The challenge of common chronic disease

The focus of public health is increasingly on chronic disorders, such as diabetes, cardiovascular disease, mental health and cancer manifesting in adulthood. Unlike monogenic (Mendelian) disorders or infectious disease, where the known primary cause permits an aetiological classification and rational approach to management, common diseases are generally multifactorial in origin with diverse genetic and environmental determinants. This contributes to the highly heterogeneous nature of such conditions, which remain an aetiological and management challenge. A multidisciplinary perspective, which includes genetic analysis, is recognised as the most appropriate approach to studying such conditions.1–3 Genetic associations are now routinely integrated with long-established epidemiological analysis of nutrition, social and other exposures through which health and disease have been traditionally explored.

Extending epidemiology beyond genetics and a single generation

For many years, parental and ancestral experience has been regarded as contributing only indirectly across generations. Clearly, such ‘cultural transmission’, which includes parental nurturing behaviour and social patterning of all kinds, does play an important role in shaping the phenotype/traits of progeny. This is an area of active research integrating information on social circumstances, skills, health and mortality, sometimes across three generations of the same lineage.4 ,5

In contrast, biological inheritance of phenotype from one generation to the next is usually regarded as resulting from transmission of genetic material (DNA) from both parents, plus ‘maternal effects’ either (i) carried within the egg cytoplasm (such as in mitochondria, proteins and RNA molecules), (ii) experienced in utero, primarily through the transplacental passage of nutrients, metabolic signals and toxins, or (iii) experienced early postnatally, via breast feeding and/or the exchange of microbiota. Additionally, the maternal genotype also contributes to the early nutritional environment of progeny through its influence on maternal metabolism during pregnancy. This is best exemplified in studies on insulin sensing and fetal growth.6 It is plausible that a maternal exposure in pregnancy could induce a ‘metabolic cascade’ to subsequent generations, whereby fetal programming could alter later adult metabolism, which, in turn, changes the physiology of the uterus receiving and programming the early embryo of the next generation7 and/or the transplacental metabolic signals to the fetus. Theoretically this process could be genotype independent. The challenges of unravelling the complexities associated with maternal transmission of exposure effects are manifold due to the multiple levels of interdependence (figure 1).

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Figure 1

Schematic pedigree diagram showing the main routes for biological transmission of the effects of exposure to the next generation(s). Left, female line; right, male line. The exposure can potentially affect the germline, the reproductive system and the soma more generally. The traditional pedigree lines (blue) show chromosomal transmission, which, in addition to the DNA, can potentially include exposure-induced epigenetic marks that escape erasure and impact on offspring development. The germline can potentially transmit exposure-induced non-coding RNAs (ncRNAs) that influence offspring development. Exposure induced metabolic changes can set up a ‘metabolic cascade’ such that changes in the reproductive tract influence early embryo programming of the offspring or change metabolic signals across the placenta. An additional maternal transmission route is the influence of the mother's microbiome on that of her child.

Until recently, paternal exposures prior to conception (other than those known to induce DNA mutations) were assumed to have little or no effect on offspring development, primarily due to the lack of a plausible biological mechanism. So ingrained was this view that confirming a lack of paternal exposure effect has been promoted as a useful ‘control’ for social confounders in the study of maternal exposure effects.8 ,9 However, several robust animal experiments exist showing transgenerational phenotypic effects of exposures via the male line.10–16 Human observational studies (reviewed in17) also support this type of non-genetic transmission.

In light of the emerging evidence for both maternal and paternal transmission of non-genetic, non-cultural effects to progeny, consideration must be given to extend traditional epidemiological methodology to include not only early-life paternal and maternal exposures, but also ancestral exposure data. The consequences of such transmission are potentially profound, particularly in relation to past assumptions underpinning much of epidemiological and genetic analyses. In transgenerational epidemiology, assuming cultural inheritance automatically when results make genetic inheritance implausible (and vice versa) is no longer acceptable.

