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The importance of being red Professor Jonathan L Rees FMedSci Dermatology, University of Edinburgh Around half the patients who attend my Dermatology clinic do so because of a set of interrelated evolutionary decisions made, say, 1¾ million years ago. Around this time, major climate change meant that our ancestors moved from the forest onto the savannah and desert. In doing this, in evolutionary terms, a number of changes were necessary. Our ancestors became dependent for food, in part, on hunting. This meant that they had to develop appropriate strategies to kill prey that over short distances could run faster than they could1 . Over long distances, say a marathon, or distances of comparable length, humans are extraordinarily good endurance runners. In a warm climate, over such distances, humans can even out perform a horse. This facility would allow them to hunt over great distances and tire their prey. The reasons humans are such good endurance runners are many, but one key feature of our physiology, is that we are extraordinarily good at losing the heat generated by muscular work. Key to this ability is between 3 and 5 million eccrine sweat glands distributed across our skin. In simple terms, these glands ultrafiltrate blood, deliver the filtrate onto the skin surface where it evaporates, cooling the skin. Our original forest dwelling ancestors had skin very different from ours. Although the evidence is indirect, most researchers believe these ancestors were covered in a dense body of hair, a skin that is ill suited to efficient eccrine sweating because the 1 Throughout this essay I tend to speak teleologically. This is merely a useful shorthand, for stylistic reasons given that the paper is an account of a talk, and such statements should not be taken literally. hair becomes matted and diminishes the cooling ability of sweat. Our ancestors, therefore, became mainly naked, but this in turn leads to another problem: how is the now naked skin to be protected against the harmful effects of ultraviolet radiation? As so often in evolution, the principle method chosen to solve this evolutionary problem was one of adapting and finding a new role for molecules that had already served other functions in biology. The substance we are concerned with is the pigment melanin, a set of related molecules that give human skin and hair much of its colour. Melanin and protection from DNA damage Melanin and a variety of other chromophores are widespread in nature. In general, the purpose of such agents is either to diminish attention (as in camouflage) or, in the case of sexual behaviour, to attract attention. Such pigments have colour because of their properties with respect to electromagnetic radiation between roughly 400 and 800nm. However, melanin in skin, despite obviously being active within this spectrum, also shows significant absorption in the shorter wavelengths between 300nm and 400nm – conventionally these wavelengths are referred to as ‘ultraviolet radiation’ (UVR). Figure 1 shows the scrotum of the blue vervet monkey and I use it to demonstrate a number of points. Although the scrotum appears blue, the actual pigment deep in the skin would appear brown if we saw it at the skin’s surface. It appears blue because of the relation between light scattering and wavelength, such that the blue end of the spectrum undergoes more scattering than the red end of the spectrum (in simple terms more blue photons bounce back from the skin). Of course, even medical students realise that in this case the pigment is not there as a 2 sun block, but as a form of sexual and social behavioural signalling. Pigment in human skin, however, appears to be principally present as a mechanism to protect skin against ultraviolet radiation. This is not the case for the pigmentation in hair, and separate explanations need to be made for at least some of the variation in hair pigmentation. Disease, as ever, highlights important rate limiting pathways in biology, and the importance of pigmentation in protecting against ultraviolet radiation can be readily seen in various clinical states. For instance, albinos are individuals who are unable to make sufficient amounts of melanin in their skin, hair and eyes due to mutations in one of the many genes that are essential for normal melanin biosynthesis. The result is that albinos, if they live in areas with significant ambient ultraviolet radiation, are prone to develop burning, blistering and secondary infection upon exposure to UVR and, in the long term, skin cancer (for the record they are also prone to several other abnormalities relating to ocular development that are still poorly understood). Another disease, that furnishes an even more striking insight into the importance of melanin, is vitiligo. Vitiligo is an acquired disorder, although there seems to be some familial component, in which the melanocytes disappear from focal areas of skin, perhaps due to some form of autoimmunity, or auto-aggression, on the part of the immune or inflammatory system. If a patient with vitiligo goes out in the sun, they are much more likely to burn in the de-pigmented area of skin, rather than the normally adjacent pigmented skin. Constitutive pigmentation, that is the pigmentation that is present in your skin prior exposure to ultraviolet radiation, is a strikingly effective sunblock. 