In a twelve-month period from 2011 to 2012, 813,200 babies were born in the UK.1 Using the rates quoted in the previous chapter, we can estimate that nearly 1,200 of these babies had Down’s Syndrome, around 270 had Edward’s Syndrome and just under 120 had Patau’s Sydrome. That’s a very small number of cots in a nursery of over three-quarters of a million babies. This is consistent with the concept that having too many copies of a chromosome is very damaging: in general we would not expect high survival rates when it occurs.
Which makes it all the more surprising to learn that about half of the babies born in that period – that’s over 400,000 children – were born with one chromosome too many. Yes, one in two of us. Even more confusingly, the extra chromosome isn’t some tiny little genetic remnant. It’s a really big chromosome. How on earth can this be, when one extra copy of a very small chromosome can cause devastating conditions such as Edward’s or Patau’s Syndromes?
The culprit here is known as the X chromosome, and it’s prevented from causing harm by a process that relies utterly on junk DNA. But before we move to exploring how this protection happens, we need to explore the nature of the X chromosome itself.
Most of the time the chromosomes in a cell are very long and stringy, and difficult to distinguish from each other. They appear like a great bundle of tangled wool when viewed under a normal light microscope. But when a cell is getting ready to divide, the chromosomes become very structured and compact, and are really discrete entities. If you know the right techniques, you can isolate all the compacted chromosomes from a nucleus, stain them with specific chemicals and examine the individual ones through a microscope. At this stage they look more like separate skeins of embroidery wool, with the centromere as the little tube of paper that holds the skeins in place.
By analysing photos of the whole complement of chromosomes in a human cell, scientists were able to identify each individual chromosome. They literally used to cut and paste the individual chromosome pictures to arrange them in order. This is how researchers discovered the causes of Down’s, Edward’s and Patau’s Syndromes, by analysing the chromosomes in cells taken from affected children.
But before identifying the underlying problems in these serious conditions, the early researchers discovered the fundamental organisation of our genetic material. They showed that the normal number of chromosomes in a human cell is 46. The exceptions are the eggs and the sperm, which each have 23. Our chromosomes are arranged in pairs, inherited equally from our mother and father. In other words, one copy of chromosome 1 from mum and one from dad. The same for chromosome 2, and for the others.
This is true for chromosome 1 up to chromosome 22. These are known as the autosomes. If we only looked at the autosomes in a cell, we would not be able to tell if the cell was from a female or a male. But this information becomes immediately apparent if we look at the last remaining pair of chromosomes, known as the sex chromosomes. Females have two identical large sex chromosomes, known as X. Males have one X chromosome and a very small chromosome, called Y. These two situations are shown in Figure 7.1.
The Y chromosome may be small, but it has an amazing impact. It’s the presence of the Y chromosome that determines the sex of the developing embryo. It only contains a small number of genes, but these are vitally important in governing gender. In fact, this is predominantly controlled by just one gene*2 which drives creation of the testes. This in turn leads to production of the hormone testosterone, which results in masculinisation of the embryo. Remarkably, a recent study has shown that just this and one other gene are sufficient not just to create male mice, but also for these mice to generate functional sperm and to father pups.3
Figure 7.1 Standard female and male karyotypes, showing all the chromosomes present in a cell. The upper panel shows a female karyotype, the lower a male one. The only difference is in the last pair of chromosomes. Females have two large X chromosomes, males have one large X and a small Y. (Wessex Regional Genetics Centre, Wellcome Images)
The X chromosome, on the other hand, is very large, containing over 1,000 genes.4 This creates a potential problem. Males only have one copy of the X chromosome and hence one copy of each of these genes. But females have double that number, so in theory could produce twice as much of the products encoded by the X chromosome as males. The trisomic conditions described in Chapter 6 demonstrated that even a 50 per cent increase in expression of the genes from a small chromosome has a hugely detrimental effect on development. How then can females tolerate a 100 per cent increase in expression of over 1,000 genes, compared with males?
