We found that a low-choline diet induced changes in lymphocyte gene expression in humans. These changes included (but were not limited to) changes in expression of genes functionally involved in folate metabolism, apoptosis, DNA damage-repair, cell cycle regulation, immune response, epigenetic regulation, and telomere maintenance, which suggested that dietary choline deficiency can alter the functionality of many pathways. Subjects who developed organ dysfunction while following a low-choline diet differed from those who did not develop organ dysfunction in their expression of many genes, including some of those related to any or all of apoptosis, the DNA integrity checkpoint, and genes of cell cycle regulation. Subjects with the PEMT (rs12325817) and MTHFD1 (rs2236225) SNPs, previously shown to predispose a person to developing organ dysfunction when fed a low-choline diet (7
), differed at baseline from those subjects without the SNP in their expression of apoptosis, the DNA damage checkpoint, and cell proliferation control genes, which suggests that they are phenotypically different even before a low-choline diet is administered.
In different comparison groups, choline deficiency induced different patterns of change (). Moreover, many of the results reported within all subjects (baseline compared with depletion, ALL SUBJECTS) could be misleading because these changes were not homogenous when subjects were classified on the basis of signs of organ dysfunction ( and ). For instance, 3 of the genes with the largest change in expression (CHEK1
, and KIF20A
) were differently expressed in the NO SIGNS group than in the SIGNS group (). CHEK1
is involved in DNA repair in human T lymphocytes (22
is required for sufficient glycogen accumulation and is normally underexpressed in whole human blood (23
regulates the transport of Golgi membranes and associated vesicles along microtubules (24
By clustering the groups according to genotype (), we found that different patterns in gene expression do indeed support this classification. For example, the previously reported (8
) protective CHDH
(318 A→C) genotype grouped close to the NO SIGNS group—those who had no clinical symptoms while following the low-choline diet—whereas the CHDH
(432 G→T) genotype, which was reported to increase susceptibility to choline deficiency (8
), grouped with the SIGNS group. Moreover, the NO SIGNS and SIGNS groups were intercalated by the ALL SUBJECTS group, which supports the heterogeneity of the previously reported responses to dietary choline deficiency. Included in the other arm from the clustering analysis were the PEMT
genotypes, which grouped around the ALL(D) group. This cluster supported our hypothesis that genetic differences account for the presence or absence of organ dysfunction in humans depleted of choline (7
). This analysis also found that the presence of the PEMT
genotypes could confer differences in the phenotypes at baseline, which suggests that different persons may have different susceptibility to dietary choline deficiency and that the risk of choline deficiency is greater in women who are carriers of the PEMT
allele [this group was closer to the ALL(D) group than to the PEMT(D) group]. Harder to interpret is the unexpected clustering of the FOLATE group (those receiving folate supplements versus those who did not) close to the CHDH
(432 G→T) genotype.
To construct a better picture of the potential functional significance of these gene expression patterns, we used GO analysis to group genes according to their functional roles. The heterogeneity of the response to a low choline diet was also shown at this level of analysis. Genes involved in folate metabolism were most affected in the carriers of the MTHFD1
(432 G→T) alleles (). A different pattern was observed for genes involved in apoptosis (), in which the MTHFD1
polymorphic allele carriers were the most affected (increases in apoptosis, induction of apoptosis, caspase activity, positive regulation of apoptosis, and decreased expression of genes involved in negative regulation of caspase activity). This result is consistent with our previous data, which showed that humans who developed organ dysfunction when fed a choline-deficient diet were more likely to have the MTHFD1
variant allele (7
) and to have increased lymphocyte DNA damage and apoptosis, as measured by caspase activity, than were those who did not develop organ dysfunction (9
). It is interesting that the same trends were present in these subjects at baseline, which suggests that their higher susceptibility to choline deficiency may be due to effects present even when subjects are consuming a normal diet. For carriers of the PEMT
polymorphic allele, the most affected genes were grouped within GO classes involved in telomere maintenance; notably, these alterations were more extensive in female PEMT
allele carriers. Telomeres are the protein-DNA structures that protect chromosome ends from being recognized as double-stranded DNA breaks, and their maintenance is important for cell longevity, normal cell cycling, and prevention of cancer (25
Our findings suggest that dietary choline deficiency may affect the homeostatic mechanisms responsible for telomere maintenance, perhaps by epigenetic changes in gene expression. The folate status of subjects had little effect on the gene expression changes seen in GO analyses.
Some genes that are regulated by DNA methylation were also identified as being changed by a low-choline diet (). In cultured human neuroblastoma cells and in rodent models, cho-line deficiency alters both global and gene-specific DNA methylation and the expression of these genes (20
). Therefore, we suggest that the observed choline deficiency–induced changes in gene expression occurred because of altered methylation in promoter regions of the genes involved. Among the genes changed in choline deficiency that are known to be regulated by gene methylation, the insulin-like growth factor 2 (IGF2
) is an imprinted gene; loss of imprinting is associated with cancer in various experimental models (as reviewed in reference 26
). Another gene identified as being changed by dietary choline and known to be regulated by methylation is telomerase reverse transcriptase (TERT
), the product of which is the protein component of a ribonucleoprotein polymerase that maintains telomere ends by addition of the telomere repeat TTAGGG, and its deregulation is involved in both cellular senescence (telomere shortening) and carcinogenesis in leukemic cells (27
In conclusion, dietary choline deficiency induced changes in gene expression profiles in human lymphocytes, and these patterns correlated with the occurrence of organ dysfunction and apoptosis in humans fed a low-choline diet. These outcomes also correlated with polymorphisms in genes that regulate folate and choline metabolism. Further studies are required to determine whether these changes are regulated by epigenetic mechanisms and to identify other populations at risk for dietary choline deficiency.