Hidden hypotheses of epigenetic studies

This 2018 UK review discussed three pre-existing conditions of epigenetic genome-wide association studies:

“Genome-wide technology has facilitated epigenome-wide association studies (EWAS), permitting ‘hypothesis-free’ examinations in relation to adversity and/or mental health problems. Results of EWAS are in fact conditional on several a priori hypotheses:

  1. EWAS coverage is sufficient for complex psychiatric problems;
  2. Peripheral tissue is meaningful for mental health problems; and
  3. The assumption that biology can be informative to the phenotype.

1. CpG sites were chosen as potentially biologically informative based on consultation with a consortium of DNA methylation experts. Selection was, in part, based on data from a number of phenotypes (some medical in nature such as cancer), and thus is not specifically targeted to brain-based, stress-related complex mental health phenotypes.

2. The assumption is often that distinct peripheral tissues are interchangeable and equally suited for biomarker detection, when in fact it is highly probable that peripheral tissues themselves correspond differently to environmental adversity and/or disease state.

3. Analyses result in general statements such as ‘neurodevelopment’ or the ‘immune system’ being involved in the aetiology of a given phenotype. Whether these broad categories play indeed a substantial role in the aetiology of the mental health problem is often hard to determine given the post hoc nature of the interpretation.”


The reviewers mentioned the statistical flaw of assuming that measured entities are interchangeable with one another in item #2. They didn’t mention that this problem also affects item #1 methodologies of averaging CpG methylation measurements in fixed genomic bins or over defined genomic regions. This was discussed in:

The reviewers offered suggestions for reducing the impacts of these three hypotheses. But doing more of the same, only better, won’t necessarily advance science.

Is it too much to ask researchers whose paychecks and reputations depend on a paradigm – such as the “biomarker” mentioned a dozen and a half times – to admit the uselessness of gathering data when the framework in which the data operates isn’t viable?

The truth about complex traits and GWAS described one relevant example of how we already know this framework, its paradigms, and its related techniques aren’t effective:

“The most investigated candidate gene hypotheses of schizophrenia are not well supported by genome-wide association studies, and it is likely that this will be the case for other complex traits as well.”

https://www.sciencedirect.com/science/article/pii/S2352250X18300940 “Hidden hypotheses in ‘hypothesis-free’ genome-wide epigenetic associations”

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A book review of “Neuroepigenetics and Mental Illness”

A 2018 online book “Neuroepigenetics and Mental Illness” was published at https://www.sciencedirect.com/bookseries/progress-in-molecular-biology-and-translational-science/vol/158/suppl/C (not freely available). Three chapters are reviewed here, with an emphasis on human studies.

“Chapter Five: Neuroepigenetics of Prenatal Psychological Stress” https://www.sciencedirect.com/science/article/pii/S1877117318300747 (not freely available)

“Chapter Eleven: Using Epigenetic Tools to Investigate Antidepressant Response” https://www.sciencedirect.com/science/article/pii/S1877117318300711 (not freely available)

“Chapter Twelve: Transgenerational Epigenetics of Traumatic Stress” https://www.sciencedirect.com/science/article/pii/S187711731830053X (not freely available)


Actually, I won’t waste my time or your time with what I planned to do. The lack of scientific integrity and ethics displayed by the book’s publisher, editor, and contributors in the below chapter spoke volumes.

How can the information in any other chapter of this book be trusted?


“Chapter Twelve: Transgenerational Epigenetics of Traumatic Stress”

This chapter continued propagating a transgenerational meme that had more to do with extending paradigms than science. The meme is that there are adequately evidenced transgenerational epigenetic inheritance human results.

As I most recently noted in Epigenetic variations in metabolism, there aren’t any published human studies that provide incontrovertible evidence from the F0 great-grandparents, F1 grandparents, F2 parents, and F3 children to confirm definitive transgenerational epigenetic inheritance causes and effects. Researchers urgently need to do this human research, and stop pretending it’s already done.

How did the book’s editor overlook what this chapter admitted?

“Literature about the inheritance of the effects of traumatic stress in humans has slowly accumulated in the past decade. However, it remains thin and studies in humans also generally lack clear “cause and effect” association, mechanistic explanations or germline assessment.”

Were the publisher and editor determined to keep the chapter heading and the reviewers determined to add another entry to their CVs in the face of this weasel-wording?

“In conclusion, although less studied from a mechanistic point of view, inter- and possibly transgenerational inheritance of the effects of traumatic stress is supported by empirical evidence in humans.”

