Identical twins have been a source of
fascination in human cultures for millennia, and this fascination
continues right into the present day. Just taking Western European
literature as one source, we can find the identical twins
Menaechmus and Sosicles in a work of Plautus from around 200 B.C.;
the re-working of the same story by Shakespeare in The Comedy of
Errors, written around 1590; Tweedledum and Tweedledee in Lewis
Carroll’s Through the Looking-Glass, and What Alice Found
There written in 1871; right up to the Weasley twins in the
Harry Potter novels of J. K. Rowling. There is something
inherently intriguing about two people who seem exactly the same as
one another.
But there is something that interests all
of us even more than the extraordinary similarities of identical
twins, and that is when we can see their differences. It’s a device
that’s been repeatedly used in the arts, from Frederic and Hugo in
Jean Anhouil’s Ring around the Moon to Beverley and Elliott
Mantle in David Cronenberg’s Dead Ringers. Taking this to
its extreme you could even cite Dr Jekyll and his alter ego Mr
Hyde, the ultimate ‘evil twin’. The differences between identical
twins have certainly captured the imaginations of creative people
from all branches of the arts, but they have also completely
captivated the world of science.
The scientific term for identical twins
is monozygotic (MZ) twins. They were both derived from the same
single-cell zygote formed from the fusion of one egg and one sperm.
In the case of MZ twins the inner cell mass of the blastocyst split
into two during the early cell divisions, like slicing a doughnut
in half, and gave rise to two embryos. And these embryos are
genetically identical.
This splitting of the inner cell mass to
form two separate embryos is generally considered a random event.
This is consistent with the frequency of MZ twins being pretty much
the same throughout all human populations, and with the fact that
identical twins don’t run in families. We tend to think of MZ twins
as being very rare but this isn’t really the case. About one in
every 250 full-term pregnancies results in the birth of a pair of
MZ twins, and there are around ten million pairs of identical twins
around the world today.
MZ twins are particularly fascinating
because they help us to determine the degree to which genetics is
the driving force for life events such as particular illnesses.
They basically allow us to explore mathematically the link between
the sequences of our genes (genotype) and what we are like
(phenotype), be this in terms of height, health, freckles or
anything else we would like to measure. This is done by calculating
how often both twins in a pair present with the same disease. The
technical term for this is the concordance rate.
Achondroplasia, a relatively common form
of short-limbed dwarfism, is an example of a condition in which MZ
twins are almost invariably affected in the same way. If one twin
has achondroplasia, so does the other one. The disease is said to
show 100 per cent concordance. This isn’t surprising as
achondroplasia is caused by a specific genetic mutation. Assuming
that the mutation was present in either the egg or the sperm that
fused to form the zygote, all the daughter cells that form the
inner cell mass and ultimately the two embryos will also carry the
mutation.
However, relatively few conditions show
100 per cent concordance, as the majority of illnesses are not
caused by one overwhelming mutation in a key gene. This creates the
problem of how to determine if genetics plays a role, and if so,
how great this role is. This is where twin studies have become so
valuable. If we study large groups of MZ twins we can determine
what percentage of them is concordant or discordant for a
particular condition. If one twin has a disease, does the other
twin also tend to develop it as well?
Figure 5.1 is a
graph showing concordance rates for schizophrenia. This shows that
the more closely related we are to someone with this disease, the
more likely we are to develop it ourselves. The most important
parts of the graph to look at are the two bars at the bottom, which
deal with twins. From this we can compare the concordance rates for
identical and non-identical (fraternal) twins. Non-identical twins
share the same developmental environment (the uterus) but
genetically are no more similar than any other pair of siblings, as
they arose from two separate zygotes as a consequence of the
fertilisation of two eggs. The comparison between the two types of
twins is important because generally speaking, the twins in a pair
(whether identical or non-identical) are likely to have shared
pretty similar environments. If schizophrenia was caused mainly by
environmental factors, we would expect the concordance rates for
the disease to be fairly similar between identical and
non-identical twins. Instead, what we see is that in non-identical
twins, if one twin develops schizophrenia, the other twin has a 17
per cent chance of doing the same. But in MZ twins this risk jumps
to nearly 50 per cent. The almost threefold higher risk for
identical versus non-identical twins tells us that there is a major
genetic component to schizophrenia.
