Folic acid during pregnancy and transposons: Turncoats?

There is a growing body of evidence that nutrition during pregnancy may influence the risk of chronic disease (including cancer) in childhood and possibly, adult life. For example, it is well-established that folic acid supplementation during early pregnancy reduces the risk of neural tube defects [Stevenson RE, et al Pediatrics 2000; 106:677-683].  There are also recent studies to suggest that folic acid supplementation may reduce the risk of childhood cancer [Thompson JR et al Lancet 2001; 358:1935-40; Olshan AF et al Epidemiol 2002; 13:575-580].  Much of this evidence, however, is from observational studies, and the biological mechanism(s) underlying these associations are largely unknown. CpG methylation is one mechanism by which certain genomic regions are silenced. Dietary factors, including methyl donors, are necessary for the synthesis of S-adenosylmethionine which is required for CpG methylation. It has been speculated that early nutrition may be important in providing methyl donors for CpG methylation. Most of the human genome displays little variability with respect to methylation levels, making this an unlikely mechanism for early nutrition to influence later disease.  Instead, research has focused on methylation and tranposable elements (including DNA transposons and retrotransposons). Transposons are common and potentially mobile sequences of DNA that are scattered throughout the genome. More than 35% of human DNA is estimated to be derived from transposons [Yoder JA et al Trends Genet 1997; 13:335-340]. Normally, transposons are highly methylated and thus silenced. Depending on where they are inserted in DNA (which appears to be random for a subset of these elements), transposons can end up silencing neighboring genes. 

An intriguing animal study comes from Waterland RA and Jirtle RL [Molecular & Cell Biology 2003; 23:5293-5300], who investigated the role of dietary supplementation of pregnant mice and the effect on coat color. The mouse agouti gene signals coat phenotype by controlling the color produced by follicular melanocytes; this transcription occurs in a promotor region of exon 2 of the agouti (A) allele. Viable agouti (Avy) mice are yellow, whereas the loss-of-function (silenced) nonagouti (a) homozygous allele mice are black. This Avy mutation occurs due to the insertion of a retrotransposon element adjacent to the gene. Expression of this gene (determined through coat phenotype) appears to depend on the level of CpG methylation of the retrotransposon. In this study, it was speculated that dietary supplementation of mice during pregnancy could change the coat color of offspring. Prior to mating, a/a (black coat) female mice were randomly assigned to the standard NIH-31 diet or the NIH-31 diet supplemented with methyl donors and cofactors including folic acid, vitamin B12, choline chloride, and betaine. They were mated with Avy/a males and supplementation was provided throughout pregnancy and lactation. Avy/a offspring were examined in both groups. Interestingly, the offspring of the supplemented dams had a coat phenotype that was shifted toward a darker or mottled coat color compared to the lighter coat color in offspring from the unsupplemented dams. The investigators also showed that the methylation status of the promoter region of the agouti gene was highly correlated with the methylation status of the adjacent transposon gene. They conclude that there is a localized epigenetic instability of methylation that arises from an interaction between the transposon and its nearby genetic region. Thus, genes that manifest a transposon region adjacent to a promoter region of DNA could be influenced by early nutrition or perhaps other environmental exposures.

COMMENT: This is a fascinating study of how in utero environmental factors (in this case maternal diet) can influence gene expression throughout the life of the offspring. In this study, the authors reported that DNA methylation (as well as coat color) was maintained throughout adulthood; thus the effects appear permanent. These findings are particularly relevant because of the demonstrated importance of folic acid in humans.  Many food stuffs are now fortified with folic acid in the United States, and folic acid is highly recommended as a supplement in pregnancy. Given the results of this study, it will be important to determine whether there may be long-term unappreciated consequences of being exposed to high levels of  folic acid in utero. For example, could silencing of transposons that are adjacent to beneficial genes (such as tumor suppressor genes) also occur? Clearly, further animal and human studies are needed to both replicate and expand on these results.  

Like father like son?

