The role of alcohol in the management of hypertension in patients in European primary health care practices - a survey in the largest European Union countries.

BACKGROUND: Even though addressing lifestyle problems is a major recommendation in most guidelines for the treatment of hypertension (HTN), alcohol problems are not routinely addressed in the management of hypertension in primary health care. METHODS: Internet based survey of 3081 primary care physicians, recruited via the mailing lists of associations for general practitioners (GPs) in France, Germany, Italy, Spain and the UK. Clinical practice, attitudes, knowledge, education and training were assessed. Logistic regression to predict screening, brief intervention and treatment for alcohol dependence in the management of hypertension were assessed. RESULTS: Overall, about one third of the interviewed GPs reported sufficient screening in cases with HTN (34.0 %, 95 % confidence interval (CI):32.1-35.8 %). One out of five GPs screened and delivered brief interventions in HTN patients with hazardous consumption (22.2 %, 95 % CI: 20.6-23.8 %) and about one in 13 GPs provided treatment for HTN patients with alcohol dependence other than advice or brief intervention (7.8 %, 95 % CI: 6.8-8.9 %). Post-graduate training and belief in their effectiveness predicted interventions. There were marked differences between countries. CONCLUSIONS: While current interventions were overall low, marked differences between countries indicate that current practices could be improved. Education and post-graduate training seems to be key in improving clinical practice of including interventions for problematic alcohol consumption and alcohol dependence in primary health care.

miRNAs can be generally associated with human pathologies as exemplified for miR-144.

BACKGROUND: miRNA profiles are promising biomarker candidates for a manifold of human pathologies, opening new avenues for diagnosis and prognosis. Beyond studies that describe miRNAs frequently as markers for specific traits, we asked whether a general pattern for miRNAs across many diseases exists. METHODS: We evaluated genome-wide circulating profiles of 1,049 patients suffering from 19 different cancer and non-cancer diseases as well as unaffected controls. The results were validated on 319 individuals using qRT-PCR. RESULTS: We discovered 34 miRNAs with strong disease association. Among those, we found substantially decreased levels of hsa-miR-144* and hsa-miR-20b with AUC of 0.751 (95% CI: 0.703-0.799), respectively. We also discovered a set of miRNAs, including hsa-miR-155*, as rather stable markers, offering reasonable control miRNAs for future studies. The strong downregulation of hsa-miR-144* and the less variable pattern of hsa-miR-155* has been validated in a cohort of 319 samples in three different centers. Here, breast cancer as an additional disease phenotype not included in the screening phase has been included as the 20th trait. CONCLUSIONS: Our study on 1,368 patients including 1,049 genome-wide miRNA profiles and 319 qRT-PCR validations further underscores the high potential of specific blood-borne miRNA patterns as molecular biomarkers. Importantly, we highlight 34 miRNAs that are generally dysregulated in human pathologies. Although these markers are not specific to certain diseases they may add to the diagnosis in combination with other markers, building a specific signature. Besides these dysregulated miRNAs, we propose a set of constant miRNAs that may be used as control markers.

Toward the blood-borne miRNome of human diseases.

In a multicenter study, we determined the expression profiles of 863 microRNAs by array analysis of 454 blood samples from human individuals with different cancers or noncancer diseases, and validated this 'miRNome' by quantitative real-time PCR. We detected consistently deregulated profiles for all tested diseases; pathway analysis confirmed disease association of the respective microRNAs. We observed significant correlations (P = 0.004) between the genomic location of disease-associated genetic variants and deregulated microRNAs.

Multivariate miRNA signatures as biomarkers for non-ischaemic systolic heart failure.

