rs4887074 is associated with nicotine dependence and modulates the effect of rs16969968 in GWAS datasets (COGEND, UW-TTURC, SAGE). 4-SNP haplotype and diplotype analyses (rs880395-rs16969968-rs1948 -rs4887074) yield nicotine dependence risk values that further differentiate those obtained with the 3-SNP model. Moreover, both the main G allele of rs16969968 and the minor G allele of rs4887074 (associated with reduced expression of CHRNB4), residing predominantly on common haplotypes that are protective, represent significant allele-specific variance QTLs, indicating that they interact with each other. CONCLUSIONS: These results indicate rs4887074 is associated with CHRNB4 expression, and along with two regulatory variants of CHRNA3 and CHRNA5, modulates the effect of rs16969968 on nicotine dependence risk. Assignable to individuals because of strong LD structures, 4-SNP haplotypes and diplotypes serve to assess the combined genetic influence of this multi-gene cluster on complex traits, accounting for complex LD relationships and tissue-specific genetic effects (CHRNA5/3) relevant to the traits analyzed. The 4-SNP haplotypes account at least in part for previous tagging SNPs, including the highly GWAS-significant rs6495308, located in a distinct pair-wise LD bin but included in protective 4-SNP haplotypes. Our approach refines and integrates the cluster's overall genetic influence, an important variable when integrating the genetics of multiple genomic loci.

The CHRNA5/CHRNA3/CHRNB4 Nicotinic Receptor Regulome: Genomic Architecture, Regulatory Variants, and Clinical Associations.

Functionally related genes often cluster into a genome region under coordinated regulation, forming a local regulome. To understand regulation of the CHRNA5/CHRNA3/CHRNB4 nicotinic receptor gene cluster, we integrate large-scale RNA expression data (brain and peripheral) from GTEx (Genotype Tissue Expression), clinical associations (GRASP), and linkage disequilibrium data (1000 Genomes) to find candidate SNPs representing independent regulatory variants. CHRNA3, CHRNA5, CHRNB4 mRNAs, and a well-expressed CHRNA5 antisense RNA (RP11-650L12.2) are co-expressed in many human tissues, suggesting common regulatory elements. The CHRNA5 enhancer haplotype tagged by rs880395 not only increases CHRNA5 mRNA expression in all tissues, but also enhances RP11-650L12.2 and CHRNA3 expression, suggesting DNA looping to multiple promoters. However, in nucleus accumbens and putamen, but not other brain regions, CHRNA3 expression associates uniquely with a haplotype tagged by rs1948 (located in the CHRNB4 3'UTR). Haplotype/diplotype analysis of rs880395 and rs1948 plus rs16969968 (a nonsynonymous CHRNA5 risk variant) in GWAS (COGEND, UW-TTURC, SAGE) yields a nicotine dependence risk profile only partially captured by rs16969968 alone. An example of local gene clusters, this nicotinic regulome is controlled by complex genetic variation, with broad implications for interpreting GWAS.

Non-linear interactions between candidate genes of myocardial infarction revealed in mRNA expression profiles.

BACKGROUND: Alterations in gene expression are key events in disease etiology and risk. Poor reproducibility in detecting differentially expressed genes across studies suggests individual genes may not be sufficiently informative for complex diseases, such as myocardial infarction (MI). Rather, dysregulation of the 'molecular network' may be critical for pathogenic processes. Such a dynamic network can be built from pairwise non-linear interactions. RESULTS: We investigate non-linear interactions represented in mRNA expression profiles that integrate genetic background and environmental factors. Using logistic regression, we test the association of individual GWAS-based candidate genes and non-linear interaction terms (between these mRNA expression levels) with MI. Based on microarray data in CATHGEN (CATHeterization in GENetics) and FHS (Framingham Heart Study), we find individual genes and pairs of mRNAs, encoded by 41 MI candidate genes, with significant interaction terms in the logistic regression model. Two pairs replicate between CATHGEN and FHS (CNNM2|GUCY1A3 and CNNM2|ZEB2). Analysis of RNAseq data from GTEx (Genotype-Tissue Expression) shows that 20 % of these disease-associated RNA pairs are co-expressed, further prioritizing significant interactions. Because edges in sparse co-expression networks formed solely by the 41 candidate genes are unlikely to represent direct physical interactions, we identify additional RNAs as links between network pairs of candidate genes. This approach reveals additional mRNAs and interaction terms significant in the context of MI, for example, the path CNNM2|ACSL5|SCARF1|GUCY1A3, characterized by the common themes of magnesium and lipid processing. CONCLUSIONS: The results of this study support a role for non-linear interactions between genes in MI and provide a basis for further study of MI systems biology. mRNA expression profiles encoded by a limited number of candidate genes yield sparse networks of MI-relevant interactions that can be expanded to include additional candidates by co-expression analysis. The non-linear interactions observed here inform our understanding of the clinical relevance of gene-gene interactions in the pathophysiology of MI, while providing a new strategy in developing clinical biomarker panels.

