Mutational analysis of FEN1 Gene in hepatocellular carcinomas

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with the highest incidence in Southeast Asia and Africa. In Korea, it accounts for an estimated 12.2% of all malignancies, with 16.4% in the male population and 6.5% in female population (1). Therefore, HCC remains a significant contributor to the world’s health burden. Heavy alcohol intake and infection with the hepatitis B virus (HBV) or hepatitis C virus (HCV) are important risk factors for HCC (2). In addition, numerous genetic abnormalities associated with HCC development have been described. Flap endonuclease 1 (FEN1) is a structure-specific and multifunctional enzyme. It plays an important role in the removal of 5′-flap during long-patch base-excision repair and Okazaki fragment processing in DNA replication (3,4). In addition, FEN1 possesses 5′ exonuclease (EXO) activity and a gapdependent endonuclease (GEN) activity (5,6). The EXO and GEN activities of FEN1 are involved in apoptotic DNA fragmentation in response to apoptotic stimuli. Because of its critical role in multiple DNA metabolic pathways, FEN1 serves as a key enzyme in maintaining the integrity of the genome. Recent studies have shown that FEN1 prevents oncogenesis that leads to unwanted genetic exchanges and eventually to a cancerous phenotype, suggesting that it may function as a tumor suppressor (7). In a murine model, deletion of both FEN1 alleles causes embryonic lethality (8). Functional deficiency of yeast RAD27, the homolog of FEN1, causes hypersensitivity to DNA-damaging agents and a marked increase in the rate of spontaneous mutation (9–11). Consistent with this phenomenon, haploinsufficiency of FEN1 results in microsatellite instability and promotes cancer progression in mice (12). Furthermore, genetic change and functional deficiency of the FEN1 gene contributes substantially to the development of cancer (4). In addition, recent studies have shown that methylation of FEN1 promoter region affects gene expression in breast cancer (13), and polymorphisms in the FEN1 gene (the –69G > A polymorphism in the FEN1 gene promoter and the 4150G > T polymorphism in intron of the FEN1 gene) were positively correlated with risk of HCC (14). Until now, functional significance of the FEN1 gene has not been described in HCC. To investigate whether genetic alterations of FEN1 are involved in hepatocellular carcinogenesis, we searched for somatic mutation of the FEN gene in HCC. Thirty-eight frozen HCCs and their corresponding surrounding non-tumor liver tissue samples in patients who underwent resection of HCC were evaluated. This study was approved by the Institutional Review Boards at the Ulsan University Hospital. Frozen tissue samples were ground to a very fine powder in liquid nitrogen. Genomic DNA was prepared using a procedure based on a protocol described previously (15). Genomic DNA samples from cancer cells and corresponding noncancerous liver tissues were amplified with six sets of primers covering the entire coding region (exon 2) of the FEN1 gene (Table 1). Numbering of DNA of the FEN1 was done with respect to the ATG start codon according to the genomic sequence of Genbank accession no. NM_004111. All cases were screened by single-strand chain polymorphism (SSCP) analysis (Mutation Detection Enhancement; FMC BioProducts, Rockland, ME, USA) of each exon for the presence of an aberrant band in tumor DNA compared to the normal DNA. After detection of a mutant allele by mobility shifts on SSCP gel, direct DNA sequencing was performed. Finally, Sequencing of aberrantly migrating bands on SSCP gel led to the identification of mutation in two (5.3%) of the specimens. The mutations were missense mutations in exon 2, which lies within the FEN1 nuclease core domain: a G to T transition at nucleotide 308 (Arg to Leu) in case no. 7 and a C to T transition at nucleotide 631 (Arg to Trp) in case no. 30 (Fig. 1A and B). The mutations were found in a patient with a HBV-positive, cirrhotic background. There was no mutation in corresponding normal DNAs of these tissues, indicating that the mutations detected in the cancer cells had arisen somatically. We repeated the experiments three times, including PCR and SSCP-sequencing, to confirm the results and found that the data were consistent. These results suggest that the mutations of the FEN1 gene maybe a rare event in the development of HCC in Korean population. Interestingly, these mutations were novel mutations detected in HCCs. It has been reported that the N-terminal and intermediate motifs are essential for nuclease activity of FEN1 proteins (16), whereas the C-terminal motif is involved in the interaction between FEN1 and proliferating cell nuclear antigen (17). Also, FEN1 mutations that