Blood RNA Samples Research Articles

Reduced TRPC6 mRNA levels in the blood cells of patients with Alzheimer's disease and mild cognitive impairment.

Transient receptor potential canonical 6 (TRPC6) inhibits β-amyloid (Aβ) production. Hyperforin, the TRPC6 agonist, reduces Aβ levels and improves cognitive performance in Alzheimer's disease (AD) models. However, it's unknown whether TRPC6 expression is changed in AD patients. In this case-control study, we measured TRPC6 expression levels in the peripheral blood cells of four independent AD sets from five hospitals and one mild cognitive impairment (MCI) set from a local community (229 AD, 70 MCI, 40 Parkinson disease and 359 controls from China, total n=698) using quantitative real-time PCR assay. We found a specific reduction of TRPC6 mRNA levels in four AD sets and one MCI set. The median TRPC6 mRNA levels were lower in the following: (1) combined AD patients than in age-matched controls (0.78 vs 1.73, P<0.001); (2) mild-to-moderate AD patients than in age-matched controls (0.81 vs 1.73, P<0.001); and (3) MCI patients than in age-matched controls (0.76 vs 1.72, P<0.001). In the receiver-operating characteristic curve analysis, the area under curve was 0.85 for combined AD, 0.84 for mild-to-moderate AD and 0.79 for MCI. In a subgroup of AD patients with brain Aβ examination, TRPC6 was associated with standardized uptake value ratio of Pittsburgh Compound B (Spearman's r=-0.49, P=0.04) and cerebrospinal fluid Aβ42 (Spearman's r=0.43, P=0.04). The TRPC6 reduction in AD patients was further confirmed in blood RNA samples from The Australian Imaging, Biomarkers and Lifestyle Flagship Study of Aging, in post-mortem brain tissues from The Netherlands Brain Bank and in induced pluripotent stem cells-derived neurons from Chinese donors. We conclude that TRPC6 mRNA levels in the blood cells are specifically reduced in AD and MCI patients, and TRPC6 might be a biomarker for the early diagnosis of AD.

Diagnostic Accuracy of a Host Gene Expression Signature That Discriminates Clinical Severe Sepsis Syndrome and Infection-Negative Systemic Inflammation Among Critically Ill Children.

SeptiCyte Lab (Immunexpress, Seattle, WA), a molecular signature measuring the relative expression levels of four host messenger RNAs, was developed to discriminate critically ill adults with infection-positive versus infection-negative systemic inflammation. The objective was to assess the performance of Septicyte Lab in critically ill pediatric patients. Prospective observational study. Pediatric and Cardiac ICUs, Seattle Children's Hospital, Seattle, WA. A cohort of 40 children with clinically overt severe sepsis syndrome and 30 children immediately postcardiopulmonary bypass surgery was recruited. The clinically overt severe sepsis syndrome children had confirmed or highly suspected infection (microbial culture orders, antimicrobial prescription), two or more systemic inflammatory response syndrome criteria (including temperature and leukocyte criteria), and at least cardiovascular ± pulmonary organ dysfunction. None (observational study only). Next-generation RNA sequencing was conducted on PAXgene blood RNA samples, successfully for 35 of 40 (87.5%) of the clinically overt severe sepsis syndrome patients and 29 of 30 (96.7%) of the postcardiopulmonary bypass patients. Forty patient samples (~ 60% of cohort) were reanalyzed by reverse transcription-quantitative polymerase chain reaction, to check for concordance with next-generation sequencing results. Postcardiopulmonary bypass versus clinically overt severe sepsis syndrome descriptors included the following: age, 7.3 ± 5.5 versus 9.0 ± 6.6 years; gender, 41% versus 49% male; Pediatric Risk of Mortality, version III, 7.0 ± 4.6 versus 8.7 ± 6.4; Pediatric Logistic Organ Dysfunction, version II, 5.1 ± 2.2 versus 4.8 ± 2.8. SeptiCyte Lab strongly differentiated postcardiopulmonary bypass and clinically overt severe sepsis syndrome patients by receiver operating characteristic curve analysis, with an area-under-curve value of 0.99 (95% CI, 0.96-1.00). Equivalent performance was found using reverse transcription-quantitative polymerase chain reaction. There was no significant correlation between the score produced by the SeptiCyte Lab test and measures of illness severity, immune compromise, or microbial culture status. SeptiCyte Lab is able to discriminate clearly between clinically well-defined and homogeneous postcardiopulmonary bypass and clinically overt severe sepsis syndrome groups in children. A broader investigation among children with more heterogeneous inflammation-associated diagnoses and care settings is warranted.