Women have an off switch
The answer is that they don’t. Females produce the same amount of X chromosome-encoded protein expression in their cells as males. They achieve this by a remarkably ingenious arrangement whereby one X chromosome is switched off in every cell. This is known as X-inactivation. Not only is it essential for human life, the process by which it occurs opened up new and totally unanticipated areas of biology that are still the subject of intense scrutiny.
One of the oddest things we have come to realise is that our cells can count the number of X chromosomes. Male cells contain an X and a Y chromosome and they never inactivate the single X. But sometimes males are born who have two X chromosomes and one Y. They are still males, because it’s the Y chromosome that drives masculinisation. But their cells inactivate the extra X, just as female cells do.
A similar thing happens in females. Sometimes females are born who have three X chromosomes in each cell. When this happens, the cells shut down two X chromosomes instead of one. The flip side of this is when females are born who only have one X chromosome. In this case, the cell doesn’t shut it off at all.
In addition to being able to count, our cells are also able to remember. When a female produces eggs, she usually only gives each egg one of each pair of chromosomes, including the X chromosome. A male produces sperm that contain either an X or a Y chromosome. When a sperm that contains an X chromosome fuses with an egg, the resulting single-cell zygote contains two X chromosomes and both are active. But very early in development, after just a few rounds of cell division, one X chromosome is inactivated in each cell of the embryo. Sometimes it’s the X that came from father, sometimes the X that came from mother. Every daughter cell that subsequently develops switches off the same X chromosome as its parental cell. This means that of the 50 trillion or so cells in the adult female body, on average about half will express the X chromosome that was provided by the egg, and the other half will express the X chromosome that was provided by the sperm.
When an X chromosome is inactivated, it adopts a very unusual physical conformation. The DNA becomes incredibly compacted. Imagine you and a friend each take hold of opposite ends of a towel. You start turning your end of the towel clockwise, and your friend does the same at the other end. Pretty quickly, the towel will start twisting in the middle, and the two of you will be pulled closer together. Now imagine that the towel is about five metres in length, but you manage to keep twisting it until it’s a dense clump of towel only a millimetre in linear length. By this stage, the towel is extraordinarily tightly wound up. Essentially, the X chromosome becomes as tightly compacted as that towel. One of the consequences is that it forms a dense structure that can be seen when looking at the nucleus of a female cell down a microscope, when all the other chromosomes are long and stringy and can’t be visualised. The condensed X chromosome is called the Barr body.
In order to try to understand how X chromosome inactivation happens, scientists studied unusual cell lines and mouse strains. These focused on examples where parts of the X chromosome had been lost, or where bits of it had been transferred to other chromosomes. Some cells that had lost part of the X chromosome were still able to inactivate one of their X chromosomes, as shown by the presence of the Barr body. But cells that had lost a different part of the X weren’t able to form Barr bodies, showing that they hadn’t inactivated a chromosome.
Where parts of the X chromosome had been transferred to other chromosomes, sometimes these abnormal chromosomes were inactivated, and other times they weren’t. It all depended on which bit of the X chromosome had been transferred.
These data enabled researchers to narrow down the region on the X chromosome that was key for inactivation. Rationally enough, they called this region the X inactivation centre. In 1991, a group reported that this region contained a gene that they called Xist.* Only the Xist gene on the inactive chromosome expressed Xist RNA.5,6 This made perfect sense, because X inactivation is an asymmetric process. In a pair of equivalent X chromosomes, one is inactivated and one is not. So it seemed consistent that this process would be driven by a scenario where one chromosome expresses a gene and the other doesn’t.
It was obvious that the next question would be to ask how Xist works and the first thing that researchers did was to try to predict the sequence of the protein that it produced. This is usually relatively straightforward. Once they had found the sequence of the Xist RNA molecule, all that the scientists had to do was run this through a simple computer program that would predict the encoded amino acid sequence. Xist RNA is very long, about 17,000 bases. Each amino acid is encoded by a block of three bases, so a 17,000-base RNA could theoretically code for a protein of over 5,700 amino acids. But when the Xist RNA sequence was examined, the longest run of amino acids was just under 300. This was despite the fact that the Xist RNA was spliced, in the way we first saw in Chapter 2, so had lost all the intervening junk sequences.
