Developmental disorders and the epigenetic clock

This 2019 UK/Canada/Germany human study investigated thirteen developmental disorders to identify genes that changed aspects of the epigenetic clock:

“Sotos syndrome accelerates epigenetic aging [+7.64 years]. Sotos syndrome is caused by loss-of-function mutations in the NSD1 gene, which encodes a histone H3 lysine 36 (H3K36) methyltransferase.

This leads to a phenotype which can include:

  • Prenatal and postnatal overgrowth,
  • Facial gestalt,
  • Advanced bone age,
  • Developmental delay,
  • Higher cancer predisposition, and, in some cases,
  • Heart defects.

Many of these characteristics could be interpreted as aging-like, identifying Sotos syndrome as a potential human model of accelerated physiological aging.

This research will shed some light on the different processes that erode the human epigenetic landscape during aging and provide a new hypothesis about the mechanisms behind the epigenetic aging clock.”

“Proposed model that highlights the role of H3K36 methylation maintenance on epigenetic aging:

  • The H3K36me2/3 mark allows recruiting de novo DNA methyltransferases DNMT3A (in green) and DNMT3B (not shown).
  • DNA methylation valleys (DMVs) are conserved genomic regions that are normally found hypomethylated.
  • During aging, the H3K36 methylation machinery could become less efficient at maintaining the H3K36me2/3 landscape.
  • This would lead to a relocation of de novo DNA methyltransferases from their original genomic reservoirs (which would become hypomethylated) to other non-specific regions such as DMVs (which would become hypermethylated and potentially lose their normal boundaries),
  • With functional consequences for the tissues.”

The researchers improved methodologies of several techniques:

  1. “Previous attempts to account for technical variation have used the first 5 principal components estimated directly from the DNA methylation data. However, this approach potentially removes meaningful biological variation. For the first time, we have shown that it is possible to use the control probes from the 450K array to readily correct for batch effects in the context of the epigenetic clock, which reduces the error associated with the predictions and decreases the likelihood of reporting a false positive.
  2. We have confirmed the suspicion that Horvath’s model underestimates epigenetic age for older ages and assessed the impact of this bias in the screen for epigenetic age acceleration.
  3. Because of the way that the Horvath epigenetic clock was trained, it is likely that its constituent 353 CpG sites are a low-dimensional representation of the different genome-wide processes that are eroding the epigenome with age. Our analysis has shown that these 353 CpG sites are characterized by a higher Shannon entropy when compared with the rest of the genome, which is dramatically decreased in the case of Sotos patients.”

https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1753-9 “Screening for genes that accelerate the epigenetic aging clock in humans reveals a role for the H3K36 methyltransferase NSD1”

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A better method of measuring neurogenesis

One of the references cited in Linking adult neurogenesis to Alzheimer’s disease was https://www.nature.com/articles/s41591-019-0375-9 “Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease” (not freely available).

This 2019 Spanish human study used improved techniques to find:

“Adult hippocampal neurogenesis (AHN), confers an unparalleled degree of plasticity to the entire hippocampal circuitry. Direct evidence of AHN in humans has remained elusive. Determining whether new neurons are continuously incorporated into the human dentate gyrus (DG) during physiological and pathological aging is a crucial question with outstanding therapeutic potential.

By combining human brain samples obtained under tightly controlled conditions and state-of-the-art tissue processing methods, we identified thousands of immature neurons in the DG of neurologically healthy human subjects up to the ninth decade of life. These neurons exhibited variable degrees of maturation along differentiation stages of AHN. In sharp contrast, the number and maturation of these neurons progressively declined as AD advanced.

These results demonstrate the persistence of AHN during both physiological and pathological aging in humans and provide evidence for impaired neurogenesis as a potentially relevant mechanism underlying memory deficits in AD that might be amenable to novel therapeutic strategies.”


The control group was 13 neurologically healthy deceased people aged 43 to 87. The AD group was 45 deceased people, distributed among the six Braak stages of the pathology, aged 52 to 97.

Transgenerational diseases caused by great-grandmother DDT exposure

This 2019 rodent study from the labs of Dr. Michael Skinner at Washington State University found:

“The exposure of a gestating female during fetal gonadal sex determination to DDT can promote the epigenetic transgenerational inheritance of obesity and disease.

Transgenerational pathologies (F3 generation) of late puberty, obesity, testis, prostate, and multiple disease were observed in the DDT lineage males. Obesity, ovarian, kidney, and multiple disease transgenerational pathologies (F3 generation) were observed in the DDT lineage females.

Epigenetic biomarkers or diagnostics provide preliminary evidence for preconception diagnosis of increased susceptibility to transgenerational disease in offspring.”


For those of us who thought DDT was discontinued:

“DDT was banned in the USA in 1973, but it is still recommended by the World Health Organization for indoor residual spray. India is by far the largest consumer of DDT worldwide.

India has experienced a 5-fold increase of type II diabetes over the last three decades with a predisposition to obesity already present at birth in much of the population. Although a large number of factors may contribute to this increased incidence of obesity, the potential contribution of ancestral toxicant exposures in the induction of obesity susceptibility requires further investigation.”