Abstract TP263: Identification and Functional Analysis of Circrna Profile in Penumbra Zones of Mice Following Middle Cerebral Artery Occlusion

Circular RNAs (circRNAs) are highly abundant in the brain, and exert regulatory roles during post-transcriptional process. Recently, deregulations of circRNA expression have been proven to be involved in hypoxia cells in vitro . However, the circRNA profiles of penumbra closely related to the prognosis of acute ischemic stroke (AIS) remain unclear. Herein, 12876 circRNAs based on circBase database were detected by microarray hybridization with blood RNA samples of Balb/c mice, including the sham group and groups of 5 min, 3 hr and 24 hr after middle cerebral artery occlusion (MCAO). Three comparison groups were set as sham vs 5 min (311 circRNAs up-regulated, 566 circRNAs down-regulated), sham vs 3 hr (513 circRNAs up-regulated, 207 circRNAs down-regulated), sham vs 24 hr (1079 circRNAs up-regulated, 1230 circRNAs down-regulated) (≥2.0 fold, p&lt;0.05). Meanwhile, using RNA-sequencing technology, we profiled circRNA expressional signatures of mice brain tissues from normal zones, penumbra and infract zones of MCAO 3 hr groups. 553 of the total 23980 circRNAs changed significantly between the differential expression profiles of penumbra vs normal and penumbra vs infract core. Among them, two circRNAs sequence were aligned with the blood data by Pairwise Sequence Alighment (SIMILARITY=100%, IDENTITY=100%). Moreover, human-mouse alignments of the two circRNAs were computed on circBase database (IDENTITY &gt;94.8% and IDENTITY=100%). Bioinformatics tools and databases were employed to explore the potential circRNAs functions. These findings are the first to identify MCAO-responsive circRNAs, which suggest potential pathological roles and may be as potential markers for penumbra progress in AIS.

Use of Blood-based mRNA profiling to Identify Biomarkers for Ovarian Cancer Screening

Purpose: To identify candidate genomic signatures for the early detection of ovarian cancer using whole bloodbased gene expression profiles. Experimental Design: We performed Affymetrix U133Plus 2.0 GeneChip microarray analyses on whole blood RNA samples obtained from 14 ovarian cancer patients and 15 age-matched, healthy women. Genes differentially expressed were identified using a parametric Welch t-test. Real-time qRT-PCR analyses were performed on RNA prepared from 96 ovarian cancer patients and 83 age-matched healthy women, using primer sets specific for 14 genes. A Mann Whitney U test assessed individual gene significance. CA125 levels were determined in the same set of samples. We used logistic regression analyses and cross validation to assess the ability of linear combinations of specific transcripts combined with CA125 to distinguish cancer from controls. Results: Microarray analyses showed that 9583 probes were significantly different in blood gene expression profiles from healthy women as compared with those from ovarian cancer patients (p<0.05). Real-time RT-PCR analyses on the 96 cases and 83 controls validated 7 genes, which showed significantly different expression levels in cases and controls. Logistic regression analyses and cross validation identified an optimal panel of markers including CA125, BRCA1, and KIAA0562, that could improve the sensitivity of CA125 alone to over 90% at 98% specificity in the detection of early stage ovarian cancer. Conclusion: Circulating blood gene expression profiles identified RNA markers that can improve the sensitivity of CA125 in the detection of early stage ovarian cancer. Further validation is warranted to confirm the clinical usefulness of these biomarkers.