AIMS: Non-ischaemic heart failure is one of the today's most prevalent cardiovascular disorders. Since modern pharmacotherapy has proved to be very effective in delaying disease progression and preventing death, imaging modalities and molecular biomarkers play an important role in early identification and clinical management as well as risk assessment of patients. The present study evaluated for the first time whole peripheral blood miRNAs as novel biomarker candidates for non-ischaemic heart failure with reduced ejection fraction (HF-REF). METHODS AND RESULTS: We assessed genome-wide miRNA expression profiles in 53 HF-REF patients and 39 controls. We could identify and validate several miRNAs that show altered expression levels in non-ischaemic HF-REF, discriminating cases from controls both as single markers or when combined in a multivariate signature. In addition, we demonstrate that the miRNAs of this signature significantly correlate with disease severity as indicated by left ventricular ejection fraction. CONCLUSION: Our data further denote that miRNAs are potential biomarkers for systolic heart failure. Since their detection levels in whole blood are also related to the degree of left ventricular dysfunction, they may serve as objective molecular tools to assess disease severity and prognosis.

Refining diagnostic microRNA signatures by whole-miRNome kinetic analysis in acute myocardial infarction.

BACKGROUND: Alterations in microRNA (miRNA) expression patterns in whole blood may be useful biomarkers of diverse cardiovascular disorders. We previously reported that miRNAs are significantly dysregulated in acute myocardial infarction (AMI) and applied machine-learning techniques to define miRNA subsets with high diagnostic power for AMI diagnosis. However, the kinetics of the time-dependent sensitivity of these novel miRNA biomarkers remained unknown. METHODS: To characterize temporal changes in the expressed human miRNAs (miRNome), we performed here the first whole-genome miRNA kinetic study in AMI patients. We measured miRNA expression levels at multiple time points (0, 2, 4, 12, 24 h after initial presentation) in patients with acute ST-elevation myocardial infarction by using microfluidic primer extension arrays and quantitative real-time PCR. As a prerequisite, all patients enrolled had to have cardiac troponin T concentrations <50 ng/L on admission as measured with a high-sensitivity assay. RESULTS: We found a subset of miRNAs to be significantly dysregulated both at initial presentation and during the course of AMI. Additionally, we identified novel miRNAs that are dysregulated early during myocardial infarction, such as miR-1915 and miR-181c*. CONCLUSIONS: The present proof-of-concept study provides novel insights into the dynamic changes of the human miRNome during AMI.

Treatment-independent miRNA signature in blood of Wilms tumor patients.

BACKGROUND: Blood-born miRNA signatures have recently been reported for various tumor diseases. Here, we compared the miRNA signature in Wilms tumor patients prior and after preoperative chemotherapy according to SIOP protocol 2001. RESULTS: We did not find a significant difference between miRNA signature of both groups. However both, Wilms tumor patients prior and after chemotherapy showed a miRNA signature different from healthy controls. The signature of Wilms tumor patients prior to chemotherapy showed an accuracy of 97.5% and of patients after chemotherapy an accuracy of 97.0%, each as compared to healthy controls. CONCLUSION: Our results provide evidence for a blood-born Wilms tumor miRNA signature largely independent of four weeks preoperative chemotherapy treatment.

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing.

The lack of efficient high-throughput methods for enrichment of specific sequences from genomic DNA represents a key bottleneck in exploiting the enormous potential of next-generation sequencers. Such methods would allow for a systematic and targeted analysis of relevant genomic regions. Recent studies reported sequence enrichment using a hybridization step to specific DNA capture probes as a possible solution to the problem. However, so far no method has provided sufficient depths of coverage for reliable base calling over the entire target regions. We report a strategy to multiply the enrichment performance and consequently improve depth and breadth of coverage for desired target sequences by applying two iterative cycles of hybridization with microfluidic Geniom biochips. Using this strategy, we enriched and then sequenced the cancer-related genes BRCA1 and TP53 and a set of 1000 individual dbSNP regions of 500 bp using Illumina technology. We achieved overall enrichment factors of up to 1062-fold and average coverage depths of 470-fold. Combined with high coverage uniformity, this resulted in nearly complete consensus coverages with >86% of target region covered at 20-fold or higher. Analysis of SNP calling accuracies after enrichment revealed excellent concordance, with the reference sequence closely mirroring the previously reported performance of Illumina sequencing conducted without sequence enrichment.