Framing future transgenerational epidemiological research

Although some have adopted the terms intergenerational, multigenerational and transgenerational to mean specific routes and numbers of generations of transmission,18 we use ‘transgenerational’ in a general sense to describe a measurable outcome in one generation (offspring) with exposures in either the mother or the father (prior to conception), or their respective parents or ancestors. ‘Transgenerational’ was first used in this context in the 1980s for the apparent transmission of the trauma suffered by Holocaust survivors to their unexposed offspring.19 This term was also adopted in early speculation about imprinted genes mediating the transmission of exposure information to the next generation20 and is used in recent reviews of the subject in mammals.15 Importantly, the mechanism of transmission must be primarily non-genetic, although mounting evidence implicates genetic factors as moderators of such effects (see below). In prenatal exposure via the mother, the fetus can be assumed to be directly exposed; and here the outcomes of interest in a ‘transgenerational’ context would be in that child's own offspring.

A priority for transgenerational epidemiology is to describe the range of measurable exposures and outcomes mediating transgenerational effects in humans. That is, what exposure at which life stage in parents, grandparents or distant ancestors is associated with a measurable phenotypic outcome in the offspring or subsequent generations? We suggest this observational research is best done in a setting where the effects of social patterning can be accounted for, thereby distinguishing cultural inheritance from a more direct biological transmission across generations. Defining exposure-sensitive periods associated with transgenerational effects will be highly informative in this regard.

Unlike much of the research in the field to date, it is important that future transgenerational epidemiology should not be restricted to the inheritance of acquired characteristics, or ‘phenotypic transmission’ to the next generation. The exposure is key, not the founder, phenotype. In fact, there need not be a measureable phenotype in the exposed (or intermediate) generation. Similarly, phenotypic outcomes should not only be assumed to be detrimental. Although the usual research design in transgenerational epidemiology is to test associations between parental or ancestral exposure and adverse outcomes in unexposed offspring, transgenerational responses/effects can also be protective ‘adaptations’, potentially lessening the risk of adverse outcomes in successive exposed generations. The latter has been demonstrated in a remarkable male line rat experiment where ancestral carbon tetrachloride-induced liver damage led to heritable, epigenetically mediated, reprogramming of hepatic healing, such that exposure in the third generation did not produce liver cirrhosis.21 Lastly, the exposures associated with transgenerational effects need not be adverse. Enriched environments can induce transgenerational responses in mice.22

In summary, a transgenerational perspective is needed if we are to better understand the determinants of major public health problems and adequately assess the value and feasibility of potential interventions. Studies in the field should not be predicated upon a particular mode of transmission from one generation to the next and should make no assumptions about the relative contributions of cultural, genetic or other molecular mechanisms of inheritance. We need to keep an open mind and design appropriate experiments for testing each potential mode of transmission independently.

Food supply

Smoking

Other paternal line exposures

Conclusions from human observations to date

It is fair to say that publicity related to recent publications31 ,39 has drawn some statistical criticism on the issue of multiple testing. Our response points to there being clear prior hypotheses (see table 1) and coherence between different studies/populations. We recognise that research conclusions that extend the public health paradigm need a higher level of evidence than normal, and further replication is needed. However, taken together the above studies demonstrate transgenerational associations between grandpaternal/paternal exposures and health outcomes that are most unlikely to be due to ‘cultural’ inheritance/social patterning or genetic inheritance in isolation. The demonstration of an exposure-sensitive period in mid-childhood prior to puberty, but not in adolescence, provides compelling evidence for some other form of transmission between generations.

Sex differences in transgenerational effects

Sex differences occur in most non-communicable diseases, including metabolic diseases, diabetes, hypertension, cardiovascular disease, psychiatric and neurological disorders and cancer, yet the potential role of non-genetic transgenerational effects in mediating this has rarely been investigated.

Mammalian experiments showing transgenerational responses have reported numerous sex-specific effects after exposure during pregnancy or on paternal exposure before breeding. These can affect offspring of both sexes,14 ,45 solely/predominantly males47 ,48 or solely/predominantly females.12 Thus, in line with the Överkalix results, the phenotypic outcomes are generally not sex-limited. The X and Y chromosomal difference between the sexes could potentially contribute to sex-specific transgenerational effects. This could include hormonal effects as well as effects that are independent of sex steroids. A recent review of sexual dimorphism49 emphasises the differential epigenetic processes in the placenta, noting the abundance of X-linked genes involved in placentogenesis and the early unequal gene expression by the sex chromosomes between males and females. Champagne's comment on sex specificity in transgenerational responses highlights the differences in epigenetic plasticity during gamete maturation.50 The large number of rodent examples of sex differences in outcomes indicates that the sex differences in outcomes observed in the few human studies (summarised above) are not unexpected. Despite this, such differences tend to be viewed with suspicion, perhaps because of the dangers of post-hoc subgroup analysis.51 However, with due attention to testing for gender interaction as part of association studies, these sex-specific effects should inform the search for mediating mechanisms in transgenerational responses.