Figure 5.1 The
concordance rates for schizophrenia. The more genetically related
two individuals are, the more likely it is that if one individual
has the disease, their relative will also develop the disorder.
However, even in genetically identical monozygotic twins, the
concordance rate for schizophrenia does not reach 100 per cent.
Data taken from The Surgeon General’s Report on Mental
Health
http://www.surgeongeneral.gov/library/mentalhealth/chapter4/sec4_1.html#etiology
Similar studies have shown that there is
also a substantial genetic component to a significant number of
other human disorders, including multiple sclerosis, bipolar
disorder, systemic lupus erythematosus and asthma. This has been
really useful in understanding the importance of genetic
susceptibility to complex diseases.
But in many ways, it’s the other side of
the question that is more interesting. It’s not the MZ twins who
both develop a specific disease who are most interesting. It’s the
MZ twins who end up with very different outcomes – one a paranoid
schizophrenic, one mentally very healthy, for example – who create
the most intriguing scientific problem. Why do two genetically
identical individuals, who in many cases have experienced very
similar environments, have such variable phenotypes? Similarly, why
is it quite rare for both MZ twins in a pair to develop type 1
diabetes? What is it, in addition to the genetic code, that governs
these health outcomes?
How epigenetics drives a wedge between
twins
One possible explanation would be that
quite randomly the twin with schizophrenia had spontaneously
developed mutations in genes in certain cells, for example in the
brain. This could happen if the DNA replication machinery had
malfunctioned at some point during brain development. These changes
might increase his or her susceptibility to a disorder. This is
theoretically possible, but scientists have failed to find much
data to support this theory.
Of course, the standard answer has always
been that discordancy between the twins is due to differences in
their environments. Sometimes this is clearly true. If we were
monitoring longevity, for example, one twin getting knocked over
and killed by a number 47 bus would certainly represent an
environmental difference. But this is an extreme scenario. Many
twins share a fairly similar environment, especially in early
development. Even so, it is certainly possible that there are
multiple subtle environmental differences that may be hard to
monitor appropriately.
But if we invoke the environment as the
other important factor in development of disease, this raises
another problem. It still leaves the question of how the
environment does this. Somehow the environmental stimuli – be these
compounds in our food, chemicals in cigarette smoke, UV rays in
sunlight, pollutants from car exhausts or any of the thousands of
molecules and radiation sources that we’re exposed to every day –
must impact on our genes and cause a change in
expression.
The majority of non-infectious diseases
that afflict most people take a long time to develop, and then
remain as a problem for many years if there is no cure available.
The stimuli from the environment could theoretically be acting on
the genes all the time in the cells that are acting abnormally,
leading to disease. But this seems unlikely, especially because
most of the chronic diseases probably involve the interaction of
multiple stimuli with multiple genes. It’s hard to imagine that all
these stimuli would be present for decades at a time. The
alternative is that there is a mechanism that keeps the
disease-associated cells in an abnormal state, i.e. expressing
genes inappropriately.
In the absence of any substantial
evidence for a role for somatic mutation, epigenetics seems like a
strong candidate for this mechanism. This would allow the genes in
one twin to stay mis-regulated, ultimately leading to a disease.
We’re only at the beginning of the investigation but some evidence
has started accumulating that suggests this may indeed be the
case.
One of the most straightforward
experiments conceptually, is to analyse if chromatin modification
patterns (the epigenome) change as MZ twins get older. In the
simplest case, we wouldn’t even need to investigate this in the
context of disease. We could start by testing a much simpler
hypothesis – that genetically identical individuals become
epigenetically non-identical as they age. If this hypothesis is
correct, this would support the idea that MZ twins can vary from
each other at the epigenetic level. This in turn would strengthen
our confidence in moving forwards to examining the role of
epigenetic changes in disease.