The ‘problem’ was that the Xist RNA was liberally scattered with sequences that don’t code for amino acids, but which act as stop signals when protein chains are being built up. We can envisage this as being a little like trying to build a tall tower out of LEGO. It is perfectly straightforward until someone hands you one of those roof bricks that doesn’t have any of the attachment nodes on the top. Once you insert this brick, your tower can’t get any bigger.
If Xist did encode a protein, it would seem very odd that a cell would go to the effort of creating an RNA that was 17,000 bases* in length just to produce a protein that could have been encoded by an RNA of about 5 per cent of that length. Researchers in the field realised relatively quickly that this wasn’t what was happening. The reality was much stranger.
DNA is found in the nucleus. It’s copied to form RNA, and messenger RNA is transported out of the nucleus to structures where it acts as a template for protein assembly. But analyses showed that Xist RNA never left the nucleus. It doesn’t encode a protein, not even a short one.7,8
Xist was in fact one of the first examples of an RNA molecule that is functional in its own terms, not as a carrier of information about a protein. It’s a great example of how junk DNA – DNA which doesn’t lead to production of a protein – is anything but junk. It’s extremely important in its own right, because without it X inactivation cannot happen.
An odd feature of Xist is not just that it doesn’t leave the nucleus. It doesn’t even leave the X chromosome that produces it. Instead, it essentially sticks to the inactive X and then spreads along the chromosome. As more and more Xist RNA is produced, it begins to spread out and cover the inactive X chromosome, in a process quaintly referred to as ‘painting’. The fact that this rather descriptive term is used is a quite good indicator that it’s something we don’t particularly understand. No one really knows the physical basis of how the Xist RNA creeps along the chromosome, like the mile-a-minute vine covering a wall. Even after more than twenty years we are still pretty hazy on how this happens. We do know that it’s not based on the sequence of the X chromosome. If the X inactivation centre is transferred on to an autosome in a cell, then the autosome can be inactivated as if it were an X.9
Although Xist is required to initiate the process of X inactivation, it has helpers that strengthen and maintain the process. As Xist paints the X chromosome, it acts as an attachment point for proteins in the nucleus. These bind to the inactivating X, and attract yet more proteins, which shut down expression even more tightly. The only gene that isn’t coated with Xist RNA and these proteins is the Xist gene itself. It remains a little beacon of expression in the chromosomal darkness of the inactive X.10
Left to right, right to left
So we have here a situation where a piece of ‘junk’ DNA – one that doesn’t code for protein – is absolutely essential for the function of half the human race. Scientists have recently discovered that this process of X inactivation requires at least one other piece of junk DNA. Confusingly, this is encoded in exactly the same place on the X chromosome as Xist. DNA, as we know, is composed of two strands (the iconic double helix). The machinery that copies DNA to form RNA always ‘reads’ DNA in one direction, which we could call the beginning and end of a specific sequence. But the two strands of DNA run in opposite directions to each other, a little like one of those funicular railways we find at older seaside and mountain resorts. This means that a particular region of DNA may carry two lots of information in one physical location, running in opposite directions to each other.
A simple example in English is the word DEER, formed by reading from left to right. We could also read the same letters from right to left and in this case we would get the word REED. Same letters, different word, different meaning.
The other key piece of junk DNA involved in X inactivation is called, rather fittingly, Tsix. This is of course Xist spelt backwards, and it is found in the same region as Xist but on the opposite strand. Tsix encodes an RNA of 40,000 bases in length, over twice the size of Xist. Like Xist, Tsix never leaves the nucleus.
Although Tsix and Xist are encoded on the same part of the X chromosome, they are not expressed together. If an X chromosome expresses Tsix, this prevents the same chromosome from expressing Xist. This means that Tsix must be expressed by the active X chromosome, unlike Xist, which is always expressed from the inactive one.
