Non-CpG DNA methylation

This 2017 Korean review compared and contrasted CpG and non-CpG DNA methylation:

“Non-CpG methylation is restricted to specific cell types, such as pluripotent stem cells, oocytes, neurons, and glial cells..accumulation of methylation at non-CpG sites and CpG sites in neurons seems to be involved in development and disease etiology.

Non-CpG methylation is established during postnatal development of the hippocampus and its levels increase over time. Similarly, non-CpG methylation is scarcely detected in human fetal frontal cortex, but is dramatically increased in later life. This increase in non-CpG methylation occurs simultaneously with synaptic development and increases in synaptic density.

In contrast, CpG methylation occurs during early development and does not increase over time.

Neurons have considerably higher levels of non-CpG methylation than glial cells..The human male ES [embryonic stem] cell line (H1) is more highly methylated than the female ES cell line (H9).

Among the different types of non-CpG methylation (CpA [adenosine], CpT [thymine], and CpC), methylation is most common at CpA sites. For instance, in human iPS [induced pluripotent stem] cells, 5mCs are found in approximately 68.31%, 7.81%, 1.99%, and 1.05% of CpG, CpA, CpT, and CpC sites, respectively.”

The reviewers’ referenced statement:

“CpG methylation occurs during early development and does not increase over time.”

was presented outside of its context. The 2013 cited study’s statement was restricted to selected areas of the rodent hippocampus:

“Consistent with a recent study of the cortex, time-course analyses revealed that CpH [non-CpG] methylation at the selected loci was established during postnatal development of the hippocampus and was then present throughout life, whereas CpG methylation was established during early development. Maturing mouse hippocampal neurons in vitro also showed a gradual increase in CpH, but not CpG, methylation over time.”

Epigenetic study methodologies improved in 2017 had more information on CpA methylation. “CpG and Non-CpG Methylation in Epigenetic Gene Regulation and Brain Function”


DNA methylation and childhood adversity

This 2017 Georgia human review covered:

“Recent studies, primarily focused on the findings from human studies, to indicate the role of DNA methylation in the associations between childhood adversity and cardiometabolic disease in adulthood. In particular, we focused on DNA methylation modifications in genes regulating the hypothalamus-pituitary-adrenal axis as well as the immune system.”

Recommendations in the review’s Epigenetics inheritance and preadaptation theory section included:

“Twin studies offer another promising design to explore the mediation effect of DNA methylation between child adversity and cardiometabolic outcomes..which could rule out heterogeneity due to genetic and familia[l]r environmental confounding.”

As it so happened, the below 2018 study provided some evidence. “The role of DNA methylation in the association between childhood adversity and cardiometabolic disease” (not freely available) Thanks to lead author Dr. Guang Hao for providing the full study.

This 2018 UK human study:

“Tested the hypothesis that victimization is associated with DNA methylation in the Environmental Risk (E-Risk) Longitudinal Study, a nationally representative 1994-1995 birth cohort of 2,232 twins born in England and Wales and assessed at ages 5, 7, 10, 12, and 18 years. Multiple forms of victimization were ascertained in childhood and adolescence (including physical, sexual, and emotional abuse; neglect; exposure to intimate-partner violence; bullying; cyber-victimization; and crime).

Hypothesis-driven analyses of six candidate genes in the stress response (

  1. NR3C1 [glucocorticoid receptor],
  2. FKBP5 [a regulator of the stress hormone system],
  3. BDNF [brain-derived neurotrophic factor],
  4. AVP [arginine vasopressin],
  5. CRHR1 [corticotropin-releasing hormone receptor 1],
  6. SLC6A4 [serotonin transporter]

) did not reveal predicted associations with DNA methylation.

Epigenetic epidemiology is not yet well matched to experimental, nonhuman models in uncovering the biological embedding of stress.”

One of the sad findings was that as the types of trauma inflicted by other people on the subjects increased, so did the percentage of subjects who hurt themselves by smoking. Two-thirds of teens who reported three or more of the seven adolescent trauma types also smoked by age 18. Self-harming behaviors other than smoking weren’t considered.

Another somber finding was:

“Childhood sexual victimization is associated with stable DNA methylation differences in whole blood in young adulthood.

These associations were not observed in relation to sexual victimization in adolescence.”

The researchers guided future studies regarding the proxy measurements of peripheral blood DNA methylation:

“The vast majority of subsequent human studies, including the present one, have relied on peripheral blood. This choice is expedient, but also scientifically reasonable given the aim of detecting effects on stress-related physical health systems that include peripheral circulating processes (immune, neuroendocrine).