Production lost due to cervical cancer in Poland in 2012.

BACKGROUND: Poland has one of the highest cervical cancer mortality rates in Europe. It is related to the problem of late diagnosis and low attendance rate in screening programs. The objective of the study has been to assess the annual production loss due to the cervical cancer morbidity and mortality in Poland in 2012. The outcomes have been to provide comprehensive information on cervical cancer's influence on population's ability to work and its overall economic burden for the society. The study has also provided the methodological framework for disease-related production losses in Polish settings. MATERIAL AND METHODS: The human capital method was used. The production losses were calculated in both monetary and quantitative terms (working days lost) due to 4 following reasons: 1) temporary disability to work, 2) permanent disability, 3) informal care, and 4) mortality. RESULTS: Cervical cancer resulted in approx. 702 964 working days lost in 2012 due to absence at work for both patients and care givers and a total number of 957 678 working days lost due to patients' mortality. The total value of production lost was assessed at 111.4 million euros. More than 66% of this value was attributed to women's mortality. CONCLUSIONS: The calculation of production lost due to cervical cancer burden provides strong evidence to support adequate health promotion and disease prevention actions. Actions promoting cervical cancer screening should be intensified including workplace health promotion activities. Med Pr 2016;67(3):289-299.

The Lyme disease as the increasing health problem in Malopolskie voivodeship compared with Poland in 1998-2014 / Borelioza jako narastajacy problem zdrowotny w województwie malopolskim na tle Polski w latach 1998-2014

OBJECTIVE: Lyme disease is one of the most known tick borne diseases in Poland caused by spirochetes of the genus Borrelia burgdorferi. Most cases of Lyme disease are diagnosed in the northeastern Poland and the south of Poland, in Slaskie, Malopolskie, Podkarpackie voivodeship. The aim of the study was to evaluate epidemiological data of Lyme disease in Malopolskie voivodeship and other voivodeships in Poland and frequency analysis of the Lyme disease as an occupational disease. METHODS: The authors analyzed prevalence from 1998 to 2014. Incidence of the Lyme disease was evaluated through review data from "Choroby zakazne i zatrucia" Bulletin and Lyme disease as an occupational disease obtained data from the Nofer Institute of Occupational Medicine in Lódz. RESULTS: It is estimated that the number of Lyme disease cases in Poland increased 18 times between 1998 and 2014 year (2,0 to 36 per 100,000 population), in the same period it was over 35 times of sudden rise in Lyme disease incidence in Malopolskie voivodeship. In years 2005-2014 the number of cases of Lyme disease as an occupational disease fluctuated with a slight upward trend both in Poland and Malopolskie voivoideship. CONCLUSIONs: In Poland number of reported cases is systematically increasing. Podlaskie and Warminsko- Mazurskie voivodeships are areas of high prevalence. Exponential increase in the number of cases is observed in southern Poland, especially in Malopolskie voivodeship from 2013.

Analyzing allele specific RNA expression using mixture models.

BACKGROUND: Measuring allele-specific RNA expression provides valuable insights into cis-acting genetic and epigenetic regulation of gene expression. Widespread adoption of high-throughput sequencing technologies for studying RNA expression (RNA-Seq) permits measurement of allelic RNA expression imbalance (AEI) at heterozygous single nucleotide polymorphisms (SNPs) across the entire transcriptome, and this approach has become especially popular with the emergence of large databases, such as GTEx. However, the existing binomial-type methods used to model allelic expression from RNA-seq assume a strong negative correlation between reference and variant allele reads, which may not be reasonable biologically. RESULTS: Here we propose a new strategy for AEI analysis using RNA-seq data. Under the null hypothesis of no AEI, a group of SNPs (possibly across multiple genes) is considered comparable if their respective total sums of the allelic reads are of similar magnitude. Within each group of "comparable" SNPs, we identify SNPs with AEI signal by fitting a mixture of folded Skellam distributions to the absolute values of read differences. By applying this methodology to RNA-Seq data from human autopsy brain tissues, we identified numerous instances of moderate to strong imbalanced allelic RNA expression at heterozygous SNPs. Findings with SLC1A3 mRNA exhibiting known expression differences are discussed as examples. CONCLUSION: The folded Skellam mixture model searches for SNPs with significant difference between reference and variant allele reads (adjusted for different library sizes), using information from a group of "comparable" SNPs across multiple genes. This model is particularly suitable for performing AEI analysis on genes with few heterozygous SNPs available from RNA-seq, and it can fit over-dispersed read counts without specifying the direction of the correlation between reference and variant alleles.

