Reply:
Authors: Zigmund Luka, M. Luz Martínez‐Chantar, Shelly C. Lu, Conrad Wager, José M. Mato
Journal: Hepatology (2009)

The letter by Chen and colleagues begins by questioning our findings in "Loss of the Glycine N-Methyltransferase Gene Leads to Steatosis and Hepatocellular Carcinoma in Mice,"1 and finishes by claiming their group is the first to demonstrate that GNMT (glycine N-methyltransferase) is a liver cancer susceptibility gene. Our group published a description of the preparation and properties of a mouse GNMT knockout in 2006.2 The strategy used was gene targeting and we replaced the first exon with part of the promoter region and intron 1 (1.90 kb) with the NEO gene (1.85 kb). In this article, we reported the complete absence of enzyme activity and enzyme protein by western blotting in 3-month-old knockout mouse livers using a polyclonal antibody. Because GNMT is a major consumer of methyl groups from S-adenosylmethionine (SAMe), this resulted in very high levels of liver SAMe. In 2007, Dr. Chen's group published an article in HEPATOLOGY titled "Glycine N-Methyltransferase−/− Mice Develop Chronic Hepatitis and Glycogen Storage Disease in the Liver".3 They described a strategy similar to that we used to generate a GNMT knockout mouse. They replaced a fragment of the Gnmt gene containing exons 1-4 and part of exon 5 with the NEO gene. This article was concerned with the phenotype of their knockout mouse model as having glycogen storage disease as stated in the title of their article. They examined knockout male and female mice at 11 weeks of age and 9 months of age but made no reference to the development of liver cancer. In 2008, we published the HEPATOLOGY study that Dr. Chen takes issue with.1 At the age of 3 months, male knockout mice developed fatty liver and fibrosis; after 8 months, all knockout mice had hepatocellular carcinoma (HCC). HCC development was a result of overmethylation of RASSF1 and SOCS2 promoters. Then, after our discovery of HCC in our GNMT−/− animals, the Chen group found that their GNMT−/− animals also developed HCC at about 17 months of age.4 Most surprising in Dr. Chen's letter is the contention that our GNMT knockout mouse is not a complete knockout based on the dubious designation of a 51-nucleotide to 52-nucleotide sequence as a gene "promoter" with no documentation to justify this. They contend that our mouse model generates a protein truncated by 73 amino acids. Our use of polyclonal antibody showed no signal smaller than that of 32 kDa (intact GNMT) upon western blotting. Moreover, if such internal start sites were indeed functional, truncated forms of GNMT would be apparent in wild-type animals. In summary, we are confused by Dr. Chen's linkage of his claim to have been the first to identify GNMT as a liver cancer susceptibility gene to the erroneous claim of technical deficiencies in our work. It should be noted here that in 1975 Dr. Kerr pointed out that GNMT was not present in fast-growing liver hepatomas and was present at a low level in slower growing tumors.5, 6 Zigmund Luka*, M. Luz Martínez-Chantar , Shelly C. Lu , Conrad Wager* §, José M. Mato , * Department of Biochemistry, Vanderbilt University, Nashville, TN, CIC bioGUNE, CIBERehd, Technology Park of Bizkaia, Bizkaia, Spain, Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University Southern California, Los Angeles, CA, § Tennessee Valley Department of Medical Affairs Medical Center, Nashville, TN.