See the comments below for an example of the poor substitutes for evidence that propagators of the transgenerational meme use to pronounce human transgenerational epigenetic inheritance a fait accompli.

Epigenetic variations in metabolism

This 2018 German review was comprehensive for its subject, epigenetic control of variation and stochasticity in metabolic disease. I’ll focus on one aspect, phenotypic variation:

“Phenotypic [Mendelian] variation can result both from gain- and loss-of-function mutations. Because of the extreme interconnectivity of cell regulatory networks, even at the cellular level, predicting the impact of a sequence variant is difficult as the resultant variation acts:

  • In the context of all other variants and
  • Their potential additive, synergistic and antagonistic interactions.

This phenomenon is known as epistasis.

∼98.5% of our genome is non-protein-coding: it is pervasively transcribed, and its transcripts can support regulatory function. Among the best functionally characterized non-coding RNAs (ncRNAs) arising from these sequences are microRNAs (miRNAs)

Environmental [non-Mendelian] variation or ‘stimuli’ occurring during critical windows of susceptibility can elicit lifelong alterations in an individual’s phenotype. Intergenerational metabolic reprogramming [in fruit flies] results from global alterations in chromatin state integrity, particularly from reduced H3K27me3 and H3K9me3 [histone] domains.

The broad variation of fingerprints in humans is thought to depend to a large degree on stochastic variation in mechanical forces. These clear examples of inducible multi-stable or stochastic variation highlight how little we know about the landscape of potential phenotypic variation itself.

Consensus estimates of heritability for obesity and T2D are ∼70% and ∼35% respectively. The remaining, unexplained component is known to involve gene–environment interactions as well as non-Mendelian players.”


Although the above graphic displays transgenerational inheritance for humans, the reviewers didn’t cite any human studies that adequately demonstrated causes for and effects of transgenerational epigenetic inheritance.

I’ve read the cited Swedish and Dutch studies. Their designs, methods, and “correlate with” / “was associated with” results didn’t provide incontrovertible evidence from the F0 great-grandparents, F1 grandparents, F2 parents, and F3 children. It’s necessary to thoroughly study each generation to confirm definitive transgenerational epigenetic inheritance causes and effects.

As noted in How to hijack science: Ignore its intent and focus on the 0.0001%, there aren’t any such published studies to cite. Researchers urgently need to do this human research, and stop using these poor substitutes [1] to pretend there are already adequately evidenced transgenerational epigenetic inheritance human results.

I downgraded the review for treating research of this and other subjects as faits accomplis. It’s opposite ends of the evidential spectrum to state “how little we know about the landscape of potential phenotypic variation,” and in the same review, speciously extrapolate animal experiments into putative human results.

https://www.sciencedirect.com/science/article/pii/S2212877818301984 “Epigenetic control of variation and stochasticity in metabolic disease”


[1] As an example of the poor substitutes for evidence, a researcher referred me to the 2013 “Transgenerational effects of prenatal exposure to the 1944–45 Dutch famine” which is freely available at https://obgyn.onlinelibrary.wiley.com/doi/full/10.1111/1471-0528.12136 as a study finding human transgenerational epigenetic inheritance.

The methods section showed:

  • The study’s non-statistical data was almost all self-reported by a self-selected sample of the F2 grandchildren, average age 37.
  • No detailed physical measurements or samples were taken of them, or of their F1 parents, or of their F0 grandparents, all of which are required as baselines for any transgenerational epigenetic inheritance.
  • No detailed physical measurements or samples were taken of their F3 children, which is the generation that may provide evidence for transgenerational findings if the previous generations also have detailed physical baselines.

The study’s researchers drew enough participants (360) such that their statistics package allowed them to impute and assume into existence a LOT of data. But the scientific method constrained them to make factual statements of what the evidence actually showed. They admitted:

“In conclusion, we did not find a transgenerational effect of prenatal famine exposure on the health of grandchildren in this study.”

Yet this study is somehow cited for evidence of human transgenerational epigenetically inherited causes and effects.

A mid-year selection of epigenetic topics

Here are the most popular of the 65 posts I’ve made so far in 2018, starting from the earliest:

The pain societies instill into children

DNA methylation and childhood adversity

Epigenetic mechanisms of muscle memory

Sex-specific impacts of childhood trauma

Sleep and adult brain neurogenesis

This dietary supplement is better for depression symptoms than placebo

The epigenetic clock theory of aging

A flying human tethered to a monkey

Immune memory in the brain

The lack of oxygen’s epigenetic effects on a fetus

A disturbance in the paradigm of child abuse

The principal way science advances is through the principle Einstein expressed as:

“No amount of experimentation can ever prove me right; a single experiment can prove me wrong.”

