Folate Deficiency Induces Genomic Uracil Misincorporation and Hypomethylation But Does Not Increase DNA Point Mutations

Abstract

Epidemiologic studies have linked nutritional folate deficiency to an increased risk of cancer, but recent trials suggest that folate supplementation does not protect against tumor formation. Our aim was to analyze the genetic and epigenetic consequences of folate deficiency and to investigate whether impairment of the uracil base excision repair pathway can enhance its effects. Wild-type mice and those deficient in uracil DNA glycosylase (Ung(-/-)) were placed on a folate-deficient diet for 8 months. We measured tumor incidence in major organs, DNA mutation rates, DNA mutation spectra, local DNA methylation, and global DNA methylation in colon epithelial cells. The experimental diet increased plasma homocysteine (60%, P< .001) and DNA uracil content (24%, P< .05) but not tumor formation. Global DNA methylation was slightly decreased in splenocytes (9.1%) and small intestinal epithelial cells (4.2%), and significantly reduced in colon epithelial cells (7.2%, P< .04). No gene-specific changes in methylation were detected at the mouse B1 element, the H19 DMR, or the Oct4 gene. By lambda CII assay and sequencing analysis of 730 mutants, we found that Ung(-/-) mice had a higher frequency of point mutations and increased C:G to T:A transitions at non-CpG sites. However, folate deficiency had no additional effect on the DNA mutation frequency or spectrum in Ung(-/-) or wild-type mice. Contradicting current concepts, these findings indicate that the effects of a low-folate diet on DNA methylation and point mutations are insufficient to promote tumor development, even in the presence of Ung deficiency.