Identification of Germline Mutations in Hereditary Nonpolyposis Colorectal Cancer Using Base Excision Sequence Scanning Analysis

Abstract

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited disease caused by loss of function of DNA mismatch repair genes. Defects in MLH1 and MSH2 account for ∼98% of the mutations in HNPCC families (1). Identification of gene carriers within these families is of great importance because surveillance may be restricted to genetically affected relatives. Identification of mutations by direct sequencing is time-consuming and not feasible in a large-scale clinical setting. Molecular screening strategies, including single-strand conformation polymorphism analysis (2), denaturing gradient-gel electrophoresis (3), constant denaturant gel electrophoresis (4), or in vitro transcription/translation assays (5), have been described and may facilitate the detection of mutations. However, these techniques often have low sensitivity with mutation detection rates of only 35–70% (6), or they are highly accurate but are technically difficult to perform (3)(4)(5). In the present study, we developed and evaluated a modified base excision sequence scanning (BESS) protocol (7) for the detection of MLH1 and MSH2 germline mutations. This simple method is based on the incorporation of dUTP into the PCR products. Subsequent digestion with uracil N -glycosylase, which releases uracil from both single-stranded and double-stranded DNA and thus creates apyrimidinic sites, and endonuclease IV, which cleaves the phosphodiester bond at these sites, generates a defined series of fragments (7)(8). Lymphocytes were prepared from whole blood of patients with HNPCC and healthy subjects using Vacutainer cell preparation tubes (Becton Dickinson). After extraction of total RNA (Tri-Star-Kit; AGS), complementary DNA synthesis was performed with reverse transcriptase (Superscript; Life Technologies) and random hexamer oligonucleotides or 2.5 μmol/L reverse primers (Table 1⇓ ). The PCR amplification was carried out in a Perkin-Elmer 9700 PCR system in a total volume of 50 μL containing 5 U of AmpliTaq Gold polymerase (Perkin-Elmer); 60 mmol/L Tris-HCl, pH …