Rapid identification of somatic mutations in colorectal and breast cancer tissues using mismatch repair detection (MRD).

Mismatch repair detection (MRD) was used to screen 93 matched tumor-normal sample pairs and 22 cell lines for somatic mutations in 30 cancer relevant genes. Using a starting amount of only 150 ng of genomic DNA, we screened 102 kb of sequence for somatic mutations in colon and breast cancer. A total of 152 somatic mutations were discovered, encompassing previously reported mutations, such as BRAF V600E and KRAS G12S, G12V, and G13D, as well as novel mutations, including some in genes in which somatic mutations have not previously been reported, such as MAP2K1 and MAP2K2. The distribution of mutations ranged widely within and across tumor types. The functional significance of many of these mutations is not understood, with patterns of selection only evident in KRAS and BRAF in colon cancer. These results present a novel approach to high-throughput mutation screening using small amounts of starting material and reveal a mutation spectrum across 30 genes in a large cohort of breast and colorectal cancers.

Multiplexed variation scanning for 1,000 amplicons in hundreds of patients using mismatch repair detection (MRD) on tag arrays.

Identification of the genetic basis of common disease may require comprehensive sequence analysis of coding regions and regulatory elements in patients and controls to find genetic effects caused by rare or heterogeneous mutations. In this study, we demonstrate how mismatch repair detection on tag arrays can be applied in a case-control study. Mismatch repair detection allows >1,000 amplicons to be screened for variations in a single laboratory reaction. Variation scanning in 939 amplicons, mostly in coding regions within a linkage peak, was done for 372 patients and 404 controls. In total, >180 Mb of DNA was scanned. Several variants more prevalent in patients than in controls were identified. This study demonstrates an approach to the discovery of susceptibility genes for common disease: large-scale direct sequence comparison between patients and controls. We believe this approach can be scaled up to allow sequence comparison in the whole-genome coding regions among large sets of cases and controls at a reasonable cost in the near future.

SNP discovery in pooled samples with mismatch repair detection.

A targeted discovery effort is required to identify low frequency single nucleotide polymorphisms (SNPs) in human coding and regulatory regions. We here describe combining mismatch repair detection (MRD) with dideoxy terminator sequencing to detect SNPs in pooled DNA samples. MRD enriches for variant alleles in the pooled sample, and sequencing determines the nature of the variants. By using a genomic DNA pool as a template, approximately 100 fragments were amplified and subsequently combined and subjected en masse to the MRD procedure. The variant-enriched pool from this one MRD reaction is enriched for the population variants of all the tested fragments. Each fragment was amplified from the variant-enriched pool and sequenced, allowing the discovery of alleles with frequencies as low as 1% in the initial population. Our results support that MRD-based SNP discovery can be used for large-scale discovery of SNPs at low frequencies in a population.