New Variants Including <i>ARG1</i> Polymorphisms Associated with C-Reactive Protein Levels Identified by Genome-Wide Association and Pathway Analysis

<div><p>C-reactive protein (CRP) is a general marker of systemic inflammation and cardiovascular disease (CVD). The genetic contribution to differences in CRP levels remains to be explained, especially in non-European populations. Thus, the aim of this study was to identify genetic loci associated with CRP levels in Korean population. We performed genome-wide association studies (GWAS) using SNPs from 8,529 Korean individuals (7,626 for stage 1 and 903 for stage 2). We also performed pathway analysis. We identified a new genetic locus associated with CRP levels upstream of <i>ARG1</i> gene (top significant SNP: rs9375813, <i>P_meta</i> = 2.85×10⁻⁸), which encodes a key enzyme of the urea cycle counteract the effects of nitric oxide, in addition to known <i>CRP</i> (rs7553007, <i>P_meta</i> = 1.72×10⁻¹⁶) and <i>HNF1A</i> loci (rs2259816, <i>P_meta</i> = 2.90×10⁻¹⁰). When we evaluated the associations between the CRP-related SNPs with cardiovascular disease phenotypes, rs9375813 (<i>ARG1</i>) showed a marginal association with hypertension (<i>P</i> = 0.0440). To identify more variants and pathways, we performed pathway analysis and identified six candidate pathways comprised of genes related to inflammatory processes and CVDs (<i>CRP, HNF1A</i>, <i>PCSK6, CD36</i>, and <i>ABCA1</i>). In addition to the previously reported loci (<i>CRP, HNF1A</i>, and <i>IL6</i>) in diverse ethnic groups, we identified novel variants in the <i>ARG1</i> locus associated with CRP levels in Korean population and a number of interesting genes related to inflammatory processes and CVD through pathway analysis.</p></div

Regional plot of the SNPs in the <i>ARG1</i> locus (up) and the LD relationship among these SNPs (down).

<p>Data are shown for the <i>ARG1</i> locus around rs9375813. Diamond-shaped dots represent -log₁₀ (<i>P</i>-values) of SNPs, and green diamond in the LD plot indicates the most significant SNP. The strength of LD relationship (<i>r</i>²) between the most strongly associated SNP and the other SNPs is presented with red color intensities based on JPT+CHB HapMap data. The light blue curve shows recombination rates drawn based on JPT+CHB HapMap data. Green bars represent the coding genes in this region.</p

Candidate CRP-associated SNPs identified by ICSNPathway analysis.

<p>*The number indicates the index of pathways that are ranked by their statistical significance (FDR) (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095866#pone-0095866-t004" target="_blank">Table 4</a>).</p>†<p>-log₁₀(<i>P</i>) in stage 1 phase of the GWAS. The ‘-’ denotes that the SNP was not present in the stage 1 phase of the GWAS.</p>‡<p>-log₁₀(<i>P</i>) for the SNP in the stage 1 phase of the GWAS, which is in LD with the SNP identified by pathway analysis.</p

Results of the genome-wide association study of serum CRP levels.

<p>Chr, chromosome; MAF, minor allele frequency; CRP, C-reactive protein; SE, standard error; β, Effect size of a minor allele on natural-log-transformed CRP; Q, p-value for Cochrane's Q statistic assessing if combining studies are homogeneous; I², I-squared index quantifying heterogeneity.</p><p>*Most significantly associated SNP in each locus based on the meta analysis results were summarized.</p>†<p>SNP positions were based on the NCBI human genome build 36.3 (hg18).</p

Association of previously reported CRP-related loci.

<p>*The most significant SNPs from each locus were shown.</p>†<p>Position is based on NCBI human genome build 36.3 (hg18).</p>‡<p>Type indicates if a SNP is genotyped or imputed. Chr, chromosome; MAF, minor allele frequency.</p

Manhattan plot showing GWAS results for serum CRP levels in 7,626 Korean subjects.

<p>The blue horizontal line (<i>P</i><10⁻⁸) denotes the general threshold for genome-wide significance. The red horizontal line (<i>P</i><10⁻⁵) denotes the threshold for selecting loci for stage 2 test. The arrow heads indicate three significant loci that passed the threshold.</p

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Background<p>Addictive use of the Internet and online games is a potential psychiatric disorder termed Internet gaming disorder (IGD). Altered microRNA (miRNA) expression profiles have been reported in blood and brain tissue of patients with certain psychiatric disorders and suggested as biomarkers. However, there have been no reports on blood miRNA profiles in IGD.</p>Methods<p>To discover IGD-associated miRNAs, we analyzed the miRNA expression profiles of 51 samples (25 IGD and 26 controls) using the TaqMan Low Density miRNA Array. For validation, we performed quantitative reverse transcription PCR with 36 independent samples (20 IGD and 16 controls).</p>Results<p>Through discovery and independent validation, we identified three miRNAs (hsa-miR-200c-3p, hsa-miR-26b-5p, hsa-miR-652-3p) that were significantly downregulated in the IGD group. Individuals with all three miRNA alterations had a much higher risk of IGD than those with no alteration [odds ratio (OR) 22, 95% CI 2.29–211.11], and the ORs increased dose dependently with number of altered miRNAs. The predicted target genes of the three miRNAs were associated with neural pathways. We explored the protein expression of the three downstream target genes by western blot and confirmed that expression of GABRB2 and DPYSL2 was significantly higher in the IGD group.</p>Conclusion<p>We observed that expressions of hsa-miR-200c-3p, hsa-miR-26b-5p, and hsa-miR-652-3p were downregulated in the IGD patients. Our results will be helpful to understand the pathophysiology of IGD.</p