Where are the human studies of this subject? Why aren’t follow-on generations’ diseases traced to the likely sources?

How many F3 great-grandchildren of women exposed to DDT during pregnancy are alive today? Millions, tens of millions?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536675 “Sperm epimutation biomarkers of obesity and pathologies following DDT induced epigenetic transgenerational inheritance of disease”

What drives cellular aging?

This 2019 US/UK human cell study by the founder of the epigenetic clock method investigated epigenetic aging:

“It is widely assumed that extension of lifespan is a result of retardation of ageing. While there is no counter-evidence to challenge this highly intuitive association, supporting empirical evidence to confirm it is not easy to acquire.

The scarcity of empirical evidence is due in part to the lack of a good measure of age that is not based on time. In this regard, the relatively recent development of epigenetic clocks is of great interest.

At the cellular level more is known, but from the perspective of what epigenetic ageing is not, rather than what it is. While we still do not know what cellular feature is associated with epigenetic ageing, we can now remove:

  • somatic cell differentiation

from the list of possibilities and place it with

  • cellular senescence,
  • proliferation and
  • telomere length maintenance,

which represent cellular features that are all not linked to epigenetic ageing.”


The study used several agents, including rapamycin, to investigate the hypotheses. Rapamycin isn’t a panacea, but:

“The ability of rapamycin to suppress the progression of epigenetic ageing is very encouraging for many reasons not least because it provides a valuable point-of-entry into molecular pathways that are potentially associated with it. Evidently, the target of rapamycin, the mTOR complex is of particular interest.

The convergence of the GWAS observation with the experimental system described here is a testament of the strength of the skin & blood clock in uncovering biological features that are consistent between the human level and cellular level. It lends weight to the emerging view that the mTOR pathway may be the underlying mechanism that supports epigenetic ageing.”

The limitation section ended with:

“It is important to note that it is inadvisable (actively discouraged) to directly extrapolate the studies here, especially in terms of the magnitude of age suppression, to potential effects of rapamycin on humans.”

https://www.aging-us.com/article/101976/text “Rapamycin retards epigenetic ageing of keratinocytes independently of its effects on replicative senescence, proliferation and differentiation”

Another important transgenerational epigenetic inheritance study

This 2019 Washington State University rodent study from Dr. Michael Skinner’s lab found:

“A cascade of epigenetic alterations initiated in the PGCs [primordial germ cells] appears to be required to alter the epigenetic programming during spermatogenesis to modify the sperm epigenome involved in the transgenerational epigenetic inheritance phenomenon.

Following fertilization there is a DNA methylation erasure to generate the stem cells in the early embryo, which then remethylate in a cell type-specific manner. The DNA methylation erasure is thought to, in part, reset deleterious epigenetics in the germline. However, imprinted gene DNA methylation sites and induced transgenerational epimutations appear to be protected from this DNA methylation erasure.

A germline with an altered epigenome has the capacity to alter the early embryo’s stem cell’s epigenome and transcriptome that can subsequently impact the epigenomes and transcriptomes of all derived somatic cells. Therefore, an altered sperm epigenome has the capacity to transmit phenotypes transgenerationally. Experiments have demonstrated that epigenetic inheritance can also be transmitted through the female germline.

Previously, the agricultural fungicide vinclozolin was found to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs) termed epimutations that help mediate this epigenetic inheritance. The current study was designed to investigate the developmental origins of the transgenerational DMRs during gametogenesis.

The current study with vinclozolin-induced transgenerational inheritance demonstrates that sperm DMRs also originate during both spermatogenesis and earlier stages of germline development, but at distinct developmental stages. This is a genome-wide analysis of epigenetic programming during gametogenesis for transgenerational sperm epimutations.”


The study’s main hypotheses were:

Following fertilization, the hypothesis is that the transgenerational epimutations modify early embryonic transcriptomes and epigenomes to re-establish the cascade for the next generation.

As the individual develops, all somatic cells have altered epigenomes and transcriptomes to promote disease susceptibility later in life.

Researchers: adopt these hypotheses, and don’t limit your study designs to the F1 children as did:

Don’t stop at the F2 grandchildren like:

Continue studies on to F3 descendants who had no direct exposure to the altering stimulus. Keep in the forefront of your research proposals that there are probably more than 10,000,000 F3 great-grandchildren of DES-exposed women just in the US.

https://www.tandfonline.com/doi/pdf/10.1080/15592294.2019.1614417?needAccess=true “Transgenerational sperm DNA methylation epimutation developmental origins following ancestral vinclozolin exposure”

The transgenerational impact of Roundup exposure

This 2019 Washington rodent study from Dr. Michael Skinner’s lab found adverse effects in the grand-offspring and great-grand-offspring following their ancestor’s exposure during pregnancy to the world’s most commonly used herbicide:

“Using a transient exposure of gestating F0 generation female rats found negligible impacts of glyphosate on the directly exposed F0 generation, or F1 generation offspring pathology. In contrast, dramatic increases in pathologies in the F2 generation grand-offspring, and F3 transgenerational great-grand-offspring were observed.

