Additional file 1: of Mutational Signatures in Cancer (MuSiCa): a web application to implement mutational signatures analysis in cancer samples

Figure S1. Somatic mutational prevalence in MuSiCa web app. Figure S2. Mutational profile representation in MuSiCa web app. Figure S3. Reconstruction of mutational profile in MuSiCa web app. Figure S4. Comparison with cancer signatures in MuSiCa web app. Figure S5. Principal component analysis in MuSiCa web app. (PDF 3039 kb

DataSheet1_Unraveling the impact of a germline heterozygous POLD1 frameshift variant in serrated polyposis syndrome.pdf

Serrated polyposis syndrome (SPS) is one of the most frequent polyposis syndromes characterized by an increased risk for developing colorectal cancer (CRC). Although SPS etiology has been mainly associated with environmental factors, germline predisposition to SPS could also be relevant for cases with familial aggregation or a family history of SPS/CRC. After whole-exome sequencing of 39 SPS patients from 16 families, we identified a heterozygous germline frameshift variant in the POLD1 gene (c.1941delG, p.(Lys648fs*46)) in a patient with SPS and CRC. Tumor presented an ultra-hypermutated phenotype and microsatellite instability. The POLD1 germline variant segregated in three additional SPS-affected family members. We attempted to create yeast and cellular models for this variant but were no viable. Alternatively, we generated patient-derived organoids (PDOs) from healthy rectal tissue of the index case, as well as from a control donor. Then, we challenged PDOs with a DNA-damaging agent to induce replication stress. No significant differences were observed in the DNA damage response between control and POLD1-Lys648fs PDOs, nor specific mutational signatures were observed. Our results do not support the pathogenicity of the analyzed POLD1 frameshift variant. One possible explanation is that haplosufficiency of the wild-type allele may be compensating for the absence of expression of the frameshift allele. Overall, future work is required to elucidate if functional consequences could be derived from POLD1 alterations different from missense variants in their proofreading domain. To our knowledge, our study presents the first organoid model for germline POLD1 variants and establishes the basis for its use as a model for disease in SPS, CRC and other malignancies.</p

Functional annotation clustering of differentially methylated genes found in multiple versus solitary tumors based on DAVID analysis.

<p>Functional annotation clustering of differentially methylated genes found in multiple versus solitary tumors based on DAVID analysis.</p

Classification of solitary and multiple tumors according to the CIMP and <i>KRAS</i> status.

<p>“(m)” indicate multiple tumors; “(s)” indicate solitary tumors; “M” indicates a β value of ≥0.1 (methylated); “U” indicates a β value of ≤0.1 (unmethylated); First panel classifies a tumor as CIMP-H or CIMP-L vs. CIMP-0; Second panel classifies a tumor as CIMP-H vs. CIMP-L/0; NA: not available.</p

Distributions of predicted risks in cases and controls.

<p>The median of risk score was 0.60 (95% CI 0.59–0.61) for cases and 0.43 (95% CI 0.42–0.45) for controls. Risk score was significantly higher in advanced adenomas and/or multiplicity.</p

Case-control association results obtained by logistic regression analyses adjusted for age and gender.

<p>Association results for cases (1,326) vs polyp-free controls (1,266). Results are based on the reported allele from previous CRC GWAS (reference number is shown). Statistically significant associations are denoted in bold (<i>P</i>-value<0.05 and multiple-comparison corrected <i>Q</i>-value<0.1).</p

Summary of the demographic and clinical characteristics of individuals included in the study.

<p>Summary of the demographic and clinical characteristics of individuals included in the study.</p

Overlap between significantly hypermethylated CpG sites in multiple and CIMP-H tumors.

<p>Blue circle shows 102 hypermethylated CpG sites found in multiple versus solitary tumors and yellow circle shows the 301 hypermethylated CpG sites in CIMP-H versus CIMP-L/0 tumors. Remarkably, 76 out of the 102 hypermethylated genes in multiple tumors were also seen to be hypermethylated in CIMP-H tumors, and are represented as an intersection.</p

Heatmap showing the 90 most significantly hypermethylated CpG sites that differentiate multiple CRCs (n = 12) with respect to solitary tumors (n = 29) based on the Infinium DNA methylation data.

<p>The DNA methylation β-values are represented by using a color scale from red (high DNA methylation) to green (low DNA methylation). Rows represent probes and columns represent tumor samples. Clinical and molecular features (group, gender, tumor location, CIMP-H and <i>KRAS</i> mutational status) are represented above the heatmap with horizontal bars.</p

Cumulative impact of the 14 selected variants on adenoma risk.

<p>Distribution of risk alleles for cases (black bars) and controls (grey bars). Upper panel and table: Plot of the ORs for cases with increasing number of risk alleles. ORs are relative to the median number of risk alleles in controls (13 risk alleles as reference group). Vertical bars correspond to 95% CI. Statistical significance is shown in the table for the different groups of multiple risk alleles.</p