p16INK4a promoter mutations are frequent in primary sclerosing cholangitis (PSC) and PSC-associated cholangiocarcinoma.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) predisposes individuals to cholangiocarcinoma; however, the molecular mechanisms involved in the carcinogenesis process remain unclear. Because p16(INK4a) inactivation has been implicated in cholangiocarcinoma, our aims were to examine PSC cholangiocytes for p16(INK4a) gene mutations.

METHODS: We studied 4 patient groups: PSC patients without cholangiocarcinoma (n = 10), patients with PSC-associated cholangiocarcinoma (n = 10), non-PSC controls (n = 10), and disease controls with primary biliary cirrhosis (n = 10). Cholangiocytes and hepatocytes were isolated from tissue sections using laser capture microdissection. Genomic DNA was extracted, and the promoter region and the 3 exons for p16(INK4a) were amplified by PCR and directly sequenced.

RESULTS: In the promoter region, 8-point mutations in 5 PSC cases and 14 mutations in 8 cholangiocarcinoma cases were observed. In exon 1, 1 PSC patient and 3 cholangiocarcinoma patients had point mutations. In contrast, no case had a mutation in exon 2 or 3. Mutations were not detected in cholangiocytes from control patients or primary biliary cirrhosis patients nor in hepatocytes from any of the groups; these data indicate that the observed base changes were disease specific and not genetic polymorphisms. Several of the promoter mutations (4 of 8) dramatically decreased promoter activity (>50% reduction in luciferase activity) in a reporter gene assay.

CONCLUSIONS: The results show that functional point mutations in the p16(INK4a) promoter region likely contribute to the initiation/progression of cholangiocarcinoma in PSC. Promoter mutations in CpG islands may function as a methylation equivalent phenomenon resulting in gene inactivation.