In 2005, a large collaborative group
headed by Professor Manel Esteller, then at the Spanish National
Cancer Centre in Madrid, published a paper in which they examined
this issue1. They made some interesting
discoveries. If they examined chromatin from infant MZ twin pairs,
they couldn’t see much difference in the levels of DNA methylation
or of histone acetylation between the two twins. When they looked
at pairs of MZ twins who were much older, such as in their fifties,
there was a lot of variation within the pair for the amount of DNA
methylation or histone acetylation. This seemed to be particularly
true of twins that had lived apart for a long time.
The results from this study were
consistent with a model where genetically identical twins start out
epigenetically very similar, and then diverge as they get older.
The older MZ twins who had led separate and different lives for the
longest would be expected to be the ones who had encountered the
greatest differences in their environments. The finding that these
were precisely the twin pairs who were most different
epigenetically was consistent with the idea that the epigenome (the
overall pattern of epigenetic modifications on the genome) reflects
environmental differences.
Children who eat breakfast are
statistically more likely to do well at school than children who
skip breakfast. This doesn’t necessarily mean that learning can be
improved by a bowl of cornflakes. It may simply be that children
who eat breakfast are more likely to be children whose parents make
an effort to get them to school every day, on time, and help them
with their studies. Similarly, Professor Esteller’s data are
correlative. They show there is a relationship between the ages of
twins and how different they are epigenetically, but they don’t
prove that age has caused the change in the epigenome. But
at least the hypothesis can remain in play.
A team led by Dr Jeffrey Craig in 2010 at
the Royal Children’s Hospital in Melbourne also examined DNA
methylation in identical and fraternal twin pairs2. They
investigated a few relatively small regions of the genome in
greater detail than in Manel Esteller’s earlier paper. Using
samples just from newborn twin pairs, they showed that there was a
substantial amount of difference between the DNA methylation
patterns of fraternal twins. This isn’t unexpected, since fraternal
twins are genetically nonidentical and we expect different
individuals to have different epigenomes. Interestingly, though,
they also found that even the MZ twins differed in their DNA
methylation patterns, suggesting identical twins begin to diverge
epigenetically during development in the uterus. Combining the
information from the two papers, and from additional studies, we
can conclude that even genetically identical individuals are
epigenetically distinct by the time of birth, and these epigenetic
differences become more pronounced with age and exposure to
different environments.
Of mice and men (and women)
These data are consistent with a model
where epigenetic changes could account for at least some of the
reasons why MZ twins aren’t phenotypically identical, but there’s
still a lot of supposition involved. That’s because for many
purposes humans are a quite hopeless experimental system. If we
want to be able to assess the role of epigenetics in the problem of
why genetically identical individuals are phenotypically different
from one another, we would like to be able to do the
following:
1. Analyse hundreds of
identical individuals, not just pairs of them;
2. Manipulate their
environments, in completely controlled ways;
3. Transfer embryos or babies
from one mother to another, to investigate the effects of early
nurture;
4. Take all sorts of samples
from the different tissues of the body, at lots of different time
points;
5. Control who mates with
whom;
6. Carry out studies on four or
five generations of genetically identical individuals.
Needless to say, this isn’t feasible
for humans.
This is why experimental animals have
been so useful in epigenetics. They allow scientists to address
really complex questions, whilst controlling the environment as
much as possible. The data that are generated in these animal
studies produce insights from which we can then try to infer things
about humans.
The match may not be perfect, but we can
unravel a surprising amount of fundamental biology this way.
Various comparative studies have shown that many systems have
stayed broadly the same in different organisms over almost
inconceivably long periods. The epigenetic machinery of yeast and
humans, for example, share more similarities than differences and
yet the common ancestor for the two species lies about one billion
years in the past3. So, epigenetic processes are
clearly fairly fundamental things, and using model systems can at
least point us in a helpful direction for understanding the human
condition.
In terms of the specific question we’ve
been looking at in this chapter – why genetically identical twins
often don’t seem to be identical – the animal that has been most
useful is our close mammalian relative, the mouse. The mouse and
human lineages separated a mere 75 million or so years ago4. 99
per cent of the genes found in mice can also be detected in humans,
although they aren’t generally absolutely identical between the two
species.
