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 Mozambiquan 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.
Lady Davis Institute for Medical Research and Jewish General Hospital, Department of Medicine, McGill University, Montreal, Canada.
G M
GM
Kirby
G
G
Batist
N
N
Fotouhi-Ardakani
H
H
Nakazawa
H
H
Yamasaki
M
M
Kew
R G
RG
Cameron
M A
MA
Alaoui-Jamali
D000328:Adult; D016604:Aflatoxin B1 / Q000009:adverse effects; D000368:Aged; D000483:Alleles; D001483:Base Sequence; D003062:Codon; D005260:Female; D016158:Genes, p53; D006509:Hepatitis B / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000737:chemistry*; D008113:Liver Neoplasms / Q000139:chemically induced / Q000235:genetics; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009073:Mozambique; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D003062:Codon; D016604:Aflatoxin B1