Effects of Lithium Monotherapy for Bipolar Disorder on Gene Expression in Peripheral Lymphocytes

Background: This study investigated the effect of lithium monotherapy on peripheral lymphocyte gene expression in bipolar disorder (BD). Method: Twenty-two medication-free bipolar subjects (11 hypomanic, 11 depressed) were started on lithium monotherapy. Closely matched healthy subjects (n = 15) were included as controls but did not receive treatment. Blood RNA samples were collected at baseline and after 2 and 8 weeks of treatment. RNA expression was measured using the Affymetrix GeneChip® Human Gene 1.0 ST Array followed by Ingenuity pathways analysis. The results for the contrast of weeks 2 and 8 were not significantly different and were combined. Results: In BD subjects, 56 genes showed significant (false discovery rate <0.1) expression changes from baseline; the effect sizes and directions for all of these were similar at weeks 2 and 8. Among these were immune-related genes (IL5RA, MOK, IFI6, and RFX2), purinergic receptors (P2RY14, P2RY2, and ADORA3) and signal transduction-related genes (CAMK1 and PIK3R6). Pathway and upstream regulator analysis also revealed that lithium altered several immune- and signal transduction-related functions. Differentially expressed genes did not correlate with week 8 clinical response, but other genes involved in protein synthesis and degradation did. Conclusion: Peripheral gene expression may serve as a biomarker of lithium effect.

Interferon α kinoid induces neutralizing anti-interferon α antibodies that decrease the expression of interferon-induced and B cell activation associated transcripts: analysis of extended follow-up data from the interferon α kinoid phase I/II study

Objective. IFN α Kinoid (IFN-K) is a therapeutic vaccine composed of IFNα2b coupled to a carrier protein. In a phase I/II placebo-controlled trial, we observed that IFN-K significantly decreases the IFN gene signature in whole blood RNA samples from SLE patients. Here, we analysed extended follow-up data from IFN-K-treated patients, in order to evaluate persistence of neutralizing anti-IFNα Abs antibodies (Abs), and gene expression profiling.Methods. Serum and whole blood RNA samples were obtained in IFN-K-treated patients included in the follow-up study, in order to determine binding and neutralizing anti-IFNα Ab titres, and perform high-throughput transcriptomic studies.Results. Neutralization studies of 13 IFNα subtypes demonstrated the polyclonal nature of the Ab response induced by IFN-K. Follow-up analyses in six patients confirmed a significant correlation between neutralizing anti-IFNα Ab titres and decrease in IFN scores compared to baseline. These analyses also revealed an inhibitory effect of IFNα blockade on the expression of B cell associated transcripts.Conclusions. IFN-K induces a polyclonal anti-IFNα response that decreases IFN- and B cell-associated transcripts.Trial registration: ClinicalTrials.gov, clinicaltrials.gov, NCT01058343

Abstract WP370: Differential Alternative Splicing of Leukocyte mRNAs After Intracerebral Hemorrhage and Ischemic Stroke of Different Etiologies