Scientists have been able to create
strains of mice in which all the individuals are genetically
identical to each other. These have been incredibly useful for
investigating the roles of non-genetic factors in creating
variation between individuals. Instead of just two genetically
identical individuals, it’s possible to create hundreds, or
thousands. The way this is done would have made even the Ptolemy
dynasty of ancient Egypt blush. Scientists mate a pair of mice who
are brother and sister. Then they mate a brother and sister from
the resulting litter. They then mate a brother and sister from
their litter and so on. When this is repeated for over twenty
generations of brother-sister matings, all the genetic variation
gets bred out, throughout the genome. All mice of the same sex from
the strain are genetically identical. In a refinement of this,
scientists can take these genetically identical mice and introduce
just one change into their DNA. They may use such genetic
engineering to create mice which are identical except for just one
region of DNA that the experimenters are most interested
in.
A mouse of a different colour
The most useful mouse model for
exploring how epigenetic changes can lead to phenotypic differences
between genetically identical individuals is called the
agouti mouse. Normal mice have hair which is banded in
colour. The hair is black at the tip, yellow in the middle and
black again at the base. A gene called agouti is essential
for creating the yellow bit in the middle, and is switched on as
part of a normal cyclical mechanism in mice.
There is a mutated version of the
agouti gene (called a) which never switches on. Mice
that only have the a, mutant version of agouti have
hair which is completely black. There is also a particular mutant
mouse strain called Avy, which
stands for agouti viable yellow. In
Avy mice, the agouti gene
is switched on permanently and the hair is yellow through its
entire length. Mice have two copies of the agouti gene, one
inherited from the mother and one from the father. The
Avy version of the gene is
dominant to the a version, which means that if one copy of
the gene is Avy and one is
a, the Avy will ‘overrule’
a and the hairs will be yellow throughout their length. This
is all summarised in Figure
5.2.
Scientists created a strain of mice that
contained one copy of Avy and one
copy of a in every cell. The nomenclature for this is
Avy/a. Since
Avy is dominant to a, you
would predict that the mice would have completely yellow hair.
Since all the mice in the strain are genetically identical, you
would expect that they would all look the same. But they don’t.
Some have the very yellow fur, some the classic mouse appearance
caused by the banded fur, and some are all shades in-between, as
shown in Figure 5.3.
Figure 5.2 Hair
colour in mice is affected by the expression of the agouti
gene. In normal mice, the agouti protein is expressed cyclically,
leading to the characteristic brindled pattern of mouse fur.
Disruption of this cyclical pattern of expression can lead to hairs
which are either yellow or black throughout their
length.
This is really odd, since the mice are
all genetically exactly the same. All the mice have the same DNA
code. We could argue that perhaps the differences in coat colour
are due to environment, but laboratory conditions are so
standardised that this seems unlikely. It’s also unlikely because
these differences can be seen in mice from the same litter. We
would expect mice from a single litter to have very similar
environments indeed.
Of course, the beauty of working with
mice, and especially with highly inbred strains, is that it’s
relatively easy to perform detailed genetic and epigenetic studies,
especially when we already have a reasonable idea of where to look.
In this case, the region to examine was the agouti
gene.
Figure 5.3
Genetically identical mice showing the extent to which fur colour
can vary, depending on expression of the agouti protein. Photo
reproduced with the kind permission of Professor Emma
Whitelaw.
Mouse geneticists knew how the yellow
phenotype was caused in Avy yellow
mice. A piece of DNA had been inserted in the mouse chromosome just
before the agouti gene. This piece of DNA is called a
retrotransposon, and it’s one of those DNA sequences that doesn’t
code for a protein. Instead, it codes for an abnormal piece of RNA.
Expression of this RNA messes up the usual control of the
downstream agouti gene and keeps the gene switched on
continuously. This is why the hairs on the
Avy mice are yellow rather than
banded.
