Additional file 3: Figure S2. of Pro-inflammatory cytokines and their epistatic interactions in genetic susceptibility to schizophrenia

Meta-analysis of TNFA rs361525 risk allele A versus G allele in schizophrenia patients in comparison to Normal control using fixed and random model. (TIF 354 kb

Additional file 1: Table S1. of Pro-inflammatory cytokines and their epistatic interactions in genetic susceptibility to schizophrenia

In silico functional prediction of relevant SNPs in this study using F-SNP program. Table S2. PCR conditions and polymorphism detection through RFLP for cytokine gene polymorphism. Table S3. List of studies included in meta-analysis. Table S4. Genotype and allele frequencies of polymorphisms that show lack of association with schizophrenia. (DOCX 41 kb

Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes

<div><p>DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (<em>Ins2</em>) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined <em>Ins2</em> exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse <em>Ins2</em> gene was methylated <em>in vitro</em> and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic DNA background. We also compared different methods for data analysis and found that the Relative Expression Ratio method is the most robust method since it incorporates both a reference value to normalize day-to-day variability as well as PCR reaction efficiencies to normalize between the methylation-specific and bisulfite-specific components of the calculations. The assay was applied in the streptozotocin-treated diabetic mouse model and detected a significant increase in circulating beta cell DNA before the rise in blood glucose level. These results demonstrate that this qMSP assay can be used for monitoring circulating DNA from insulin-producing cells, which will provide the basis for development of assays to detect beta cell destruction in early T1DM.</p> </div

Comparison of the average and standard deviation of three methods of quantification of unmethylated DNA levels using qMSP.

<p>n is the number of replicates at each time point and represents data from at least 4 mice.</p>*<p>P-value calculated by Student's <i>t</i>-test for the difference between each time point and pre-treatment value. SD is the standard deviation. N/A = not applicable.</p

Rationale for selection of the primers that differentiate between methylated and unmethylated CpG.

<p>A) Schematic illustration of the mouse <i>Ins2</i> gene with promoter region (blue), exon 1 (yellow), intron 1 (white), and exon 2 (green) showing the positions of CpG sites and the primers used in this study. Black arrows represent the bisulfite-specific primers (BSP) that amplify both methylated and unmethylated DNA. Red arrows represent methylation-specific primers (MSP) that amplify unmethylated but not methylated DNA. B) Gel electrophoresis (3% agarose) of PCR products amplified by reactions using different primer sets and the cloned <i>Ins2</i> gene as template. The clone was methylated (M) or sham methylated (N) and bisulfite-treated prior to use in the reactions. NTC means non-template control. TSS indicates the transcription starting site.</p

Oligonucleotides used in this study.

<p>Underline indicates extra nucleotides added at 5′-end of the primer.</p

Reproducibility of the qMSP assay.

<p>The variability of the qMSP results at different time points as analyzed using Relative Expression Ratio (RER) compared with Demethylation Index (DI) at days 1, 6, 7, and 14 post-STZ treated mice. The data display the percent coefficient of variation (%CV) with standard deviation (SD) of the variation at each point representing data from at least 4 mice. The statistical significance at each time point was calculated by Two-Way ANOVA to compare RER and DI and the significance level indicated by asterisks (*, P<0.05; **, P<0.01). ns =  non significant.</p

The amplification efficiency of qMSP and qBSP standard curves.

<p>MSP = methylation-specific PCR, BSP = bisulfite-specific PCR, Slope = slope of the standard curve, R² = the square of the correlation coefficient of the standard curve.</p

Statistical variation of qMSP standard curve.

*<p>n = 12; SD = standard deviation, %CV = percent coefficient of variation [(SD/Cq average) ×100].</p

Beta cell-specific demethylation of exon 2 of the mouse <i>Ins2</i> gene.

<p>A) Schematic illustration of the mouse <i>Ins2</i> gene with exon 1 (yellow), intron 1 (white), and exon 2 (green) with the positions of the CpG sites indicated. B) Methylation pattern of mouse <i>Ins2</i> exon 2 showing the percentage of methylated (solid box) and unmethylated (empty box) CpG sites obtained from the 60% mouse beta cell fraction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047942#pone.0047942-Kuroda1" target="_blank">[21]</a>, NIT-1 mouse insulinoma cell line, and other mouse tissues (liver, spleen, kidney, brain, muscle, and blood). Each pattern results from 17, 9, and 23 clones of pancreatic beta cell fraction, NIT-1, and other tissues, respectively. TSS indicates the transcription start site.</p