Subject(s)
Biomarkers, Tumor/genetics , Class I Phosphatidylinositol 3-Kinases/genetics , Colorectal Neoplasms/genetics , Mutation , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/pathology , Exons , Genetic Predisposition to Disease , Humans , Microsatellite Instability , PhenotypeABSTRACT
AIMS: We sought to use PCR followed by high-resolution melting analysis to develop a single closed-tube screening panel to screen for Lynch syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter and BRAF mutation. METHODS: For MSI testing, five mononucleotide markers (BAT25, BAT26, BCAT25, MYB, EWSR1) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n=99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS) were tested. RESULTS: All the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. The MLH1 promoter and BRAF were tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation while 41/44 cases with MSI showed full methylation. The three cases with MSI and a non-methylated pattern had aberrations in MSH2 and MSH6 expression. BRAF mutation was detected in 61% of MSI cases and 11% of MSS cases.Finally, 12 cases were blind screened by using the whole panel as a single test. Of these, five were identified as MSS, four as MSI/non-LS and three as MSI/possible LS. These results were concordant with the previous data. CONCLUSION: We describe the Nottingham Lynch Syndrome Test (N_LyST). This is a quick, simple and cheap method for screening for Lynch syndrome.
Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/diagnosis , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , DNA Methylation , DNA Mutational Analysis/methods , Gene Expression Profiling/methods , Microsatellite Instability , MutL Protein Homolog 1/genetics , Mutation , Polymerase Chain Reaction , Proto-Oncogene Proteins B-raf/genetics , Genetic Predisposition to Disease , HCT116 Cells , Humans , Phenotype , Predictive Value of Tests , Promoter Regions, Genetic , Reproducibility of Results , WorkflowABSTRACT
Currently, short DNA segments of sub-100 bp can be sequenced either directly by next-generation sequencing and pyrosequencing, which are expensive, or indirectly, via Sanger sequencing combined with the cumbersome and failure-prone plasmid cloning. To circumvent these issues, we have generated a novel sequencing-purposed PCR assay using long-tailed primers (squirrel primers) to Sanger sequence directly sub-100 bp genomic amplicons. Squirrel primers, 40-65 nt in length, were used to amplify 51-93 bp long genomic sequences of KRAS exons 2 and 3, BRAF exon 15, PI3K catalytic subunit alpha exon 20, and phosphatase and tensin homolog exon 3 from colorectal cancer (CRC) cell lines and preamplified clinical CRC samples with known mutation status by PCR. Following this, a short second pair of primers that bind at the 5' region of the long tails was used for sequencing on the 3130 × l ABI Prism Genetic Analyzer. The sequencing data were analyzed via FinchTV software. High-quality sequencing data were obtained from 51 to 93 bp long genomic sequences with our novel PCR assay, with capture of all of the target sequences in all of the samples in both the forward and reverse directions and confirmation of the mutation status of the CRC samples. Whereas the sequencing quality was independent of the template type, it showed a squirrel primer tail length-dependent pattern. Our novel PCR assay for direct and targeted Sanger sequencing of short genomic segments has potential applications in focused molecular/genetic profiling of cancer in research and diagnostics fields in which fragmented DNA, such as circulating tumor DNA and archival tissue DNA, are used as starting templates.