Transcriptome studies have used 3’-biased microarrays to study regulated genes and pathways in the blood of ischemic stroke (IS) patients, missing a majority of transcript isoforms due to alternative splicing (AS). AS is implicated in many diseases but is unexplored with respect to IS and intracerebral hemorrhage (ICH). Thus, we hypothesized AS differs between 1) IS, ICH and vascular risk factor control (VRFC) subjects; 2) three main causes of IS (Cardioembolic, Large Vessel and Lacunar), ICH and VRFC. For Aim1, differential alternative splicing (DAS) in whole blood of IS vs ICH vs VRFC males was studied on Affymetrix exon junction HTA 2.0 arrays (n=36, 12 per group). For Aim 2, we investigated DAS between Cardioembolic, Large Vessel and Lacunar IS etiologies, and ICH and VRFC using paired-end RNA-Seq (Illumina Solexa, 200M 2X100bp reads per sample) on 20 whole blood RNA samples (n=4 males per group). DAS was assessed with 1-way-ANOVA (FDR p&lt;0.05). Significant differential exon-usage was calculated between each group (p&lt;0.0005; fold change&gt; |1.2|). Differential usage of 90 exons separated the IS, ICH and VRFC groups. Affected genes included HGF (hepatic growth factor), involved in thrombosis and angiogenesis, and NOS1 (nitric oxide synthase 1), implicated in stroke risk. DAS among ICH, three main causes of IS, and VRFC was displayed by 412 genes. Differential expression of 308 exons separated these 5 groups. Several genes with DAS belonged to NF-kB and PPAR signaling pathways, SRA1, steroid receptor RNA activator 1, and Akt1, all of which have been implicated in experimental ICH pathogenesis. ICH activates NF-kB transcription factor which perpetuates inflammation. PPAR-gamma, via inhibition of NF-kB and induction of antioxidative pathways, reduces inflammation and oxidative stress and stimulates phagocytosis-mediated hematoma cleanup. In conclusion, our results provide first evidence for DAS in whole blood in ICH vs IS, and vs different IS etiologies. These data indicate disease specific immune response and could yield biomarkers for ICH and various IS causes. Future studies in a larger cohort are needed to validate results.

Transcriptomic Analysis of the Host Response and Innate Resilience to Enterotoxigenic Escherichia coli Infection in Humans.

Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection.

Investigating CD11c expression as a potential genomic biomarker of response to TNF inhibitor biologics in whole blood rheumatoid arthritis samples.

IntroductionGene expression profiling is rapidly becoming a useful and informative tool in a much needed area of research. Identifying patients as to whether they will respond or not to a given treatment before prescription is not only essential to optimise treatment outcome but also to lessen the economic burden that such drugs can have on healthcare resources. In rheumatoid arthritis (RA), there is of yet no genetic/genomic biomarker which can accurately predict response to TNF inhibitor biologics prior to treatment, despite much interest in this area. Multiple studies have reported findings on potential candidate genes; however, due to relatively small sample sizes or lack of sufficient validation, results have been disappointingly inconsistent. The aim of this research was to further explore the predictive value of a previously reported association between CD11c expression and response to the TNF inhibitor biologics, adalimumab and etanercept.MethodsReal-time qPCR was performed using whole blood RNA samples obtained from seventy-five rheumatoid arthritis patients about to commence treatment with a TNF inhibitor biologic drug, whose response status was determined at 3-month follow-up using the EULAR classification criteria. Relative quantification of CD11c using the comparative CT method outputted differential expression between good-responders and non-responders as a fold-change.ResultsRelative expression of CD11c in patients receiving TNF inhibitor biologics yielded a decrease of 1.025 fold in good-responders as compared to non-responders (p-value = 0.36). Upon stratification of patients dependent upon the specific drug administered, adalimumab or etanercept, similar findings to the full cohort were observed, decreases of 1.015 (p-value = 0.33) and 1.032 fold (p-value = 0.13) in good-responders compared to non-responders, respectively.ConclusionThe results from this study reveal that CD11c expression does not correlate with response to TNF inhibitor biologics when tested for within pre-treatment whole blood samples of rheumatoid arthritis patients.

MicroRNA circulating in the early aftermath of motor vehicle collision predict persistent pain development and suggest a role for microRNA in sex-specific pain differences.

