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.