Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems

Impact of polyols on Oral microbiome of Estonian schoolchildren

BackgroundOral microbiome has significant impact on both oral and general health. Polyols have been promoted as sugar substitutes in prevention of oral diseases. We aimed to reveal the effect of candies containing erythritol, xylitol or control (sorbitol) on salivary microbiome.MethodsNinety children (11.30.6years) consumed candies during 3years. Microbial communities were profiled using Illumina HiSeq 2000 sequencing and real-time PCR.ResultsThe dominant phyla in saliva were Firmicutes (39.1%), Proteobacteria (26.1%), Bacteroidetes (14.7%), Actinobacteria (12%) and Fusobacteria (6%). The microbiome of erythritol group significantly differed from that of the other groups. Both erythritol and xylitol reduced the number of observed bacterial phylotypes in comparison to the control group. The relative abundance of the genera Veillonella, Streptococcus and Fusobacterium were higher while that of Bergeyella lower after erythritol intervention when comparing with control. The lowest prevalence of caries-related mutans streptococci corresponded with the lowest clinical caries markers in the erythritol group.ConclusionsDaily consumption of erythritol, xylitol or control candies has a specific influence on the salivary microbiome composition in schoolchildren. Erythritol is associated with the lowest prevalence of caries-related mutans streptococci and the lowest levels of clinical caries experience.Trial registration p id=Par5 ClinicalTrials.gov Identifier NCT01062633

Fibrinogen beta variants confer protection against coronary artery disease in a Greek case-control study

<p>Abstract</p> <p>Background</p> <p>Although plasma fibrinogen levels are related to cardiovascular risk, data regarding the role of fibrinogen genetic variation in myocardial infarction (MI) or coronary artery disease (CAD) etiology remain inconsistent. The purpose of the present study was to investigate the effect of <it>fibrinogen A (FGA)</it>, <it>fibrinogen B (FGB) </it>and <it>fibrinogen G (FGG) </it>gene SNPs and haplotypes on susceptibility to CAD in a homogeneous Greek population.</p> <p>Methods</p> <p>We genotyped for rs2070022, rs2070016, rs2070006 in <it>FGA </it>gene, the rs7673587, rs1800789, rs1800790, rs1800788, rs1800787, rs4681 and rs4220 in <it>FGB </it>gene and for the rs1118823, rs1800792 and rs2066865 SNPs in <it>FGG </it>gene applying an arrayed primer extension-based genotyping method (APEX-2) in a sample of CAD patients (n = 305) and controls (n = 305). Logistic regression analysis was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), before and after adjustment for potential confounders.</p> <p>Results</p> <p>None of the <it>FGA </it>and <it>FGG </it>SNPs and <it>FGA, FGB, FGG </it>and <it>FGA-FGG </it>haplotypes was associated with disease occurrence after adjustment. Nevertheless, rs1800787 and rs1800789 SNPs in <it>FGB </it>gene seem to decrease the risk of CAD, even after adjustment for potential confounders (OR = 0.42, 95%CI: 0.19-0.90, p = 0.026 and OR = 0.44, 95%CI:0.21-0.94, p = 0.039, respectively).</p> <p>Conclusions</p> <p><it>FGA </it>and <it>FGG </it>SNPs as well as <it>FGA, FGB, FGG </it>and <it>FGA-FGG </it>haplotypes do not seem to be important contributors to CAD occurrence in our sample. On the contrary, <it>FGB </it>rs1800787 and rs1800789 SNPs seem to confer protection to disease onset lowering the risk by about 50% in homozygotes for the minor alleles.</p

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy

Background: CCAAT enhancer-binding protein epsilon (C/EBP epsilon) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBP epsilon is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. Objective: The aim of this study was to molecularly characterize the effects of C/EBP epsilon transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. Methods: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. Results: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. Conclusion: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBP epsilon. Mutated C/EBPe acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBP epsilon. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.Peer reviewe