3 Melanin absorption within the area of the electromagnetic spectrum I have been discussing is greatest at a little over 300nm. What does radiation of 300nm do to skin if we overcome the natural protection afforded by pigment? Figure 2 is the author’s back following a single ‘excess’ dose of radiation at around 311nm. Given a large enough dose, erythema develops starting at around 6-8 hours, peaking at perhaps 8-24 hours and thereafter diminishing. What is the erythema due to? DNA damage. We know this because it is possible to measure DNA damage following ultraviolet radiation and to show that as the damage is repaired, the amount of inflammation diminishes. Furthermore, patients with a rare disorder, xeroderma pigmentosum, show an abnormal sensitivity to ultraviolet radiation because they lack the normal enzymatic system to repair ultraviolet radiation induced DNA damage. If I was to compare individuals with very pale skin, with those with very dark skin, the amount of ultraviolet radiation I might need to increase blood flow (used as a proxy for DNA damage), measured with assays that are robust to confounding from melanin pigmentation, might vary by a hundred-fold. Melanin is an astonishingly effective sunblock. From Mendel’s peas to red hair In the 19th Century, associations between skin colour, geography and ancestry were noted. Descriptive maps of variation in skin colour were published. The Victorians were natural historians par excellence and, of course, great travellers and explorers. The irony, however, is that the simplifying insight necessary to try and relate variation in skin colour with both geography and ancestry came not from the great explorers but from an individual who literally left the safety of his garden on only a handful of occasions. This is of course the monk, Gregor Mendel. 4 Mendel did not appear to his teachers at least a strikingly incisive student. He belonged to a teaching order and was therefore sent to the University of Vienna, but he failed the necessary exams to become a teacher. As has been said of him before, he returned to the monastery at Brno, aged 31, a failure. It also seems apparent that Mendel had a streak of dissent running through his character. The teaching of biology at that time was forbidden by the abbot, but what did Mendel spend the rest of his life doing – asking and answering one of the central questions of that age and the present age: what is the nature of inheritance? Mendel worked with garden peas initially, and later with bees. He made two extraordinary hypotheses. The first is that particular characteristics are a reflection of the inheritance of what he called two particles – what we could call two genes – one inherited from the mother and one from the father. Second, that if the particles were not the same, then one was dominant to the other (and the other therefore recessive). This was an extraordinary and brave hypothesis. Most scientists of the day were convinced that heredity worked by some form of averaging. (Presumably they had observed examples contrary to this view but, as sometimes happens, ignored the evidence of their eyes). Mendel published his work in the Journal of the Brno Natural History Society and achieved instant oblivion—at least at first. His views were too radical and the evidence he produced not deemed substantial enough to overturn the then current dogma. He attempted to publicise his findings, but to not avail. Things did not stay that way forever however. 5 Mendel’s work was rediscovered by a variety of workers around the turn of the 20th Century and just a few years later, germane to my purpose here tonight, people noted that if you study red hair in families, then it approximates to a Mendelian autosomal recessive. By this, I mean that if two individuals who do not exhibit the trait have children, but are ‘carriers’ then about one in four of those children will have the trait (in this case red hair). I say approximately because just as with Mendel’s peas, the numbers are not always perfect, but using this simple rule, we will be right to say 8 times out of 10. The value of models: from peas to mouse fancy We have grown so inured to the use of model systems and the need to exploit simplicity that we might need to pinch ourselves to realise the power of a paradigm that can go from wrinkling of peas through to explaining a visible human visible trait. However, advance in the genetics of red hair has relied not just on peas but also on a much closer relative, the mouse. Even before Mendel undertook his work people began to collect mice with unusual coat colours. The common mouse of our childhood is an albino mouse, not a mouse as we would find it in the wild, instead one that because of a spontaneous mutation has been selected (by man) because of its lack of pigmentation. By selective breeding and swapping of mice with different pigmentary characteristics (and the amazing richness of human vision to detect variation in coat colour), a large number of mice with spontaneous mutations effecting the pigmentary system have been identified. This collection of mice, when married with the technical revolution that occurred