But whole blood is heterogeneous, and although cell-type composition can be evaluated and controlled, as in the present study, it does raise the question of whether peripheral blood is a problematic surrogate tissue for research on the epigenetics of stress.

Comparisons of methylomic variation across blood and brain suggest that blood-based EWAS may yield limited information relating to underlying pathological processes for disorders where brain is the primary tissue of interest.”

The comment on “epigenetic epidemiology” overstated the study’s findings because the epigenetic analysis, although thorough, was limited to peripheral blood DNA methylation. Other consequential epigenetic effects weren’t investigated, for example, histone modifications and microRNA expression.

One unstated limitation was that the DNA methylation analyses were constrained by budgets. Studies like The primary causes of individual differences in DNA methylation are environmental factors point out restrictions in the methodology:

“A main limitation with studies using the Illumina 450 K array is that the platform only covers ~1.5 % of overall genomic CpGs, which are biased towards promoters and strongly underrepresented in distal regulatory elements, i.e., enhancers.

WGBS [whole-genome bisulfite sequencing] offers single-site resolution CpG methylation interrogation at full genomic coverage.

Another advantage of WGBS is its ability to access patterns of non-CpG methylation.”

I’d expect that in the future, researchers with larger budgets would reanalyze the study samples with WGBS and other DNA methylation techniques.

The researchers started and ended the study presenting their view of human “embedding of stress” as a fact rather than a paradigm. Epigenetic effects of early life stress exposure compared and contrasted this with another substantiated view.

An opportunity to advance science was missed regarding intergenerational and transgenerational epigenetic inheritance. Wouldn’t the parents’ blood samples and histories – derived from administering the same questionnaires their twins answered at age 18 – likely provide some causal evidence for the children’s observed effects? And lay the groundwork for hypotheses about aspects of future grandchildren’s physiologies and behaviors? “Analysis of DNA Methylation in Young People: Limited Evidence for an Association Between Victimization Stress and Epigenetic Variation in Blood” (not freely available) Thanks to coauthor Dr. Helen Fisher for providing the full study.

Can researchers make a difference in their fields?

The purpose and finding of this 2017 UK meta-analysis of human epigenetics and cognitive abilities was:

“A meta-analysis of the relationship between blood-based DNA methylation and cognitive function.

We identified [two] methylation sites that are linked to an aspect of executive function and global cognitive ability. The latter finding relied on a relatively crude cognitive test..which is commonly used to identify individuals at risk of dementia.

One of the two CpG sites identified was under modest genetic control..there are relatively modest methylation signatures for cognitive function.”

The review’s stated limitations included:

“It is, of course, possible that a reliable blood-based epigenetic marker of cognitive function may be several degrees of separation away from the biological processes that drive cognitive skills.

There are additional limitations of this study:

  • A varying number of participants with cognitive data available for each test;
  • Heterogeneity in relation to the ethnicity and geographical location of the participants across cohorts; and
  • Relating a blood-based methylation signature to a brain-based outcome.

A 6-year window [between ages 70 and 76] is possibly too narrow to observe substantial changes in the CpG levels.”

All of these limitations were known before the meta-analysis was planned and performed. Other “possible” limitations already known by the 47 coauthors include those from Genetic statistics don’t necessarily predict the effects of an individual’s genes.

The paper referenced studies to justify the efforts, such as one (cited twice) coauthored by the lead author of A problematic study of DNA methylation in frontal cortex development and schizophrenia:

“Epigenome-wide studies of other brain-related outcomes, such as schizophrenia, have identified putative blood-based methylation signatures.”

Was this weak-sauce meta-analysis done just to plump up 47 CVs? Why can’t researchers investigate conditions that could make a difference in their fields?

Was this meta-analysis done mainly because the funding was available? I’ve heard that the primary reason there are papers like the doubly-cited one above is that the US NIMH funds few other types of research outside of their biomarker dogma.

The opportunity costs of this genre of research are staggering. Were there no more productive topics that these 47 scientists could have investigated?

Here are a few more-promising research areas where epigenetic effects can be observed in human behavior and physiology:

I hope that the researchers value their professions enough to make a difference with these or other areas of their expertise. And that sponsors won’t thwart researchers’ desires for difference-making science by putting them into endless funding queues. “Meta-analysis of epigenome-wide association studies of cognitive abilities”

Make consequential measurements in epigenetic studies

The subject of this 2017 Spanish review was human placental epigenetic changes:

“39 papers assessing human placental epigenetic signatures in association with either

  • (i) psychosocial stress,
  • (ii) maternal psychopathology,
  • (iii) maternal smoking during pregnancy, and
  • (iv) exposure to environmental pollutants,

were identified.

