MTHFR regulatory effects on methylation of CG05575921 in response to smoking: Effects are also discernable using MTHFR expression

To the Editor: In this letter, we present novel research replicating and extending recently reported findings by Beach et al. (2017a) using a new sample of young adult African Americans and a focus on methylene tetrahydrofolate reductase (MTHFR) expression to show generalization across a different method of indexing MTHFR availability. Beach et al. (2017a) suggested that, because MTHFR is the rate-limiting enzyme in the methyl cycle, a pathway critical for folate availability, it is potentially a source of individual differences in efficiency of methylation repair activities (Stover, 2009), and so degree of demethylation at cg05575921 in response to smoking. Reduced MTHFR availability, whether indexed via greater methylation or lower gene expression, should enhance demethylation in response to smoking. Conversely, greater MTHFR availability, whether indexed by lower methylation or greater gene expression, should blunt the impact of smoking on demethylation of cg05575921. Providing initial support for this hypothesis, Beach et al. (2017a) showed that level of methylation of MTHFR (i.e., mMTHFR) moderated the impact of smoking on CG05575921 demethylation in two independent samples (N5293; N5368) of African American young adults. Those with greater mMTHFR showed greater demethylation at cgCG05575921 in response to smoking. Level of demethylation of CG05575921 in response to smoking is of particular interest because it captures a key element in the biological regulation of the response to smoking. In brief, change in methylation status at cg05575921 in response to smoking is directly related to the role of AHRR, the gene on which cg05575921 is located, in response to the dioxin and poly aromatic hydrocarbons found in tobacco smoke (Talhout et al., 2011). The initial response to these toxins is a robust cellular response that includes activation of the aryl hydrocarbon receptor (AHR), and in turn, activation of CYP1A1, CYP1A2, and CYP1B1. However, because the unrestrained activity of the xenobiotic response system is potentially dangerous, and so under tight regulatory control, a second nuclear receptor, AHRR, serves as a feedback modulator of the effects of AHR activation, competing for the obligate heterodimer partner of AHR, the aryl hydrocarbon nuclear translocator, and for DNA binding motifs for AHR. The enhancer region of AHRR that includes cg05575921 plays a key role in increased AHRR transcription (Zeilinger et al., 2013). Notwithstanding the clear importance of demethylation at cg05575921 in the response to smoking, several potential concerns can be raised regarding the generalizability of the moderating role of MTHFR availability reported by Beach et al. (2017a). In particular, the initial report focused on mMTHFR to index MTHFR availability. Although the authors suggested that mMTHFR may influence MTHFR availability via effects on MTHFR expression, they did not directly examine MTHFR expression as a moderator of smoking’s effects. If Beach et al. (2017a) account of MTHFR’s role in regulating demethylation at cg05575921 in response to smoking is correct, however, individual differences in level of MTHFR expression should provide an alternative approach to index MTHFR availability, and so should also moderate smoking’s effect on cg05575921. Gene expression, in general, would be expected to show a similar, but reversed, pattern compared to that seen usingMTHFRmethylation. That is, lower levels ofMTHFR expression, should predict greater demethylation at cg05575921 in response to smoking. Conversely, greater MTHFR expression should predict a blunted impact of smoking on demethylation of cg05575921. Accordingly, using a new young adult sample of similar racial composition (African American), sample size (n5334), and sex balance (N5117 males; N5217 females), we conducted a stringent conceptual replication, examining the moderating effect of MTHFR expression on the extent to which self-reported smoking resulted in demethylation at cg05575921. As we did for the prior analyses, we also present analyses including only cotinine verified nonsmokers, thereby replicating findings in a subsample that excludes probable under reporters of smoking. In addition to potential concerns regarding lack of direct examination of MTHFR expression as an index of MTHFR availability, there also are potential concerns regarding lack of controls for the possible impact of secondhand smoke exposure or family history of smoking on methylation in general, and demethylation at cg05575921 in particular. Levels of cotinine are elevated in passive smokers, and so gene expression may also be sensitive to secondhand smoke (Benowitz, Hukkanen, & Jacob, 2009), suggesting the value of controlling secondhand smoke effects or other smoking-related family effects that may influence demethylation of cg05575921. Controlling for these effects may qualify or eliminate apparent moderating effects of MTHFR availability.