High throughput detection of microsatellite instability by denaturing high-performance liquid chromatography
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Published on: September 25, 2018
Kai-feng Pan1, Wanguo Liu, You-Yong Lu
1Department of Cancer Epidemiology, Peking University School of Oncology, Beijing Institute for Cancer Research, Beijing Cancer Hospital, Beijing, China.
Abstract:
Microsatellite instability (MSI) is a hallmark of the DNA replication error phenotype, due to the inactivation of mismatch repair genes. MSI has been implicated in colon and many other gastrointestinal cancers. MSI usually can be analyzed by PCR amplification of microsatellite markers followed by electrophoresis and detected using autoradiography or fluorescence techniques. We report here a novel method for high-throughput detection of MSI using denaturing high performance liquid chromatography (DHPLC). Amplification of two mononucleotide markers (BAT25 and BAT26) by polymerase chain reaction (PCR) is followed by DHPLC analysis to display alteration in the length of repetitive sequences. These two markers were tested in 84 colorectal cancer samples confirmed to be 44 MSI-H and 40 MSI-L or MSS, previously defined by multiple microsatellite markers and/or by immunohistochemical analyses of MLH1 and MSH2 proteins. Among 44 MSI-H samples, sequence variations in BAT26 and BAT25 were detected in 44 (100%) and 43 (98%), respectively, while no sequence variation in the two markers was detected in 40 MSI-L or MSS samples. A total of 96 gastric cancers and their matched normal tissues were then analyzed for MSI-H using this method. Sequence variations in BAT26 and BAT25 were detected in nine (9.4%) samples. Seven of the nine cases were shown unstable at both BAT26 and BAT25; one each was unstable at BAT26 or BAT25. These results were confirmed by autoradiography analyses. Together, our results demonstrate high sensitivity and specificity of DHPLC in the analysis of sequence variations in BAT25 and BAT26 to determine MSI status. This simple, efficient, and high-throughput approach will facilitate analysis of MSI in large sample sets of any cancers.
Insights
A new method using denaturing high performance liquid chromatography (DHPLC) accurately detects microsatellite instability (MSI) in colorectal and gastric cancers. This high-throughput approach offers a sensitive and specific way to analyze MSI status in large patient cohorts.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Microsatellite instability (MSI) is a key indicator of DNA replication errors, often linked to mismatch repair gene inactivation.
- MSI is frequently observed in colorectal and other gastrointestinal cancers, impacting disease progression and treatment response.
- Traditional MSI detection methods, such as PCR followed by electrophoresis, can be time-consuming and less efficient for large-scale studies.
Purpose of the Study:
- To introduce and validate a novel, high-throughput method for detecting MSI using denaturing high performance liquid chromatography (DHPLC).
- To assess the sensitivity and specificity of DHPLC for analyzing microsatellite sequence variations in cancer samples.
- To evaluate the utility of DHPLC for determining MSI status in colorectal and gastric cancer.
Main Methods:
- Polymerase chain reaction (PCR) amplification of two mononucleotide microsatellite markers (BAT25 and BAT26).
- Analysis of PCR products using denaturing high performance liquid chromatography (DHPLC) to detect alterations in repetitive sequences.
- Validation of DHPLC results against established methods (multiple microsatellite markers, immunohistochemistry, autoradiography) in 84 colorectal and 96 gastric cancer samples.
Main Results:
- DHPLC accurately identified MSI in 100% (44/44) of MSI-H colorectal cancer samples using BAT26 and 98% (43/44) using BAT25.
- No sequence variations were detected in MSI-L or MSS colorectal cancer samples (40/40) using either marker.
- DHPLC detected MSI in 9.4% (9/96) of gastric cancer samples, with results confirmed by autoradiography.
Conclusions:
- DHPLC offers a highly sensitive and specific method for detecting sequence variations in BAT25 and BAT26 to determine MSI status.
- This DHPLC-based approach is efficient, simple, and suitable for high-throughput analysis of MSI in extensive cancer sample sets.
- The method facilitates broader application of MSI testing in various cancer types, aiding in diagnosis and research.
