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On 25th March 2015, 530 years after his death, King Richard III of England will be interred in Leicester Cathedral. This remarkable ceremony is only taking place because of the success of DNA analysis in identifying his skeletal remains. So what sort of genes might a king be expected to have? Or, more prosaically, how do you identify a long dead corpse from its DNA? Several methods were used, and in particular the deduction of the skeleton’s probable hair and eye colour raises some interesting questions about future trends in forensic DNA analysis.

Richard III is one of England’s best known kings, largely due to the famous play of William Shakespeare in which he is portrayed as an evil villain. He only reigned for two years and was killed at the age of 32 at the battle of Bosworth in 1485. According to the historical records he was unceremoniously buried at Greyfriars Friary in Leicester. At some stage knowledge of the exact location of Richard’s burial was lost. But in 2012 excavations under a car park at the probable site of the former friary yielded “skeleton 1″. Suspicion of his royal identity was excited by the fact that the skeleton had a severely bent spine causing the right shoulder to be higher than the left. This well-known deformity of Richard was mentioned in a contemporary source, as well as by Shakespeare. Furthermore, the skeleton was male, the age was about right, it had evidently been killed in battle, and the radiocarbon date was consistent with death in 1485.

This was all very suggestive, but it was the DNA analysis that really proved the case. The work was led by a team at the University of Leicester, with participation by many other UK and European centres. It is important to note that this was not the normal type of forensic DNA identification, which relies on comparing a set of highly variable DNA markers to a database. Such analysis is fine so long as your suspect is in the database, but it is no use for identifying a long dead individual who is not in any database.

By far the best evidence for the identity of Richard III comes from the analysis of his mitochondrial DNA. Mitochondria are bodies found in every cell, responsible for the production of energy. They have their own DNA which is passed down the generations only through the female line. Barring the occasional new mutation, the DNA sequence of mitochondrial DNA should be identical from mother to daughter down a particular female line of descent. Like their sisters, males also carry the mitochondrial DNA of their mothers, but they do not pass it down to their own offspring.

Richard will have shared mitochondrial DNA with his sister, Anne of York. Two complete female lines of descent were traced back to Anne of York, one of 17 generations down to Michael Ibsen, a resident of London, and the other of 19 generations down to Wendy Duldig, formerly of New Zealand. Complete sequencing of their mitochondrial DNA showed a 100% match between skeleton 1 and of Michael Ibsen, and a single base change compared to Wendy Duldig. One change over this period of time is quite likely to be a new mutation. The sequence family (haplogroup) to which the mitochondrial DNA sequence belongs is a fairly rare one, so few other people in England in 1485 would have shared it and in fact the team has systematically ruled out all the other males of the period who might have shared it because of a common female lineage with Richard III. So this match is highly significant and is the best piece of evidence that the “skeleton 1″ is indeed King Richard.

Also applied was a newer method which is a technique for predicting the hair and eye colour of someone from their DNA. The most important variants affecting hair colour are mutations of a gene called MC1R, which encodes a cell surface receptor for a hormone. Individuals carrying variants of the MC1R gene with reduced function are likely to have red or blond hair rather than the normal dark hair. The pigmentation of the iris of the eye depends significantly on a gene called OCA2, encoding a protein which transports tyrosine into cells. Again variants of reduced function give less pigmented eyes, meaning that the colour is blueish rather than brownish. Recently a Dutch group created a forensic test based on variants at 24 genetic loci, of which 11 are in the MC2R gene and the rest in 12 other positions including the OCA2 gene. Identification of these 24 variants yields a fairly accurate prediction of hair and eye colour, and in the case of skeleton 1 the prediction was for blue eyes and blond hair. The existing portraits of Richard III all date from some time after his death but the older ones do indeed show light-coloured eyes and reddish-brown hair, an appearance which is consistent with the prediction.

These two types of analysis indicate two rather different senses in which we use the word “gene”. The sequence variants of the mitochondrial DNA, like those used in normal forensic identification, do not, in general, affect the characteristics of the individuals carrying them. The DNA changes often lie outside actual genes, in the regions of DNA between genes. They are better described as “markers” than as “genes”. But the hair and eye colour analysis is based at least partly on actual gene variants that might be expected to generate those visible characteristics.

How much further might this kind of analysis be pushed? Could the height, facial features or skin colour of a crime suspect be deduced from their DNA? The essential issue is the number of gene variants in the population that affect a feature. If it is relatively small, as with hair and eye colour, then prediction is possible. If it is very large, as for height, then it is not possible, because most of the variants affecting height have too small effects to be detectable. Most of the human characteristics that have been studied in this way have turned out to depend on a very large number of variants of small effect. So, contrary to popular perception, there are real limits to what is possible in terms of prediction of bodily features from DNA data. There will doubtless be some other features that are predictable, and these may eventually include skin colour. But unless a completely new approach is invented, it is unlikely that we shall ever see an identikit picture of a suspect generated from DNA at the crime scene.

