<i>CDKN2A</i> and <i>IKZF1</i> SNP allele proportions in tumor DNA relative to genomic control copy number.

<p>Stacked histograms showing tumor DNA copy number of (A) <i>CDKN2A</i> and (B) <i>IKZF1</i> SNPs relative to a genomic control locus (<i>SLC24A3</i>). Black and grey bars represent the proportions of normalized SNP copy number accounted for by the risk and protective alleles respectively. White bars represent the difference between <i>CDKN2A</i>/<i>IKZF1</i> SNP copy number and the genomic control gene copy number. SMART-ddPCR was used to measure copy number of SNP risk/protective alleles, as well as the genomic control locus, in 35 leukemia bone marrow (tumor) DNA samples for <i>CDKN2A</i> (SNP rs3731249) and 75 tumor DNA samples for <i>IKZF1</i> (SNP rs4132601). Samples are grouped into those with allelic imbalance (AI) and those without AI, and arranged in order of normalized gene copy number relative to the genomic control.</p

Risk allele proportions at genomic loci with somatic gain (<i>i</i>.<i>e</i>. hyperdiploid chromosomes).

<p>Allelic copy number was measured in constitutional DNA and leukemia bone marrow (tumor) DNA from HeH ALL patients heterozygous for ALL-associated SNPs on chromosomes frequently gained in HeH ALL: <i>CEBPE</i> SNP rs2239633 (A), <i>ARID5B</i> SNP rs7089424 (B), <i>PIP4K2A</i> SNP rs10764338 (C), and <i>GATA3</i> SNP rs3824662 (D). Risk allele proportions are displayed as a fraction of the total allelic copy number measured in each patient using ddPCR. Each subject was assayed in duplicate, and error bars represent the standard error of the mean (some error bars not visible due to their range falling within boundaries of the data point). Upper/lower thresholds of allelic imbalance (AI) were +/- 3 SDs from the mean allelic proportion from repeat measurements in constitutional DNA samples (white squares). For rs2239633, 19 tumor samples showed AI favoring the risk allele versus 13 patients with AI favoring the protective allele (P = 0.19). For rs7089424, 20 tumor samples showed AI favoring the risk allele versus 15 patients with AI favoring the protective allele (P = 0.25). For rs10764338, 4 tumor samples showed AI favoring the risk allele versus 5 patients with AI favoring the protective allele (P = 0.50). For rs3824662, 10 tumor samples showed AI favoring the risk allele versus 9 patients with AI favoring the protective allele (P = 0.50). Data points clustering at ~0.66 and ~0.33 represent a 3:2 or 2:3 risk:protective allele proportion due to chromosomal copy number shifting from diploid (n = 2) to triploid (n = 3). Data points at ~0.75 represents a 3:1 risk:protective allele proportion due to a diploid to tetraploid (n = 4) shift in chromosome ploidy. Data points at 1 and 0 likely represent HeH ALL that has arisen via near-haploidy, leading to chromosomal LOH (Paulsson <i>et al</i>. 2005) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143343#pone.0143343.ref031" target="_blank">31</a>].</p

Candidates for tumor PAI: recurrent SCNA loci from TCGA that overlap cancer-associated SNPs (NHGRI GWAS Catalog) identified in matching tumor types.

<p>SNP associations and tumor types highlighted in bold indicate those whereby cancer type of SNP associations matches tumor type in which recurrent SCNAs were identified.</p><p>* Chromosomal locations based on human genome build hg19.</p><p>** Cancer type of SNP association loci that overlap SCNA regions (SNPs retrieved from January 2015 version of NHGRI GWAS catalog).</p><p>‡ Tumor type in which recurrent SCNAs were detected in TCGA.</p><p>ALL = acute lymphoblastic leukemia; BLCA = bladder; BRCA = breast; CLL = chronic lymphoblastic leukemia; CRC = colorectal; GBM = glioblastoma multiforme; HNSC = head and neck squamous cell carcinoma; KIRC = kidney renal cell carcinoma; LAML = acute myeloid leukemia; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; OV = serous ovarian carcinoma; UCEC = endometrial (uterine).</p><p>Candidates for tumor PAI: recurrent SCNA loci from TCGA that overlap cancer-associated SNPs (NHGRI GWAS Catalog) identified in matching tumor types.</p

Risk allele proportions at genomic loci with somatic loss.