Targeted high throughput sequencing of a cancer-related exome subset by specific sequence capture with a fully automated microarray platform.

Sequence capture methods for targeted next generation sequencing promise to massively reduce cost of genomics projects compared to untargeted sequencing. However, evaluated capture methods specifically dedicated to biologically relevant genomic regions are rare. Whole exome capture has been shown to be a powerful tool to discover the genetic origin of disease and provides a reduction in target size and thus calculative sequencing capacity of >90-fold compared to untargeted whole genome sequencing. For further cost reduction, a valuable complementing approach is the analysis of smaller, relevant gene subsets but involving large cohorts of samples. However, effective adjustment of target sizes and sample numbers is hampered by the limited scalability of enrichment systems. We report a highly scalable and automated method to capture a 480 Kb exome subset of 115 cancer-related genes using microfluidic DNA arrays. The arrays are adaptable from 125 Kb to 1 Mb target size and/or one to eight samples without barcoding strategies, representing a further 26 - 270-fold reduction of calculative sequencing capacity compared to whole exome sequencing. Illumina GAII analysis of a HapMap genome enriched for this exome subset revealed a completeness of >96%. Uniformity was such that >68% of exons had at least half the median depth of coverage. An analysis of reference SNPs revealed a sensitivity of up to 93% and a specificity of 98.2% or higher.

Targeted next-generation sequencing by specific capture of multiple genomic loci using low-volume microfluidic DNA arrays.

We report a flexible method for selective capture of sequence fragments from complex, eukaryotic genome libraries for next-generation sequencing based on hybridization to DNA microarrays. Using microfluidic array architecture and integrated hardware, the process is amenable to complete automation and does not introduce amplification steps into the standard library preparation workflow, thereby avoiding bias of sequence distribution and fragment lengths. We captured a discontiguous human genomic target region of 185 kb using a tiling design with 50mer probes. Analysis by high-throughput sequencing using an Illumina/Solexa 1G Genome Analyzer revealed 2150-fold enrichment with mean per base coverage between 4.6 and 107.5-fold for the individual target regions. This method represents a flexible and cost-effective approach for large-scale resequencing of complex genomes.

Tests of rRNA hybridization to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be predicted.

Hybridization of rRNAs to microarrays is a promising approach for prokaryotic and eukaryotic species identification. Typically, the amount of bound target is measured by fluorescent intensity and it is assumed that the signal intensity is directly related to the target concentration. Using thirteen different eukaryotic LSU rRNA target sequences and 7693 short perfect match oligonucleotide probes, we have assessed current approaches for predicting signal intensities by comparing Gibbs free energy (DeltaG degrees) calculations to experimental results. Our evaluation revealed a poor statistical relationship between predicted and actual intensities. Although signal intensities for a given target varied up to 70-fold, none of the predictors were able to fully explain this variation. Also, no combination of different free energy terms, as assessed by principal component and neural network analyses, provided a reliable predictor of hybridization efficiency. We also examined the effects of single-base pair mismatch (MM) (all possible types and positions) on signal intensities of duplexes. We found that the MM effects differ from those that were predicted from solution-based hybridizations. These results recommend against the application of probe design software tools that use thermodynamic parameters to assess probe quality for species identification. Our results imply that the thermodynamic properties of oligonucleotide hybridization are by far not yet understood.

Optimization of candidate-gene SNP-genotyping by flexible oligonucleotide microarrays; analyzing variations in immune regulator genes of hay-fever samples.

