engleski

APC gene mutation detection by submerged gel electrophoresis of PCR fragments

Purpose of study: Familial adenomatous polyposis (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 ehe adenomatous polyposis coli (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-base-pair 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 sequence, 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 polyps per square cm on the colonic mucosa), 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 DNA was isolated from EDTA blood. Polymerase chain reaction (PCR) was performed using specific pair of primers. PCR products were analysed by electrophoresis on a Spredex EL 300 gels. Result: After only 85 min PCR fragments of 91 and 86 bp (5 bp deletion in codon 1309) are completely resolved on a Spredex EL 300 gel. Besides the 91 bp and 86 bp fragments, in heterozygous sample there is a heteroduplex band above the 91 bp fragment. Conclusion: Electrophoresis on a Spredex gels is a simple and rapid method for determination of the most frequent germ-line mutations in the APC gene.

FAP; APC; germ-line mutation; mutation detection

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