Use of mononucleotide repeat markers for detection of microsatellite instability in mouse tumors

Abstract

Tumors lacking DNA mismatch repair activity (MMR) from patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or those with sporadic colorectal cancer can be identified by the presence of high levels of instability in repetitive sequences known as microsatellites (MSI). The assessment of MSI phenotype in human tumors helps to establish a clinical diagnosis and is accomplished with a reference panel of five mononucleotide repeats. By contrast, detection of MSI in mouse tumors has proven to be problematic and lack of a uniform set of markers for classification of MSI has impeded comparison of results between studies. We tested for MSI in intestinal tumors from MMR-deficient mice with four mononucleotide repeats with polyA(24-37) tracts and three new markers with extended polyA(59-67) tracts. All seven markers were sensitive to MSI in MMR-deficient tumors, but those with extended mononucleotide tracts displayed larger deletions, which were easily distinguishable from the germline alleles. With a panel of the five most sensitive and specific mononucleotide repeats, a high level of MSI was detected in 100% of MMR-deficient tumors, but not in tumors with MMR activity. This novel panel is an improvement over existing combinations of mono- and dinucleotide repeat markers and should facilitate MSI screening and standardize results from different studies.

Detection of Microsatellite Instability in Colorectal Cancer Using an Alternative Multiplex Assay of Quasi-Monomorphic Mononucleotide Markers

“…13,14,19,20 Although adequate in the great majority of circumstances, analysis of additional repeats may be needed in some cases, especially in individuals of African origin. 21,22 Five additional mononucleotide markers (PTHL3, SEC63, HPDMPK, U79260, and CAT25) were selected after in-depth review of the literature. 15,16 Two of these selected markers (SEC63 and CAT25) were identified as quasi-monomorphic and, similar to BAT26 and BAT25, highly sensitive to somatic deletions in MSI-H tumors.…”

Mouse Model of Colonic Adenoma-Carcinoma Progression Based on Somatic Apc Inactivation

“…Analysis for loss of heterozygosity of Apc alleles was done by multiplex PCR using Apc-P3 primer 5 ¶-GTTCTGTATCATG-GAAAGATAGGTGGTC-3 ¶, Apc-P4 primer 5 ¶-CACTCAAAACGCTTTTGAG-GGTTGATTC-3 ¶, Apc-P5 primer 5 ¶-GAGTACGGGGTCTCTGTCTCAGTGAA-3 ¶, targeted allele (580S), deleted allele (580D), and wild-type allele amplified as 314-bp (P3 and P4), 258-bp (P3 and P5), and 226-bp (P3 and P4) PCR products, respectively (15) Microsatellite stability analysis. Microsatellite analysis of mouse tumors was done as described using six microsatellite repeat markers previously shown to be informative in tumors from DNA mismatch repairdeficient mice (20)(21)(22): A33, GA29, D7Mit91, D17Mit123, mBat-26, and mBat-37.…”

“…To assess microsatellite instability in the CPC;Apc tumors, we studied three mononucleotide repeat and three dinucleotide repeat markers that have previously been shown to manifest instability in a subset of tissues and tumors from mice defective for mismatch repair (20)(21)(22). Of 66 CPC;Apc tumors analyzed with these six markers, one tumor showed variant alleles at one mononucleotide repeat marker (data not shown).…”

Microsatellite instability in radiation-induced murine tumours; influence of tumour type and radiation quality

“…SP-PCR analysis was then performed on X-ray-induced AML samples using mBat-59. The PCR protocol used was as reported by Bacher et al (2005). Separation and detection of the amplified fragments was performed using an ABI PRISM 3100 Genetic Analyser (Applied Biosystems, Foster City, CA, USA) ( Figure 2).…”