BACKGROUND: Genetic variants in immune regulator genes have been associated with numerous diseases, including allergies and cancer. Increasing evidence suggests a substantially elevated disease risk in individuals who carry a combination of disease-relevant single nucleotide polymorphisms (SNPs). For the genotyping of immune regulator genes, such as cytokines, chemokines and transcription factors, an oligonucleotide microarray for the analysis of 99 relevant SNPs was established. Since the microarray design was based on a platform that permits flexible in situ oligonucleotide synthesis, a set of optimally performing probes could be defined by a selection approach that combined computational and experimental aspects. RESULTS: While the in silico process eliminated 9% of the initial probe set, which had been picked purely on the basis of potential association with disease, the subsequent experimental validation excluded more than twice as many. The performance of the optimized microarray was demonstrated in a pilot study. The genotypes of 19 hay-fever patients (aged 40-44) with high IgE levels against inhalant antigens were compared to the results obtained with 19 age- and sex-matched controls. For several variants, allele-frequency differences of more than 10% were identified. CONCLUSION: Based on the ability to improve empirically a chip design, the application of candidate-SNP typing represents a viable approach in the context of molecular epidemiological studies.

Another side of genomics: synthetic biology as a means for the exploitation of whole-genome sequence information.

The successful completion of the Human Genome Project and other sequencing projects opened the door for another quantum jump in science advancement. The most important public sequence databases are doubling in size every 18 months. By revealing the genetic program of many organisms, these efforts endow biologists with the ability to study the basic information of life in toto as an initial step toward a comprehensive understanding of the complexity of entire organisms. We review the area of synthetic biology, defined as the making and use of biosystems founded on the chemical synthesis of the coding DNA (and potentially RNA). The recent developments discussed here introduce a rich source of oligonucleotides to the field: in situ synthesised microarrays, which in fact represent nothing else but matrix nucleic acid synthesisers. With this new way of producing the oligonucleotides used in the making of synthetic genes in a very cost-effective manner, the field of synthetic biology can be expected to change dramatically in the next decade. Synthetic genes will then be the tools of choice to obtain any sequence at any time in any laboratory.

Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling.

Here we describe a novel microarray platform that integrates all functions needed to perform any array-based experiment in a compact instrument on the researcher's laboratory benchtop. Oligonucle otide probes are synthesized in situ via a light- activated process within the channels of a three-dimensional microfluidic reaction carrier. Arrays can be designed and produced within hours according to the user's requirements. They are processed in a fully automatic workflow. We have characterized this new platform with regard to dynamic range, discrimination power, reproducibility and accuracy of biological results. The instrument detects sample RNAs present at a frequency of 1:100 000. Detection is quantitative over more than two orders of magnitude. Experiments on four identical arrays with 6398 features each revealed a mean coefficient of variation (CV) value of 0.09 for the 6398 unprocessed raw intensities indicating high reproducibility. In a more elaborate experiment targeting 1125 yeast genes from an unbiased selection, a mean CV of 0.11 on the fold change level was found. Analyzing the transcriptional response of yeast to osmotic shock, we found that biological data acquired on our platform are in good agreement with data from Affymetrix GeneChips, quantitative real-time PCR and--albeit somewhat less clearly--to data from spotted cDNA arrays obtained from the literature.

High-throughput qRT-PCR validation of blood microRNAs in non-small cell lung cancer.

Validation of biomarkers is essential to advance the translational process to clinical application. Although there exists an increasing number of reports on small non-coding RNAs (microRNAs) as minimally-invasive markers from blood, serum or plasma, just a limited number is verified in follow-up studies. We used qRT-PCR to evaluate a known miRNA signature measured from blood that allowed for separation between patients with non-small cell lung cancer (NSCLC), COPD and healthy controls.From the data of our previous microarray studies we selected a panel of 235 miRNAs related to lung cancer and COPD. We observed a high concordance between the AUC values of our initial microarray screening and the qRT-PCR data (correlation of 0.704, p < 10-16). Overall, 90.3% of markers were successfully validated. Among the top markers that were concordant between both studies we found hsa-miR-20b-5p, hsa-miR-20a-5p, hsa-miR-17-5p, and hsa-miR-106a-5p. The qRT-PCR analysis also confirmed that non-small cell lung cancer patients could be accurately differentiated from unaffected controls: a subset of five markers was sufficient to separate NSCLC patients from unaffected controls with accuracy of 94.5% (specificity and sensitivity of 98% and 91%). Beyond differentiation from controls, we also succeeded in separating NSCLC patients from patients with COPD. MiRNAs that were identified as relevant for the separation between lung cancer and COPD by both qRT-PCR and the array-based studies included hsa-miR-26a-5p, hsa-miR-328-3p and hsa-miR-1224-3p. Although for differentiation between NSCLC patients from COPD patients more markers were required, still high accuracy rates were obtained (5 markers: 78.8%; 10 markers: 83.9%; 50 markers: 87.6%).

