Human Molecular Genetics Advance Access originally published online on April 5, 2007
Human Molecular Genetics 2007 16(11):1335-1342; doi:10.1093/hmg/ddm082
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Epigenetic defects of hepatocellular carcinoma are already found in non-neoplastic liver cells from patients with hereditary haemochromatosis
1 Institute of Pathology, Medizinische Hochschule Hannover and 2 Department of Disease and Stress Biology, John Innes Centre, Colney Lane, Norwich NRE 7UH, UK, 3 Department of Gastroenterology, Hepatology, and Endocrinology, 4 Department of Visceral and Transplantation Surgery and 5 Institute of Virology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany
* To whom correspondence should be addressed at: Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Tel: +49 5115324501; Fax: +49 5115325799; Email: lehmann.ulrich{at}mh-hannover.de
Received November 21, 2006; Revised March 5, 2007; Accepted March 26, 2007
Gene silencing through aberrant CpG island methylation is a frequent epigenetic defect in hepatocellular carcinoma (HCC). However, nothing is known as yet whether aberrant hypermethylation occurs already in non-neoplastic liver cells from patients with hereditary haemochromatosis who have a clearly elevated risk for developing HCC. Therefore, quantitative real-time PCR-based methylation analysis of six genes frequently hypermethylated in HCC (RASSF1A, cyclinD2, p16INK4a, GST
1, SOCS-1, APC) was performed for liver biopsies from patients with hereditary haemochromatosis. For genotyping of the HFE gene restriction enzyme analysis and PyrosequencingTM were used. Transcriptional repression of hypermethylated genes was assessed using real-time RT–PCR. Eighty-four percent of all samples with severe hepatic iron overload and a mutated HFE gene (but without HCC) had at least one gene hypermethylated. All six genes tested were affected by aberrant hypermethylation, albeit to a different extent: RASSF1A 55%, cyclinD2 45%, p16INK4a 32%, GST1 10%, SOCS-1 6%, APC 8%. Concomitant transcriptional down-regulation was shown for RASSF1A, cyclinD2, GST1 and SOCS-1. Biopsies from haemochromatosis patients showed significantly more aberrant hypermethylation than normal liver tissue or benign liver tumours (P < 0.001) and also to a higher degree. This effect is independent of patient age, cirrhosis or hepatitis infection. This is the first report demonstrating that longstanding severe iron overload is frequently associated with epigenetic defects characteristic of HCC, which reflects the increased risk of these lesions to progress to HCC. Thus, changes in DNA methylation patterns are an early event preceding morphological alterations of malignant transformation and represent promising targets for early detection.