Aims
Fifty percent of colorectal cancers show elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and are associated with inflammation, metastasis, and poor patient outcome. EMAST
Background & aims
Fifty percent of colorectal cancers show elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and are associated with inflammation, metastasis, and poor patient outcome. EMAST
Conclusions
Tetranucleotide frameshifts show a deletion bias and undergo more than 1 deletion event via intermediates, with insertions converted into deletions. Tetranucleotide markers added to traditional microsatellite instability panels will be able to determine both EMAST and classic microsatellite instability, but needs to be assessed by multiple markers to account for mutational behavior and intermediates.
Methods
We constructed plasmids containing native (AAAG)18 and altered-length ([AAAG]15 and [AAAG]12) human D9S242 locus that placed enhanced green fluorescent protein +1 bp/-1 bp out-of-frame for protein translation and stably transfected into DNA mismatch repair-deficient cells for clonal selection. We used flow cytometry to detect enhanced green fluorescent protein-positive cells to measure mutational behavior.
Results
Frameshift mutation rates were 31.6 to 71.1 × 10-4 mutations/cell/generation and correlated with microsatellite length (r2 = 0.986, P = .0375). Longer repeats showed modestly higher deletion over insertion rates, with both equivalent for shorter repeats. Accumulation of more deletion frameshifts contributed to a distinct mutational bias for each length (overall: 77.8% deletions vs 22.2% insertions), likely owing to continual deletional mutation of insertions. Approximately 78.9% of observed frameshifts were 1 AAAG repeat, 16.1% were 2 repeats, and 5.1% were 3 or more repeats, consistent with a slipped strand mispairing mutation model. Conclusions: Tetranucleotide frameshifts show a deletion bias and undergo more than 1 deletion event via intermediates, with insertions converted into deletions. Tetranucleotide markers added to traditional microsatellite instability panels will be able to determine both EMAST and classic microsatellite instability, but needs to be assessed by multiple markers to account for mutational behavior and intermediates.