Results from this study provide compelling evidence that DNAm of genes associated with myelination, when measured in buccal cells, may serve as predictive markers for WMV in a normative pediatric cohort. Importantly, results demonstrate that the relationship between WMV and peripheral epigenetics is contingent upon the developmental phase, such that this relationship appears to subside after early childhood, underscoring the intricate interplay between epigenetic modification and neurodevelopment. Furthermore, our approach provides evidence that the variance in DNAm across the gene body (as gauged by PCA), alongside individual CpG methylation levels throughout the gene and in flanking non-coding regions, are both instrumental metrics of DNA methylation given their capacity to prognosticate anatomical phenotypes downstream. Such findings should inform future research to further probe our understanding of the biological functionality of DNA methylation beyond island regions.
Myelin-related genes play diverse and important roles in white matter development and maintenance. For example, the MOG gene plays a crucial role in the structural component of the myelin sheath through its creation of the myelin oligodendrocyte glycoprotein36–38. Genetic variations of MOG have been associated with decreased total WMV in children with obsessive-compulsive disorder38. Oligodendrocyte cell body size has been related to increased expression of OLIG1 and MOG mRNA levels in post-mortem brain samples from adults with Major Depressive Disorder37. Notably, MYRF expression has been found to proliferate during the premyelinating and myelinating stages of oligodendrocyte development39. Concordantly, MYRF is known to activate the expression of genes related to myelin structure, such as MOG, MBP, MAG, and PLP40. MYRF, therefore, is not only crucial in promoting the maturation of oligodendrocytes, but it is also important for the maintenance of oligodendrocytes even after development and maturation are complete. Koenning and colleagues demonstrated this facet of expression through the ablation of MYRF in knock-out mice, causing degeneration of myelin sheaths in the adult CNS and decreased expression of PLP, MAG, MBP, and MOG41. Despite MYRF importance across the lifespan, our data suggests that peripheral DNA methylation of this gene, among others, is more reflective of WMV in young, but not older, children. Future studies are needed to characterize how the relationship between peripheral DNA methylation of myelin-related genes and WMV may change throughout the life course.
Results from this study highlight the importance of DNA methylation patterns across the gene body, as opposed to levels of DNA methylation at specific CpG sites. Using PCA analyses, we show that CpG site methylation levels from all gene regions (i.e., island, shore, shelf, and open sea) correlate, as can be seen by their loading together on the first principal component (Table 3), indicating that both intragenic CpG methylation levels and variance can predict WMV. Importantly, our group has previously used this method and found associations between parenting styles and epigenetics of hypothalamic-pituitary-adrenal axis genes35 and immune genes34 in a pediatric cohort. Not only did we find that social exposures predicted PC1s, we further demonstrated that PC1 predicts diurnal cortisol slope35 and physical health34, highlighting the exposure to epigenome to physiology pathway. Importantly, we further confirmed the impact of environment by controlling for genetics in a monozygotic twin difference design, in which the co-twin difference in the first principal components of dopaminergic genes predicted the co-twin difference in cognitive function33. Taken together, gene-level DNA methylation summary scores that capture variance across the gene body appear to be an effective strategy for assessing relationships with social exposures, behavior, and physiology.
Most studies of DNAm in relation to brain and behavior have assumed that functionally important DNAm will occur in promoters and that most DNAm underlying brain and behavioral health occur in CpG islands. However, prior research suggests that CpG methylation in shores is also anti-correlated with gene expression levels43,44 and that the methylation status of CpGs in the gene body can correlate with gene expression in the absence of DNAm methylation changes in promoter regions45. A meta-analysis of genome-wide epigenomic and gene expression data from cell lines describes a bell-shape distribution, i.e., the lowest levels of intragenic DNA methylation correspond to both the lowest and highest expressed genes, while the highest methylation levels are associated with genes expressed at intermediate levels46. Additionally, intragenic methylation levels may also affect downstream biology by regulating alternative transcripts, alternative splicing, non-coding RNAs, and transposable elements47. The importance of intragenic DNAm is consistent with cancer studies43,44,48 and a more recent study implicating DNAm at CpG shores in the onset of Alzheimer’s disease49. Thus, we call for more studies to incorporate the assessment of intragenic CpG sites within studies of early life stress exposure, brain, and behavior.