The combination of sex-specific transmissions and outcomes in the Överkalix study are consistent with X and Y chromosome transmission and have led to hypotheses in which the non-recombining part of the Y chromosome could more easily retain epigenetic marks in the gametes52 or carry a genomic stress ‘sensor’ where DNA damage triggers a non-coding RNA response.17 However, it should be noted that some sex-specific transgenerational effects in mice are incompatible with XY transmission.50 ,53 Nevertheless, it is highly plausible that transgenerational responses are one of the determinants of common non-communicable diseases and also of some of the sex differences. Only a transgenerational extension of epidemiological research can address this question.

Variable outcomes across the generations from ancestral exposure

Interestingly, the phenotypic effect of an ancestral exposure can vary across generations. This may reflect a propensity for one sex to transmit but not manifest the transgenerational effect.53 Using unpredictable maternal absence in neonatal mice as a stress-inducing exposure, Franklin et al found transgenerational effects through the male line that included transmission by intermediate males who were not themselves overtly affected.54 This model has been explored further in terms of sperm non-coding RNAs55 (see below). In contrast, other studies have shown that transgenerational effects may become restricted over multiple generations. For example, in a study of maternal high-fat diet exposure in mice, there was an increase in body size and reduced insulin sensitivity that persisted across two generations via both maternal and paternal lineages. However, an examination of third-generation progeny revealed that only females displayed the increased body size phenotype, and this effect was only transmitted via the paternal lineage.56 As highlighted by these authors, an emerging phenomenon is that some traits extinguish via one or both lineages, whereas others persist across generations, suggesting that divergent mechanisms of transmission may be involved at the same time.

The majority of animal experiments have tended to restrict phenotyping of descendants to the system that was disturbed by the ancestral exposure, for example, dietary exposures with metabolic dysfunction, stress with behavioural outcomes. Given our present state of ignorance, such limited analysis is unwise. Most human multigenerational cohorts have a range of measures that would allow studies to address the question of whether outcomes in descendants are limited to the system being challenged by the exposures or can be of a more generic character. Interestingly, a recent mouse study using a stress-inducing exposure found both behavioural and metabolic disturbances in descendants.55

Transmission between generations

As noted above (figure 1), transmission of exposure information down the maternal line may have several different routes with potential to confound any specific mechanism. Thus, proof-of-principle studies often focus on the male line, even if the initial exposure is during fetal development via the pregnant mother/dam. In the latter situation, the exposed pregnant individual is usually termed F0.

Irrespective of the exposure type and timing, a prerequisite for transgenerational effects beyond the second generation is the involvement of germline transmission, which may occur in several ways. First, by directly impacting on the developing primordial germ cells early in utero, as has been reported in mice.57 In this instance, resulting gametes produced later in life might be anticipated to carry a transmissible ‘memory’ of the original exposure via the mother that can be passed on to subsequent generations. Alternatively, any effect transmitted to the F2 generation via F1 gametes might arise purely in response to the altered development or metabolism of the F1 generation arising from the F0 exposure (see Aiken and Ozanne7 for a discussion of such ‘propagational programming’). These are two distinct processes, each of which might mediate the effects of an F0 exposure on the F2 and subsequent generations, independently of non-gametic maternal effects. It is worth noting that even male gametic transmission does not necessarily require the molecular ‘memory’ of the original exposure to pass through meiosis. There are tight cell–cell junctions between Sertoli-cells and postmeiotic spermatids that theoretically could act as a route for ‘message delivery’ to the zygote and beyond,58 and recent work has shown an influence of seminal fluid in conjunction with uterine factors on offspring metabolic development.59

