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Tetranucleotide Microsatellite Mutational Behavior Assessed in Real Time: Implications for Future Microsatellite
Maide Ö Raeker1, Jovan Pierre-Charles1, John M Carethers2
1Division of Gastroenterology and Hepatology, University of Michigan, Ann Arbor, Michigan; Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan.
Cellular and Molecular Gastroenterology and Hepatology
|January 27, 2020
Summary
Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in colorectal cancer are linked to poor outcomes. This study models tetranucleotide frameshifts, revealing a deletion bias and informing diagnostic marker development.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Fifty percent of colorectal cancers exhibit elevated microsatellite alterations at selected tetranucleotide repeats (EMAST).
- EMAST is associated with inflammation, metastasis, and poor patient prognosis.
- EMAST arises from interleukin 6-induced DNA mismatch repair protein displacement, affecting microsatellite stability.
Purpose of the Study:
- To develop cell models for real-time assessment of tetranucleotide frameshifts.
- To investigate the mutational behavior of tetranucleotide repeats in DNA mismatch repair-deficient cells.
Main Methods:
- Constructed plasmids with native and altered-length human D9S242 tetranucleotide repeats.
- Utilized enhanced green fluorescent protein (eGFP) reporter system for frameshift detection.
- Employed flow cytometry to quantify mutation rates and behavior in transfected cells.
Main Results:
- Frameshift mutation rates ranged from 31.6 to 71.1 × 10^-4 mutations/cell/generation, correlating with microsatellite length.
- Observed a deletion bias (77.8% deletions vs. 22.2% insertions) in tetranucleotide frameshifts.
- Identified that most frameshifts involved one AAAG repeat, consistent with slipped strand mispairing.
Conclusions:
- Tetranucleotide frameshifts exhibit a deletion bias and can involve multiple deletion events.
- Tetranucleotide markers can complement traditional microsatellite instability panels for EMAST detection.
- Accurate EMAST assessment requires multiple markers to account for complex mutational behavior.