Members of the scientific community and of the public should be satisfied that the scientific process is working well when hypotheses are discarded due to nonconfirming evidence. Researchers should strive to develop evidence that rejects paradigms, and be lauded for their efforts.

The opposite took place with this 2018 commentary on two studies where the evidence didn’t confirm current biases. I curated one of these studies in DNA methylation and childhood adversity.

The commentators’ dismissive tone was set in the opening paragraph:

“Is early exposure to adversity associated with a genetic or an epigenetic signature? At first glance, two articles in this issue -..and the other from Marzi et al., who measured genome-wide DNA methylation in a prospective twin cohort assessed at age 18 – appear to say that it is not.”

The two commentators, one of whom was a coauthor of Manufacturing PTSD evidence with machine learning, went on to protect their territory. Never mind the two studies’ advancement of science that didn’t coincide with the commentators’ vested interests.


My main concern with the study was that although the children had been studied at ages 5, 7, 10, 12, and 18, the parents had never been similarly evaluated! The researchers passed up an opportunity to develop the parents as a F0 generation for understanding possible human transgenerational inherited epigenetic causes and effects.

The study focused on the children’s intergenerational epigenetic effects. However, animal studies have often demonstrated transgenerational effects that skip over the F1 generation children!

For example:

https://ajp.psychiatryonline.org/doi/pdf/10.1176/appi.ajp.2018.18020156 “Considering the Genetic and Epigenetic Signature of Early Adversity Within a Biopsychosocial Framework” (not freely available)

The hypothalamus and aging

This 2018 Korean review discussed aspects of the hypothalamus and aging:

“A majority of physiological functions that decline with aging are broadly governed by the hypothalamus, a brain region controlling development, metabolism, reproduction, circadian rhythm, and homeostasis. In addition, the hypothalamus is poised to connect the brain and the body so that the environmental information affecting aging can be transmitted through the hypothalamus to affect the systematic aging of the peripheral organs.

The hypothalamus is hypothesized to be a primary regulator of the process of aging of the entire body. This review aims to assess the contribution of hypothalamic aging to the age-related decline in body functions, particularly from the perspective of:

  • energy homeostasis,
  • hormonal balance,
  • circadian rhythm, and
  • reproduction,

and to highlight its underlying cellular mechanisms with a focus on:

  • nutrient sensing
  • inflammation,
  • loss of stem cell,
  • loss of proteostasis, and
  • epigenetic alterations.”


The reviewers didn’t consider aging to be an “unintended consequence” of development. This perspective was found in a reference to A study of DNA methylation and age:

“Aging is not and cannot be programmed. Instead, aging is a continuation of developmental growth, driven by genetic pathways.

Genetic programs determine developmental growth and the onset of reproduction. When these programs are completed, they are not switched off.

Aging has no purpose (neither for individuals nor for group), no intention. Nature does not select for quasi-programs. It selects for robust developmental growth.”

The epigenetic clock theory of aging cited the same author, and modified his point to say:

“The proposed epigenetic clock theory of ageing views biological ageing as an unintended consequence of both developmental programmes and maintenance programmes.”

This review’s opposite paradigm was:

“The hypothalamus is hypothesized to be a primary regulator of the process of aging.”

Almost all of the details discussed were from rodent studies.


I favor the “unintended consequence” explanation of aging. As detailed in How to cure the ultimate causes of migraines? and its references, the hypothalamus is a brain structure that lacks feedback mechanisms for several of its activities.

This structure develops shortly after conception and has an active prenatal role. The hypothalamus plays its part in getting us developed and ready to reproduce, with several feedback loops being evolutionarily unnecessary.

The hypothalamus perfectly illustrates the point of:

“When these programs are completed, they are not switched off.”

Should hypothalamic activity not winding down when its developmental role is over be interpreted to construe a role that has some other meaning or purpose as we age?

https://www.sciencedirect.com/science/article/pii/S0047637418300502 “Role of hypothalamus in aging and its underlying cellular mechanisms” (not freely available)

The lack of oxygen’s epigenetic effects on a fetus

This 2018 Loma Linda review subject was gestational hypoxia:

“Of all the stresses to which the fetus and newborn infant are subjected, perhaps the most important and clinically relevant is that of hypoxia. This review explores the impact of gestational hypoxia on maternal health and fetal development, and epigenetic mechanisms of developmental plasticity with emphasis on the uteroplacental circulation, heart development, cerebral circulation, pulmonary development, and the hypothalamic-pituitary-adrenal axis and adipose tissue.

