Regulatory potential at type 2 diabetes-associated SNPs at the <i>CDC123/CAMK1D</i> locus.

<p>A) The 11 SNPs in high LD (r²≥.7, EUR) with GWAS index SNP rs12779790. Arrows indicate the two SNPs that overlap islet, liver, and HepG2 open chromatin and epigenomic marks and that are located near to HepG2 ChIP-seq peaks; these two SNPs were tested for allele-specific transcriptional activity. B) DNase hypersensitivity peaks identified in two pooled islet samples from the ENCODE Consortium. C) FAIRE peaks identified in one representative islet sample from the ENCODE Consortium. D) H3K4me1 histone modifications from the Roadmap Epigenomics Consortium. E) FOXA1 and FOXA2 ChIP-seq peaks and signal from ENCODE. Image is taken from the UCSC genome browser, February 2009 (GRCh37/hg19) assembly (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004633#pgen.1004633-Fujita1" target="_blank">[51]</a>. The 5′ end of <i>CAMK1D</i> begins after position 12,390,000.</p

Alleles of rs11257655 differentially bind FOXA proteins in rat 832/13 insulinoma cells, mouse MIN6 insulinoma cells and human HepG2 hepatoma cells.

<p>EMSA using 832/13 (A), MIN6 (B) and HepG2 (C) nuclear extract shows differential protein-DNA binding of rs11257655 alleles. The probe containing risk allele rs11257655 -T shows allele-specific protein binding (arrows a–e) compared to the probe containing non-risk allele C. Excess unlabeled probe containing the T allele (T-comp) more efficiently competed away allele-specific bands than unlabeled probe for the C allele (C-comp). Incubation of 832/13 and HepG2, nuclear extract with FOXA1/FOXA2 antibodies disrupt the DNA-protein complex formed with T allele-containing DNA probe (arrow a, d, e) and result in band supershifts (asterisks). Incubation of MIN6 nuclear extract with FOXA2 antibody decreases the DNA-protein complex formed with T allele-containing DNA probe (arrow b) and results in a band supershift. To enhance visualization of protein complexes, free biotin-labeled probe is not shown. (D) DNA affinity-capture identified differential binding of FOXA2 at rs11257655 alleles in 832/13 cells. (E) The T allele of rs11257655 is predicted as a FOXA1 and FOXA2 consensus core-binding motif.</p

rs11257655-T allele shows increased binding to FOXA1 and FOXA2 in human islets.

<p>FOXA1 (A) and FOXA2 (B) ChIP in human islets shows enrichment at rs11257655 compared to IgG control. Islets containing one copy of the rs11257655-T allele show 7.2-fold greater FOXA1 enrichment and 4.2-fold greater FOXA2 enrichment. rs11257655 CT heterozygotes are more significantly enriched than rs11257655 CC homozygotes for FOXA1 (one-sided <i>t</i>-test, <i>P</i> = .06) and FOXA2 (one-sided <i>t</i>-test, <i>P</i> = .026). A negative control region 28 kb downstream of rs11257655 was not enriched in FOXA1- and FOXA2- bound chromatin (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004633#pgen.1004633.s003" target="_blank">Figure S3A and S3B</a>). Error bars represent standard error of two to three islets for each represented genotype.</p

Association Studies with Imputed Variants Using Expectation-Maximization Likelihood-Ratio Tests

<div><p>Genotype imputation has become standard practice in modern genetic studies. As sequencing-based reference panels continue to grow, increasingly more markers are being well or better imputed but at the same time, even more markers with relatively low minor allele frequency are being imputed with low imputation quality. Here, we propose new methods that incorporate imputation uncertainty for downstream association analysis, with improved power and/or computational efficiency. We consider two scenarios: I) when posterior probabilities of all potential genotypes are estimated; and II) when only the one-dimensional summary statistic, imputed dosage, is available. For scenario I, we have developed an expectation-maximization likelihood-ratio test for association based on posterior probabilities. When only imputed dosages are available (scenario II), we first sample the genotype probabilities from its posterior distribution given the dosages, and then apply the EM-LRT on the sampled probabilities. Our simulations show that type I error of the proposed EM-LRT methods under both scenarios are protected. Compared with existing methods, EM-LRT-Prob (for scenario I) offers optimal statistical power across a wide spectrum of MAF and imputation quality. EM-LRT-Dose (for scenario II) achieves a similar level of statistical power as EM-LRT-Prob and, outperforms the standard Dosage method, especially for markers with relatively low MAF or imputation quality. Applications to two real data sets, the Cebu Longitudinal Health and Nutrition Survey study and the Women’s Health Initiative Study, provide further support to the validity and efficiency of our proposed methods.</p></div

Rejection Sampling vs. Dosage Approximation for Estimation.

<p>MAF: Minor allele frequency.</p><p>MSE: Mean square error.</p><p>Rejection Sampling vs. Dosage Approximation for Estimation.</p

Computing Time: Mixture Method vs EM-LRT-Prob.

<p>The computing time of the Mixture method and our proposed EM-LRT-Prob method is displayed across a range of sample sizes. For each sample size, computing time is averaged across 2,000 simulated datasets.</p

Q–Q Plot for Null Variants with Low Imputation Quality in the CLHNS Study.

<p>The observed (Y-axis) vs. expected (X-axis) –log₁₀[<i>p</i>-values] are shown for 1,135 SNPs in the CLHNS data set. These SNPs are considered to be under the null hypothesis (true <i>p</i>-value >5×10⁻⁶), and all have low imputation quality (<i>R</i>²<0.3).</p

Spearman Correlation with Gold Standard <i>P</i>-values.

<p>The Spearman correlation (Y-axis) between gold standard <i>p</i>-values and <i>p</i>-values from different methods is displayed across a spectrum of MAF and <i>R²</i>.</p

One-sample T-test for Type I Error.

<p>*: <i>P</i>-value <5E-4.</p><p>One-sample T-test for Type I Error.</p

Associated Variants with <i>R²</i>≤0.3 in the CLHNS Study.

<p>*: Coordinates are in genome build 37.</p><p>Bold with †: The most significant <i>p</i>-value among the four methods.</p><p>Bold without †: The second most significant <i>p</i>-values among the four methods.</p>#<p>: Truth was established by regressing phenotype on true genotypes.</p><p>Associated Variants with <i>R²</i>≤0.3 in the CLHNS Study.</p