This mutually exclusive expression of Tsix and Xist is of critical importance at a point in early development. The X chromosome in the egg has lost any of the protein marks that show it was inactivated (if it was the inactive version) and the X chromosome in the sperm had never been inactivated anyway. Following fusion and six or seven rounds of cell division, there will be a hundred or so cells in the embryo. At this stage, each cell in the female embryo switches off one of its two X chromosomes randomly. This requires a fleeting but intense physical relationship between the pair of X chromosomes in a cell. For just a couple of hours the two X chromosomes are physically associated in a brief encounter that ends with one being inactivated. The association is only over a small region of the X chromosome – the X inactivation centre, which codes for both Xist and TsiX RNA.11
A fleeting moment lasts forever
This is the mother of all one-night stands. In those two hours, chromosomal decisions get made which are then maintained for the rest of life. Not just during foetal development, but right up until the woman dies, even if that is more than a hundred years later. And it affects not just the hundred or so cells, but the trillions that come after them, because the same X chromosome is inactivated in all daughter cells.
It’s still not entirely clear what happens during the hours of X chromosome intimacy in early development. The current theory is that there is a reallocation of junk RNA between the two chromosomes, such that one ends up with all the Xist and becomes the inactive X. We don’t know how, but it’s possible that one chromosome expresses slightly more or less of Xist or another key factor. We do know that the process begins just as levels of Tsix start to drop. It may be that once its levels fall below a certain critical threshold, Xist can start getting expressed from one of the X chromosomes.
Gene expression tends to have what’s known as a stochastic component, by which we simply mean there’s a bit of random variability in the levels. If one of the chromosomes is expressing a slightly higher amount of one or more key factors, this may be sufficient to build a self-amplifying network of proteins and RNA molecules. Because the inequality in expression is essentially stochastic (due to random ‘noise’) the inactivation will also be essentially random across the hundred or so cells.
Here’s a possible way of visualising this. Imagine you get home late one evening and you have a hankering for melted cheese on two slices of toast. Just as you start to make this delicious supper, you realise you don’t have much cheese in the fridge. What do you do? Make two rounds where neither really contains enough cheese to be satisfying? Or concentrate all of it on one slice, so that you get the dairy hit you are craving? Most people probably choose the latter, and in a way this is what the pair of X chromosomes do during the phase when random inactivation is taking place in the embryo. Evolution has favoured a process whereby, rather than each have a sub-critical amount of a key factor, the factor migrates to the chromosome that has slightly more to begin with. The more you have, the more you get.
X inactivation is entirely dependent on ‘junk’ DNA, and really gives the lie to that terminology. The process is absolutely essential in female mammals for normal cell function and a healthy life. It also has consequences in various disease states. Full-blown Fragile X syndrome of mental retardation, which we encountered in Chapter 1, only affects boys. This is because the gene is carried on the X chromosome. Women have two X chromosomes. Even if one of their chromosomes carries the mutation, enough protein is produced from the other (normal) one to avoid the worst of the symptoms. But males only possess one X chromosome and one Y chromosome, which is very small and doesn’t carry many genes apart from the sex determining ones. Consequently, there is no compensatory normal Fragile X gene in males who carry a mutation on their X chromosome. If their sole X chromosome carries the Fragile X expansion, they can’t produce the protein and so they develop symptoms.
This is also true of a whole range of genetic disorders where the mutated gene is carried on the X chromosome. Boys are more likely to have symptoms of an X-linked genetic disorder than girls, because the boys can’t compensate for a faulty gene on their single X chromosome. Relevant medical conditions range from relatively mild issues such as red–green colour blindness to much more severe diseases. These include haemophilia B, the blood clotting disorder. Queen Victoria was a carrier of this condition and one of her sons (Leopold) was a sufferer and died at the age of 31 from a brain haemorrhage. Because at least two of Victoria’s daughters were also carriers, and the royal families of Europe tended to inter-marry, this mutation was passed on to various other dynasties, most famously the Romanov line in Russia.12
Although women carrying the mutation that causes haemophilia only produce 50 per cent of the normal amounts of the clotting factor, this is enough to protect them from symptoms. This is partly because this clotting factor is released from cells and circulates in the bloodstream, where it reaches high enough levels for protection against bleeds, no matter where they happen.
