ALLELE-SPECIFIC PCR ANALYSIS OF P53 CODON-249 AGT TRANSVERSION IN LIVER-TISSUES FROM PATIENTS WITH VIRAL-HEPATITIS

AGG to AGT mutations in codon 249 of the p53 tumor-suppressor gene are frequently observed in hepatocellular carcinomas (HCC) from areas whe re exposure to aflatoxin B-1 (AFB) occurs. We developed a sensitive al lele-specific polymerase chain reaction (AS-PCR) assay to detect this point mutation in non-neoplastic human liver tissues. Three oligonucle otide primers, I specific for the mutant allele and 2 specific for the wild-type allele were used. The mutant allele primer differed from th e wild-type allele due to a G-to-T transversion in its terminal 3' nuc leotide. The first stage involved amplification of exon 7 of p53 follo wed by a selective amplification of mutant codon 249 sequences. This m ethod allowed for the detection of a mutant codon 249 allele in the pr esence of as many as 10(5) copies of the wild-type allele and was 100- fold more sensitive than the restriction fragment length polymorphism- PCR technique. We have applied this AS-PCR protocol to examine codon 2 49 AGT transversion in tumor and matched non-tumor liver samples from North American patients with hepatitis and from Mozambiquan patients e xposed to AFB. Mutations were detected in 5 of 6 samples of non-neopla stic liver from Mozambican patients, all of whom were HBsAg- or HBcAg- poritive and AFB-exposed. In contrast, no mutations were detected in n on-neoplastic liver from North American patients with either HBV- or H CV-derived hepatitis and cirrhosis. This procedure is a simple and pow erful approach for screening p53 codon 249 AGT mutation in heterogeneo us non-neoplastic hepatocyte populations. (C) 1996 Wiley-Liss, Inc.