Their findings revealed placental tissue as a unique source of epigenetic variability that does not correlate with epigenetic patterns observed in maternal or newborn blood.

Each study’s confounders were summarized by a column in Table 1. Some of the reviewers’ comments included:

“33 out of 39 papers reviewed (85%) reported significant associations between either placental DNA methylation or placental miRNA expression and exposure to any of the risk factors assessed. However, the methodological heterogeneity present throughout the studies reviewed does not allow meta-analytic exploration of reported findings.

Heterogeneity regarding the origin of biological tissues analyzed confounds the replicability and validity of reported findings and their potential synthesis.”

Sponsors and researchers really have to take their work seriously if the developmental origins of health and disease hypothesis can advance to a well-evidenced theory. Study designers should:

  1. Sample consequential dimensions. “There were no studies examining histone modifications.” Why were there no human studies in this important category of epigenetic changes in the placenta, the “barrier protecting the fetus”?
  2. Correct methodological deficiencies in advance. Eliminate insufficiencies like “Once collected, processing and storage of placental samples also differed across studies and was not reported in all of them.”
  3. Stop using convenient but non-etiologic proxy assays such as global methylation. How can a study advance the DOHaD hypothesis if everyone knows ahead of time that its outcome will be yet another finding that epigenetic changes “are associated with” non-causal factors?
  4. Forget about non-biological measurements like educational attainment per Does a societal mandate cause DNA methylation?.

Every human alive today has observable lasting epigenetic effects caused by environmental factors during the earliest parts of our lives. Isn’t this sufficient rationale to expect serious efforts by research sponsors and designers? “The impact of prenatal insults on the human placental epigenome: A systematic review” (click the Download PDF link to read the paper)

Epigenetics research and evolution

This 2017 UK essay was a longish review of how epigenetics and other research has informed evolutionary theory:

“There are several processes by which directed evolutionary change occurs—targeted mutation, gene transposition, epigenetics, cultural change, niche construction and adaptation.

Evolution is an ongoing set of iterative interactions between organisms and the environment..Directionality is introduced by the agency of organisms themselves.”

A few takeaway items concerned:

“It is of course the functional phenotype that is ‘seen’ by natural selection. DNA sequences are not directly available for selection other than through their functional consequences.

..the comparative failure of genome-wide association studies to reveal very much about the genetic origins of health and disease. This is one of the most important empirical findings arising from genome sequencing.

Environmental epigenetic impacts on biology and disease include:

  • Worldwide differences in regional disease frequencies
  • Low frequency of genetic component of disease as determined with genome wide association studies (GWAS)
  • Dramatic increases in disease frequencies over past decades
  • Identical twins with variable and discordant disease frequency
  • Environmental exposures associated with disease
  • Regional differences and rapid induction events in evolution

The above list was from the cited 2016 review “Developmental origins of epigenetic transgenerational inheritance”

I was especially interested in the points about behavior’s role in evolution:

“Differential mutation rates are not essential to enable organisms to guide their own evolution.

If organisms have agency and, within obvious limits, can choose their lifestyles, and if these lifestyles result in inheritable epigenetic changes, then it follows that organisms can at least partially make choices that can have long-term evolutionary impact.”

These discussions provided support for the central question of The PRice “equation” for individually evolving: Which equation describes your life?:

“Applying the “How does a phenotype influence its own change?” question to a person:

How can a person remedy their undesirable traits – many of which are from their ancestral phenotype – and acquire desirable traits?” “Was the Watchmaker Blind? Or Was She One-Eyed?”

Epigenetic study methodologies improved in 2017

Let’s start out 2018 paying more attention to advancements in science that provide sound empirical data and methodology. Let’s ignore and de-emphasize studies and reviews that aren’t much more than beliefs couched in models and memes, whatever their presumed authority.

Let sponsors direct researchers to focus on ultimate causes of diseases. Let’s put research of treatments affecting causes ahead of those that only address symptoms.

Here are two areas of epigenetic research that improved in 2017.

