Seasonal variation in TP53 R249S-mutated serum DNA with aflatoxin exposure and hepatitis B virus infection

Abstract

Background: Chronic hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) exposure are etiological factors for hepatocellular carcinoma (HCC) in countries with hot, humid climates. HCC often harbors a TP53 (tumor protein p53) mutation at codon 249 (R249S). In chronic carriers, 1762T/1764A mutations in the HBV X gene are associated with increased HCC risk. Both mutations have been detected in circulating cell-free DNA (CFDNA) from asymptomatic HBV carriers.

Objective: We evaluated seasonal variation in R249S and HBV in relation to AFB1 exposure.

Methods: R249S was quantitated by mass spectrometry in CFDNA in a cross-sectional survey of 473 asymptomatic subjects (237 HBV carriers and 236 noncarriers) recruited in three villages in the Gambia over a 10-month period. 1762T/1764A HBV mutations were detected by quantitative polymerase chain reaction. In addition, the HBV S gene was sequenced in 99 subjects positive for HBV surface antigen (HBsAg).

Results: We observed a seasonal variation of serum R249S levels. Positivity for R249S and average concentration were significantly higher in HBsAg-positive subjects surveyed during April-July (61%; 5,690 ± 11,300 R249S copies/mL serum) than in those surveyed October-March [32% and 480 ± 1,030 copies/mL serum (odds ratio = 3.59; 95% confidence interval: 2.05, 6.30; p < 0.001)]. Positivity for HBV e antigen (HBeAg) (a marker of HBV replication) and viral DNA load also varied seasonally, with 15-30% of subjects surveyed between April and June HBeAg positive, compared with < 10% surveyed during other months. We detected 1762T/1764A mutations in 8% of carriers, half of whom were positive for R249S. We found HBV genotype E in 95 of 99 HBsAg-positive subjects.

Conclusion: R249S is detectable in CFDNA of asymptomatic subjects. Evidence of temporal and quantitative variations suggests an interaction among AFB1 exposure, HBV positivity, and replication on TP53 mutation formation or persistence.

Figure 1

Box and whisker plots for the distribution of serum concentrations of TP53 R249S in HBsAg-negative (A) and -positive (B) subjects during higher and lower AFB₁ exposure periods. Boxes extend from the 25th to the 75th percentile; horizontal lines within the boxes represent the median; whiskers extend 1.5 times the length of the interquartile range above and below the 75th and 25th percentiles, respectively; and diamonds represent outliers. Four outliers > 40,000 copies/mL serum are not represented: one in March for HBsAg-negative subjects, and one in April and two in June for HBsAg-positive subjects. The number of individuals sampled (2–51 subjects/month) and the distribution of HBsAg-positive subjects also positive for the 1762^T/1764^A double mutation are given.

Figure 2

Serum concentrations (mean ± SD) of TP53 R249S in HBsAg-positive and ‑negative subjects, grouped by seasonal AFB₁ exposure. *p < 0.001 compared with HBsAg-negative subjects with lower AFB₁ exposure, and compared with HBsAg-positive subjects with higher AFB₁ exposure, by Student’s t-test.

Figure 3

Seasonal variation of HBeAg positivity (HBeAg+) and HBV DNA negativity (HBV–) in HBsAg-positive subjects. The number of individuals sampled each month are given. The value for November 2002 was not considered because the sample size was too small.