in recombinant DNA technology in the 1970’s and 80’s meant that it now became possible to understand the relation between DNA sequence and the ‘particles’ or genes that Mendel had hypothesised. The strategy was clear: if you can identify 6 genes in the mouse at the level of the DNA, it would provide a striking and simple way to find genes that might have analogous function in humans. In the area of human pigmentation, this has been an amazingly successful approach. Most of what we know about the genetics of human pigmentation has relied on work in the mouse (and more recently zebrafish). The Melanocortin 1 Receptor (MC1R) Early in the 1990’s, Roger Cone’s group identified the locus that underpinned a variety of different coat colours in mouse. The locus, known as ‘extension’, harboured a gene subsequently christened the melanocortin 1 receptor (mc1r). In the mouse at least, a hormone, melanocyte stimulating hormone (MSH), interacted with the mc1r receptor which is situated on the outer cell membrane of melanocytes. (Melanocytes are the neural crest derived cells in skin that synthesis melanin which is then passed into the more numerous surrounding keratinocytes). Melanin is a complex substance and although we do not understand its chemistry in detail, we know that it is useful to consider the amount and type of melanin as being a key determinant of coat colour. If the signalling described through the mc1r is functional, then brown or very dark melanin is produced. If however, the signalling pathway acting via the mc1r is impaired then the ratio of brown or black melanin to red or yellow melanin is reduced. In a series of experiments, Cone was able to show that mc1r was a key control factor in this pathway: if the receptor was functioning brown melanin was produced, if the receptor was mutated then the production of more red or yellow melanin was favoured. 7 For reasons that are not clear, despite Roger Cone and colleagues obviously wondering about the human relevance of their work they were unable to successfully take the work into man. By contrast, along with Tony Thody in Newcastle and Ian Jackson in the MRC Human Genetics Unit here in Edinburgh, we were able to quickly show that people with red hair and pale skin also seemed to have changes in the human melanocortin 1 receptor (MC1R). The story, however, was not as clear-cut as we had wished. Earlier, I alluded to the fact that red hair is inherited as an autosomal recessive – or at least you can model it this way and be right most of the time. If this was the case we had expected to find one variant on each chromosome, one which is inherited from the mother and one which is inherited from the father. By contrast, what we found was that some red haired individuals seem to only have one sequence change on one allele with the other allele being root sequence, whereas other individuals with red hair seemed to have two or three different variants. Since we know the individuals only had a pair of chromosomes, this meant that they had more than one change on the same allele. We were surprised by this, but in many ways perhaps we shouldn't have been. Subsequently we, and many other workers around the world, have shown that there are over 100 variants of the MC1R, which occur at varying frequencies, particularly in North European populations. However, the large number of variants means that tidying up the story required a number of different approaches. For instance, if we went back and repeated the family studies done at the turn of the century, and sequenced the MC1R of these individuals, we could make sense of an autosomal recessive model, but only if we considered some variants to be important and some not. Worse still, the evidence for the activity of some variants was ambivalent – we weren’t certain how to interpret what they showed. One solution 8 to this was to take particular variants out of individuals and transfect them into cells and see how well the receptor worked. This is a very standard biochemical assay and when we did this we could show that different variants of the gene worked to varying degrees. What we were seeing was not an all or nothing phenomenon, but pardon the pun, shades of red. Ian Jackson was able to perhaps perform an even neater demonstration of this when he was able to take human genes, put them back into mice as BAC transgenics, and show that different human variants had different functional characteristics on a uniform genetic background. Again, we were seeing shades of red rather than a simple binary phenotype. When we study humans with respect to red hair, of course, we are turning what we have to believe is a continuous trait into a binary state. However, depending on how you look and classify individuals, you can see that the relationship between the MC1R and phenotype was slightly more complicated than we would have expected from Mendel’s autosomal recessive pattern. As already mentioned, different changes in the gene have different functional effects in terms of the amount of melanin. Furthermore, if you looked at individuals with different shades of red hair, say between carrot and strawberry blonde and auburn, you found that auburns were much more likely to only have one variant allele, whereas people with carrot coloured