<p>Allelic copy number was measured in constitutional DNA and leukemia bone marrow (tumor) DNA from ALL patients heterozygous for <i>CDKN2A</i> tagging SNP rs3731217 (A), and <i>IKZF1</i> SNP rs4132601 (B). Risk allele proportions are displayed as a fraction of the total allelic copy number measured in each patient using ddPCR. Each subject was assayed in duplicate, and error bars represent the standard error of the mean (some error bars not visible due to their range falling within boundaries of the data point). Upper/lower thresholds of allelic imbalance (AI) were +/- 3 SDs from the mean allelic proportion from repeat measurements in constitutional DNA samples (white squares). For rs3731217, 11 tumor samples showed AI favoring the risk allele versus 6 patients with AI favoring the protective allele (P = 0.17). For rs4132601, 17 tumor samples showed AI favoring the risk allele versus 12 patients with AI favoring the protective allele (P = 0.23).</p

Comparison between deletion gene copy number measurements made by SMART-ddPCR and MLPA.

<p>Copy number measurements were available from ddPCR and MLPA assays for SNPs at the two deletion genes <i>CDKN2A</i> (SNP rs3731249) and <i>IKZF1</i> (SNP rs4132601) in 27 and 75 tumor DNA samples respectively. (A) High correlation (R2 = 0.91) between the combined deletion gene copy number measurements made by ddPCR and MLPA. (B) Bland-Altman plot displaying the difference between measurements made in the same individual against their mean, as measured by two different methodologies (<i>i</i>.<i>e</i>. ddPCR and MLPA). There is very close agreement between the copy number measurements made by the two assays, as demonstrated by the narrow limits of agreement (-0.170 to 0.138) either side of the observed average agreement (-0.016).</p

Summary of the childhood ALL-associated SNPs investigated and the corresponding tumor DNA allelic imbalance results.

<p>* Number of heterozygous samples (for each SNP) with available bone marrow (i.e. tumor) DNA.</p><p>‡ % of HeH ALL samples with gains of that chromosome, based on data from Paulsson <i>et al</i>. (2010) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143343#pone.0143343.ref021" target="_blank">21</a>] and Dastugue <i>et al</i>. (2013) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143343#pone.0143343.ref022" target="_blank">22</a>].</p><p>† High hyperdiploid samples only.</p><p>Significant p-values highlighted in bold.</p><p>Summary of the childhood ALL-associated SNPs investigated and the corresponding tumor DNA allelic imbalance results.</p

GSV analysis of candidate obesity loci.

<p>SNP genotyping data from 3 separate cohorts were analysed using the cnvHap algorithm, and GSVs were identified that corresponded to the GSVs under investigation. For GSVs identified in at least one individual, association with obesity status (excluding overweight individuals from the analysis) was tested according to Fisher’s exact test. For GSVs identified in at least 3 members of the NFBC1966 cohort, association with log₁₀BMI as a quantitative trait was tested by 2-way ANOVA with gender as the second covariate. Italics denote a direction of effect opposite to that in the original report.</p>a<p>refers to the Illumina Human CNV370 array.</p>b<p>re-calculated after excluding individuals with deletions of the neighbouring 16p11.2 obesity-associated region (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058048#pone.0058048.s006" target="_blank">Text S1</a>).</p

Procedure for identification of GSVs.

<p>Following data export and QC, GSV calling was carried out using the cnvHap algorithm. Illustrative data for 3 GSV loci (shaded) show all positive GSV calls (black) together with examples of calls not meeting the necessary criteria (grey); probes at which copy number changes were identified are also indicated (circles).</p

Reduced BMI in carriers of deletions in the <i>FOXP2</i> region.

<p>Deletions within <i>FOXP2</i> are shown relative to selected tracks from the UCSC browser (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>) for the corresponding region of chromosome 7: <i>FOXP2</i> coding transcripts (UCSC Genes); histone modifications H3K4Me1, H3K4Me3, H3K27Ac (ENCODE Regulation); and binding by transcription factor NF-κB (ENCODE TFBS). Multiple additional transcription factors bind at the apparent NF-κB binding site. The minimum extent of each predicted deletion, the probes at which copy number changes were identified and the BMI for carriers of each deletion are as shown. Grey shading indicates the region previously associated with BMI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058048#pone.0058048-Glessner1" target="_blank">[16]</a>.</p