ABSTRACT: The one-carbon metabolism pathway is strongly implicated in the development of hepatocellular
carcinoma (HCC) in patients with non-alcoholic fatty liver disease (NAFLD). The incidence of HCC has
increased in the US, and about a third of all HCC cases diagnosed in the US do not have a history of viral
hepatitis or alcohol abuse, but these cases of HCC often have clinical or biochemical features of NAFLD.
Indeed, NAFLD-related HCC cases are expected to increase further in the US. Functionally, the one-carbon
metabolism pathway regulates DNA methylation through metabolism of one-carbon nutrients, such as choline,
methionine and folate, to form S-adenosylmethionine (SAM). SAM is the universal methyl donor used for DNA
methylation; the formation of 5-methylcytosine (5mC). Following the transfer of methyl groups from SAM for
methylation, SAM is converted to S-adenosylhomocysteine (SAH), which is a potent inhibitor of the activities of
DNA methyltransferases (DNMTs) and thus suppresses DNA methylation. Hence, while SAM promotes
methylation, SAH inhibits methylation, and the SAM/SAH ratio reflects a “hepatic methylation index” because
both SAM and SAH are formed primarily in the liver. Additionally, recent data show that 5mC is not a static
DNA mark, it is converted to 5-hydroxymethylcytosine (5hmC) by TET enzymes. 5hmC is now recognized as a
unique epigenetic modification to DNA that is distinct from 5mC. However, both 5mC and 5hmC are involved in
gene regulation and both have been implicated in the progression of established HCC, but it is unclear which
of these DNA marks is most relevant to HCC development. The central hypothesis of this proposal is that
individual differences in hepatic methylation index or epigenetic modifications to DNA influence susceptibility to
HCC in isolation or in concert with genetic variants in one-carbon, DNMT or TET genes. The Specific Aims are:
(1) to identify common and rare genetic risk variants in the one-carbon metabolism pathway associated with
HCC risk. (2) To assess the association between hepatic methylation index (SAM/SAH ratio) and HCC risk,
and identify one-carbon gene modifiers of the association. (3) To assess the association between epigenetic
modifications to DNA (5mC and 5hmC) and HCC risk, and evaluate interaction by one-carbon, DNMT, and
TET genes. Findings from this research are expected to yield novel insights into the one-carbon metabolism
pathway as a previously under-recognized, potentially modifiable contributor to HCC and improve strategies for
the prevention, risk stratification, and early detection of HCC. The activities outlined in this application are
designed to extend the applicant’s expertise in genetic and molecular epidemiology, and acquire new skills in
epigenetics, metabolomics, and bioinformatics. Members of the mentoring team have complementary expertise
that spans these disciplines. Overall, the cross-disciplinary, integrative molecular epidemiologic approach of
this proposal and the career development activities that include formal coursework and “hands-on” workshops
will enable the applicant to acquire the needed competencies to foster his transition to independence.