hair were more likely to have two variant alleles. It’s well known that people with red hair tend to freckle more. Freckles are a focal over production of melanin and are a response to environmental ultraviolet radiation acting on a genetic susceptibility to ultraviolet radiation. What we found was that if you counted the number of freckling sites (as a proxy for the number of freckles and sensitivity), individuals with the most freckling sites tended to have two variant alleles, individuals with no variant alleles had far fewer freckling sites, but individuals who just carried one variant allele and who didn't have red hair, were somewhere in between. This could 9 be called a dosage or a heterozygote effect. This gradation of phenotype was also evident when you looked at hair colour. Some men have red beards, but the scalp hair is not red. Such persons were more likely to carry one variant allele than those individuals who did not develop red beards, nor have red hair. It is also possible to extract melanin from hair chemically and estimate the ratio of the different sorts of melanin, eumelanin (brown or black melanin) and pheomelanin (red or yellow melanin). If you do this, you find that individuals who do not have red hair, but who harbour one variant allele, are much more likely to have higher ratios of pheomelanin to eumelanin, than individuals who do not harbour any variant alleles. Again, there seems to be a dosage effect. To return to skin, using a variety of assays based on exposure to ultraviolet radiation, and objective measurement of tanning, we can show that individuals with one variant allele, although they do not usually have red hair, are more sun sensitive than individuals who also do not have red hair but do not carry a variant allele. The message here is easily obscured in the detail but is, in reality, quite straightforward. In the 19th Century, the person who provided the simplifying hypothesis that allowed insight into human pigment genetics was Mendel with, what we now name after him, simple Mendelian patterns of inheritance. This model approximates well in the human but it is not sufficient. Instead, we know that the alleles are not all functionally equivalent, that is that sequence changes lead to variation in activity on a continuous scale and that it is possible to achieve the same functional defect by either having two alleles, perhaps of moderate impairment, or one with more major impairment. Finally, whether the trait one describes is classed as an autosomal recessive or not, depends on how one is assessing that trait. So, even within a simple quasi-Mendelian trait, considerable diversity can be generated simply be having a range of 10 alleles that have various degrees of functional activity. Of course, the MC1R is not the only gene involved in human pigmentation. Current evidence suggested at least over 60 genes may influence human pigmentation and a nice example of this is shown by a group of children we were able to study with Colin McKenzie in the West Indies. These children come from a group that would describe themselves as ethnically black but refer to themselves as ‘Red Ibos’. Although these children’s skin is very dark, they have bright red hair and if you study the melanins in their hair, the levels are very similar to the ones we see in North European redheads. When you sequence the MC1R you find variants found in Northern Europe, but also some variants we hadn’t seen before in Northern Europe. So even although we assume that many of the other genes which determine overall pigmentation levels are different in these individuals (as shown by their dark skin) the MC1R effects on hair seems more penetrant than those on skin. Our evidence for this is incomplete, as we haven’t been able to study these children in detail, but their hair phenotype seems much more in keeping with what we see in Northern Europeans than their skin. We suspect that their skin is lighter than it would have been if they did not carry two MC1R alleles but we haven’t been able to test this objectively. So What! We subsequently showed in populations studies that particular variants of the MC1R are risk factors for melanoma and other skin cancers, with odds ratios varying between 2 and 4. This is in keeping with what we would expect based on our knowledge of the relation between skin colour and cancer risk. We also know that even in those without red hair, say in southern European populations, that the risk of melanoma is increased in those carrying a single variant MC1R allele. In the round however such discoveries are of limited use, if any, in the clinic. 