<i>BTG1</i> microdeletion occurrence within cytogenetic subgroups of ALL.

<p><i>BTG1</i> deletion status was determined using MLPA.</p>a<p>Because of missing values, numbers do not always add up to 722 BCP-ALL cases. Data was available for 637 cases on hyperdiploidy; 513 cases for <i>ETV6-RUNX1</i>; 648 cases for <i>BCR-ABL1;</i> 649 cases for <i>MLL</i>-rearranged.</p>b<p>The ‘other’ subgroup encompasses cases negative for <i>ETV6-RUNX1</i> or <i>BCR-ABL1</i> translocations, <i>MLL</i>-rearrangement and/or hyperdiploidy. This group includes 10 cases with <i>E2A-PBX1</i> translocation, of which none harbor a <i>BTG1</i> deletion.</p>c<p>Subgroup unknown includes all cases in which no data is available in one or more cytogenetic classifications.</p>d<p>Fisher's exact test was used when sample groups were small.</p

Multiple <i>BTG1</i> deletion-positive clones are present in specific BCP-ALL subtypes.

<p>(A) Recurrence of multiclonal <i>BTG1</i> deletions. A sensitive PCR method was used to screen for eight different deletion breakpoints (deletion I–VIII) in <i>BTG1</i> MLPA deletion positive (+) cases (n = 65), and to screen for the three most frequent deletion breakpoints (deletion III, V and VIII) in <i>BTG1</i> MLPA deletion negative (−) cases (n = 89). (B) <i>BTG1</i> deletion frequency in the two major cytogenetic subgroups of BCP-ALL (Hyperdiploid and <i>ETV6</i>-<i>RUNX1</i>). Presence of a <i>BTG1</i> deletion in the predominant clone was determined by MLPA on the entire cohort of BCP-ALL cases (n = 722), and was compared to deletions detected as a minor clone in MLPA-negative cases (n = 89) by deletion-spanning PCR. Distributions are similar, being depleted from hyperdiploid cases and enriched in <i>ETV6</i>-<i>RUNX1</i>-positive cases as compared to the total group.</p

Expression of <i>BTG1</i> truncated read-through transcripts in BCP-ALL cells with <i>BTG1</i> deletions.

<p>(A) Schematic representation of the wild-type human <i>BTG1</i> gene, existing of two partly coding exons, and five different <i>BTG1</i> transcripts due to <i>BTG1</i> gene deletions. Exons are represented by black (coding) or white (non-coding) bars. Indicated are the RT-PCR primers that were used to detect expression of the wild-type <i>BTG1</i> transcript (primers A and B), or one of the <i>BTG1</i> truncated read-through transcripts for deletion II (primers A and C), deletion III (pimers A and D), deletion IV (primers A and E), deletion V (primers A and F), or deletion VIII (primers A and G). (B) RT-PCR analyses on total RNA isolated from the BCP-ALL cell lines Nalm6 and RS4;11 (<i>BTG1</i> wild-type) and REH, SUP-B15 and 380, each with distinct monoallelic <i>BTG1</i> deletions. (C) RT-PCR analyses on primary BCP-ALL samples in which a single <i>BTG1</i> deletion (Pt1, Pt2, Pt3 and Pt5), multiple <i>BTG1</i> deletions (Pt4 and Pt6) or no <i>BTG1</i> deletions were detected with genomic PCR (Pt7). Type of deletions (III, V, or VIII) and outcome of MLPA (p: deletion-positive; n: deletion-negative) are indicated. <i>BTG1</i> read-through transcripts were verified by sequencing (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002533#pgen.1002533.s007" target="_blank">Table S5</a>), except for Pt3-deletion V, which was an unrelated DNA sequence. (D) Quantitative real-time RT-PCR data representing relative expression levels of <i>BTG1</i> measured 5′ (primers exon 1/2) and 3′ of the <i>BTG1</i> breakpoint hotspot (primers exon 2). Expression levels were normalized to <i>HPRT</i> levels, and compared to the expression level in Nalm6, which was set to 1. The data shown represent the average of two independent cDNA reactions and triplicate qRT-PCR reactions.</p

Increased levels of H3K4me3 at the <i>BTG1</i> locus in BCP-ALL versus T-ALL cell lines.

<p>(A) Quantitative real-time RT-PCR data representing relative expression levels of <i>BTG1</i> in T-ALL cell lines HSB2, Jurkat and KARPAS45, and BCP-ALL cell lines RS4;11, Nalm6 and CCRF-SB (<i>HPRT</i> normalized and related to HSB2 expression levels). Data shown are the average of two independent cDNA reactions and triplicate qRT-PCR reactions. (B and C) Percentage recovery after ChIP performed with H3K4me3 antibody (B) or H3K9/14Ac antibody (C) on T-ALL (HSB2, Jurkat, KARPAS45) and BCP-ALL (RS4;11, Nalm6 and CCRF-SB) cell lines. Real-time quantitative PCR was performed with primers specific for the region 1 kb upstream of the transcription start site (−1 kb prom), directly flanking the transcription start site (prox prom), the second exon near the breakpoint hotspot (exon 2) and towards the end of the 3′untranslated region (3′UTR) at the second (and last) exon of the human <i>BTG1</i> gene. Values represent two independent ChIP experiments. Student's <i>t</i>-test was performed to assess differences between the average recovery of T-ALL versus BCP-ALL samples. Asterisk (*) indicates a p-value<0.05.</p

Authentic RSSs and candidate RSSs flanking breakpoints of <i>BTG1</i> microdeletions.