There are, however, circumstances wherein the presence of two X chromosomes in a woman doesn’t guarantee protection from an X-linked disorder. Rett Syndrome is a devastating neurological disease which presents in some ways as a really extreme form of autism. Baby girls appear to be perfectly healthy when born and they reach all the normal developmental milestones for the first six to eighteen months of life. But after that, they begin to regress. They lose any spoken language skills they have developed. They also develop repetitive hand actions, and lose purposeful ones such as pointing. The girls suffer serious learning disability for the rest of their lives.13
Rett Sydrome is caused by mutations in a protein-coding gene on the X chromosome.*,14 Affected females have one normal copy of this gene, and one version which is mutated and can’t produce functional protein. Assuming random X inactivation, we expect that on average half of the cells in the brain will express normal amounts of the protein, and there will be no expression from the other ones. It is obvious from the clinical presentation that there are severe problems if half the brain cells can’t express this protein.
Rett Syndome pretty much only affects girls. This is unusual for an X-linked disorder, where girls are usually carriers and boys are affected. This might make us wonder how boys are protected from the effects of a Rett mutation. But the reality is that they are not. The reason we almost never find boys who are affected by Rett Syndrome is because affected male embryos don’t develop properly and the foetuses don’t survive to term.
Never underestimate luck, good or bad
Scientists are trained to think about many things during our education and careers. But something we are rarely asked to ponder is the role played by luck. Even when we do, we usually dress it up with terms like ‘random fluctuations’ or ‘stochastic variation’. And that’s a shame, because sometimes ‘luck’ is probably a better description.
Duchenne muscular dystrophy is a severe muscle wasting disease, which we first met in Chapter 3. Boys with this disorder are fine initially but during childhood their muscles begin to degenerate, in a characteristic pattern. For example, in the legs the thigh muscles begin to waste first. The boys develop very large calves as their bodies try to compensate, but after a while these muscles also wither. The children are usually wheelchair users by their teens and the average life expectancy is only 27 years of age. The early mortality is caused to a large extent by the eventual destruction of the muscles involved in breathing.15
Duchenne muscular dystrophy is caused by a mutation in a gene on the X chromosome that encodes a large protein called dystrophin.16 This protein seems to act as a sort of shock absorber in muscle cells. Because of the mutation, males can’t produce functional protein and this ultimately leads to destruction of the muscle. Carrier females will usually produce 50 per cent of the normal amounts of functional dystrophin protein. This is generally sufficient, because of an odd anatomical feature. As we develop, individual muscle cells fuse to create a large super-cell with lots of individual nuclei in it. This means each super-cell has access to multiple copies of the necessary genes, in all the different nuclei. So the muscles of carrier females overall contain enough dystrophin protein for normal activity, instead of one cell with enough, and one cell with none.
There was an unusual case of a woman with all the classic symptoms of Duchenne muscular dystrophy. This is very rare but there are ways we could predict this would happen. One possibility would be if her mother was a carrier and her dad was a Duchenne sufferer who survived long enough to father a child. If that was the case she would definitely have inherited a mutated gene from her father (because he would only possess one – affected – X chromosome). There would be a one in two chance that any egg produced by her carrier mother also contained a mutated dystrophin gene. If that scenario had occurred, neither of her X chromosomes would have a normal copy of the gene, and she wouldn’t be able to produce the necessary protein.
But the doctors treating this patient had taken a family history and they knew that her father didn’t have Duchenne muscular dystrophy, so another explanation was necessary. Sometimes mutations arise quite spontaneously when eggs or sperm are produced. The gene that codes for dystrophin is very large, so just by chance it is at relatively high risk of mutation compared with most other genes in the genome. That’s because mutation is essentially a numbers game. The bigger the gene, the more likely it is that it may mutate. So, one mechanism by which a female could inherit Duchenne muscular dystrophy is if she inherits a mutated chromosome from her carrier mother, and a new mutation in the sperm that fertilised the egg.
This would normally seem like quite a good bet for explaining why this female patient had developed this disorder. There was only one problem. The patient had a sister. A twin sister. An identical twin sister, derived from the very same egg and sperm. And her twin sister was absolutely healthy. No symptoms of Duchenne muscular dystrophy at all. How on earth could two women who were genetically absolutely identical differ so much with respect to a genetically inherited disorder?
