Discussion
lymphoma, TET2
LYMPHOMA
We report here the long-term effects of dietary supplementation with 1 g of vitamin C on methylation, hydroxymethylation, and gene expression in individuals carrying a germline loss-of-function mutation in Unexpectedly, vitamin C supplementation also resulted in decreased genomic hydroxymethylation in the unaffected mutation carriers. This seemed to contradict studies in cultured cells where hydroxymethylation levels were increased in response to vitamin C [TET enzymes catalyze multiple reaction steps and collaborate with other enzymes to complete the demethylation cycle and restore unmodified cytosine. Thus, the hydroxymethylation levels alone do not fully depict TET activity nor the further TDG/BER-mediated reaction steps involved in replication-independent active demethylation. As such, 5-hmC does not lead to active demethylation and a substantial proportion undergoes replication-dependent dilution [Using long-read nanopore sequencing combined with methylation calling [Interestingly, TET2 can yield 5-fC and 5-caC iteratively in a single encounter with 5-mC containing DNA, without releasing a 5-hmC intermediate [As expected, vitamin C supplementation resulted in changes at DNA methylation and hydroxymethylation level rather than causing extensive transcriptional changes. However, we observed a decreased number of differentially expressed genes between the healthy Limitations of the study include a small sample size, precluding the use of more sophisticated statistical models, such as models accounting for age and sex. Our cytosine demethylation model ignores the cell cycle, and our data allow only steady-state observations. Baseline plasma or intracellular levels of vitamin C were not measured, introducing unknown individual variations. In contrast to the unaffected mutation carriers, two mutation carriers previously diagnosed with lymphoma did not show vitamin C-driven methylation changes. Older age, chemotherapy, and in particular the low baseline expression of 