 So, I want to thank you. I have no relevant relationships with industry, but as a federal government employee, I want to thank you for support of NIH with your tax dollars. So I think we're going to have to lower the lights a bit so we can see these pictures. That's better. So here's a child who had a severe burn with minimal sun exposure. You can see the blistering on her face. She's 15 months old. This is in Chicago. And if you look at the side, you can see where she was wearing a hat. And this lets you know that it was due to sun exposure. The family, the doctor that I got the slide from, Dr. Nancy Esterly, was a very good pediatric dermatologist. She diagnosed XP or Xeroderma pigmentosum. And the family then went to People Magazine. And they had, the people did this story about the two children. By that time, Jamie and Sherry Harrison live in the shadows, prisoners of a genetic disorder that makes sunlight a killer, hidden in curtain rooms, playing outside only at night. The girls and their anxious parents have made the darkness a place where life and love can survive. Here's their moving powerful story. This is very good for selling magazines, and it's even more important than Elizabeth Taylor, as we see. But almost nothing about that is correct. These children don't have to be in the dark. They have to be protected from ultraviolet radiation that they can be indoors with lights on, and they only need the candles if it's their birthday. On the other hand, this child had very severe sunburn, minimal exposure of all places in Scotland. And the parents were falsely accused of child abuse. This child also had the zero derma pigment test. And some of the patients are so severely sensitive to light that you can get this. But Dr. Ferguson, dermatologist, saved the day and was able to make the correct diagnosis. Not all of the patients actually burn so severely. Some of them freckle without burn. And here's a child with, she was two years old, and she had marked freckling all over her face. Before the age of two was very unusual and makes one think of some genetic abnormality, possibly a zero derma. So this was in September. And if you notice, she had a small lesion on her lip. And then by the next month, this large mass grew in that place, a rapidly growing tumor. And in fact, this child was in daycare. And the daycare provider had seen an article about XP and told the parents to go get a checked out from the dermatologist. And in fact, this was either a squamous cell cancer or a rapidly growing carotid oacanthoma. So you see two forms of zero derma, the patients that are exquisitely sun sensitive and burning on minimal sun exposure, acute burning. And the others who don't have that, but they tan normally and freckle. All of them are subject to increased cancer risk. And that's because they have a deficiency in DNA repair. Now there are many different DNA repair systems. There's base excision repair that cuts out parts of the damage. A nucleotide excision repair, mismatch repair, which in the abnormal cases gives you a colon cancer recombination repair, replication fork arrest. So we're focusing on this one nucleotide excision repair or NER, which has two forms. You'll see gene-specific and global. But the diseases that I'll mention now are zero derma pigmentosum, trichothyodistrophy, and coccane syndrome. And so why do we study these rare human genetic disease? Well, one is to care for the affected patients, although it's rare that patients do need doctors to help them. The other is to discover mechanisms of disease. The third is to assess treatment methods using small numbers of patients at high risk of cancer, and then I'll be also talking about future direction. So this is the remainder of the talk. We'll start about the care for the affected patients. And so we're comparing zero derma to coccane syndrome and trichothyodistrophy. So the XP patients are sun sensitive, although not all of them burn minimal sun exposure. However, they all have a very high risk of developing cancer. They can have ocular abnormalities. Any part of the body that's exposed to ultraviolet radiation can show these abnormalities. Some of them have neurologic abnormalities. They have a defect in nucleotide excision repair. So this is mostly due to an environment and a small component of a developmental abnormality. So here's the appearance of a patient we had with what the dermatologists call porculoderma. That's due to radiation exposure or sun exposure of increased pigment, decreased pigment, atrophy, and dilated blood vessels, telangitasia. This you see in farmers or sailors after many years of sun exposure, but this unusual picture is from a girl who's only 11 years old. And so this is a disease of premature aging. And you can see the areas of the body that are doubly covered, like the buttocks don't show the abnormality. And here's skin cancers on several of the patients. They get the same kind of cancers as in the general population. They'll get basal cell cancers, squamous cell cancers, and melanomas, but at a monthly thousand fold increase frequency. Also they get tumors and clouding of the cornea, tumors of the anterior surface of the eye. So they have premature photo aging of the epidermis. So this also is a disease of premature aging. So here's a summary of zero derma. It's equally distributed among males and females. It's found all over the world, even in dark skinned people. I'll show you some pictures later. Frequency is about one in 20,000 in Japan, and it's more rare in the United States, about one in a million. So here's some of our experience at NIH. We've been studying patients with S.P. since 1971. The oldest patient's about 75. The sun sensitivity or freckling has its onset very early in life, under the age of two. The skin cancer has a mediated onset of nine years, and in the past death occurred 38 years earlier than in the U.S. general population. However, some early diagnosis and sun protection may prolong life. So why is it, what's the situation in Japan? Well there's a founder mutation. S.P. is more frequent in Japan than in the United States. 