That still doesn’t answer the question of
why genetically identical Avy/a
mice had variable coat colour. The answer to this has been shown to
be due to epigenetics. In some
Avy/a mice the CpG sequences in
the retrotransposon DNA have become very heavily methylated. As we
saw in the previous chapter, DNA methylation of this kind switches
off gene expression. The retrotransposon no longer expressed the
abnormal RNA that messed up transcription from the agouti
gene. These mice were the ones with fairly normal banded mouse coat
colour. On other genetically identical
Avy mice, the retrotransposon was
unmethylated. It produced its troublesome RNA which messed up the
transcription from the agouti gene so that it was switched
on continuously and the mice were yellow. Mice with in-between
levels of retrotransposon methylation had in-between levels of
yellow fur. This model is shown in Figure
5.4.
Here, DNA methylation is effectively
working like a dimmer switch. When the retrotransposon is
unmethylated, it shines to its fullest extent, producing lots of
the abnormal RNA. The more the retrotranposon is methylated, the
more its expression gets turned down.
The agouti mouse has provided a
quite clear-cut example of how epigenetic modification, in this
case DNA methylation, can make genetically identical individuals
look phenotypically different. However, there is always the fear
that agouti is a special case, and maybe this is a very
uncommon mechanism. This is particularly of concern because it’s
proved very difficult to find an agouti gene in humans – it
seems to be in that 1 per cent of genes we don’t share with our
mouse neighbours.
Figure 5.4
Variations in DNA methylation (represented by black circles)
influence expression of a retrotransposon. The variation in
expression of the retrotransposon in turn affects expression of the
agouti gene, leading to coat colour variability between genetically
identical animals.
There is another interesting condition
found in mice, in which the tail is kinked. This is called
Axin-fused and it also demonstrates extreme variability between
genetically identical individuals. This has been shown to be
another example where the variability is caused by differing levels
of DNA methylation in a retrotransposon in different animals, just
like the agouti mouse.
This is encouraging as it suggests this
mechanism isn’t a one off, but kinked tails still don’t really
represent a phenotype that is of much concern to the average human.
But there’s something we can all get on board with: body weight.
Genetically identical mice don’t all have the same body
weight.
No matter how tightly scientists control
the environment for the mice, and especially their access to food,
identical mice from inbred mouse strains don’t all have exactly the
same body weight. Experiments carried out over many years have
shown that only about 20–30 per cent of the variation in body
weights can be attributed to the post-natal environment. This
leaves the question of what causes the other 70–80 per cent of
variation in body weight5. Since it isn’t being caused
by genetics (all the mice are identical) or by the environment,
there has to be another source for the variation.
In 2010, Professor Emma Whitelaw, the
terrifically enthusiastic and intensely rigorous mouse geneticist
working at the Queensland Institute of Medical Research, published
a fascinating paper. She used an inbred strain of mice and then
used genetic engineering to create subsets of animals which were
genetically identical to the starting stock, except that they only
expressed half of the normal levels of a particular epigenetic
protein. She performed the genetic engineering independently in a
number of mice, so that she could create separate groups of
animals, each of which was mutated in a different gene coding for
epigenetic proteins.
When Professor Whitelaw analysed the body
weights of large numbers of the normal or mutated mice, an
interesting effect appeared. In a group of normal inbred mice, most
of the animals had relatively similar body weights, within the
ranges found in many other studies. In the mice with low levels of
a certain epigenetic protein, there was a lot more variability in
the body weights within the group. Further experiments published in
the same paper assessed the effects of the decreased expression of
these epigenetic proteins. Their decreased expression was linked to
changes in expression levels of selected genes involved in
metabolism6, and increased variability in
that expression. In other words, the epigenetic proteins were
exerting some control over the expression of other genes, just as
we might expect.
Emma Whitelaw tested a number of
epigenetic proteins in her system, and found that only a few of
them caused the increased variation in body weight. One of the
proteins that had this effect was Dnmt3a. This is one of the
enzymes that transfers methyl groups to DNA, to switch genes off.
The other epigenetic protein that caused increased variability in
body weight was called Trim28. Trim28 forms a complex with a number
of other epigenetic proteins which together add specific
modifications to histones. These modifications down-regulate
expression of genes near the modified histones and are known as
repressive histone modifications or marks. Regions of the genome
that have lots of repressive marks on their histones tend to become
methylated on their DNA, so the Trim28 may be important for
creating the right environment for DNA methylation.