Featured image credit: Stained glass, by VeteranMP. CC-BY-SA 3.0 via Wikimedia Commons

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This month, it was reported that scientists at Harvard University have successfully made insulin-secreting beta cells from human pluripotent stem cells. This is an important milestone towards a “stem cell therapy” for diabetes, which will have huge effects on human medicine.

Diabetes is a group of diseases in which the blood glucose is too high. In type 1 diabetes, the patients have an autoimmune disease that causes destruction of their insulin-producing cells (the beta cells of the pancreas). Insulin is the hormone that enables glucose to enter the cells of the tissues and in its absence the glucose remains in the blood and cannot be used. In type 2 diabetes the beta cells are usually somewhat defective and cannot adapt to the increased demand often associated with age and/or obesity. Despite the availability of insulin for treating diabetes since the 1920s, the disease is still a huge problem. If the level of blood glucose is not perfectly controlled it will cause damage to blood vessels and this eventually leads to various unpleasant complications including heart failure, stroke, kidney failure, blindness, and gangrene of limbs. Apart from the considerable suffering of the affected patients, the costs of dealing with diabetes is a huge financial burden for all health services. The prevalence of type 2 diabetes in particular is rising in most parts of the world and the number of patients is now counted in the hundreds of millions.

To get perfect control of blood glucose, insulin injections will never be quite good enough. The beta cells of the pancreas are specialised to secrete exactly the correct amount of insulin depending on the level of glucose they detect in the blood. At present the only sources of beta cells for transplantation are the pancreases taken from deceased organ donors. However this has enabled a clinical procedure to the introduced called “islet transplantation”. Here, the pancreatic islets (which contain the beta cells) are isolated from one or more donor pancreases and are infused into the liver of the diabetic patient. The liver has a similar blood supply to the pancreas and the procedure to infuse the cells is surgically very simple. The experience of islet transplants has shown that the technique can cure diabetes, at least in the short term. But there are three problems. Firstly the grafts tend to lose activity over a few years and eventually the patients are back on injected insulin. Secondly the grafts require permanent immunosuppression with drugs to avoid rejection by the host, and this can lead to problems. Thirdly, and most importantly, the supply of donor pancreases is very limited and only a tiny fraction of what is really needed.

This background may explain why the production of human beta cells has been a principal objective of stem cell research for many years. If unlimited numbers of beta cells could be produced from somewhere then at least the problem of supply would be solved and transplants could be made available for many more people. Although there are other potential sources, most effort has gone into making beta cells from human pluripotent stem cells (hPSC). These resemble cells of the early embryo: they can be grown without limit in culture, and they can differentiate into most of the cell types found in the body. hPSC comprise embryonic stem cells, made by culturing cells directly from early human embryos; and also “induced pluripotent stem cells” (iPSC), made by introducing selected genes into other cell types to reprogram them to an embryonic state. The procedures for making hPSC into beta cells have been designed based on the knowledge obtained by developmental biologists about how the pancreas and the beta cells arise during normal development of the embryo. This has shown that there are several stages of cell commitment, each controlled by different extracellular signal substances. Mimicking this series of events in culture should, theoretically, yield beta cells in the dish. In reality some art as well as science is required to create useful differentiation protocols. Many labs have been involved in this work but until now the best protocols could only generate immature beta cells, which have a low insulin content and do not secrete insulin when exposed to glucose. The new study has developed a protocol yielding fully functional mature beta cells which have the same insulin content as normal beta cells and which secrete insulin in response to glucose in the same way. These are the critical properties that have so far eluded researchers in this area and are essential for the cells to be useful for transplantation. Also, unlike most previous procedures, the new Harvard method grows the cells as clumps in suspension, which means that it is capable of producing the large number of cells required for human transplants.

These cells can cure diabetes in diabetic mice, but when will they be tried in humans? This will depend on the Food and Drug Administration (FDA) of the USA. The FDA has so far been very cautious about stem cell therapies because they do not want to see cells implanted that will grow without control and become cancerous. One thing they will insist on is extremely good evidence that there are absolutely none of the original pluripotent cells left in the transplant, as they would probably develop into tumours. This highlights the fact that the treatment is not really “stem cell therapy” at all, it is actually “differentiated cell therapy” where the transplanted cells are made from stem cells instead of coming from organ donors. The FDA will also much prefer a delivery method which will enable the cells to be removed, something which is not the case with current islet transplants. One much discussed possibility is “encapsulation” whereby the cells are enclosed in a semipermeable membrane that can let nutrients in and insulin out but will not allow cells to escape. This might also enable the use of immunosuppressive drugs to be avoided, as encapsulation is also intended to provide a barrier against the immune cells of the host.

Stem cell therapy has been hyped for years but with the exception of the long established bone marrow transplant it has not yet delivered. An effective implant which is easy to insert and easy to replace would certainly revolutionize the treatment of diabetes, and given the importance of diabetes worldwide, this in itself can be expected to revolutionize healthcare.

Featured image credit: A colony of embryonic stem cell. Public Domain via Wikimedia Commons

The post Stem cell therapy for diabetes appeared first on OUPblog.

        

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