55% of the Japanese patients have a defect in the gene called XPA, and most of them have the exact same splice mutation. So this was identified by Dr. Kyoji Tanaka at Osaka University. That was actually the first of the genes of XP that were found. So we did a study in collaboration with our Japanese colleagues, and we did haplotype analysis of the XPA families, and we found an area of the DNA surrounding the mutation that was common to all the families. And if you do the appropriate calculations, that means that this arose in the population about 100 generations ago. So if a generation is 20 years, that means it arose 2,000 years ago. This gene for Xeroderma has been in the Japanese population for many years. Japan is an island and they tend to have children with their relatives, or at least people from that same ethnic group. So then we did another study in collaboration with the people in Hiroshima, looking to see the frequency of this mutation in the general population. It's about 1% of the population. And so this means that the frequency of the XP patients is 1 in 22,000, and this rate, if representative, implies that there are about a million carriers of the XPA-founded mutation in the Japanese population. I don't know that it has advantage and may actually have a disadvantage. We're doing a study now with the epidemiologist at NIH to see if the asymptomatic heterozygotes have an increased cancer risk. There's some suggestions for other diseases, such as a taxiotelangitasia, that the carriers have increased cancer risk. And so we're doing that now here in the United States. And we're hoping maybe to be able to do it with our Japanese colleagues. No. It's there. So we looked at the location of the skin cancers in the US population and in the XP patients. If you look at the basal cell and squamous cell cancers, these are primarily caused by sun exposure. And the predominant area in the general population is on the face, head, and neck. And in the XP patients, it's also mostly on the face, head, and neck. So we'll see here. So there's a similar site distribution of non-melanoma skin cancer in XP patients and normals. Now, melanoma is a more dangerous type of tumor. It can spread and metastasize throughout the body. That occurs more commonly on other areas than on the face. And here we see a patient who had Xeroderma with actually dozens of melanomas on her legs and her extremities. And the distribution in the general population of melanomas is spread out more uniformly over the body. And similarly in the XP patients, it's more spread out over the trunk and extremities. So again, there's a similar site distribution of melanoma skin cancer in XP patients and in normals. However, the age of onset of the non-melanoma skin cancer, we said, is a median age of nine or so. And in the general population, it's 67. So there's a 58-year reduction in age of onset of skin cancer in the XP patients. And they have a DNA repair defect, so that says that you people sitting here with normal DNA repair can thank that for 58 years more sun exposure before you get your first skin cancer, median age 67. Now melanoma is different. The median age of the melanoma in the XP patients is 22. In the general population, it's 55. So in the general population, melanoma occurs at an earlier, at a younger age than non-melanoma cancer. But in the XP, it's at a later age, although there's a 33-year reduction here. So there's a 10,000-fold increase in skin cancer in the non-melanoma cancer. And the melanoma induction mechanism is different from the non-melanoma skin cancer mechanism, we can tell, because of these different age of onset. So by studying the rare diseases, you can learn about factors that are involved in induction of these skin cancers. Now, XP patients in the general population, the people who get cancer are the ones that burn easily and tan poorly. Well, in the XP, it's somewhat different in that the people who never burned get cancer at a younger age than the people who always or sometimes burn. And this seems to be a paradox, but as I showed you before, the patients who burn really burn. And so the child that I showed you at the beginning, where the parents were accused of child abuse, when they figured out it was due to sun exposure, they would keep the children out of the sun even before the diagnosis of Xeroderma was made. And so we think that the reason may be that they are related to early sun protection because of the severity of the sunburn of the affected children. And that's why the ones that burn are well protected and get tumors at a later age than the ones that never burn. Now, not all of the patients have defective nucleotide excision repair. We see the patients, we start XP and then we started numbering from one. And BE is Bethesda. So this was the fourth patient that we saw. This was actually working with Dr. J. Robbins in the dermatology branch at the time. And the first three patients had defective DNA repair, but his was normal. So he was called XP variant. But that doesn't mean it's a mild disease. In fact, he had basal, he had squamous cell cancer here. And he had a, there's a nodule of melanoma on his scalp. And he died of a metastatic melanoma involving most of the organs of his body. He has a defect in another gene, a bypass polymerase called polymerase eta. And he died of age 27 of metastatic melanoma. That was in 1971. And at NIH, the pathologists never throw anything out. And so when it became interesting to look at the molecular basis of the melanomas, we were able to go back to the pathology department and ask them to pull the blocks. And then we cut the blocks again and did DNA sequencing. And we found p-tenth of this tumor suppressor gene mutations in the metastatic melanoma gene lesions of the patient. Interesting thing about it, we looked at half a dozen melanoma metastases and everyone had a different p-tenth mutation. In fact, they had multiple mutations and none of them were