engleski

Submerged gel electrophoresis of PCR fragments-new method for APC gene mutation detection

Goal of study: The goal of this study was to determine the usefulness of a new method for APC gene mutation detection. FAP is an autosomal dominant inherited disease, affecting 1 in 2000 people. Patients with FAP develop hundreds to thousands of adenomatous polyps in the colon and rectum during their second or third decades and one or more of them progress to cancer if left without surgical treatment. Because FAP patients have a very high risk of colorectal cancer, identification of the individual risk in family members is important to prevent cancer deaths. The method of providing such accurate presymptomatic diagnosis is to determine whether a family member has inherited the particular germ-line mutation of the APC gene carried by the affected parent. The majority of deletions and insertions in the APC gene were observed at positions containing repeated sequences within the coding sequence, and all caused frameshift mutations, which resulted in stop codons downstream. The most frequent germ-line mutations were found to occur at codon 1307-1311 all of which include the AAAAG 5 bp deletion. The frequency of this mutation reaches 24% of the total mutations previously detected. The second most frequently mutated codon 1060-1063, also contains the repeated, AAAACAAAA. Other deletions also occured in the region within or near the repeated sequence (codon 1156 and 1546). Variation in phenotypic expression of disease gene has been observed in FAP patients. This variation includes profuse and sparse types in respect to the number of colorectal polyps and differences in the frequency of extracolonic manifestations. The profuse type FAP patients develop more than 5000 adenomatous polyps ( 10 or more polyp per square cm on the colonic mucossa), and the sparse type develop fewer than 5000 of polyps. By comparing the positions of germ-line mutation of the APC gene in these two types, it was suggested that FAP patients with germ-line mutation between codon 1250 and 1464 tend to have the profuse phenotype. Method: Genomic DNAs were isolated from EDTA blood of patients and their relatives. PCR was performed using specific pair of primers. PCR products were analysed by RFLP method and electrophoresis on a Spreadex EL 300 gels. Result: After only 130 min, PCR fragments of 91 and 86 pb (5 bp deletion in codon 1309) were completely resolved on a Spredex EL 300 gels. Besides the 91 bp and 86 bp fragments, in samples containing mutation, ther was a heteroduplex band above the 91 bp fragment. Conclusion: Electrophoresis using Spredex gels provides a simple and rapid method for determination of the most frequent germ-line mutations in the APC gene.

FAP; APC gene mutation

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