Overview of the somatic mutation dataset.

<p>Overview of the somatic mutation dataset.</p

Mutation accumulation and TFBS motif disruption in cancer compared to control sites and polymorphism data.

<p>The X-axis shows the ratio of the number of substitutions in functional, relative to control sites in cancer, divided by the corresponding numbers for 1KG polymorphisms, i.e. (cancer_functional/cancer_control)/ (1KG_functional/1KG_control). Values > 1 indicate an excess of mutations in functional binding sites in cancer, correcting for the amount of variability that is tolerated at these sites at the population level. The Y-axis shows the corresponding ratio for the reduction in PWM-score. Values < 1 indicate that the matrix score is reduced to a greater extent in functional, relative to control sites in cancer compared to 1KG polymorphisms. The color and shape of the data points indicate the significance of their departure from random expectation. Note that some motifs were excluded from this plot because the <i>p</i>-value for the difference in PWM-score reduction could not be calculated (full list in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006207#pgen.1006207.s019" target="_blank">S1 Dataset</a>).</p

CTCF-binding sites are often mutated when acting as modulators of chromatin structure.

<p>CTCF-motifs are highly enriched inside loop anchor points and across domain boundaries (A, C). The number of substitutions in CTCF-motifs is increased for motifs that are located in loop anchor points (B); for domain boundaries, no significant increase in substitution rate was observed (D).</p

Somatic mutation and polymorphism patterns within TF binding sites.

<p>Substitution counts across all binding sites for each of three motifs, selected from the full list of 118 motifs (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006207#pgen.1006207.s008" target="_blank">S8 Fig</a> for similar plots for all motifs tested). For comparison, substitution counts at control sites and 1KG high frequency polymorphism counts are shown in the panels below. (A) Substitution counts for CTCF: MA0139.1. (B) Substitution counts for USF1: MA0093.2. (C) Substitution counts for ZBTB33: MA0527.1.</p

Functional binding sites are enriched for somatic mutations that are deleterious to motif binding potential.

<p>(A) In the 1KG dataset, high frequency polymorphisms (>5% minor allele frequency) are depleted at functional binding sites compared to control sites (Wilcoxon test; <i>p</i>-value = 0.003), whereas the opposite trend is observed for somatic mutations (Wilcoxon test; <i>p</i>-value = 6.522e-09). (B) There are more somatic substitutions in functional, relative to control sites in cancer compared to 1KG polymorphisms (Wilcoxon test; <i>p</i>-value = 3.652e-10). (C) The relative change of the PWM-score is lower at functional sites compared to control sites in 1KG (Wilcoxon test; <i>p</i>-value = 8.442e-05), whereas the PWM-score introduced by somatic mutations is indistinguishable between functional and control sites. Each of the 118 binding motifs contributes one data point to the plots in this Figure.</p

Sizing the repeat in all available family members with gene specific repeat primed PCR.

<p>Alleles were sized by gene specific repeat primed PCR. As expansions containing over 300 repeated units cannot be reliably sized using this technique, we indicated these alleles as “>300”. Subject A.II.4 is marked with an asterisk as she is mosaic for a 134-bp deletion taking away the entire CGG repeat in combination with a largely expanded allele, and this in addition to a normal sized allele.</p

Southern blot analysis of the <i>AFF3</i> CGG repeat in all available members of the three families and two unrelated control individuals (C).

<p>DNA restriction fragments obtained from blood samples of all available members of family A (lanes 1–4), B (lanes 5–6) and C (lanes 7–9) where blotted with a specific 32P-labeled probe (chr2:100088460–100089451; hg19) after digestion with <i>Hind</i>III. Lanes 10 and 11 contain DNA restriction fragments from two unrelated control samples (represented as diamonds) after digestion with <i>Hind</i>III. In addition to a normal size allele fragment of 4.4 kb, individuals CII.1, CI.2, BII.1, BI.2, AII.4, AII.3 and AIII.1 show additional fragments of a larger size, indicating repeat expansion. These expanded fragments were not present in the controls and individuals CI.1 and AI.1. A 1 kb length marker is presented at the left of the figure.</p

FISH analysis of the FRA2A fragile site.

<p><b>A.</b> The BAC-clone 549H5 (labelled green) spans the fragile site FRA2A. <b>B.</b> FISH analysis using the 10 kb L10K (labelled green; chr2: 100721983–100733233; hg19) and 18 kb L18K (labelled red; chr2: 100700447–100718834; hg19) PCR-generated probes, targeted to map either side of FRA2A. The additional telomeric FISH signal on the red channel (red arrow chr2: 110520380–110538822 and chr2:111347822–111366260; hg19) is the result of a 24 kb low copy repeat (24 kb LCR pink text) encompassing L18K. <b>C.</b> A schematic representation of the position of the LCR.</p

Allele frequencies of the FRA2A associated CGG repeat in a population of 100 control individuals.

<p>PCR amplification of the repeat and subsequent sequencing in 200 control chromosomes revealed that it is highly polymorphic with a length ranging from 3 to 20 copies. The most commonly found allele contains eight repeated units.</p

Clinical findings in FRA2A carriers.

<p>Clinical findings in FRA2A carriers.</p