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 in item #2 the statistical flaw of assuming that measured entities are interchangeable with one another. 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.

Measuring epigenetic changes at a single-cell level

This 2018 Canadian cell study described the development of a single-cell protocol to:

“Profile primitive hematopoietic cells of mouse and human origin to identify epigenetically distinct subpopulations. Deep sampling of the CpG content of individual HSCs allowed for the near complete reconstitution of regulatory states from epigenetically defined subpopulations of HSCs and revealed a high level of redundancy of CpG methylation states within these phenotypically defined hematopoietic cell types.

Hematopoietic stem cells (HSCs) are functionally defined cells that display evidence of extensive self-renewal of their ability to generate mature blood cells for the lifetime of the organism and following transplantation into myelosuppressed permissive hosts. Most of the epigenetic measurements underpinning these observations represent consensus values experimentally derived from thousands of cells partially enriched in HSCs or their progeny, thus failing to discern distinct epigenetic states within HSCs.

Current analytical strategies for single-cell DNA methylation measurements average DNA methylation in fixed genomic bins or over defined genomic regions. However, inference across cells (as well as sequence context) assumes homogeneity across cells, which is at cross-purposes with the generation of single-cell molecular measurements through the potential to mask rare subpopulations.

We identified donor as a significant source of consistent epigenetic heterogeneity, which was reduced but not eliminated by correcting for personal genetic variants. This observation is consistent with previous reports that showed genetic diversity as related to but not accountable for all DNA methylation differences and suggests that in utero environmental differences may be encoded within the HSC compartment.”


The study advanced science not only by measuring single-CpG methylation within each HSC but also by producing another data point “that in utero environmental differences may be encoded within the HSC compartment.”

The paragraph with “assumes homogeneity across cells” bold text provided another example of the statistical analysis flaw that gives individually inapplicable results per Group statistics don’t necessarily describe an individual. The above graphic of human hematopoietic phenotypes demonstrated that the researchers have potentially solved this problem by measuring individual cells.

The researchers discussed another aspect of the study that’s similar to the epigenetic clock methodology:

“Phenotype-specific methylation signatures are characterized by extensive redundancy such that distinct epigenetic states can be accurately described by only a small fraction of single-CpG methylation states. In support of such a notion, the unique components of a DNA methylation “age” signature are contained in ∼353 CpGs sites, presumably representing a random sample of a total age signature that involves many more sites not detected using the reduced representation strategies from which these signatures have been derived.”

Also, in The epigenetic clock theory of aging the originator of the epigenetic clock characterized HSCs as an effective intervention against epigenetic aging:

“In vivo, haematopoietic stem cell therapy resets the epigenetic age of blood of the recipient to that of the donor.”

https://www.cell.com/stem-cell-reports/article/S2213-6711(18)30308-4/fulltext “High-Resolution Single-Cell DNA Methylation Measurements Reveal Epigenetically Distinct Hematopoietic Stem Cell Subpopulations”

Allergies and epigenetic histone modifications

This 2018 German review provided short summaries of 44 studies on the contribution of histone modifications to allergies. An overall summary of their search results was:

“There are at least two levels at which the role of histone modifications is manifested.

  • One is the regulation of cells that contribute to the allergic inflammation (T cells and macrophages) and those that participate in airway remodeling.
  • The other is the direct association between histone modifications and allergic phenotypes.

Inhibitors of histone-modifying enzymes may potentially be used as anti-allergic drugs. Furthermore, epigenetic patterns may provide novel tools in the diagnosis of allergic disorders.”


This type of search is what’s expected of researchers who will perform either:

  • A meta-analysis of studies selected from the search results; or
  • Their own study.

These reviewers didn’t indicate that they were proceeding along either path.

The review was fine for the purpose of presenting current studies of the subject. But the review was just the preparatory stage of research.

https://aacijournal.biomedcentral.com/articles/10.1186/s13223-018-0259-4 “Histone modifications and their role in epigenetics of atopy and allergic diseases”

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

Transgenerational epigenetic effects of maternal obesity during pregnancy

This 2018 Belgian review subject was in part the transgenerational epigenetic effects of maternal obesity during pregnancy. The subject was tailored for the journal in which it appeared, Atherosclerosis, so other transgenerationally inherited epigenetic effects weren’t reviewed:

“The transgenerational impact of these alterations in methylation patterns are only shown in animal studies with HFD [high-fat diet] animals. In this respect the paternal influence also comes forward.

Alterations in methylation at the spermatozoa of male rats fed with a HFD were shown in combination with transgenerational metabolic effects, mainly on the female offspring. Methylation alterations in spermatozoa were also found in the male offspring of dams fed with HFD during their pregnancy. Consequent effects on the phenotype were again only shown in female offspring (until third generation).

A transgenerational inheritance through the female germline by mitochondrial inheritance has been suggested. A recent, small study in humans found altered mitochondrial functioning in the male offspring of overweight woman. A finding that has been confirmed in mice studies with a persistence of this transfer of aberrant oocyte mitochondria into the third generation.

The identification of a number of alterations in active cardiovascular microRNA species in the offspring of animals with obesity offer promising perspectives for the future.”

Evidence for transgenerational aspects of in utero programming included two studies I hadn’t previously curated:

  1. https://www.cell.com/cell-reports/fulltext/S2211-1247(16)30663-5 “Maternal Metabolic Syndrome Programs Mitochondrial Dysfunction via Germline Changes across Three Generations” (2016)
  2. https://www.sciencedirect.com/science/article/pii/S221287781500232X “High-fat diet reprograms the epigenome of rat spermatozoa and transgenerationally affects metabolism of the offspring” (2016)

https://www.atherosclerosis-journal.com/article/S0021-9150(18)30328-9/fulltextIn utero programming and early detection of cardiovascular disease in the offspring of mothers with obesity”