Descriptive epidemiology, pedigree analysis, and molecular investigations have all been used to investigate genes that predispose to childhood (and adult) cancer.  Another strategy for identifying heritable risk factors is to examine cancer incidence among the parents of children with cancer.  Cancer incidence and also non-cancer mortality among such parents can also help to identify environmental risk factors for childhood cancer. A previous study [Olsen JH et al. 1995 NEJM 333: 1594-1599] found no overall excess of cancer among parents, but suffered from outdated cancer classifications of both parents and children.  In a more recent study [Pang D et al. 2003 CEBP 12, 538-534] researchers in the UK gathered information from the National Health Service Central Register on cancer incidence and all-cause mortality for 2,288 mothers and 2,204 fathers of the 2,604 children diagnosed with solid tumors between 1954-1996 and reported to the population-based Manchester Children’s Tumor Registry.  Childhood leukemias were excluded from the study since the evidence for familial clustering of these malignancies is weaker than for solid tumors. All childhood tumors were coded to the International Classification of Disease for Oncology, 7th edition, using diagnostic slides and clinical records. Parents’ person time at risk for cancer from birth of the parent until the earliest of: diagnosis with cancer, date last known to be alive, date of emigration from the UK, or the closing date of the study.  Person time at risk for mortality began at the birth of the child and ended at death, emigration, age 85 years, or the end of the study.  The resulting cancer incidence and mortality rates were compared to the rates of the UK as a whole to produce Standardized Incidence Ratios (SIRs) and Standardized Mortality Ratios (SMRs), respectively, for mothers and fathers separately and in combination.  The researchers conducted several different analyses as were appropriate for testing their three prior hypotheses:

“First, there may be excesses of specific cancers in parents of children with certain tumors because of mutations in the TP53, NF1, NF2, patched, and Rb1 genes.  Second, there may be excesses of carcinoma of the lung, leukemia, and non-Hodgkin’s lymphoma (NHL) in the parents as a result of exposure to cigarette smoking, hydrocarbons, and agrochemicals. Third, the pattern of mortality from non-cancer causes among the parents of children with cancer may provide indications of lifestyle factors that could increase or decrease the risk of childhood cancer.”

The first analysis examined specific diagnoses among parents of children with any solid tumor.  There were significant excesses of central nervous system (CNS) tumors (p  < 0.05) in mothers and fathers combined, breast carcinoma in the mother (p < 0.05), and bone and soft tissue sarcomas (p < 0.01) and retinoblastoma (p < 0.001) in both fathers and mothers.  The second analysis reversed the first by examining the incidence of overall cancer among parents of children with specific diagnoses. There were significant excesses of cancer among mothers of children with primitive neuroectodermal tumors (p < 0.05), osteosarcoma (p < 0.05), gonadal germ cell tumors (GCT, p < 0.01), and skin cancer (p < 0.01); among fathers of children with retinoblastoma (p < 0.05); and among mothers and fathers combined of children with retinoblastoma (p < 0.01) and gonadal GCTs (p < 0.01). Next, the researchers grouped childhood tumors into those known to be associated with the TP53 or Rb1 genes and those not known to be and looked at the incidence of breast cancers, sarcomas, CNS tumors, and overall cancer separately for each group.  Among the parents of children with TP53 or Rb1 associated tumors, there were significant excesses of breast carcinomas in mothers  (p < 0.01), sarcomas (p < 0.05) and all cancer (p < 0.05) in fathers, and breast carcinoma (p < 0.01), sarcoma (p < 0.01), and all cancers (p < 0.01) combined in mothers and fathers. Among parents of children with TP53 or Rb1 unassociated tumors, there were, unexpectedly, significant excesses of CNS tumors in fathers (p < 0.05) and in mothers and fathers combined (p < 0.05). Lastly, the researchers compared mortality among parents, regardless of the type of tumor their child had, to that of the entire UK population.  There were no significant associations with mortality from all causes, any cancer, specific cancers, or with a variety of non-cancers causes of death, with the exceptions of nervous system diseases and suicide, both of which were significantly less common among parents of children with solid tumors (p < 0.05 and p < 0.01, respectively).

COMMENT: This study, with its population-based registration of both parental and child cancers and its individual review of childhood cancers, was method-ologically very sound.  The specific parental cancers found to be in excess among parents of children with solid tumors were completely to be expected based on previous reports of their associations with predisposing genes.  The excess of cancers among parents of children with gonadal GCTs was novel but compelling given that it was a highly significant finding in a well-constructed study.  It may be the researchers have uncovered a new multiple cancer syndrome, though independent studies are needed to confirm this.  Finally, the finding of fewer suicides among parents of children with cancer was surprising, since childhood cancer is devastating for all involved, but reassuring if it is not due to chance. Thus, perhaps it is possible that after such tragedy occurs, parents find a new appreciation for life. 

C3 Quarterly Newsletter
Children's Cancer Research Fund
Epidemiology Research Unit
Division of Pediatric Epidemiology
Clinical Research
University of Minnesota
420 Delaware St. SE, Box 422
Minneapolis, MN 55455
pedsepi@umn.edu

Editors: 
Stella M. Davies, MD, PhD, and Julie A. Ross, PhD