TP53 R249S mutation, genetic variations in HBX and risk of hepatocellular carcinoma in The Gambia.

In regions with high prevalence of chronic hepatitis B virus (HBV) infection and dietary aflatoxin B(1) (AFB(1)) exposure, hepatocellular carcinomas (HCCs) often contain TP53 mutation at codon 249 (R249S). Furthermore, a C-terminal truncated HBx protein expressed from hepatocyte integrated HBV is associated with HCC development. This study evaluates the association between R249S and HBX status in relation to HCC in West African population. HBX (complete or 3'-truncated) and HBS genes were assessed by PCR in cell-free DNA (CFDNA) from plasma of subjects recruited in a hospital-based case-control study (325 controls, 78 cirrhotic patients and 198 HCC cases) conducted in The Gambia. These samples had been previously analyzed for R249S and HBV serological status. Complete HBX sequence was frequently detected in CFDNA of HCC-R249S positive (77%, 43/56) compared with HCC-R249S-negative cases (44%, 22/50). Conversely, the proportion of 3'-truncated HBX gene was significantly higher in HCC-R249S negative than positive cases (34%, 17/50, compared with 12%, 7/56) (χ(2) = 12.12; P = 0.002; distribution of R249S negative and positive according to HBX status). Occult HBV infection (detected by PCR) was present in 24% of HCC previously considered as negative by HBV serology. Moreover, HBV mutation analysis revealed that double mutation at nucleotides 1762(T)/1764(A) was associated with diagnosis of cirrhosis or HCC {cirrhosis: odds ratio (OR): 9.50 [95% confidence interval (CI) 1.50-60.11]; HCC: OR: 11.29 [95% CI 2.07-61.47]}. These findings suggest that in HCC from The Gambia, complete HBX sequences are often associated with the presence of TP53 R249S mutation.

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

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.

Effects of the TP53 p.R249S mutant on proliferation and clonogenic properties in human hepatocellular carcinoma cell lines: interaction with hepatitis B virus X protein.

Aflatoxin B(1) (AFB(1)) is a risk factor for hepatocellular carcinoma (HCC) in many low-resource countries. Although its metabolites bind at several positions in TP53, a mutation at codon 249 (AGG to AGT, arginine to serine, p.R249S) accounts for 90% of TP53 mutations in AFB(1)-related HCC. This specificity suggests that p.R249S confers a selective advantage during hepatocarcinogenesis. Using HCC cell lines, we show that p.R249S has lost the capacity to bind to p53 response elements and to transactivate p53 target genes. In p53-null Hep3B cells, stable transfection of p.R249S or of another mutant, p.R248Q, did not induce significant changes in cell proliferation and survival after cytotoxic stress. In contrast, in a cell line that constitutively expresses both p.R249S and the hepatitis B virus antigen HBx (PLC/PRF/5), silencing of either p.R249S or HBx by RNA interference slowed down proliferation, with no additive effects when both factors were silenced. Furthermore, the two proteins appear to form a complex. In human HCC samples, mutation at codon 249 did not correlate with p.R249S protein accumulation or HBx truncation status. We suggest that p.R249S may contribute to hepatocarcinogenesis through interaction with HBx, conferring a subtle growth advantage at early steps of the transformation process, but that this interaction is not required for progression to advanced HCC.