11 By contrast, the odds ratios are much higher for the association between particular combinations of MC1R alleles and red hair (running in the region of 40 to 50). Is this of any use? Well, one obvious use is in forensics. Red hair was the first visible human phenotypic trait that could be detected by examination of scene of crime DNA. With certain caveats, it is now possible to predict with a reasonable degree of accuracy, what the likelihood is of an individual either having red hair or having had a history of red hair or of having very pale freckled skin. There is, however, another “forensic” use of understanding variation in DNA sequence. A couple of years ago, a group from Spain were able to sequence the MC1R of some Neanderthal DNA. Neanderthal’s are a distant relative of modern man on a different evolutionary path. Whatever else we know about the Neanderthals, we now believe it is highly likely that some of them had red hair. If the Neanderthals were on a distinct line from modern man, the question arises what can we say from studying MC1R sequence about our own more recent evolution. In Figure 3 is shown a photograph of a mother, her albino son and a dermatologist, Dr Sandy McBride. The picture was taken in Tanzania where an outreach establishment of the British Association of Dermatology, promotes training in Dermatology and provision of care. The mother’s skin you can see is well adapted to this climate. This is an area of the world close to the equator and with therefore high ambient ultraviolet radiation. By contrast, her son who must carry two recessive alleles because he is an albino (one from the mother and one from the father) is clearly at a disadvantage. He will burn in the sun, this will make schooling difficult, and he 12 will have a number of other disabling features, including visual impairment. His skin is clearly not matched to the environment he finds himself in. The interesting question relates to Dr McBride. She has red hair but in what way is she adapted to her usual environment in the British and Irish Isles? Out of Africa As I mentioned earlier, there are more than 100 sequence variants in the MC1R, a comparatively small gene. Along with Dr Rosalind Harding from the Department of Anthropology and Statistics in Oxford, we were able to study the diversity of MC1R in relation to place of birth and by implication ancestory. When one looks at MC1R sequence in Africa, there is very little sequence variation and what it seen is of a form that doesn’t usually alter the protein sequence of the receptor. By contrast, out of Africa, there is considerable diversity. How can we interpret this data? By using leverage from the known sequence of the chimpanzee, we can hypothesis what the sequence was of a common ancestor. What seems to have happened is that diversity of the MC1R has, in effect, not been tolerated (thinking in evolutionary terms) in Africa, whereas out of Africa diversity has occurred. We can think of this in two ways. The first is that nature is fairly indifferent to what happens to the MC1R out of Europe, or second, that there has been a selection for diversity on the MC1R out of Africa. The evidence on this topic is far from clear and different groups have interpreted the same data in slightly different ways. Whereas there does seem to be constraint on the gene in Africa, we are still uncertain whether active selection has taken place on the MC1R in Europe, or whether the changes we see could just be due to chance. It is important to emphasise here that this pattern of diversity, with a greater diversity being seen in non-African populations is unusual. 13 For many genes, if not most, the majority of diversity is in Africa, because man’s roots go back longer in Africa than elsewhere —we are all literally a subset of African peoples. If the data from the MC1R is slightly ambiguous, there seems to be more supportive evidence from study of other pigment genes that selection for lightening of skin may have taken place as humans moved out of Africa. The changes we see in the MC1R allow us to date some of these changes and they are compatible with what we know in terms of movement of peoples over the last 50,000-100,000 years or so. The likely reason for selection for lightening of skin relates to a key aspect of cutaneous biology, namely that vitamin D is synthesised in skin in response to ultraviolet radiation. In areas of high ambient ultraviolet radiation, and even dark skin, the amount of ultraviolet radiation appears to be sufficient for good health. As however humans have migrated further northwards, for many months a year at least, ultraviolet radiation is more scant. This would not be an issue if the human diet was rich in Vitamin D. However, evidence even today suggests that many of the population have Vitamin D levels that may be lower than those necessary for full health. For instance, shown below (Figure 4) is a picture of a deformed pelvis secondary to Vitamin D deficiency. The mother would have had rickets and would have died during child labour, simply because the child’s head would not be able to move through the pelvis. As humans moved out of Africa, our diet again changed, and with the advent of agriculture, became predominantly cereal based. This is a diet that may support a relatively high density of humans compared with hunter-gatherers, but unless supplemented by a range of particular food stuffs, or pharmacological supplementation, puts individuals at risk of rickets, if sun exposure is minimal. If this hypothesis is correct then a gradual lightening of skin will have been an advantage in terms of Vitamin D biosynthesis. Furthermore, red hair may just be 14 one other way to actively lighten your skin colour, rather than any selection taking place on red hair per se, rather than light skin. We don’t know this is the case and it remains a hypothesis. Conclusions I started this essay by pointing out that there were changes