Codon 249 mutation in exon 7 of p53 gene in plasma DNA: maybe a new early diagnostic marker of hepatocellular carcinoma in Qidong risk area, China

Abstract

Aim: One of the characteristics of hepatocellular carcinoma (HCC) in Qidong area is the selective mutation resulting in a serine substitution at codon 249 of the p53 gene (1, 20), and it has been identified as a "hotspot" mutation in heptocellular carcinomas occurring in populations exposed to aflatoxin and with high prevalence of hepatitis B virus carriers (2,3,9, 10,16,24). We evaluated in this paper whether this "hotspot" mutation could be detected in cell-free DNA circulating in plasma of patients with hepatocellular carcinoma and cirrhosis in Qidong, China, and tried to illustrate the significance of the detection of this molecular biomarker.

Methods: We collected blood samples from 25 hepatocellular carcinoma patients, 20 cirrhotic patients and 30 healthy controls in Qidong area. DNA was extracted and purified from 200 microl of plasma from each sample. The 249(Ser) p53 mutation was detected by restriction digestion analysis and direct sequencing of exon-7 PCR products.

Results: We found in exon 7 of p53 gene G-T transversion at the third base of codon 249 resulting 249(Arg) - 249(Ser) mutation in 10/25 (40 %) hepatocellular carcinoma cases, 4/20 (20 %) cirrhotics, and 2/30 (7 %) healthy controls. The adjusted odds ratio for having the mutation was 22.1(95 % CI, 3.2-91.7) for HCC cases compared to controls.

Conclusion: These data show that the 249(Ser) p53 mutation in plasma is strongly associated with hepatocellular carcinoma in Qidong patients. We found this mutation was also detected, although it was at a much lower frequency, in plasma DNA of Qidong cirrhotics and healthy controls; We consider that these findings, together with the usual method of HCC diagnosis, will give more information in early diagnosis of HCC, and 249(Ser) p53 mutation should be developed to a new early diagnostic marker for HCC.

Figure 1

The electrophoresis map of PCR products. Lane 1: DNA Molecular weight markers. Lane 2-9: PCR products of partial samples.

Figure 2

Mutation at coden 249 was identified by restriction digestion. Lane 1: negative control, Lane 2: positive control, Lane 3-9: HCC samples, Lane 10: DNA molecular weight markers.

Figure 3

Sequencing results (partial electropherogram). A: Wild type sequence shows codon 249 AGG; B: Mutation sequence shows codon 249 AGG→AGT transversion; C: Mutation sequence shows codon 249 AGG→AGC transversion; D: A→C transver-sion at the second base of codon 258.