Fetal vitamin B-12: a modulator of folate-dependent homocysteine remethylation?

ation levels were strikingly different among the assays, ranging from ;0% for GATA3 and STAT3 to as high as 70% and 100% for LINE1 and FOXP3, respectively (our Table 3) (5). These observations strongly rule out the existence of incomplete bisulfite converted DNA, which would otherwise result in increased methylation levels in all genomic regions with no genes exhibiting methylation levels as low as 0% methylation. We are thus confident that our methylation assays were quantitatively sensitive and that the impact of different biases could be ruled out. With regard to the impact of the outliers, as one can see from our Figure 1A, even after including additional 61 samples in the analysis the scatter for the nonsmokers is markedly greater than for smokers. Whereas the reason for this observation is unknown (one can speculate that it is the result of smoking status), a single outlying sample cannot result in significantly lower methylation of LINE-1 sequences of the entire group, especially considering the number of samples in each group. Furthermore, the numbers of atopic mothers in the experimental groups were provided in our Table 1 (n 1⁄4 72 in the control group compared with n 1⁄4 70 in the x-3 PUFA group) (5), and thus the proportion of atopic mothers was indeed balanced. We also confirmed that interaction between maternal smoking and x-3 PUFA on LINE-1 methylation was significant when adjusted for sex, gestational duration, BMI, and batch of laboratory analyses. This result supports x-3 PUFA involvement in LINE-1 methylation changes depending on smoking status. We appreciated Burdge’s point with regard to the need to take our study a step further in mechanistic terms. Indeed, one limitation of our study is the lack of experiments to show a causal link between changes in LINE-1 methylation and T cell differentiation/ function. Because we had a limited amount of frozen CBMCs, there was a technical challenge in performing functional tests and thus we were not able to carry out mechanistic experiments with the use of CBMC samples. Because x-3 PUFA-supplemented and control groups were well balanced for all main covariates (including maternal age, height, weight, BMI, educational level, socioeconomic level, maternal smoking during pregnancy, paternal smoking status, sex, birth weight, and gestational duration), these are unlikely to be confounding factors. Therefore, the main findings of our study are unlikely to be influenced by confounding factors and technical biases.