These experiments suggested that certain
epigenetic proteins act as a kind of dampening field. ‘Naked’ DNA
is rather prone to being switched on somewhat randomly, and the
overall effect is like having a lot of background chatter in our
cells. This is called transcriptional noise. The epigenetic
proteins act to turn down the volume of this random chat. They do
this by covering the histones with modifications that reduce the
genes’ expression. It’s likely that different epigenetic proteins
are important for suppressing different genes in some tissues
rather than in others.
It’s clear that this suppression isn’t
total. If it were, then all inbred mice would be identical in every
aspect of their phenotype and we know this isn’t the case. There is
variation in body weight even in the inbred strains, it’s just that
there’s even more variation in the mice with the depressed levels
of the epigenetic proteins.
This sophisticated balancing act, in
which epigenetic proteins dampen down transcriptional noise but
don’t entirely repress gene expression, is a cellular compromise.
It leaves cells with enough flexibility of gene expression to be
able to respond to new signals – be these hormones or nutrients,
pollutants or sunlight – but without the genes being constantly
ready to fire up just for the heck of it. Epigenetics allows cells
to perform the difficult compromise between becoming (and
remaining) different cell types with a variety of functions, and
not being so locked into a single pattern of gene expression that
they become incapable of responding to changes in their
environment.
Something that is becoming increasingly
clear is that early development is a key period when this control
of transcriptional noise first becomes established. After all, very
little of the variation in body weight in the original inbred
strains could be attributed to the post-natal environment (just
20–30 per cent). Interest is increasing all the time in the role of
a phenomenon called developmental programming, whereby events
during foetal development can impact on the whole of adult life,
and it is increasingly recognised that epigenetic mechanisms are
what underlie a major proportion of this programming.
Such a model is entirely consistent with
Emma Whitelaw’s work on the effects of decreased levels of Dnmt3a
or Trim28 in her mouse studies. The body weight effects were
apparent when the mice were just three weeks old. This model is
also consistent with the fact that decreased levels of Dnmt3a
resulted in the increased variability in body weight, but decreased
levels of the related enzyme Dnmt1 had no effect in Emma Whitelaw’s
experiments. Dnmt3a can add methyl groups to totally unmethylated
DNA regions, which means it is responsible for establishing the
correct DNA methylation patterns in cells. Dnmt1 is the protein
that maintains pre-established methylation patterns on DNA. It
seems that the most important feature for dampening down gene
expression variability (at least as far as body weight is
concerned) is establishing the correct DNA methylation patterns in
the first place.
The Dutch Hunger Winter
Scientists and policy-makers have
recognised for many years the importance of good maternal health
and nutrition during pregnancy, to increase the chances that babies
will be born at a healthy weight and so be more likely to thrive
physically. In more recent years, it’s become increasingly clear
that if a mother is malnourished during pregnancy, her child may be
at increased risk of ill-health, not just during the immediate
post-birth infancy, but for decades. We’ve only recently begun to
realise that this is at least in part due to molecular epigenetic
effects, which result in impaired developmental programming and
life-long defects in gene expression and cellular
function.
As already highlighted, there are
extremely powerful ethical and logistical reasons why humans are a
difficult species to use experimentally. Tragically, historical
events, terrible at the time, conspire to create human scientific
study groups by accident. One of the most famous examples of this
is the Dutch Hunger Winter, which was mentioned in the
Introduction.
This was a period of terrible hardship
and near-starvation during the Nazi fuel and food blockade of the
Netherlands in the last winter of the Second World War. Twenty-two
thousand people died and the desperate population ate anything they
could find, from tulip bulbs to animal blood. The dreadful
privations of the population created a remarkable scientific study
population. The Dutch survivors were a well-defined group of
individuals all of whom suffered just one period of malnutrition,
all of them at exactly the same time.