A role for epigenetics in transgenerational responses

Epigenetic events are an essential mediator of cellular differentiation and therefore development in multicellular organisms. However, the underlying dynamism and responsiveness to subtle subcellular cues that are a hallmark of epigenetic change during development also make the developing epigenetic profile susceptible to external influence, with the potential to ‘program’ the underlying genome towards an altered phenotype in later life.60 The labile nature of some epigenetic marks to environmental influence has made epigenetic processes an appealing mechanism for transgenerational responses. In fact, transgenerational epigenetic inheritance has been widely documented in prokaryotes, fungi, plants and animals, via both the male and female germline. In at least some instances (eg, in Caenorhabditis elegans), initial transmission down the female line can ‘spread’ to encompass paternal transmission in subsequent generations.61

In mammals, data demonstrating the erasure of nearly all epigenetic marks twice during the life course (early postzygotically and in the developing primordial germ cells in early development) have been difficult to reconcile with an epigenetic mode of transgenerational effects. Nevertheless, for decades it has been known that mammals have a set of imprinted genes; those that are selectively silenced in either the maternal or paternal germline during gamete formation, such that only one allele is expression following fertilisation. Genomic imprinting establishes the principle of transgenerational epigenetic inheritance—the gene is active or silent depending on epigenetic marks placed in the parental generation that survive erasure as they pass to the offspring.62 The parent-of-origin-dependent gene expression reflects a robust, evolved response to differences in cellular conditions between egg and sperm development. Imprinted genes escape one of the two phases of genome demethylation between generations, namely in the preimplantation embryo soon after fertilisation.63 Importantly, the epigenetic marks that regulate some imprinting regions have been demonstrated to show interindividual, tissue-specific, age-specific and environmentally sensitive variation.25 ,64–72

Recent mouse experiments have also reported a small percentage of loci that escape demethylation at the second phase during primordial gonadal cell differentiation in the early embryo. These are largely associated with repeat sequences, often adjacent to transposable DNA elements.73 Interestingly, there is growing evidence that some repeat-based transposable elements have gene enhancer activity.74 It is also now clear that many non-imprinted, non-repetitive genomic loci remain subject to epigenetic control in both sperm and eggs. This applies to both DNA methylation75 ,76 and histone modification profiles.77 ,78 Further, emerging data exist that demonstrate intergametic variation in epigenetic profile, supporting a role for environmental and/or genetic influence in determining the epigenetic state transmitted to the next generation.79 ,80

Of particular note is a recent study in mice that provides a plausible mechanism for transgenerational inheritance of environmental information, linking behavioural, neuroanatomical and epigenetic processes. Using an olfactory fear conditioning protocol, wherein an odour (acetophenone) is paired with a mild foot-shock, Dias et al noted a behavioural sensitivity to acetophenone in the next two generations of animals, despite having no exposure to this odour. This was associated with structural changes in the olfactory nervous system accompanying the fear conditioning and distinct hypomethylation of the Olfr151 gene (known to be activated by acetophenone) in sperm of conditioned F0 males and F1-naive offspring.16 The extent to which other specific environmental exposures elicit similarly specific epigenetic changes in gametes, and the extent to which these are retained during postzygotic development, remains unclear but warrants further investigation.

Another recent study in a non-genetic prediabetes mouse model provides further compelling evidence for gametic transmission of environmentally induced epigenetic change.81 In this model, offspring of prediabetic fathers show glucose intolerance and insulin resistance in association with distinct changes in gene expression and DNA methylation patterns in pancreatic islets, including reproducible changes in methylation of insulin signalling genes. Interestingly, the sperm of prediabetic fathers have extensive changes in methylation patterns in sperm, many of which overlapped with that of pancreatic islets in offspring.81

Non-coding RNAs (ncRNAs) as mediators of transgenerational responses

There is evidence for a role of sperm-derived RNAs in mediating paternal transgenerational effects, with several classes of RNA recently identified in sperm that are likely to be essential factors in male fertility, including many Y-linked ncRNAs.82 Such RNAs have the potential to play roles in early postfertilisation development.83