New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Natalizumab restores aberrant miRNA expression profile in multiple sclerosis and reveals a critical role for miR-20b.

OBJECTIVE: To identify microRNAs (miRNAs) regulated by anti-α4 integrin monoclonal antibody therapy (natalizumab) in the peripheral blood of patients with relapsing-remitting (RR) multiple sclerosis (MS) and to confirm their role in experimental settings in vivo. METHODS: In a longitudinal study of 17 RR-MS patients, we investigated blood miRNA expression profiles at baseline and after 1 year of natalizumab therapy by microarray technique and quantitative PCR validation. We compared the baseline expression profiles of these patients to those of 18 age- and sex-matched healthy controls. We confirmed the contribution of resulting candidate miRNAs in an animal model of MS, experimental autoimmune encephalomyelitis (EAE) induced by adoptive transfer of proteolipid protein (PLP)139-151-activated lymphocytes in SJL/J mice or by active immunization of miR-106a∼363-deficient C57BL/6 mice (or wildtype litter mates) with myelin oligodendrocyte glycoprotein (MOG)35-55. RESULTS: Our longitudinal analysis revealed that miR-18a, miR-20b, miR-29a, and miR-103 were upregulated and predominantly expressed by CD4(+) T cells, whereas miR-326 was downregulated upon natalizumab treatment. A comparison of untreated RR-MS patients at baseline with healthy controls revealed that the four natalizumab-upregulated targets were initially downregulated in MS. All confirmed targets showed disease-dependent expression in splenocytes of mice suffering from EAE. Genetic deletion of the miRNA cluster miR-106a∼363 (containing natalizumab-regulated miR-20b) resulted in a more severe EAE course and an in vivo upregulation of the miR-20b target genes rorgt, stat3, and vegfa. INTERPRETATION: Our study indicates that natalizumab restores dysregulated miRNA patterns in MS and reveals the contribution of miR-20b in autoimmune demyelination in vivo.

Impact of depressive mood on relapse in patients with inflammatory bowel disease: a prospective 18-month follow-up study.

OBJECTIVE: There is evidence of an interaction between psychological factors and activity of inflammatory bowel disease (IBD). We examined the influence of depressive mood and associated anxiety on the course of IBD over a period of 18 months in a cohort of patients after an episode of active disease. METHODS: In this prospective, longitudinal, observational study, 60 patients (37 women and 23 men) with clinically inactive IBD (Crohn disease, n = 47, 78%; ulcerative colitis, n = 13, 22%) were enrolled after a flare of disease. Psychological status, health-related quality of life (HRQOL), and disease activity were evaluated at baseline and then every 3 months for a period of 18 months by means of clinical and biological parameters, the Beck Depression Inventory (BDI), the Spielberger State-Trait Anxiety Inventory, the Inflammatory Bowel Disease Questionnaire, the Perceived Stress Questionnaire, and the Rating Form of Inflammatory Bowel Disease Patients Concerns. RESULTS: At baseline, depression (BDI > or = 13 points) was found in 17 of 60 (28%) patients. Thirty-two patients (59%) experienced at least one relapse during the 18 months of follow-up. Regression analysis showed a significant correlation between BDI scores at baseline and the total number of relapses after 12 (p <.01) and 18 months (p <.01) of follow-up. Furthermore, depression scores at baseline correlated with the time until the first recurrence of the disease (p <.05). Anxiety and low HRQOL were also related with more frequent relapses during follow-up (p <.05 and p <.01, respectively). CONCLUSIONS: Psychological factors such as a depressive mood associated with anxiety and impaired HRQOL may exert a negative influence on the course of IBD. Therefore, assessment and management of psychological distress should be included in clinical treatment of patients with IBD.

