Comprehensive genome- and transcriptome-wide analyses of mutations associated with microsatellite instability in Korean gastric cancers

Abstract

Microsatellite instability (MSI) is a critical mechanism that drives genetic aberrations in cancer. To identify the entire MS mutation, we performed the first comprehensive genome- and transcriptome-wide analyses of mutations associated with MSI in Korean gastric cancer cell lines and primary tissues. We identified 18,377 MS mutations of five or more repeat nucleotides in coding sequences and untranslated regions of genes, and discovered 139 individual genes whose expression was down-regulated in association with UTR MS mutation. In addition, we found that 90.5% of MS mutations with deletions in gene regions occurred in UTRs. This analysis emphasizes the genetic diversity of MSI-H gastric tumors and provides clues to the mechanistic basis of instability in microsatellite unstable gastric cancers.

Figure 1.

The landscape of MS mutations in human gastric cancer. (A) Graphical representation of six gastric cancer genomes in a Circos plot (Krzywinski et al. 2009). Remarkable differences in the number of MS mutations were identified in three MSI-H cell lines, with mapping depth and gene density displaying similar patterns. (B) Venn diagram depicting the dispersion of MS mutations in CDS and UTRs of genes between three MSI-H and sum of three MSS gastric cancer cell lines from whole-genome sequencing. The overlapping regions indicate the number of length alterations in gene regions that the cell lines have in common. The number of novel genes is displayed in parentheses. (C) Comparisons between mutated repeat tracts (mutated MS) and all existed repeat tracts (all MS) in gene regions in which both contain five or more mononucleotides in length (A/T and G/C) are displayed as ratios. (D) Length distribution of mononucleotide repeat instabilities in gene regions found in one, two, or all three MSI-H cell lines.

Figure 2.

Refinement of repeat sequence tract instabilities within gene regions in gastric cancer. (A–C) Total deletion counts in gene regions from whole-genome sequencing of six gastric cancer cell lines (A), RNA sequencing of 18 gastric cancer cell lines (B), and RNA sequencing of 16 pairs of gastric cancer and normal matched control tissues (C). (D–F) Deletion frequency of MSIs that contain mononucleotide repeats in human gastric cancer, identified by whole-genome sequencing (D) and RNA sequencing (E,F). Red bars indicate MSI-H cell lines (E) or tissues (F).

Figure 3.

Analysis of mononucleotide MS mutations associated-genes in gastric cancer. (A–C) Deletions of specific genes carrying mononucleotide MSs were analyzed by whole-genome sequencing (A), and RNA sequencing in gastric cancer cell lines (B) and primary tissues (C).

Figure 4.

mRNA expression is dysregulated by the deletion of MSs in UTRs. (A) The number of mononucleotide repeat tracts in genes that correlate with down-regulated mRNA expression were identified by comparing the results of whole-genome sequencing with those of RNA sequencing in three MSI-H cell lines. (B) mRNA transcript levels of MGLL with MSIs within their 3′ UTR in MSI-H cell lines were measured by Q-PCR. The levels of mRNA expression quantified by RNA sequencing are shown. (C) A schematic of the luciferase gene construct is displayed above. Significant deletion of polyA in the 3′ UTR causing decreased mRNA stability was confirmed via a reporter assay in MKN-1 cell line.