BackgroundMolecular mediators influencing the transition from acute to persistent musculoskeletal pain following common stress exposures such as motor vehicle collision (MVC) remain poorly understood. In this exploratory, proof of concept study, we compared circulating microRNA (miRNA) expression profiles in the early aftermath of MVC among individuals who did and did not subsequently develop persistent pain. Blood RNA samples were obtained from African American individuals (n = 53) who presented to the emergency department after MVC and were discharged to home after evaluation. The presence or absence of severe pain in the axial region, the most common and morbid region in which post-MVC pain occurs, was assessed 6 weeks following MVC via standardized questionnaire. miRNA expression was determined using miRNA-sequencing; nonparametric analyses were used to compare miRNA expression levels among individuals with and without persistent pain.ResultsThirty-two mature miRNA were differentially expressed (p < 0.05) in those with and without severe axial pain at 6 weeks. miR-135a-5p, a regulator of the serotonin receptor that is known to be stress-responsive, differed most significantly between groups (p = 3 × 10−4). This miRNA, and miR-3613-3p (p = 0.001) survived correction for multiple testing (FDR = 0.15) in this small sample. Interestingly, differentially expressed miRNA were enriched for X chromosome location. In secondary analyses, the eight X chromosome miRNA were (a) more significantly associated with axial pain in women than men, (b) expressed more highly in the peripheral blood of women than men, and (c) predicted in pathway analyses (DIANA miRPath v 2.0) to regulate neuronal and neuroendocrine pathways previously implicated in various pain pathologies.ConclusionsThese results show that circulating miRNA predict persistent severe axial pain after MVC and suggest that they may be involved in the pathogenesis of post-traumatic musculoskeletal pain. However, further studies are needed to determine if these miRNA play a direct causal role.Electronic supplementary materialThe online version of this article (doi:10.1186/s12990-015-0069-3) contains supplementary material, which is available to authorized users.

Whole blood microarray analysis of pigs showing extreme phenotypes after a porcine reproductive and respiratory syndrome virus infection

BackgroundThe presence of variability in the response of pigs to Porcine Reproductive and Respiratory Syndrome virus (PRRSv) infection, and recent demonstration of significant genetic control of such responses, leads us to believe that selection towards more disease resistant pigs could be a valid strategy to reduce its economic impact on the swine industry. To find underlying molecular differences in PRRS susceptible versus more resistant pigs, 100 animals with extremely different growth rates and viremia levels after PRRSv infection were selected from a total of 600 infected pigs. A microarray experiment was conducted on whole blood RNA samples taken at 0, 4 and 7 days post infection (dpi) from these pigs. From these data, we examined associations of gene expression with weight gain and viral load phenotypes. The single nucleotide polymorphism (SNP) marker WUR10000125 (WUR) on the porcine 60 K SNP chip was shown to be associated with viral load and weight gain after PRRSv infection, and so the effect of the WUR10000125 (WUR) genotype on expression in whole blood was also examined.ResultsLimited information was obtained through linear modeling of blood gene differential expression (DE) that contrasted pigs with extreme phenotypes, for growth or viral load or between animals with different WUR genotype. However, using network-based approaches, molecular pathway differences between extreme phenotypic classes could be identified. Several gene clusters of interest were found when Weighted Gene Co-expression Network Analysis (WGCNA) was applied to 4dpi contrasted with 0dpi data. The expression pattern of one such cluster of genes correlated with weight gain and WUR genotype, contained numerous immune response genes such as cytokines, chemokines, interferon type I stimulated genes, apoptotic genes and genes regulating complement activation. In addition, Partial Correlation and Information Theory (PCIT) identified differentially hubbed (DH) genes between the phenotypically divergent groups. GO enrichment revealed that the target genes of these DH genes are enriched in adaptive immune pathways.ConclusionThere are molecular differences in blood RNA patterns between pigs with extreme phenotypes or with a different WUR genotype in early responses to PRRSv infection, though they can be quite subtle and more difficult to discover with conventional DE expression analyses. Co-expression analyses such as WGCNA and PCIT can be used to reveal network differences between such extreme response groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1741-8) contains supplementary material, which is available to authorized users.

