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

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>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

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

Clinical characteristics of the discovery and replication cohorts with mismatch-repair proficient colorectal cancer.

<p>Clinical characteristics of the discovery and replication cohorts with mismatch-repair proficient colorectal cancer.</p

The <i>HMMR</i> locus and breast cancer risk in <i>BRCA1</i> mutation carriers.

<p>(<b>A</b>) Forest plots showing rs299290 HRs and 95% CIs (retrospective likelihood trend estimation) for participating countries (relatively small sample sets are not shown) ordered by sample size. Left and right panels show results for <i>BRCA1</i> and <i>BRCA2</i> mutation carriers, respectively. The sizes of the rectangles are proportional to the corresponding country/study precision. (<b>B</b>) The rs299290-containing region, including the genes, variation and regulatory evidence mentioned in HMECs. Exons are marked by black-filled rectangles and the direction of transcription is marked by arrows in the genomic structure. The chromosome 5 positions (base pairs (bp)) and linkage disequilibrium structure from Caucasian HapMap individuals are also shown.</p

Gene expression interactions in breast cancer survival.

<p>(<b>A</b>) Kaplan–Meier survival curves based on categorization of <i>HMMR</i> (probe NM_012484) and <i>AURKA</i> (NM_003600) expression in tertiles (low, medium or high expression). For simplicity, only the tertiles for “high” <i>AURKA</i> are shown. The tumours with high expression levels for both genes were not those with the poorest prognosis. (<b>B</b>) Kaplan–Meier survival curves based on categorization of <i>HMMR</i> (NM_012484) and <i>TUBG1</i> (NM_016437) expression in tertiles (low, medium or high expression). For simplicity, only the tertiles for “high” <i>HMMR</i> are shown. The cases with high expression levels for both genes were those with the poorest prognosis.</p

Potential GxG associated with breast cancer risk in <i>BRCA1/2</i> mutation carriers.

<p>*Each estimate is derived from the interaction term of a Cox regression model.</p><p>Potential GxG associated with breast cancer risk in <i>BRCA1/2</i> mutation carriers.</p

Mapping of the 17q21 locus.

<p><i>Top 3 panels:</i> P-values of association (−log₁₀ scale) with ovarian cancer risk for genotyped and imputed SNPs (1000 Genomes Project CEU), by chromosome position (b.37) at the 17q21 region, for <i>BRCA1</i>, <i>BRCA2</i> mutation carriers and combined. Results based on the kinship-adjusted score test statistic (1 d.f.). <i>Fourth panel:</i> Genes in the region spanning (43.4–44.9 Mb, b.37) and the location of the most significant genotyped SNPs (in red font) and imputed SNPs (in black font). <i>Bottom panel:</i> Pairwise r² values for genotyped SNPs on iCOG array in the 17q21 region covering positions (43.4–44.9 Mb, b.37).</p

Study design for selection of the SNPs and genotyping of <i>BRCA1</i> samples.

<p>GWAS data from 2,727 <i>BRCA1</i> mutation carriers were analysed for associations with breast and ovarian cancer risk and 32,557 SNPs were selected for inclusion on the iCOGS array. A total of 11,705 <i>BRCA1</i> samples (after quality control (QC) checks) were genotyped on the 31,812 <i>BRCA1</i>-GWAS SNPs from the iCOGS array that passed QC. Of these samples, 2,387 had been genotyped at the SNP selection stage and are referred to as “stage 1” samples, whereas 9,318 samples were unique to the iCOGS study (“Stage 2” samples). Next, 17 SNPs that exhibited the most significant associations with breast and ovarian cancer were selected for genotyping in a third stage involving an additional 2,646 <i>BRCA1</i> samples (after QC).</p