Novel Genes Associated with Colorectal Cancer Are Revealed by High Resolution Cytogenetic Analysis in a Patient Specific Manner

<div><p>Genomic abnormalities leading to colorectal cancer (CRC) include somatic events causing copy number aberrations (CNAs) as well as copy neutral manifestations such as loss of heterozygosity (LOH) and uniparental disomy (UPD). We studied the causal effect of these events by analyzing high resolution cytogenetic microarray data of 15 tumor-normal paired samples. We detected 144 genes affected by CNAs. A subset of 91 genes are known to be CRC related yet high GISTIC scores indicate 24 genes on chromosomes 7, 8, 18 and 20 to be strongly relevant. Combining GISTIC ranking with functional analyses and degree of loss/gain we identify three genes in regions of significant loss (ATP8B1, NARS, and ATP5A1) and eight in regions of gain (CTCFL, SPO11, ZNF217, PLEKHA8, HOXA3, GPNMB, IGF2BP3 and PCAT1) as novel in their association with CRC. Pathway and target prediction analysis of CNA affected genes and microRNAs, respectively indicates TGF-β signaling pathway to be involved in causing CRC. Finally, LOH and UPD collectively affected nine cancer related genes. Transcription factor binding sites on regions of >35% copy number loss/gain influenced 16 CRC genes. Our analysis shows patient specific CRC manifestations at the genomic level and that these different events affect individual CRC patients differently.</p></div

Counts of copy number gains and losses per subject.

<p>Counts of copy number gains and losses per subject.</p

LOH and copy neutral LOH (Uniparental Disomy) events.

<p><b>A:</b> Segmental UPD events spanning multiple microRNA regions in chromosome 4 in patient 9F. <b>B:</b> LOH event in chromosome 17 of the same sample above. LOH is associated with copy number variations. <b>C:</b> Target Prediction analysis of miRNAs exhibit the probable involvement of known signaling pathways in colorectal cancer. Y-axis represents the numerical score indicative of predictive value. <b>D:</b> TPM3 and MUC1 are affected by UPD events in patient 7F. <b>E:</b> MYC region sustain LOH events and copy gains in patient 10F.</p

Chromosomes 18, 20,7 and 8 loss and gain.

<p>Chromosomes 18, 20,7 and 8 loss and gain.</p

CNAs affecting transcription factor binding sites that may affect CRC related genes.

<p><b>A:</b> TFBS in regions where the copy number changes in excess of +/−35% are marked on chromosomes 7 (gain), 14 (loss), 20 (short arm gain (males), loss (females), long arm gain (both sexes)), 21 (loss) and X (somewhat similar to chromosome 20). The marking is by the gene names corresponding to these TFBS. <b>B:</b> Association of TFBS genes with significant functions and <b>C:</b> pathways. Cancer and TGF-β signaling were statistically the most significant associations for these affected genes.</p

Net copy number changes.

<p><b>A: Percentage of copy number gains and losses in tumor samples compared with their normal corresponding tissue for all chromosomes in fifteen study subjects.</b> Losses are marked in Red below the baseline whereas gains are represented in Blue bars above the baseline. Circled chromosome numbers (12 and 14) reflect events of loss and gain solely observed in our dataset. <b>B: Gender wise copy number changes in context of overall CNAs:</b> The slight loss in the short arm of chromosomes 4, throughout chromosome 6 and 10 are frequent in the female group.</p

Chromosome wide GISTIC results.

<p><b>A:</b> Amplitude of gains and losses reveal 144 genes of different GISTIC scores across all chromosomes (numbered accordingly). The spikes in the grayed out regions of enrichment correspond to GISTIC scores for genes in these regions. <b>B: Molecular network of genes affected by significant copy number aberrations in tumor samples.</b> These events were located on chromosomes 7, 8, 18 and 20. 11 of 24 genes show no association with the colorectal cancer function. This is in conformation with the GISTIC analysis except for the G-score of TSHZ1. Solid lines between nodes indicate direct molecular interaction between connected genes with respect to CRC. Functions are indicated by shapes: enzymes (diamonds), cytokines (squares), kinases (triangles), transcription factors (horizontal ovals), transmembrane receptors (vertical ovals), transporters (trapezoids), and other (circles).</p