Abstract O.29: Transcriptional Profiling Discriminates Complete and Incomplete KD from Human Adenovirus

Background: The diagnosis of Kawasaki disease (KD) is often difficult to distinguish from HAdV. Objective: 1) To characterize the specific transcriptional profiles of KD patients versus acute HAdV infection 2) To determine whether the molecular distance to health (MDTH) score (a molecular score that reflects the perturbation derived from whole genome transcriptional analysis) correlates with response to therapy. Methods: Whole blood RNA samples collected in Tempus tubes were analyzed using Illumina chips and GeneSpring software 7.4 from 76 pediatric patients with complete KD, 13 with incomplete KD, and 19 patients with HadV, and 20 age- and sex-matched healthy controls (HC). We used class comparison algorithms (Mann-Whitney p&lt; 0.01, Benjamini-Hochberg, and 1.25- fold change filter) and modular analysis to define the KD profiles; class prediction algorithm was used to identify genes that best differentiate KD and HAdV. Results: Statistical group comparisons identified 7,899 genes differentially expressed in 39 complete KD patients versus HC (KD biosignature). This signature was validated in another 37 patients with complete KD and in 13 patients with incomplete KD. Modular analysis in children with complete KD demonstrated overexpression of inflammation, neutrophils, myeloid cell, coagulation cascade, and cell cycle genes. The class prediction algorithm identified 25-classifier genes that differentiated children with KD vs HAdV infection in two independent cohorts of patients with 92% (95% CI [73%-99%]) sensitivity and 90% [67%-98%] specificity. MDTH scores in KD patients significantly correlated with the baseline c-reactive protein (R=0.29, p=0.008) and was four fold higher than in children with HAdV (p&lt;0.01). In addition, KD patients that remained febrile 36 hours after treatment with IVIG (non-responders) demonstrated higher baseline, pre-treatment MDTH values compared with responders [12,290 vs. 5572 respectively; p=0.009]. Conclusion: Transcriptional signatures can be used as a tool to discriminate between KD and HAdV infection, and may also provide prognostic information.

Type of Renal Replacement Therapy (Hemodialysis versus Peritoneal Dialysis) Does Not Affect Cytokine Gene Expression or Clinical Parameters of Renal Transplant Candidates.

Patients with renal failure suffer from immune disturbances, caused by uremic toxins and influenced by dialysis treatment. The aim of the present study was to reveal whether type of dialysis modality (hemodialysis, HD, versus peritoneal dialysis, PD) differentially affects the immunocompetence, particularly the expression of genes involved in the immune response. Material. 87 renal transplant candidates (66 HD, 21 PD) were included in the study. Methods. The peripheral blood RNA samples were obtained with the PAXgene Blood system just before transplantation. The gene expression of CASP3, FAS, TP53, FOXP3, IFNG, IL2, IL6, IL8, IL10, IL17, IL18, LCN2, TGFB1, and TNF was assessed with real-time PCR on custom-designed low density arrays (TaqMan). Gene expression data were analyzed in relation to pretransplant clinical parameters. Results. The mean expression of examined genes showed no significant differences between PD and HD with the exception of FAS, expression of which was 30% higher in PD patients compared to the HD group. There was nonsignificantly higher expression of proinflammatory cytokines in the PD group. The clinical inflammatory parameters (CRP, albumin, cholesterol, and hemoglobin levels) did not differ between the groups. Conclusion. Type of renal replacement therapy exerts no differential effect on cytokine gene expression or inflammatory clinical parameters.

Standard of hygiene and immune adaptation in newborn infants

The prevalence of immune-mediated diseases, such as allergies and type 1 diabetes, is on the rise in the developed world. In order to explore differences in the gene expression patterns induced in utero in infants born in contrasting standards of living and hygiene, we collected umbilical cord blood RNA samples from infants born in Finland (modern society), Estonia (rapidly developing society) and the Republic of Karelia, Russia (poor economic conditions). The whole blood transcriptome of Finnish and Estonian neonates differed from their Karelian counterparts, suggesting exposure to toll-like receptor (TLR) ligands and a more matured immune response in infants born in Karelia. These results further support the concept of a conspicuous plasticity in the developing immune system: the environmental factors that play a role in the susceptibility/protection towards immune-mediated diseases begin to shape the neonatal immunity already in utero and direct the maturation in accordance with the surrounding microbial milieu.

Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7

DNA methylation is a hallmark of genomic imprinting and differentially methylated regions (DMRs) are found near and in imprinted genes. Imprinted genes are expressed only from the maternal or paternal allele and their normal balance can be disrupted by uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair exclusively from only either the mother or the father. Maternal UPD for chromosome 7 (matUPD7) results in Silver-Russell syndrome (SRS) with typical features and growth retardation, but no gene has been conclusively implicated in SRS. In order to identify novel DMRs and putative imprinted genes on chromosome 7, we analyzed eight matUPD7 patients, a segmental matUPD7q31-qter, a rare patUPD7 case and ten controls on the Infinium HumanMethylation450K BeadChip with 30 017 CpG methylation probes for chromosome 7. Genome-scale analysis showed highly significant clustering of DMRs only on chromosome 7, including the known imprinted loci GRB10, SGCE/PEG10, and PEG/MEST. We found ten novel DMRs on chromosome 7, two DMRs for the predicted imprinted genes HOXA4 and GLI3 and one for the disputed imprinted gene PON1. Quantitative RT-PCR on blood RNA samples comparing matUPD7, patUPD7, and controls showed differential expression for three genes with novel DMRs, HOXA4, GLI3, and SVOPL. Allele specific expression analysis confirmed maternal only expression of SVOPL and imprinting of HOXA4 was supported by monoallelic expression. These results present the first comprehensive map of parent-of-origin specific DMRs on human chromosome 7, suggesting many new imprinted sites.

Circulating microRNA Profile throughout the Menstrual Cycle

Normal physiological variables, such as age and gender, contribute to alterations in circulating microRNA (miRNA) expression levels. The changes in the female body during the menstrual cycle can also be reflected in plasma miRNA expression levels. Therefore, this study aimed to determine the plasma miRNA profile of healthy women during the menstrual cycle and to assess which circulating miRNAs are derived from blood cells. The plasma miRNA expression profiles in nine healthy women were determined by quantitative real time PCR using Exiqon Human Panel I assays from four time-points of the menstrual cycle. This platform was also used for studying miRNAs from pooled whole blood RNA samples at the same four time-points. Our results indicated that circulating miRNA expression levels in healthy women were not significantly altered by the processes occurring during the menstrual cycle. No significant differences in plasma miRNA expression levels were observed between the menstrual cycle time-points, but the number of detected miRNAs showed considerable variation among the studied individuals. miRNA analysis from whole blood samples revealed that majority of miRNAs in plasma are derived from blood cells. The most abundant miRNA in plasma and blood was hsa-miR-451a, but a number of miRNAs were only detected in one or the other sample type. In conclusion, our data suggest that the changes in the female body during the menstrual cycle do not affect the expression of circulating miRNAs at measurable levels.

The splice site variant rs11078928 may be associated with a genotype-dependent alteration in expression of GSDMB transcripts