An understanding of the specific hypoxia-induced environmental and epigenetic adaptations linked to specific organ systems will enhance the development of target-specific inhibition of DNA methylation, histone modifications, and noncoding RNAs that underlie hypoxia-induced phenotypic programming of disease vulnerability later in life.

A potential stumbling block to these efforts, however, relates to timing of the intervention. The greatest potential effect would be accomplished at the critical period in development for which the genomic plasticity is at its peak, thus ameliorating the influence of hypoxia or other stressors.

With future developments, it may even become possible to intervene before conception, before the genetic determinants of the risk of developing programmed disease are established.”

Table 3 “Antenatal hypoxia and developmental plasticity” column titles were Species | Offspring Phenotypes of Disorders and Diseases | Reference Nos.

Hypoxia phenotypes


This review was really an ebook, with 94 pages and 1,172 citations in the pdf file. As I did with Faith-tainted epigenetics, I read it with caution toward recognizing the influence of the sponsor’s biases, and any directed narrative that ignored evidence contradicting the narrative, and any storytelling.

See if you can match the meaning of the review’s last sentence (“intervene before conception” quoted above) with the meaning of any sentence in its cited reference Developmental origins of noncommunicable disease: population and public health implications.

One review topic that was misconstrued was transgenerational epigenetic inheritance of hypoxic effects. The “transgenerational” term was used inappropriately by several of the citations, and no cited study provided evidence for gestational hypoxic effects through the  F2 grandchild and F3 great-grandchild generations.

One omitted topic was gestational hypoxic effects of caffeine. The first paper that came up for my PubMed search of “caffeine pregnancy hypoxia” was an outstanding 2017 Florida rodent review Long-term consequences of disrupting adenosine signaling during embryonic development that had this paragraph and figure:

“One substance that fetuses are frequently exposed to is caffeine, which is a non-selective adenosine receptor antagonist. We discovered that in utero alteration in adenosine action leads to adverse effects on embryonic and adult murine hearts. We find that cardiac A1ARs [a type of adenosine receptor] protect the embryo from in utero hypoxic stress, a condition that causes an increase in adenosine levels. 

After birth in mice, we observed that in utero caffeine exposure leads to abnormal cardiac function and morphology in adults, including an impaired response to β-adrenergic stimulation. Recently, we observed that in utero caffeine exposure induces transgenerational effects on cardiac morphology, function, and gene expression.”

The timing of in utero caffeine treatment leads to differences in adult cardiac function, gene expression, and phenotype. Exposure to caffeine from E6.5–9.5 leads the F1 generation to develop dilated cardiomyopathy with decrease % FS and increased Myh7 expression. In utero caffeine exposure from E10.5–13.5 leads to a hypertrophic cardiomyopathy in the F2 generation along with increased % FS and decreased Myh7 expression

Why was this review and its studies omitted? It was on target for both gestational hypoxia and transgenerational epigenetic inheritance of hypoxic effects!

It was alright to review smoking, cocaine, methamphetamine, etc., but the most prevalent drug addiction – caffeine – couldn’t be a review topic?


The Loma Linda review covered a lot, but I had a quick trigger due to the sponsor’s bias. I started to lose “faith” in the reviewers after reading the citation for the review’s last sentence that didn’t support the statement.

My “faith” disappeared after not understanding why a few topics were misconstrued and omitted. Why do researchers and sponsors ignore, misrepresent, and not continue experiments through the F3 generation to produce evidence for and against transgenerational epigenetic inheritance? Where was the will to follow evidence trails regardless of socially acceptable beverage norms?

The review acquired the taint of storytelling with the reviewers’ assertion:

“..timing of the intervention. The greatest potential effect would be accomplished at the critical period in development for which the genomic plasticity is at its peak, thus ameliorating the influence of hypoxia or other stressors.”

Contradictory evidence was in the omitted caffeine study’s graphic above which described two gestational critical periods where an “intervention” had opposite effects, all of which were harmful to the current fetus’ development and/or to following generations. Widening the PubMed link’s search parameters to “caffeine hypoxia” and “caffeine pregnancy” returned links to human early life studies that used caffeine in interventions, ignoring possible adverse effects on future generations.

This is my final curation of any paper sponsored by this institution.

https://www.physiology.org/doi/abs/10.1152/physrev.00043.2017 “Gestational Hypoxia and Developmental Plasticity” (not freely available) Thanks to coauthor Dr. Xiang-Qun Hu for providing a copy.