Think back to those hundred or so cells that undergo X inactivation during early embryonic development. Just by chance, about 50 per cent of them will switch off one X chromosome, and the remainder will switch off the other one. The same pattern of X inactivation is passed on to all the daughter cells throughout life.
The sister with Duchenne muscular dystrophy was simply incredibly unlucky during this stage. Just by sheer chance, all the cells that would ultimately give rise to muscle switched off the normal copy of the X chromosome. This was the one inherited from her father. This meant that the only X chromosome switched on in her muscle cells was the faulty one from her carrier mother. So none of the affected twin’s muscle cells were able to express dystrophin and she developed the symptoms normally only seen in males.
When her genetically identical twin was developing, however, some of the cells that would give rise to muscle switched off the normal X chromosome and some switched off the mutated one. This meant that her muscles expressed enough dystrophin to keep them healthy, and she was an asymptomatic carrier, just like her mother.17
It is quite extraordinary to think that this was all caused by a simple fluctuation in the distribution of Xist, a long bit of RNA derived from junk DNA. The fluctuation lasted no more than a couple of hours, and occurred over a distance considerably less than one-millionth of the diameter of a human hair. Yet it was the difference between winning and losing in the health lottery.
Luck can be patchy
It is perhaps even stranger to think that some of the cat lovers among us look at, and stroke, the consequences of X inactivation every day. Tortoiseshell or calico cats (depending on which side of the Atlantic you’re reading this book) are the ones with the distinctive patterns of orange and black. These coat colours occur in patches. The gene that controls the coat colour comes in two forms. An individual X chromosome carries either the orange version or the black version.
If the X chromosome carrying the black version is inactivated, the orange version on the other chromosome will be expressed and vice versa. When the cat embryo is at the size of a hundred cells or so, one or other X chromosome will be inactivated in each cell. And just as in all the other examples, all the daughter cells will switch off the same X chromosome. Eventually, some of these daughter cells will give rise to the cells that create pigment in the fur. As more and more of these cells divide and develop, they stay close to each other. This means that daughter cells tend to be clustered in patches. Because of the pattern of X inactivation in the daughter cells, this will lead to patches of orange fur and patches of black fur. This process is shown in Figure 7.2.
Figure 7.2 Schematic showing how patches of orange or black fur develop in female tortoiseshell cats depending on random X chromosome inactivation. The genes for fur colour lie on the X chromosome. If the black version is on the chromosome that is inactivated in a cell during early development, all descendants of that cell will only express the orange gene. The situation is reversed if the X chromosome carrying the orange gene is inactivated.
In 2002 scientists demonstrated beautifully just how random the process of X inactivation really is, by cloning a calico cat. They took cells from an adult female cat, and carried out the standard (but still fiendishly tricky) process of cloning. To do this, they removed the nucleus from the adult cat cell and put it into a cat egg whose own chromosomes they’d removed. This egg was implanted into a surrogate cat mother, and a lively and beautiful female kitten was born. And she didn’t look anything like the genetically identical cat of which she was a clone.18
When this procedure is used to clone animals, the egg treats the new nucleus as if it was the real product of an egg fusing with a sperm. It strips off as much information as possible from the DNA, taking it back to its basic genetic sequence. This doesn’t happen as effectively as in a real egg and sperm, which is one of the reasons why the success rate of this type of cloning is still very low. But sometimes it does work, as was the case here, and a cloned animal is born.
When the nucleus from the mother cat was put inside a cat egg, the egg caused changes to the chromosomes. One of these changes was the removal of the inactivating proteins on one of the X chromosomes, and the switching off of Xist expression. So for a short period in early development, both copies of the X chromosome were active. As the embryo developed, it went through the normal process at around the 100-cell stage of randomly inactivating an X chromosome in each cell. The pattern of X inactivation was passed on to daughter cells in the standard way, and the kitten thereby developed a different distribution of orange and black fur from its clonal ‘parent’.
The moral of this story? If you have a calico cat you think is exceptionally beautiful, take lots of videos, lots of photos and if you want to be very weird about it, call in a taxidermist when she dies. But if you are ever approached by a door-to-door travelling cloner, just send them on their way.