BackgroundMany genetic variants have been associated with susceptibility to complex traits by genome wide association studies (GWAS), but for most, causal genes and mechanisms of action have yet to be elucidated. Using bioinformatics, we identified index and proxy variants associated with autoimmune disease susceptibility, with the potential to affect splicing of candidate genes. PCR and sequence analysis of whole blood RNA samples from population controls was then carried out for the 8 most promising variants to determine the effect of genetic variation on splicing of target genes.ResultsWe identified 31 splice site SNPs with the potential to affect splicing, and prioritised 8 to determine the effect of genotype on candidate gene splicing. We identified that variants rs11078928 and rs2014886 were associated with altered splicing of the GSDMB and TSFM genes respectively. rs11078928, present in the asthma and autoimmune disease susceptibility locus on chromosome 17q12-21, was associated with the production of a novel Δ exon5-8 transcript of the GSDMB gene, and a separate decrease in the percentage of transcripts with inclusion of exon 6, whereas the multiple sclerosis susceptibility variant rs2014886, was associated with an alternative TFSM transcript encompassing a short cryptic exon within intron 2.ConclusionsOur findings demonstrate the utility of a bioinformatic approach in identification and prioritisation of genetic variants effecting splicing of their host genes, and suggest that rs11078928 and rs2014886 may affect the splicing of the GSDMB and TSFM genes respectively.

AB0289 Can cd11c expression successfully predict response to etanercept in rheumatoid arthritis patients?

BackgroundThe treatment of rheumatoid arthritis (RA) with biologic drugs such as anti-tumor necrosis factor alpha (TNF) has significantly improved the clinical outcome in patients; however, 30-40% of patients fail to...

Increased sensitivity of next generation sequencing-based expression profiling after globin reduction in human blood RNA

BackgroundTranscriptome analysis is of great interest in clinical research, where significant differences between individuals can be translated into biomarkers of disease. Although next generation sequencing provides robust, comparable and highly informative expression profiling data, with several million of tags per blood sample, reticulocyte globin transcripts can constitute up to 76% of total mRNA compromising the detection of low abundant transcripts. We have removed globin transcripts from 6 human whole blood RNA samples with a human globin reduction kit and compared them with the same non-reduced samples using deep Serial Analysis of Gene Expression.ResultsGlobin tags comprised 52-76% of total tags in our samples. Out of 21,633 genes only 87 genes were detected at significantly lower levels in the globin reduced samples. In contrast, 11,338 genes were detected at significantly higher levels in the globin reduced samples. Removing globin transcripts allowed us to also identify 2112 genes that could not be detected in the non-globin reduced samples, with roles in cell surface receptor signal transduction, G-protein coupled receptor protein signalling pathways and neurological processes.ConclusionsThe reduction of globin transcripts in whole blood samples constitutes a reproducible and reliable method that can enrich data obtained from next generation sequencing-based expression profiling.

CCAAT‐enhancer‐binding protein‐beta expression in vivo is associated with muscle strength

Declining muscle strength is a core feature of aging. Several mechanisms have been postulated, including CCAAT/enhancer-binding protein-beta (C/EBP-β)-triggered macrophage-mediated muscle fiber regeneration after micro-injury, evidenced in a mouse model. We aimed to identify in vivo circulating leukocyte gene expression changes associated with muscle strength in the human adult population. We undertook a genome-wide expression microarray screen, using peripheral blood RNA samples from InCHIANTI study participants (aged 30 and 104). Logged expression intensities were regressed with muscle strength using models adjusted for multiple confounders. Key results were validated by real-time PCR. The Short Physical Performance Battery (SPPB) score tested walk speed, chair stand, and balance. CEBPB expression levels were associated with muscle strength (β coefficient = 0.20560, P = 1.03*10(-6), false discovery rate q = 0.014). The estimated handgrip strength in 70-year-old men in the lowest CEBPB expression tertile was 35.2 kg compared with 41.2 kg in the top tertile. CEBPB expression was also associated with hip, knee, ankle, and shoulder strength and the SPPB score (P = 0.018). Near-study-wide associations were also noted for TGF-β3 (P = 3.4*10(-5) , q = 0.12) and CEBPD expression (P = 9.7*10(-5) , q = 0.18) but not for CEBPA expression. We report here a novel finding that raised CEBPB expression in circulating leukocyte-derived RNA samples in vivo is associated with greater muscle strength and better physical performance in humans. This association may be consistent with mouse model evidence of CEBPB-triggered muscle repair: if this mechanism is confirmed, it may provide a target for intervention to protect and enhance aging muscle.