Overview of all SNPs analyzed in this study with T1D disease association.

<p>SNPs which mark their respective haplotype are marked in bold.</p

SNP pull-down for TFAP2 and SP1.

<p>A: Coomassie stained and anti-TFAP2 immunostained blot. TFAP2 enrichment with the SELEX-derived binding site can be visualized by Western blot, but not within the context of other protein bands in Coomassie staining. B: SNP pull-down for TFAP2. The transcription factor binds to its SELEX sequence (C variant), but a single nucleotide exchange abrogates binding (A variant). Performing proteome-wide detection for differentially binding transcription factors, only TFAP2 is enriched at the wild-type sequence observed in the individual mass spectrum of the TFAP2 peptide LSLLSSTSK²⁺ and by comparison of all SILAC ratios of proteins detected in the experiment. C: SNP pull-down for rs509813. Applying PWAS, we identify SP1 together with SP3, which both bind to the same DNA motif. ZNF148, was also detected as a significant interaction partner in our proteome-wide screen, although not bioinformatically predicted.</p

Schematic diagram of SNP pull-down.

<p>Synthetic oligonucleotides containing the SNP are phosphorylated, polymerized and subsequently strand-specifically desthiobiotin-labeled. The immobilized DNA fragments are incubated with either light or heavy extract. After removal of unbound proteins, bead fractions are combined and DNA-protein complexes are eluted with biotin. The eluate is precipitated, digested and analyzed by single-run, high resolution, quantitative mass spectrometry. Specific interaction partners result in a ratio different from 1∶1, demonstrating specific enrichment at one variant of the single nucleotide polymorphism.</p

Functional analysis for SNP rs12722508.

<p>A: Immunostaining with an antibody against endogenous RUNX1 validates differential binding between the A- and G-allele of rs12722508, input refers to nuclear extract incubated with either rs12722508 allele. B: mRNA levels are reduced upon esiRNA knock-down with mean and s.d. of triplicates; C: changes in firefly luciferase activity in mock-transfection and upon knock-down of TFAP4, RUNX1, CREB and TFAP4. Knock-down of RUNX1 results in an activation with an allele-specific difference (<i>P</i> = 0.016) demonstrating the functional consequence of differential RUNX1 binding between the two alleles of rs12722508.</p

Schematic representation of a two-dimensional interaction plot.

<p>While specific outliers are found in the upper left (variant A) or the lower right (variant G) quadrant, most proteins cluster around the origin as they are binding to both variants equally. Contaminants have a SILAC ratios lower than 1 even when labels are switched and thus are grouped in the lower left quadrant.</p

Interaction analysis for fine-mapped T1D SNPs.

<p>Two-dimensional interaction plots reveal the specific binding of transcription factors. A: CREB1 and TFAP4 are enriched on rs12722522*C while this allele resembles a perfect match to the CREB consensus binding motif. B: rs12722508 is bound by several transcription factors (upper panel) among them the RUNX1-CBFB heterodimer, although the sequence around rs12722508*A does not match perfectly the transcription factor consensus motif (lower panel); C: Quantitative SNP pull-down screen identifies LEF1 to bind around 8 times stronger to rs41295061*A.</p

Manhattan plot for the FBAT-CNV P-values.

<p>The y-axis shows the distribution of –log₁₀(p) where p is the FBAT-CNV test association test P-value for all CNV loci passing quality control filters (Methods). The x-axis shows chromosomes numbered from 1 (left) to X (right).</p

Analysis of association of <i>PTPN22</i> ‘haplotype 6–10’ group (<i>rs12144309</i>: C&gt;T) with rheumatoid arthritis.

<p>1. Cases top line, controls bottom line. The NZ controls deviated mildly from HWE (<i>P</i> = 0.02).</p><p>2. Imputed genotypes were taken from <a href="http://www.wtccc.org.uk" target="_blank">www.wtccc.org.uk</a>.</p><p>3. The Mantel-Haenszel combined OR = 0.90 [0.82–0.98], <i>P</i> = 0.021. The Breslow-Day test for heterogeneity <i>P</i> = 0.90.</p

Quantile-quantile plot comparing the expected versus the observed distribution of the FBAT-CNV P-values.

<p>These plots show the distribution of -2log₁₀(p), which is, under the null, distributed as chi-square with 2 degrees-of-freedom. IgG/TCR loci are discussed elsewhere and not included in these plots. A – N = 3,286 CNVs that passed quality controls and were tested for association. Loci overlapping the MHC region are marked in blue. Loci mapping to, or in strong LD with, the <i>INS</i> VNTR region are marked in red. B – N = 3,214 CNVs passed quality controls and did not overlap or tagged the <i>INS</i> VNTR and the MHC region. C – N = 448 VNTRs targeted by the CGH array that passed quality controls. <i>INS</i> VNTR CNV regions are marked in red as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004367#pgen-1004367-g003" target="_blank">Figure 3A</a>.</p

Top ten T1D associated CNVs after removing known loci and technical artifacts.

<p>The last column lists the genes for which at least one exon overlaps the defined CNV region. P-value refers to the FBAT association test for autosomal CNVs, and to the FBAT-X association test otherwise.</p