<p>Mismatches from consensus are underlined;</p>a<p>RSSs flanking V(D)J gene segments;</p>b<p>Sequence shown is in reverse complement orientation;</p>c<p>Functional cryptic RSSs at proto-oncogene breakpoints <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002533#pgen.1002533-Marculescu1" target="_blank">[18]</a>.</p

<i>BTG1</i> deletions in relapsed cases.

a<p>Determined by sequence analysis of breakpoint spanning PCR product.</p>b<p>The deletion breakpoint could not be detected (N.D.) using the eight breakpoint-spanning PCR assays (I–VIII).</p>c<p>The breakpoint does not cluster within <i>BTG1</i> exon 2, but is located 2 kb downstream in the 3′UTR.</p>d<p>Homozygous deletion.</p

Variants in candidate CRC susceptibility genes identified by whole-exome sequencing and targeted re-sequencing.

<p>Variants in candidate CRC susceptibility genes identified by whole-exome sequencing and targeted re-sequencing.</p

Rare variants in <i>LRP6</i> in three cases.

<p>(A) Distribution of missense <i>LRP6</i> variants identified in the CRC discovery cohort (red dots). Somatic <i>LRP6</i> mutations identified in colorectal tumors [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref024" target="_blank">24</a>] are indicated with grey (missense) and black (protein truncating) dots. Structural domains include the β-propeller domains that are used to form the receptor complex (pink bars), and the transmembrane domain (purple). (B) 3D protein structure of the β-propeller domains of LRP6 with the positions of the identified missense variants in red. Insets show conservation of the region in which the missense variants (indicated with the red box) are located with, underneath, close ups of the local 3D protein structure with mutant (red) and wild-type (green) residues. The mutant residue at position 239 is predicted to disturb the protein structure (project HOPE; <a href="http://www.cmbi.ru.nl/hope/" target="_blank">http://www.cmbi.ru.nl/hope/</a>). The mutant residue at position 789 is much smaller than the wild-type residue and may disturb the binding of Dickkopf-1. Residue 867 is located on the surface of the protein and the mutant residues are not expected to disturb protein structure, but may influence protein binding. (C) Immunofluorescence analyses of LRP6 wild-type and mutant proteins showing similar subcellular localizations. (D) LRP6 protein expression levels normalized to β-actin are similar between wild-type and mutant LRP6. (E) TOPflash analyses of wild-type and mutant LRP6 to determine their effects on the WNT signaling pathway. Luciferase activity was normalized to control and wild-type constructs. Both p.N789S and p.T867A mutants reveal a significant increase in activation compared to the wild-type LRP6 protein. Experiments were performed three times in triplicate. **<i>P</i> <0.001; error bars represent the standard error of the mean.</p

Study design, variant filtering and candidate gene prioritization.

<p>Whole-exome sequencing was performed on germline DNA of 55 early-onset CRC cases. The exome data were first filtered for quality and frequency, followed by filtering for protein truncating and highly conserved missense variants. Next, we removed all known loss-of-function-tolerant genes from this list and searched for known and novel CRC predisposing gene variants.[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref012" target="_blank">12</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref013" target="_blank">13</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref057" target="_blank">57</a>] An additional filtering was applied to identify genes that were affected by two or more potentially pathogenic variants and to remove genes that are frequently affected by protein-truncating or highly conserved missense variants in healthy controls. The remaining set of recurrent variants was filtered for (i) genes recurrently affected by protein truncating variants; (ii) cancer driver genes in CRC [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref023" target="_blank">23</a>]; (iii) genes identified as CRC susceptibility genes in mice [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref029" target="_blank">29</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref030" target="_blank">30</a>] and involved in cancer-related KEGG pathways [hsa04310 (WNT signaling), hsa04350 (TGF-beta signaling), hsa03430 (mismatch repair), hsa03410 (base excision repair), hsa03420 (nucleotide excision repair), map03450 (non-homologous end-joining), hsa03460 (Fanconi anemia), and hsa05200 (pathways in cancer)] (iv) genes identified in CRC GWAS studies [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref005" target="_blank">5</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref007" target="_blank">7</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref032" target="_blank">32</a>]. Genes that remained after these filter steps were selected for re-sequencing in a replication cohort of 174 CRC cases. CRC: colorectal cancer; VUS: variant of unknown significance.</p

Rare variants in <i>PTPN12</i> encoding PTP-PEST in four cases.

<p>(A) Distribution of missense variants identified in the CRC discovery cohort (red dots) and CRC replication cohort (blue dots) in PTP-PEST. Somatic <i>PTPN12</i> mutations identified in colorectal tumors [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005880#pgen.1005880.ref024" target="_blank">24</a>] are indicated with black (missense) and green (protein truncating) dots. The kinase domain is shown in yellow and the proline-rich-regions are shown in orange. (B) Amino acid conservation of the three regions with missense variants (indicated with the red box) among 11 species, and the 3D protein structure of the kinase domain with the p.A105V variant. The close up shows the structural difference between the mutant (red) and wild-type (green) residue. The mutant residue at position 105 is bigger and may cause bumps during protein folding. The mutant residue at position 522 is smaller, which can result in loss of interactions. The mutant residue at position 684 is more hydrophobic than the wild-type residue, this may disturb correct protein folding.</p