Single-cell transcriptome analysis of endometrial tissue

STUDY QUESTION: How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER: By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY: Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION: The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603 commonly expressed genes were detected, with 241 significantly differentially expressed genes. Of these, 231 genes were up- and 10 down-regulated in cultured cells, respectively. In addition, we performed a gene ontology analysis of the differentially expressed genes and found that these genes are mainly related to cell cycle, translational processes and metabolism. LIMITATIONS, REASONS FOR CAUTION: Although CD9-positive single epithelial cells sorting was successfully established in our laboratory, the amount of transcriptome data per individual epithelial cell was low, complicating further analysis. This step most likely failed due to the high dose of RNases that are released by the cells' natural processes, or due to rapid turnaround time or the apoptotic conditions in freezing- or single-cell solutions. Since only the cells from the late-secretory phase were subject to more focused analysis, further studies including larger sample size from the different time-points of the natural menstrual cycle are needed. The methodology also needs further optimization to examine different cell types at high quality. WIDER IMPLICATIONS OF THE FINDINGS: The symbiosis between clinical biopsy and the sophisticated laboratory and bioinformatic protocols described here brings together clinical diagnostic needs and modern laboratory and bioinformatic solutions, enabling us to implement a precise analytical toolbox for studying the endometrial tissue even at the single-cell level.Peer reviewe

Nasal upregulation of CST1 in dog-sensitised children with severe allergic airway disease

Background: The clinical presentation of children sensitised to dog dander varies from asymptomatic to severe allergic airway disease, but the genetic mechanisms underlying these differences are not clear. The objective of the present study was to investigate nasal transcriptomic profiles associated with dog dander sensitisation in school children and to reveal clinical symptoms related with these profiles. Methods: RNA was extracted from nasal epithelial cell brushings of children sensitised to dog dander and healthy controls. Blood sample analyses included IgE against dog dander, dog allergen molecules, other airborne and food allergens, basophil activation and white blood cell counts. Clinical history of asthma and rhinitis was recorded, and lung function was assessed (spirometry, methacholine provocation and exhaled nitric oxide fraction). Results: The most overexpressed gene in children sensitised to dog dander compared to healthy controls was CST1, coding for Cystatin 1. A cluster of these children with enhanced CST1 expression showed lower forced expiratory volume in 1 s, increased bronchial hyperreactivity, pronounced eosinophilia and higher basophil allergen threshold sensitivity compared with other children sensitised to dog dander. In addition, multi-sensitisation to lipocalins was more common in this group. Conclusions: Overexpression of CST1 is associated with more severe allergic airway disease in children sensitised to dog dander. CST1 is thus a possible biomarker of the severity of allergic airway disease and a possible therapeutic target for the future treatment of airborne allergy.Peer reviewe

Characterization of extracellular vesicles produced by single human embryos at early stages of development

Background: Extracellular vesicles (EVs) are recognized as potent vehicles for intercellular communication. To date, there is little information available regarding the role of EVs during the early stages of human embryonic development. The aim of this study was to develop techniques for the recovery of EVs secreted by a single human embryo in an in vitro culture system. The EVs were characterized according to size, concentration and electrical surface properties (zeta potential), in order to understand the role of EVs production in human embryos for determination of their quality at early stages of development. Methods: Human embryos were produced by in vitro fertilization (IVF) for 24 h in fertilization medium, cultured individually for 48 h (3 days) in cleavage medium and additionally 48 h in blastocyst medium (day 5). Conditioned media, at days 3 and 5 post-IVF, was collected and EVs were isolated using a series of centrifugations and size-exclusion chromatography. The size, concentration and zeta potential of EVs were characterized using a nanoparticle tracking analysis. Results: Using this method of isolation, we were able to collect and characterize EVs produced by a single human embryo. Analysis confirmed the presence of EVs at early stages of development, with the concentration of EVs being higher in early blastocysts (day 5), as compared to 4-8 cell-stage embryos (day 3). Moreover, already at day 3, we were able to discriminate between embryos that were properly developing and those that were later visually determined as degrading at day 5. The data indicates that embryos following normal development at day 3 but degrading at later stages (day 5) were producing significantly higher number of EVs (with size range of 100-160 nm) compared with those developing properly at day 3 and later progressing to early blastocysts at day 5. Summary/conclusion: In conclusion, we have developed a sensitive protocol for the isolation of EVs from human embryos cultured individually. We have demonstrated that human embryos secrete EVs in varying amounts and sizes during the early stages of their development. Further investigations are needed to establish EV characteristics of early human embryo as a quality marker for human clinical embryology

Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome

Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49–96%) corresponded to rRNA-, coding- and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics

Profiling of extracellular small RNAs highlights a strong bias towards non-vesicular secretion

The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R2 = 0.69–0.87), quantitatively an outstanding 96.2–99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future