One of the first aspects to be studied
was the effect of the famine on the birthweights of children who
had been in the womb during the famine. If a mother was well-fed
around the time of conception and malnourished only for the last
few months of the pregnancy, her baby was likely to be born small.
If, on the other hand, the mother suffered malnutrition for the
first three months of the pregnancy only (because the baby was
conceived towards the end of this terrible episode), but then was
well-fed, she was likely to have a baby with normal body weight.
The foetus ‘caught up’ in body weight, because foetuses do most of
their growing in the last few months of pregnancy.
But here’s the thing – epidemiologists
were able to study these groups of babies for decades and what they
found was really surprising. The babies who were born small stayed
small all their lives, with lower obesity rates than the general
population. Even more unexpectedly, the adults whose mothers had
been malnourished only early in their pregnancy had higher obesity
rates than normal. Recent reports have shown a greater incidence of
other health problems as well, including certain aspects of mental
health. If mothers suffered severe malnutrition during the early
stages of pregnancy, their children were more likely than usual to
develop schizophrenia. This has been found not just in the Dutch
Hunger Winter cohort but also in the survivors of the monstrous
Great Chinese Famine of 1958 to 1961, in which millions starved to
death as a result of Mao Tse Tung’s policies.
Even though these individuals had seemed
perfectly healthy at birth, something that had happened during
their development in the womb affected them for decades afterwards.
And it wasn’t just the fact that something had happened that
mattered, it was when it happened. Events that take place in
the first three months of development, a stage when the foetus is
really very small, can affect an individual for the rest of their
life.
This is completely consistent with the
model of developmental programming, and the epigenetic basis to
this. In the early stages of pregnancy, where different cell types
are developing, epigenetic proteins are probably vital for
stabilising gene expression patterns. But remember that our cells
contain thousands of genes, spread over billions of base-pairs, and
we have hundreds of epigenetic proteins. Even in normal development
there are likely to be slight variations in the expression of some
of these proteins, and the precise effects that they have at
specific chromosomal regions. A little bit more DNA methylation
here, a little bit less there.
The epigenetic machinery reinforces and
then maintains particular patterns of modifications, thus creating
the levels of gene expression. Consequently, these initial small
fluctuations in histone and DNA modifications may eventually become
‘set’ and get transmitted to daughter cells, or be maintained in
long-lived cells such as neurons, that can last for decades.
Because the epigenome gets ‘stuck’, so too may the patterns of gene
expression in certain chromosomal regions. In the short term the
consequences of this may be relatively minor. But over decades all
these mild abnormalities in gene expression, resulting from a
slightly inappropriate set of chromatin modifications, may lead to
a gradually increasing functional impairment. Clinically, we don’t
recognise this until it passes some invisible threshold and the
patient begins to show symptoms.
The epigenetic variation that occurs in
developmental programming is at heart a predominantly random
process, normally referred to as ‘stochastic’. This stochastic
process may account for a significant amount of the variability
that develops between the MZ twins who opened this chapter. Random
fluctuations in epigenetic modifications during early development
lead to nonidentical patterns of gene expression. These become
epigenetically set and exaggerated over the years, until eventually
the genetically identical twins become phenotypically different,
sometimes in the most dramatic of ways. Such a random process,
caused by individually minor fluctuations in the expression of
epigenetic genes during early development also provides a very good
model for understanding how genetically identical
Avy/a mice can end up with
different coat colours. This can be caused by randomly varying
levels of DNA methylation of the
Avy retrotransposon.
Such stochastic changes in the epigenome
are the likely reason why even in a totally inbred mouse strain,
kept under completely standardised conditions, there is variation
in body weight. But once a big environmental stimulus is introduced
in addition to this stochastic variation, the variability can
become even more pronounced.
A major metabolic disturbance during
early pregnancy, such as the dramatically decreased availability of
food during the Dutch Hunger Winter, would significantly alter the
epigenetic processes occurring in the foetal cells. The cells would
change metabolically, in an attempt to keep the foetus growing as
healthily as possible despite the decreased nutrient supply. The
cells would change their gene expression to compensate for the poor
nutrition, and the patterns of expression would be set for the
future because of epigenetic modifications to the genes. It’s
probably no surprise that it was the children whose mothers had
been malnourished during the very early stages of pregnancy, when
developmental programming is at its peak, who went on to be at
higher risk of adult obesity. Their cells had become epigenetically
programmed to make the most of limited food supply. This
programming remained in place even when the environmental condition
that had prompted it – famine – was long over.
