The gist of the Guardian article was that “life experience can affect the genes of subsequent generations,” with a tone enthusiastically selling trans-generational epigenetic inheritance, helped by quotations from the study authors (or, more likely, the public relations department of their institution), including “The gene changes in the children could only be attributed to Holocaust exposure in the parents,” and “To our knowledge, this provides the first demonstration of transmission of pre-conception stress effects resulting in epigenetic changes in both the exposed parents and their offspring in humans.”

So what were these conclusions based on? The cohort was 32 Holocaust survivors and 8 Jewish control individuals, and 22 offspring of the Holocaust survivors and 9 Jewish controls, matched for age, sex, a pertinent FKBP5 genotype and other phenotypic parameters, the design was cross-sectional, the biological sample used was peripheral blood leukocytes, themolecular assay was bisulphite pyrosequencing of 6 CG dinucleotides at an intronic enhancer of the FKBP5 gene, combining the observations in three ‘bins’ of 2, 3 and 1 CG and averaging the DNA methylation values observed in the bins with ≥1 CG before making comparisons. The analysis involved modelling any potential contributors that suggested themselves to the authors, including the FKBP5 rs1360780 genotype, and a number of psychological measures, looking for DNA methylation changes that could be attributed to the direct or parental Holocaust exposure alone.

I really don’t like criticising colleagues, a tendency I’m guessing we all share in science. Giving negative feedback on what is clearly in this case an over-interpreted study should always cause us to think sympathetically about the good intentions of the authors. However, if a study is over-interpreted, and is gaining media traction, and influencing other scientists to pursue similarly poor studies, there needs to be some sort of critical response. So, with apologies to the study authors, I offer the following feedback.

The starting hypothesis is not explicitly stated, but can be inferred to be as follows – people who have survived the trauma of the Holocaust are distinctive for having cells in their bodies that change DNA methylation, and their offspring, not having been exposed to the Holocaust, also have changes in DNA methylation in their cells, making them distinctive compared with other people who are not offspring of Holocaust survivors. What is assumed based on the shared change in DNA methylation is that the parent changed DNA methylation, that this occurred in somatic and germ line cells, and that the offspring inherited the DNA methylation change, a trans-generational inheritance of this epigenetic regulator.

So what exactly did they find in this study? Fundamentally, they found that while the first two bins of DNA methylation values measured for >1 CG showed no difference between groups, the DNA methylation values of the single CG in the third bin were different in the Holocaust survivors themselves compared with controls, and in their offspring compared with controls.

So does this prove the starting hypothesis? The answer is no, the study is pretty typical of all epigenetics studies today for being uninterpretable, for the following reasons:

a. Confounding molecular processes are not studied. I describe these molecular processes in a recent PLOS Genetics Biologue, listing transcription through the locus being tested and the cis effects of DNA sequence variability between individuals upon DNA methylation as major influences that need to be tested. The control group was matched for ethnicity (’Jewish’) but there is clearly enough genetic diversity within an ethnic group to drive DNA methylation differences through methylation quantitative trait loci (mQTLs). All of the effects observed could be secondary to mQTLs or, at this intronic locus, polymorphic transcription of the FKBP5 gene between the individuals tested. Without knowing the effects of these influences, you can’t assume the changes of DNA methylation are independent of these influences in a way that addresses the starting hypothesis.

b. The influence of cell subtype composition is unstudied. Since Andy Houseman demonstrated the effects of leukocyte subtypes on DNA methylation in 2012, the epigenetics field has started to pay more attention to this issue. The peripheral blood tested in the Holocaust study is undoubtedly variable in terms of cell subtypes between individuals; the question is whether the locus tested is differentially DNA methylated between leukocyte subtypes and that there is enough of a systematic shift in proportion of one of these differentially DNA methylated cell subtypes into the test or control group to influence the DNA methylation patterns observed.

In fact, a critical look at the results strongly suggest this to be the major influence occurring in the Holocaust study. The DNA methylation values at the discriminatory CG dinucleotide are between 50-60%. Such intermediate DNA methylation can only be due to 50-60% of the alleles (probably cells) in the population studied being methylated at that locus, and 40-50% being unmethylated. The presence of intermediate DNA methylation indicates that the testing has been performed on what we call a meta-epigenome, or a collection of epigenomes, with mosaicism for the individual specific cell types and their characteristic DNA methylation patterns. To shift from 50-60% DNA methylation has to be due to an altered proportion of cells within the population having DNA methylation at that locus. While this could indeed reflect a specific cell type changing its epigenetic pattern, it has first to be excluded that the change is not merely reflective of cell subtype proportional changes, and that no cell type within the population has, in fact, changed its DNA methylation pattern at all.

Knowing what cell subtype has changed in proportion to cause the DNA methylation difference would be quite interesting in terms of the question being asked by the authors of the study, but would not address the starting hypothesis.

Without wishing to beat this study to death, the fact that the DNA methylation difference that characterises the exposed parents is an increase while that characterising the offspring is adecrease is difficult to reconcile with any sort of heritability mechanism. 

The story being told by the Holocaust study is indeed fascinating as a scientific possibility, and will no doubt prompt others to pursue similar studies. Unfortunately, the story is typical of many in the field of epigenetics, with conclusions drawn based on uninterpretable studies. While there have been high-profile reviews that emphasise the weakness of the evidence for trans-generational epigenetic heritability, especially in humans, these have not been enough to steer people towards the rigourous studies needed to test this attractive but poorly-founded idea. Every week there are uninterpretable epigenetics studies published, the Holocaust study is merely one of many, these authors are merely following prevailing beliefs in over-interpreting their data. However, every such study damages the ‘brand’ of epigenetics a little more. If we want human disease epigenetics to be sustainable as a field of research, we have got to start to do substantially better in designing, executing, interpreting, reviewing and funding these studies.