Analysis of the hereditary pancreatitis-associated cationic trypsinogen gene mutations in exons 2 and 3 by enzymatic mutation detection from a single 2.2-kb polymerase chain reaction product

Background: Hereditary pancreatitis is associated with at least 2 mutations in the cationic trypsinogen gene. The purpose of the present study is to test the utility of T4 endonuclease VII for the detection of cationic trypsinogen R117H mutations. In addition, the possibility of screening for R117H, N21I, and A8V mutations in a single 2.2-kb polymerase chain reaction (PCR) product usingT4 endonuclease VII was investigated. Methods: Twenty-nine DNAs from control patients and patients with known cationic trypsinogen R117H, A8V, or N21I mutations were selected from the ongoing hereditary pancreatitis study of the Midwest Multicenter Pancreatic Study Group. The samples were coded and randomized, and a 911-bp sequence containing exon 3 or a 2,212-bp sequence containing exons 2 and 3 were amplified by PCR using fluorescent-labeled primers. The PCR products were digested with T4 endonuclease VII and screened for mutations on an automated DNA sequencer. Results: In all cases with a mutation, a cleavage fragment on the direct and/or complementary DNA strand could easily be visualized, and its approximate size correlated with the predicted location of the known mutations within the PCR product. When the code for affected status was broken, there was 100% correlation between previous DNA sequence or restriction fragment length polymorphism findings and the T4 endonuclease VII digestion results for all 29 DNAs. Conclusion: T4 endonuclease VII accurately identified the known cationic trypsinogen gene mutations in exons 2 and 3. Enzymatic mutation detection appears to be an accurate and useful method for screening individuals for known trypsinogen gene mutations and may be useful in identifying previously unidentified mutations within large regions of interest.