Conditional entropy in variation-adjusted windows detects selection signatures associated with expression quantitative trait loci (eQTLs).

BACKGROUND: Over the past 50,000 years, shifts in human-environmental or human-human interactions shaped genetic differences within and among human populations, including variants under positive selection. Shaped by environmental factors, such variants influence the genetics of modern health, disease, and treatment outcome. Because evolutionary processes tend to act on gene regulation, we test whether regulatory variants are under positive selection. We introduce a new approach to enhance detection of genetic markers undergoing positive selection, using conditional entropy to capture recent local selection signals. RESULTS: We use conditional logistic regression to compare our Adjusted Haplotype Conditional Entropy (H|H) measure of positive selection to existing positive selection measures. H|H and existing measures were applied to published regulatory variants acting in cis (cis-eQTLs), with conditional logistic regression testing whether regulatory variants undergo stronger positive selection than the surrounding gene. These cis-eQTLs were drawn from six independent studies of genotype and RNA expression. The conditional logistic regression shows that, overall, H|H is substantially more powerful than existing positive-selection methods in identifying cis-eQTLs against other Single Nucleotide Polymorphisms (SNPs) in the same genes. When broken down by Gene Ontology, H|H predictions are particularly strong in some biological process categories, where regulatory variants are under strong positive selection compared to the bulk of the gene, distinct from those GO categories under overall positive selection. . However, cis-eQTLs in a second group of genes lack positive selection signatures detectable by H|H, consistent with ancient short haplotypes compared to the surrounding gene (for example, in innate immunity GO:0042742); under such other modes of selection, H|H would not be expected to be a strong predictor.. These conditional logistic regression models are adjusted for Minor allele frequency(MAF); otherwise, ascertainment bias is a huge factor in all eQTL data sets. Relationships between Gene Ontology categories, positive selection and eQTL specificity were replicated with H|H in a single larger data set. Our measure, Adjusted Haplotype Conditional Entropy (H|H), was essential in generating all of the results above because it: 1) is a stronger overall predictor for eQTLs than comparable existing approaches, and 2) shows low sequential auto-correlation, overcoming problems with convergence of these conditional regression statistical models. CONCLUSIONS: Our new method, H|H, provides a consistently more robust signal associated with cis-eQTLs compared to existing methods. We interpret this to indicate that some cis-eQTLs are under positive selection compared to their surrounding genes. Conditional entropy indicative of a selective sweep is an especially strong predictor of eQTLs for genes in several biological processes of medical interest. Where conditional entropy is a weak or negative predictor of eQTLs, such as innate immune genes, this would be consistent with balancing selection acting on such eQTLs over long time periods. Different measures of selection may be needed for variant prioritization under other modes of evolutionary selection.

RNA sequencing of transcriptomes in human brain regions: protein-coding and non-coding RNAs, isoforms and alleles.

BACKGROUND: We used RNA sequencing to analyze transcript profiles of ten autopsy brain regions from ten subjects. RNA sequencing techniques were designed to detect both coding and non-coding RNA, splice isoform composition, and allelic expression. Brain regions were selected from five subjects with a documented history of smoking and five non-smokers. Paired-end RNA sequencing was performed on SOLiD instruments to a depth of >40 million reads, using linearly amplified, ribosomally depleted RNA. Sequencing libraries were prepared with both poly-dT and random hexamer primers to detect all RNA classes, including long non-coding (lncRNA), intronic and intergenic transcripts, and transcripts lacking poly-A tails, providing additional data not previously available. The study was designed to generate a database of the complete transcriptomes in brain region for gene network analyses and discovery of regulatory variants. RESULTS: Of 20,318 protein coding and 18,080 lncRNA genes annotated from GENCODE and lncipedia, 12 thousand protein coding and 2 thousand lncRNA transcripts were detectable at a conservative threshold. Of the aligned reads, 52 % were exonic, 34 % intronic and 14 % intergenic. A majority of protein coding genes (65 %) was expressed in all regions, whereas ncRNAs displayed a more restricted distribution. Profiles of RNA isoforms varied across brain regions and subjects at multiple gene loci, with neurexin 3 (NRXN3) a prominent example. Allelic RNA ratios deviating from unity were identified in > 400 genes, detectable in both protein-coding and non-coding genes, indicating the presence of cis-acting regulatory variants. Mathematical modeling was used to identify RNAs stably expressed in all brain regions (serving as potential markers for normalizing expression levels), linked to basic cellular functions. An initial analysis of differential expression analysis between smokers and nonsmokers implicated a number of genes, several previously associated with nicotine exposure. CONCLUSIONS: RNA sequencing identifies distinct and consistent differences in gene expression between brain regions, with non-coding RNA displaying greater diversity between brain regions than mRNAs. Numerous RNAs exhibit robust allele selective expression, proving a means for discovery of cis-acting regulatory factors with potential clinical relevance.