The transgenerational pathologies observed include prostate disease, obesity, kidney disease, ovarian disease, and parturition (birth) abnormalities:

  1. Prostate disease in approximately 30% of F3 generation glyphosate lineage males, a three-fold increase in disease rate over controls.
  2. A transgenerational (F3 generation) obese phenotype was observed in approximately 40% of the glyphosate lineage females and 42% of the glyphosate lineage males.
  3. An increased incidence of kidney disease observed in the F3 generation glyphosate lineage females affecting nearly 40% of females.
  4. A significant increase in ovarian disease observed in the F2 [48% vs. 21% for controls] and F3 [36% vs. 15% for controls] generation glyphosate lineage females.
  5. During the gestation of F2 generation mothers with the F3 generation fetuses, dramatic parturition abnormalities were observed in the glyphosate lineage. The frequency of unsuccessful parturition was 35%. To further investigate the parturition abnormalities an outcross of F3 generation glyphosate lineage males with a wildtype female was performed. There were parturition abnormalities observed with a frequency of 30%.

Classic and current toxicology studies only involve direct exposure of the individual, while impacts on future generations are not assessed. The ability of glyphosate and other environmental toxicants to impact our future generations needs to be considered, and is potentially as important as the direct exposure toxicology done today for risk assessment.”


Why isn’t coverage of this study the top story of the world’s news organizations? Is what’s reported more important than reliable evidence of generational consequences to environmental experiences?

Current toxicology practices are a scientific disgrace:

  • What are the hypotheses of practices that only test effects on somatic cells, that don’t look for generational effects of germ cell modifications?
  • Are they selected for their relative convenience instead of chosen for their efficacy?

Why don’t sponsors fund and researchers perform human studies of transgenerational epigenetic inheritance? For example, from Burying human transgenerational epigenetic evidence:

“From the late 1930s through the early 1970s, DES was given to nearly two million pregnant women in the US alone.

Fourth [F3] generation effects of prenatal exposures in humans have not been reported.

Zero studies of probably more than 10,000,000 F3 great-grandchildren of DES-exposed women just here in the US!

There will be abundant human evidence to discover if sponsors and researchers will take their fields seriously.

https://www.nature.com/articles/s41598-019-42860-0.pdf “Assessment of Glyphosate Induced Epigenetic Transgenerational Inheritance of Pathologies and Sperm Epimutations: Generational Toxicology”

Epigenetic transgenerational inheritance mechanisms that lead to prostate disease

This 2019 Washington rodent study found:

“Ancestral exposure to the toxicant vinclozolin induces an epigenetic transgenerational increase in susceptibility to prostate pathology in F3 [male great-grandchildren] generation rats. These results are in agreement with previous studies which found a transgenerational increase in rates of prostatic epithelial atrophy, cystic hyperplasia, and prostatitis in the transgenerational F3 and F4 [male great-great-grandchildren] generations after exposure of F0 [great-great-grandmother] generation pregnant rats to vinclozolin. These effects were accompanied by transgenerational changes in mRNA expression in F3 generation ventral prostate epithelial cells.

A number of previous transgenerational studies have shown no ventral prostate histopathology or disease detected. Therefore, observations suggest ancestral exposure specificity in the ability to induce the transgenerational inheritance of prostate disease.

There was also no increase in prostate histopathology in the directly exposed F1 [male children] or F2 [male grandchildren] generation vinclozolin lineage rats compared to controls.

prostate pathology

The mechanism by which epigenetic transgenerational inheritance affects prostate epithelium involves control of gene expression by DNA methylation and lncRNAs. It will be necessary to determine the exact gene targets of these epigenetic modifications to determine further mechanisms.

Future studies need to investigate if similar mechanisms are at work in human males who have adult-onset BPH or prostate cancer. Ancestral exposures to toxicants and epigenetic transgenerational inheritance may contribute to the development of prostate disease in men today.”


The study’s above bolded sentence added to the evidence that epigenetic effects may skip generations. A study by the same group, Epigenetic transgenerational inheritance of ovarian disease, found in females:

There was no increase in ovarian disease in direct fetal exposed F1 or germline exposed F2 generation vinclozolin or DDT lineage rats compared to controls.

A disturbance in the paradigm of child abuse referenced other studies that found generation-skipping effects.

Serious researchers are closer to discovering evidence for precise mechanisms of epigenetic transgenerational inheritance. It’s well past time that other researchers performing studies like Burying human transgenerational epigenetic evidence take their work seriously enough to truly investigate human evidence for epigenetic transgenerational inheritance.

What are more important funding priorities than such human studies?

https://www.nature.com/articles/s41598-019-38741-1 “Environmental Toxicant Induced Epigenetic Transgenerational Inheritance of Prostate Pathology and Stromal-Epithelial Cell Epigenome and Transcriptome Alterations: Ancestral Origins of Prostate Disease”