Sanger sequencing of MMR genes in a one-plate system

Abstract

Both incidence and mortality of colorectal cancer (CRC) in Romania have shown a continuous increase during the last decades. Hereditary Non-Polyposic Colorectal Cancer (HNPCC), also known as Lynch syndrome, is mainly attributable to mismatch repair (MMR) genes MSH2, MSH6, and MLH1. Individuals carrying germ-line mutations of these genes present high lifetime risk of colorectal and other cancers, compared to non-carriers. Oncogenetics is developed worldwide nowadays, for identifying hereditary predisposition to cancer and offering appropriate clinical follow-up to patients and mutation carriers in Lynch families. Molecular oncogenetic diagnosis in Lynch syndrome is based on complete Sanger sequencing of entire MMR genes, which is time and resources consuming, therefore needing an appropriate and adapted optimization. Conventional sequencing requires a sufficient number of available samples to be processed simultaneously, which increases the waiting time for diagnostic results. Complete analysis for only one patient meets difficult technical problems due to the complex co-amplification of all gene regions of interest within the same conditions, therefore increasing the costs and reducing the cost-effectiveness of the test. Here we present an original and robust technical protocol for sequencing the entire MSH2, MSH6, and MLH1 coding sequence for one patient in a single PCR plate. Our optimized and verified system overcomes all technical problems and offers a quick, robust, and cost-effective possibility to personalize molecular oncogenetic diagnosis in Lynch syndrome.

Do we really need genetic tests in current clinical practice?

“…Sanger sequencing is largely used for the identification of somatic or germline mutations in cancer. In this respect, a new, robust, fast and cost-effective protocol for sequencing mismatch repair (MMR) genes was recently developed by Negura et al (17). Hereditary Nonpolyposis Colorectal Cancer (HNPCC), also known as Lynch syndrome, is mainly attributable to germline mutations in the MSH2, MSH6, and MLH1 genes.…”

“…Hereditary Nonpolyposis Colorectal Cancer (HNPCC), also known as Lynch syndrome, is mainly attributable to germline mutations in the MSH2, MSH6, and MLH1 genes. The one plate system proposed will allow to personalize molecular oncogenetic diagnosis in Lynch syndrome in Romania (17).…”

The utility of molecular genetic techniques in craniosynostosis cases associated with intellectual disability

“…In the last decades, molecular genetic techniques have been developed to confirm the genetic etiology of many genetic disorders (1,2). Nowadays, the genetic etiology of the most frequent genetic disorders has been reported, and a causative gene mutation is identified in approximately 24% of patients with craniosynostosis (1).…”

Simultaneous FLT3, NPM1 and DNMT3A mutations in adult patients with acute myeloid leukemia – case study

“…The detailed protocols used with locally adapted and optimized conditions are mentioned in Table I. In order to confirm the mutations found by RFPL-PCR (FLT3-D835 and DNMT3A), we performed sequencing as previously described [16].…”