Recent studies examining DNA methylation
patterns in the Dutch Hunger Winter survivors have shown changes at
key genes involved in metabolism. Although a correlation like this
doesn’t prove cause-and-effect, the data are consistent with
under-nutrition during the early developmental period changing the
epigenomic profile of key metabolic genes7.
It’s important to recognise that even in
the Dutch Hunger Winter cohort, the effects that we see are not
all-or-nothing. Not every individual whose mother had been
malnourished early in pregnancy became obese. When scientists
studied the population they found an increased likelihood of
adult obesity. This is again consistent with a model where random
epigenetic variability, the genotypes of the individuals and early
environmental events, and the responses of the genes and cells to
the environment combine in one great big complicated – and as yet
not easily decipherable – equation.
Severe malnutrition is not the only
factor that has effects on a foetus that can last a lifetime.
Excessive alcohol consumption during pregnancy is a leading
preventable cause of birth defects and mental retardation (foetal
alcohol syndrome) in the Western world8. Emma Whitelaw used the
agouti mouse to investigate if alcohol can alter the
epigenetic modifications in a mouse model of foetal alcohol
syndrome. As we have seen, expression of the
Avy gene is epigenetically
controlled via DNA methylation of a retrotransposon. Any stimulus
that alters DNA methylation of the retrotransposon would change
expression of the Avy gene. This
would affect the colour of the fur. In this model, fur colour
becomes a ‘read-out’ that indicates changes in epigenetic
modifications.
Pregnant mice were given free access to
alcohol. The coat colour in the pups from the alcohol-drinking
mothers was compared with the coat colour of the pups from pregnant
mice that didn’t have access to booze. The distribution of coat
colours was different between the two groups. So were the levels of
DNA methylation of the retrotransposon, as predicted. This showed
that the alcohol had led to a change in the epigenetic
modifications in the mice. Disruption of epigenetic developmental
programming may lead to at least some of the debilitating and
lifelong symptoms of foetal alcohol syndrome in children of mothers
who over-use alcohol during pregnancy.
Bisphenol A is a compound used in the
manufacture of poly-carbonate plastics. Feeding bisphenol A to
agouti mice results in a change in the distribution of coat
colour, suggesting this chemical has effects on developmental
programming through epigenetic mechanisms. In 2011 the European
Union outlawed bisphenol A in drinking bottles for
babies.
Early programming may also be one of the
reasons that it’s been very difficult to identify the environmental
effects that lead to some chronic human conditions. If we study
pairs of MZ twins who are discordant for a specific phenotype, for
example multiple sclerosis, it may be well nigh impossible to
identify an environmental cause. It may simply be that one of the
pair was exceptionally unlucky in the random epigenetic
fluctuations that established certain key patterns of gene
expression early in life. Scientists are now mapping the
distribution of epigenetic changes in concordant and discordant MZ
twins for a number of disorders, to try to identify histone or DNA
modifications that correlate with the presence or absence of
disease.
Children conceived during famines and
mice with yellow coats have each clearly taught us remarkable
things about early development, and the importance of epigenetics
in this process. Oddly enough, these two disparate groups have one
other thing to teach us. At the very beginning of the 19th century,
Jean-Baptiste Lamarck published his most famous work,
Philosophie Zoologique. He hypothesised that acquired
characteristics can be transmitted from one generation to the next,
and that this drives evolution. As an example, a short-necked
giraffe-like animal that elongated its neck by constant stretching
would pass on a longer neck to its offspring. This theory has been
generally dismissed and in most cases it is simply wrong. But the
Dutch Hunger Winter cohort and the yellow mice have shown us that
startlingly, the heretical Lamarckian model of inheritance can,
just sometimes, be right on the money, as we are about to
see.