Hisayoshi Nakazawa

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 where exposure to aflatoxin B1 (AFB) occurs. We developed a sensitive allele‐specific polymerase chain reaction (AS‐PCR) assay to detect this point mutation in non‐neoplastic human liver tissues. Three oligonucleotide primers, 1 specific for the mutant allele and 2 specific for the wild‐type allele were used. The mutant allele primer differed from the wild‐type allele due to a G‐to‐T transversion in its terminal 3′ nucleotide. The first stage involved amplification of exon 7 of p53 followed by a selective amplification of mutant codon 249 sequences. This method allowed for the detection of a mutant codon 249 allele in the presence of as many as 105 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 249 AGT transversion in tumor and matched non‐tumor liver samples from North American patients with hepatitis and from Mozambiquan patients exposed to AFB. Mutations were detected in 5 of 6 samples of non‐neoplastic liver from Mozambican patients, all of whom were HBsAg‐ or HBcAg‐positive and AFB‐exposed. In contrast, no mutations were detected in non‐neoplastic liver from North American patients with either HBV‐ or HCV‐derived hepatitis and cirrhosis. This procedure is a simple and powerful approach for screening p53 codon 249 AGT mutation in heterogeneous non‐neoplastic hepatocyte populations.