Association Between Prediagnostic Serum 25-Hydroxyvitamin D Concentration and Glioma.

There are no previous studies of the association between prediagnostic serum vitamin D concentration and glioma. Vitamin D has immunosuppressive properties; as does glioma. It was, therefore, our hypothesis that elevated vitamin D concentration would increase glioma risk. We conducted a nested case-control study using specimens from the Janus Serum Bank cohort in Norway. Blood donors who were subsequently diagnosed with glioma (n = 592), between 1974 and 2007, were matched to donors without glioma (n = 1112) on date and age at blood collection and sex. We measured 25-hydroxyvitamin D [25(OH)D], an indicator of vitamin D availability, using liquid chromatography coupled with mass spectrometry. Seasonally adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated for each control quintile of 25(OH)D using conditional logistic regression. Among men diagnosed with high grade glioma >56, we found a negative trend (P = .04). Men diagnosed ≤ 56 showed a borderline positive trend (P = .08). High levels (>66 nmol/L) of 25(OH)D in men >56 were inversely related to high grade glioma from ≥2 yr before diagnosis (OR = 0.59; 95% CI = 0.38, 0.91) to ≥15 yr before diagnosis (OR = 0.61; 95% CI = 0.38,0.96). Our findings are consistent long before glioma diagnosis and are therefore unlikely to reflect preclinical disease.

Missing heritability of common diseases and treatments outside the protein-coding exome.

Genetic factors strongly influence risk of common human diseases and treatment outcomes but the causative variants remain largely unknown; this gap has been called the 'missing heritability'. We propose several hypotheses that in combination have the potential to narrow the gap. First, given a multi-stage path from wellness to disease, we propose that common variants under positive evolutionary selection represent normal variation and gate the transition between wellness and an 'off-well' state, revealing adaptations to changing environmental conditions. In contrast, genome-wide association studies (GWAS) focus on deleterious variants conveying disease risk, accelerating the path from off-well to illness and finally specific diseases, while common 'normal' variants remain hidden in the noise. Second, epistasis (dynamic gene-gene interactions) likely assumes a central role in adaptations and evolution; yet, GWAS analyses currently are poorly designed to reveal epistasis. As gene regulation is germane to adaptation, we propose that epistasis among common normal regulatory variants, or between common variants and less frequent deleterious variants, can have strong protective or deleterious phenotypic effects. These gene-gene interactions can be highly sensitive to environmental stimuli and could account for large differences in drug response between individuals. Residing largely outside the protein-coding exome, common regulatory variants affect either transcription of coding and non-coding RNAs (regulatory SNPs, or rSNPs) or RNA functions and processing (structural RNA SNPs, or srSNPs). Third, with the vast majority of causative variants yet to be discovered, GWAS rely on surrogate markers, a confounding factor aggravated by the presence of more than one causative variant per gene and by epistasis. We propose that the confluence of these factors may be responsible to large extent for the observed heritability gap.

Alert microorganisms isolated from patients hospitalized in Malopolskie province in 2010-2012.

OBJECTIVE: Healthcare centers undertake supervisory activities to control health care-associated infections (HCAIs) by elaborating procedures, identifying alert microorganisms and analyzing data collected. The aim of the study was to analyze the prevalence of alert microorganisms in hospital wards in 2010-2012. MATERIAL AND METHODS: Legislation which is in force since several years introduced the principles of health care-associated infections control and reporting system. Analysis was based on annual reports on alert microorganisms provided by 19 District Sanitary and Epidemiological Stations from Malopolskie province. The data discuss positive tests results for alert microorganisms in patients who stayed in hospitals supervised by the Sanitary and Epidemiological Stations. RESULTS: Compared to 2010-2011, the number of tests per hospital bed in 2012 was lower, amounting to 24 (2010 - 44, 2011 - 34). Of these tests, the majority was performed in the following wards: transplantology (2010 - 339, 2011 - 354, 2012 - 330), burn care (2010 - 354, 2011 - 148, 2012 - 113) and ICUs for adults (2010 - 155, 2011 - 157, 2012 - 140). In 2010-2012, an increase in the number of positive test results for extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL+) and Clostridium difficile as well as slight decrease in the number of positive test results for other alert microorganisms were noted. The highest number of microorganisms was identified in neonatal and neonatal pathology (Enterobacteriaceae ESBL+); pediatric and infectious diseases (Rotavirus); infectious diseases (Rotavirus, C.difficile); burn care (Acinetobacter baumanii, Pseudomonas sp.) and ophthalmic and hemodialysis wards (MRSA). CONCLUSION: Irrespective of a decrease in the number of tests per hospital bed in 2012, a high number of positive test results for alert microorganisms was observed. It suggests the necessity for wider application of collected data as to improve monitoring of infections and reduce resulting threats.