Improved methodologies enabled DNA methylation studies of adenine, one of the four bases of DNA, to advance, such as this 2017 Wisconsin/Minnesota study N6-methyladenine is an epigenetic marker of mammalian early life stress:

“6 mA is present in the mammalian brain, is altered within the Htr2a gene promoter by early life stress and biological sex, and increased 6 mA is associated with gene repression. These data suggest that methylation of adenosine within mammalian DNA may be used as an additional epigenetic biomarker for investigating the development of stress-induced neuropathology.”

Most DNA methylation research is performed on the cytosine and guanine bases.

Other examples of improved methodologies were discussed in this 2017 Japanese study Genome-wide identification of inter-individually variable DNA methylation sites improves the efficacy of epigenetic association studies:

“A strategy focusing on CpG sites with high DNA methylation level variability may attain an improved efficacy..estimated to be 3.7-fold higher than that of the most frequently used strategy.

With ~90% coverage of human CpGs, whole-genome bisulfite sequencing (WGBS) provides the highest coverage among the currently available DNAm [DNA methylation] profiling technologies. However, because of its high cost, it is presently infeasible to apply WGBS to large-scale EWASs [epigenome-wide association studies], which require DNAm profiling of hundreds or thousands of subjects. Therefore, microarrays and targeted bisulfite sequencing are currently practicable for large-scale EWASs and thus, effective strategies to select target regions are essentially needed to improve the efficacy of epigenetic association studies.

DNAm levels measured with microarrays are invariable for most CpG sites in the study populations. As invariable DNAm signatures cannot be associated with exposures, intermediate phenotypes, or diseases, current designs of probe sets are inefficient for blood-based EWASs.”

How to cure the ultimate causes of migraines?

Most of the spam I get on this blog comes in as ersatz comments on The hypothalamus couples with the brainstem to cause migraines. I don’t know what it is about the post that attracts internet bots.

The unwanted attention is too bad because the post represents a good personal illustration of “changes in the neural response to painful stimuli.” Last year I experienced three three-day migraines in one month as did the study’s subject. This led to me cycling through a half-dozen medications in an effort to address the migraine causes.

None of the medications proved to be effective at treating the causes. I found one that interrupted the progress of migraines – sumatriptan, a serotonin receptor agonist. I’ve used it when symptoms start, and the medication has kept me from having a full-blown migraine episode in the past year.

1. It may be argued that migraine headache tendencies are genetically inherited. Supporting personal evidence is that both my mother and younger sister have migraine problems. My father, older sister, and younger brother didn’t have migraine problems. Familial genetic inheritance usually isn’t the whole story of diseases, though.

2. Migraine headaches may be an example of diseases that are results of how humans have evolved. From Genetic imprinting, sleep, and parent-offspring conflict:

“..evolutionary theory predicts: that which evolves is not necessarily that which is healthy.

Why should pregnancy not be more efficient and more robust than other physiological systems, rather than less? Crucial checks, balances and feedback controls are lacking in the shared physiology of the maternal–fetal unit.

Both migraine causes and effects may be traced back to natural lacks of feedback loops. These lacks demonstrate that such physiological feedback wasn’t evolutionarily necessary in order for humans to survive and reproduce.

3. Examples of other processes occurring during prenatal development that also lack feedback loops, and their subsequent diseases, are:

A. Hypoxic conditions per Lack of oxygen’s epigenetic effects are causes of the fetus later developing:

  • “age-related macular degeneration
  • cancer progression
  • chronic kidney disease
  • cardiomyopathies
  • adipose tissue fibrosis
  • inflammation
  • detrimental effects which are linked to epigenetic changes.”

B. Stressing pregnant dams per Treating prenatal stress-related disorders with an oxytocin receptor agonist caused fetuses to develop a:

  • “defect in glutamate release,
  • anxiety- and depressive-like behavior,

and abnormalities:

  • in social behavior,
  • in the HPA response to stress, and
  • in the expression of stress-related genes in the hippocampus and amygdala.”

1. What would be a treatment that could cure genetic causes for migraines?

I don’t know of any gene therapies.

2. What treatments could cure migraines caused by an evolved lack of feedback mechanisms?

We humans are who we have become, unless and until we can change original causes. Can we deal with “changes in the neural response to painful stimuli” without developing hopes for therapies or technologies per Differing approaches to a life wasted on beliefs?

3. What treatments could cure prenatal epigenetic causes for migraines?

The only effective solution I know of that’s been studied in humans is to prevent adverse conditions like hypoxia from taking place during pregnancy. The critical periods of our physical development are over once we’re adults, and we can’t unbake a cake.

Maybe science will offer other possibilities. Maybe it will be necessary for scientists to do more than their funding sponsors expect?