Analysis of DNA-microarrays produced by inverse in situ oligonucleotide synthesis.

5'-Phosphoramidites protected by 2-nitrophenylethyl (NPE) and 2-(4-nitrophenyl)ethoxy carbonyl (NPEOC) functions were employed for in situ synthesis of oligonucleotides in 5'-->3' direction on flat glass surfaces. By this inverse synthesis format, the oligonucleotides are attached to the solid support via their 5'-ends while the free 3'-hydroxyl groups are available as substrates for enzymatic reactions such as elongation by polymerases, thereby adding another feature to the portfolio of chip-based applications. Having a fluorescence dye present at the first base during synthesis, the quality of the oligonucleotides was analysed quantitatively by capillary electrophoresis after release from the solid support. With about 95% yield per condensation, it was found to be equivalent to synthesis results achieved on CPG support. The chip-bound oligonucleotides could be extended enzymatically upon hybridisation of a DNA-template. Surprisingly, however, only 63% of the oligonucleotides were elongated in polymerase reactions, while oligonucleotides that were released from the support behaved normally in standard PCR amplifications. This rate of 63% nevertheless compares favourably with an extension rate of only 50%, which was achieved under identical conditions, if pre-fabricated oligonucleotides of identical sequence had been spotted to the glass support.

Microfluidic primer extension assay.

MicroRNAs (miRNAs) are a new class of biomarkers. They represent a group of small, noncoding RNAs that regulate gene expression at the posttranslational level by degrading or blocking translation of messenger RNA (mRNA) targets. miRNAs are important players when it comes to regulating cellular functions and in several diseases, including cancer (Cancer Res 66:7390-7394, 2006; Nature 435:834-838, 2005). So far, miRNAs have been extensively studied in tissue material. Only recently, it was found that miRNAs also exist in a broad range of body fluids (Clin Chem 56:1733-1741, 2010). A major challenge still is the efficient and specific detection of miRNAs. The short length of miRNAs, with only 17-27 base pairs, comes with technical difficulties for analysis. Furthermore, individual miRNAs, especially members of a miRNA family (e.g., the let-7 family), show high sequence homology, with sequences differing by as little as a single base pair. Although miRNAs are abundant in higher copy numbers compared to mRNAs, miRNAs lack a common feature like a poly-A tail that eases detection in a complex background of other RNA species. Besides qPCR, in situ hybridization, and next-generation sequencing, microarrays are versatile tools for high-throughput analysis of already known miRNAs (PLoS One 12:e9685, 2010; Nat Genet 38:S2-S7, 2006; Nature Methods 50:298-301, 2010). Different assay formats have been proposed for expression analysis of miRNAs on microarrays, of which most employed prelabeled RNA molecules. As a modification, the so-called RAKE assay was developed that combined the use of unlabeled RNA with on-chip enzymatic labeling by exonuclease cleavage and polymerase primer extension (RNA 12:187-191, 2006; Nature Methods 1:155-161, 2004; Genome Res 16:1289-1298, 2006).Here, we describe a simple method for detection of miRNAs based on a combination of stringent hybridization and enzymatic primer extension on a microfluidic microarray starting from total RNA material, without the need for enrichment, amplification, or labeling of the native RNA samples (N Biotechnol 25:142-149, 2008). This assay can be used with starting material as low as 30 ng of total RNA. We have used this technique extensively for identifying specific sets of miRNAs (miRNA signatures) for diagnosis of cancer and cardiovascular or inflammatory diseases from blood samples of patients (Br J Cancer 103:693-700, 2010; BMC Cancer 9:353, 2009; PLoS One 4:e7440, 2009; BMC Cancer 10:262, 2010; Basic Res Cardiol 106(1):13-23, 2011).