in skin physiology in response to climatic change leading to changes in diet. I have also pointed out that further changes in diet, particularly the adoption of cereal based diets, may again have had implications for cutaneous and general health. We know that exposure to ultraviolet radiation increases the risk of skin cancer, and that melanin is a very effective natural sun block. I have argued, along with the conventional hypothesis of the day, that skin lightening allows a larger number of photons through and allows increased Vitamin D biosynthesis. On the other hand, we know that ultraviolet radiation causes DNA damage and that there is a dose relationship between exposure to ultraviolet radiation and skin cancer. At the other extreme we know that if an individual receives little Vitamin D then that person is at risk of rickets. Still, today, even with a fairly broad diet available, many people appear Vitamin D deficient, and we know from the history of patients who are particularly sun sensitive, (such as in some forms of porphyria) and who therefore avoid the sun, that they are at particular risk from vitamin D deficiency. What remains unclear, and critical to broad statements about sun exposure or avoidance of sun, is what levels of vitamin D are associated with optimum health. We know that severe deficiency leads to bone disease, but there is increasing suspicion – but not proof – that what were once considered reasonable levels of vitamin D may, in actual fact, be sub-optimal. At present, this issue is not resolved. Practically, its worth remembering that it 15 is always possible to take additional vitamin D orally which would seem to get around the problem of harmful effects of ultraviolet radiation on skin. The picture I have sketched is a standard interpretation of what we know about skin and the relation with UV ultraviolet radiation and vitamin D biosynthesis. It remains largely a hypothesis as its difficult in this sort of historical subject to get absolute proof. There are some odd things, however, still unexplained. It seems to this author at least since time immemorial that people have suggested that those with red hair are different in all sorts of ways from those without red hair. In general, most of these claims, that do not involve the skin, have not stood the test of time. However, several years ago, Jeff Mogil and colleagues, working in Canada, were trying to indentify genes that influence pain susceptibility in mice. They tracked down a locus and subsequently demonstrated that the locus in question was the mc1r and that mc1r status seemed a determinant of sensitivity to pain, and sensitivity to opiates (given to relieve pain). Subsequently, human studies have been in keeping with some of this earlier work, although the picture does not seem completely clear. How does this relate to pigmentation and MC1R? The answer is that we don’t know. Whereas we think that we have a clear understanding of what MC1R does in skin, the data on extra-cutaneous effects of MC1R is minimal and what there is, is confusing. We know that the ligand for MC1R, melanocyte stimulating hormone, has a number of other actions, but many of these we can explain via a number of receptors that are related, but distinct, from the MC1R. Time will tell us more Oscar Wilde was not known for his scientific quotes, but a quotation from the ‘Picture of Dorian Gray’ is apposite here. “It is only shallow people who do not judge by appearances. The true mystery of the world is the visible, not the invisible.” 16 Whilst, I am not convinced that Oscar Wilde has got this completely right, I hope I have been able to convince you that at least some of the mystery of the visible has fallen prey to the power of modern pigment genetics, but that some invisible aspects may still be lurking beneath our current explanations. Acknowledgements: I have had the good fortune to collaborate on much of the work described above with Professor Ian Jackson, MRC Human Genetics Unit, Edinburgh; with Professor Tony Thody, University of Newcastle; Dr Rosalind Harding, University of Oxford; and Professor Brian Diffey, University of Newcastle. Much of the work in my laboratory over the last 15-16 years or so has been supported by The Wellcome Trust, the MRC, the Leech Trust and the Foundation for Skin Research (Edinburgh). Further reading If you go to www.reestheskin.eu and follow the links to ‘red hair’ and ‘red hair for the non experts’ there are pdf of reviews written by me in the Annual Review of Genetics, and the American Journal of Human Genetics. 17 Legends Figure 1. The scrotum of the blue vervet monkey. The ‘blue’ colour is due to deep lying ‘brown’ melanin in the skin, such that scattering is shifted to the blue end of the visible spectrum. Figure 2. The author demonstrates what happens to skin if the natural barriers to ultraviolet radiation are breached. The resulting erythema reflects DNA damage and disappears as that DNA is damaged. Figure 3. A mother(left) with her albino son(centre), together with the red-haired Dr Sandy McBride (right) in Moshi, Tanzania. Figure 4. A deformed pelvis secondary to vitamin D deficiency (rickets). The birth canal is grossly narrowed and would lead to maternal and infant death 18 Figure 1 Figure 2 Figure 3 Figure 4