Prior research has documented consistent trajectories of both DNAm and myelination development in early life. Specifically, both DNAm and myelination development are more rapid in early life compared to later in life50,51. Both myelination and DNAm follow a characteristic pattern of change50,51 and are thought to be especially sensitive to environmental impacts during early life51,52. As such, both systems mature alongside the development of various complex behavioral outcomes17,53, and alterations in both are consistently found across neurodevelopmental, psychiatric, and neurodegenerative disorders. Here, we identified a developmental window in which peripheral epigenetic signatures are able to predict WMV. Specifically, these results suggest that myelin-related gene DNAm measured in buccal cells may serve as predictors for WMV in infancy and early childhood but not in middle or late childhood. These results are interesting as the developmental window maps well onto the rapid development and plasticity of both myelination and DNAm. However, we are careful to note the possibility that later events, such as adolescence or trauma, may introduce another opportunity for peripheral signatures to predict brain structure. Future research with longitudinal cohorts consisting of a tight age range could further evaluate these questions.
Generally, at age-variable CpG sites, DNAm levels tend to decrease with age while also increasing in between-person variability50. However, much of the prior research has assessed DNAm change over time using linear models, but a recent study demonstrated that quadratic models may better fit the change over time in early development29. Here, assessing myelination-related gene DNAm, our results are consistent with rapid non-linear change and increased between-person variability after the first year of life29,50,54. Further, because we used principal components, we show that variance across a gene, not just levels, of DNAm increases with age. Such results suggest that quadratic and linear statistical models should be compared for the best model fit in epigenetic change across age, particularity in early development.
There are limitations to this study that are important to note. Notably, the inclusion of another peripheral sample would have improved this study, as it is plausible that another tissue type may more accurately predict brain structure metrics. Importantly, array-based methods of measuring DNAm cannot differentiate between methylation and hydroxymethylation, which play differing roles in transcription regulation. Because our study did not measure downstream implications of DNAm, such as RNA or protein levels, we cannot ascertain the functional impact of DNAm on gene transcription. Additionally, our study is limited by a cross-sectional design and would benefit from reproduction in a longitudinal cohort. However, our cross-sectional cohort is more diverse than typical neuroimaging55 and genomic studies56, consisting of 32% non-White and 20% Hispanic participants. While beyond the scope of this study, future studies should assess if racial, ethnic, or other social detriments to health, such as socio-economic status, moderate the relationships between WMV and peripheral epigenetics. Lastly, because this is a self-selected healthy population from a single geographical location, it is unknown if the results will generalize to other populations or ages.
Synoptically, these results highlight a strong relationship between the peripheral DNAm of myelin-related genes and WMV in young children but not older children. We demonstrate low variability in myelin-related epigenetic variance at birth, which rapidly increases non-linearly with age when measured in a peripheral sample. Potentially, increased variance with age is at least partially reflecting individual differences in social determinants of health known to impact the epigenome in development, such as socioeconomic status, nutrition, toxin exposures, and stress. Importantly, we also demonstrate that peripheral epigenetic profiles of myelin-related genes are highly correlated with profiles measured in the brain from an independent cohort. Taken together, these results suggest that peripheral myelinating-epigenetic profiles may serve as indicators of developmental milestones or developmental delays due to either genetic or environmental impacts on WMV development29,54. These results corroborate previous evidence that the peripheral epigenome can predict brain structure/function and behavior. The question of whether peripheral tissues can accurately reflect the epigenetics of brain tissue is an ongoing research question and needs to be continuously examined. Our study, however, suggests that the well-known “tissue issue” may be less of an issue after all within certain systems, and perhaps a more complex and further refined composite of peripheral DNAm may one day serve as a biomarker of overall brain health.