Phenotypic differences between two Gnmt−/− mouse models for hepatocellular carcinoma
Authors: Yi‐Ming Arthur Chen, Yi‐Jen Liao, Shih‐Ping Liu, Ting‐Fen Tsai
Journal: Hepatology (2009)

We would like to express some technical concerns about the results presented in the article by Mato and coworkers in the April 2008 issue of HEPATOLOGY.1 We have identified the gene for glycine N-methyltransferase (GNMT) as a liver cancer susceptibility gene, generated Gnmt−/− mice, and reported on their tendencies toward chronic hepatitis, glycogen storage disease, and liver cancer.2-6 Although the Gnmt−/− mouse models described by Dr. Mato's team and our group share similar strain (129 substrain/B6) backgrounds, they have at least three significant differences in terms of phenotype: (1) Dr. Mato's group reported that their male Gnmt−/− mice developed hepatocellular carcinoma (HCC) at 8 months old, whereas our Gnmt−/− mice did not develop HCC until a minimum of 14 months of age (up to 24 months); (2) we showed that seven of seven (100%) female Gnmt−/− mice develop HCC, whereas Dr. Mato did not mention whether their female Gnmt−/− mice had developed HCC; and (3) Dr. Mato's team described global DNA hypermethylation in the liver tissues of their Gnmt−/− mice using capillary electrophoresis, whereas we used both [3H]methyl group labeling and a commercially available enzyme immunoassay to identify global DNA hypomethylation in liver tissues from both male and female Gnmt−/− mice.1, 6 Published reports indicate that global DNA hypomethylation and promoter gene cytosine-guanosine dinucleotide hypermethylation are both present in human HCC.7 However, with the exception of Dr. Mato's report, no one has stated that gene knockout mice can develop HCC as early as 8 months of age. One possible explanation for the differences between these two Gnmt−/− models is the completeness of the Gnmt gene section being knocked out. The Gnmt gene has six exons; we knocked out exons 1-4 and part of exon 5,4 whereas Dr. Mato and his collaborator, Dr. Wagner, only knocked out exon 1.8 Results from a nucleotide sequence analysis indicate that the disrupted allele generated in Dr. Wagner's model contained three potential promoters in intron 1 (Fig. 1). In addition, four potential in-frame ATG codons were located downstream from the potential promoters (three in intron 1 and one in exon 2). Targeted modification of mouse Gnmt gene locus. The disrupted allele generated in Dr. Wagner's model contains three potential promoters in intron 1, and four potential start codons in the downstream promoter region and exon 2. Filled arrows point to start codons. Open triangles indicate three potential promoter sites in intron 1. The full-length mouse Gnmt contains 293 amino acids (aa); an incomplete knockout strategy may generate an artificial truncated form containing 220 amino acids. This type of disrupted gene may generate a truncated form of GNMT that lacks the N-terminal 73 amino acids. Previous reports show (1) Gnmt uses its N-terminal 20 amino acids to form a tetramer in the cytosol and (2) the presence of a phosphorylation site in serine-9 of the Gnmt protein.9, 10 We feel there is a need to clarify whether Dr. Mato's Gnmt−/− mice generated such a truncated form of Gnmt, which may cause it to act as a dominant-negative protein, thereby implying its involvement in tumorigenesis. If this is the case, then the mouse model they generated is not a complete Gnmt−/− mouse model. In summary, we believe we are the first group to demonstrate that GNMT is a liver cancer susceptibility gene. Although both teams showed HCC development in Gnmt−/− mice, without further data, we suspect that Dr. Mato's model is an incomplete Gnmt knockout model. Yi-Ming Arthur Chen* , Yi-Jen Liao , Shih-Ping Liu , Ting-Fen Tsai§, * Department of Microbiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Molecular Medicine Program, Institute of Public Health, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Center for Neuropsychiatry, China Medical University and Hospital, Taichung, Taiwan, § Faculty of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.