The specific RNA content of sperm has been assessed in response to specific environmental exposures. Diet-induced paternal obesity in mice alters microRNA (miRNA) content and sperm methylation status in male mice in association with altered metabolic function in two subsequent generations of progeny.84 Chronic stress in male mice results in altered miRNA content of sperm in association with reduced HPA stress axis responsiveness in the progeny.85 Recently, exploiting their transgenerational model of unpredictable maternal absence in neonatal mice as a stress-inducing exposure, Mansuy's group have examined the associated miRNA changes in sperm. Injection of sperm RNAs from exposed males into fertilised wild-type oocytes reproduced the behavioural and metabolic alterations in the resulting offspring. Interestingly, affected F2 mice had normal sperm miRNAs, yet their F3 offspring still inherited the behavioural phenotype.55 In humans, the small RNA component of sperm is altered in male smokers, including miRNAs predicted to target the epigenetic regulators.86 As a whole, the emerging picture suggests that the RNA content of sperm is sensitive to environmental exposures, with the potential to influence early embryonic development, including the establishment of the postzygotic epigenome. This is speculated to be an initiating mechanism for transmission of impaired metabolic health to future generations via the male lineage.84

A modifying role for genetics in mediating transgenerational effects

Genetic variation will clearly impact to a variable extent on the apparent heritability of some epigenetic marks as evidenced by multigenerational familial studies87 and exemplified at one extreme by MLH1 germline epimutation analysis. Originally thought to be solely due to inheritance of a ‘pure’ epigenetic variant (DNA hypermethylation) across generations,88 in some cases this is genetically driven, being erased in the germline but faithfully re-established early postzygotically in association with a proximal genetic variant or haplotype.89 ,90 Epigenetic responses to exposures and the propensity to be transmitted to the next generation(s) are likely to have evolved under selective pressure. As such one can expect variation in ‘responsiveness’ to a specific exposure to be underpinned by genetic differences. Compelling data in support of this have recently emerged, highlighting the potential of genetic variation in both cis (regions on the same chromosome) and trans (different chromosome) to mediate the effects of environmental exposures on the neonatal epigenetic profile in humans.91 As such, any study of environmentally driven epigenetic variation transmitted transgenerationally needs to considered on a background of genetic variation as a modifier of this effect. For example, although genetic differences may contribute directly to the phenotype in a specific environment, in a classic G×E manner, the additional layer of epigenetic regulation has the potential to make such effects more nuanced on certain genetic backgrounds. Alternatively, some transgenerational environmentally induced epigenetically mediated effects may be exacerbated in association with other ‘risk’ genotypes. Finally, some transgenerational phenotypic effects are likely to be completely independent of genotype.

Different transmitting mechanisms within the same lineage

As described above, environmental exposures can potentially initiate transgenerational effects through altered miRNA in sperm, but some aspects of the phenotype can be transmitted to later generations in the absence of the original miRNA change.55 This separation of initiation and later transmission has been explored using experimental genetic changes. For example, experimental genetic effects in rodents can set up a developmental environment that promotes transgenerational responses in the absence of the genetic change, for example, by changing weight and food intake in subsequent generations.92 It is plausible that ‘random’ somatic mutations leading to substantial mosaicism might induce epigenetic or ncRNA changes that in turn initiate transgenerational effects, or exposures can induce DNA damage as the first step, for example, stress-related DNA damage.93 Further, recent data in mice have revealed that genetic variation in the genes regulating one carbon metabolism can manifest as distinct and reproducible phenotypic variation across multiple systems and generations.94 Importantly, these defects were dependent upon the genotypes of the maternal grandparents. Embryo transfer experiments revealed that haploinsufficiency in mice leads to two distinct, separable phenotypes: adverse effects on their wild-type daughters' uterine environment, leading to growth defects in wild-type grandprogeny, and the appearance of congenital malformations independent of maternal environment that persist for five generations, likely through transgenerational epigenetic inheritance.94

Biological samples

Many cohorts are establishing DNA banks, but it is also worth bearing in mind that neonatal blood spots (Guthrie cards), collected and stored for many decades in some countries, can be a valuable resource for capturing neonatal epigenome and other data.97 In light of the role of miRNAs, discussed above, the collection of cell-free circulating miRNAs is likely to be a valuable additional resource.98 In terms of ‘future proofing’, serious consideration should be given to storing ‘living genomes’ in the form of EBV-transformed lymphoblastoid cell lines or other viable cells derived from primary tissues (eg, induced pluripotent stem cells). The value of such cells in assessing epigenome associations with early-life experience is an active research area, including how to deal with possible culture-induced genetic and epigenetic changes. If such approaches prove useful, this will offer considerable opportunities for life course and transgenerational epidemiology.