common to each other. So that attests to the genetic instability in serodermic metastase and particularly in the melanomas. We didn't have the primary tumor of this patient, so we don't know where the first one was. But it does offer a challenge. If we wanted to use some of the more sophisticated new treatments that are targeted at specific mutations, we couldn't do that in this patient because he had many different mutations, even in the same gene, in the tumors. Now, not all of the patients that we've seen were very rare children. And here's a man who came to us. He had these pictures. When he was 19 years old, he was in the U.S. Navy in the South Pacific. There he is. He's actually a Greek-American ancestry. So his skin was slightly dark to begin with. By the time we saw him, he'd had dozens of melanomas and we were able to diagnose seroderma. And then we followed him for a number of years. And he ultimately lived till 72 and he died of a schwannoma, a nervous system tumor. So not all of the XP patients are the children that we thought about before. So if you see adults with multiple skin cancers, you do want to think of the diagnosis of seroderma. So our conclusions at this point. DNA repair plays a major role in prevention of melanoma and non-melanoma skin cancer in the general population. Sunlight exposure is responsible for the induction of melanoma as well as non-melanoma skin cancer. But the mechanism of induction of melanomas is different from that of non-melanoma skin cancer in the XP patient. We don't know what it is, but we can tell from this epidemiology that they are different. Now, any part of the body that's exposed to ultraviolet radiation shows these abnormalities. So they can get a tumor of the conjunctiva. This is a seven-year-old boy. It's an XP variant. And why is that? Ultraviolet radiation is absorbed by the cornea and the lens, and visible light is visible because it reaches the retina. And so if you look at where the damage is in the XP patients, you can see it's the cornea, the conjunctiva, the cornea, and also of the lids, but not of the retina. So just by close examination of the eyes, you can get a good idea of which wavelengths are important and the damage. And it's the ultraviolet radiation. I mentioned before, zero-dermicurs over the world, all over the world, and even in dark-skinned people. These were two brothers from Africa that came to us. Here's one was 23. The other was 17. This is his eye. He had a large lesion on the eye. We were very concerned. It was the squamous cell cancer, the ophthalmologist. One of them was opting to actually remove the eye, but looked a little more closely. We were able to actually realize what the changes were just on the surface. And they were able to very carefully remove that, and now he actually has very good vision. The other thing is this lesion here that we were concerned with what? What did this look like after all this talk of melanoma? We were working with Dr. Tom Horniak, who is now at the University of Maryland. He was here in the clinical center. He used a dermatoscope, which is a hand-held magnifier. He said, nope, that's not a melanoma. That is what we call a pigmented basal cell carcinoma, which was lucky because he had dozens of these lesions. It's easier to treat a basal cell carcinoma than it is a melanoma. So that's a pigmented basal cell carcinoma. So not everything that's pigmented is a melanoma. That's why you want to go to a good dermatologist. This patient actually was very unusual because on the tip of his tongue, he had this lesion that was growing. And it was at the base of it was a squamous cell cancer. Again, we had this discussion with the oral pathologists, and some of them wanted to cut out his whole tongue. Dr. Jaime Berhim, who was there at the time, and now at the University of Maryland, said, no, just remove that and treat the base with a liquid nitrogen. And he did that, and he's doing very well. Most tumors of the tongue don't occur on the tip of the tongue. They occur on the back of the tongue. It's on the more interior surface, and it's by people who are much older and who were chewing tobacco or beetle nuts. And it has a very different appearance. This, and they grow fairly rapidly, and they metastasize. We checked this patient for metastatic lesions. He did not have that. It was just on the tip of the tongue. We were wondering what might have caused that. And so we did a literature review. And checked with the lock horns. And it says, last summer, she burned her tongue. Talking so much. So these boys were a dark skin. They were out in Africa, and we think that that tumor of the tip of the tongue was due to a sun exposure, in fact. And this has been seen in a number of other zero demo patients. So the tip of the tongue is an area that we wouldn't have anticipated to be a sun exposed area. But if you look closely in some of these patients, you'll see changes on the lips and the tongue, the tip of the tongue that show this sun damage. And they may be licking their lips when they're outdoors. So the median age of death in the XP patients was 38-year reduction in comparison to the US population. This is a study that we did with the epidemiologist and with Portia Bradford, who was now a dermatologist, but she was a fellow with the genetic epidemiology branch with Margaret Tucker and Elisa Goldstein, who did this review of all our XP patients from 1971. Now, about 25% of the patients have a progressive neurologic degeneration. So here's a patient that we saw at age four. We've been able to follow these patients for many years. At 17, she was here. She'd had dozens of skin cancers on her face at that point, but she was fairly neurologically intact. However, by 41, she was severely impaired. She couldn't walk or talk or feed herself or dress herself. And the reason for that is that this is what her brain was at age 20. And by the time she was 41, there was a massive atrophy and the enlargement of the ventricles. And the overall size of her brain had shrunk to that of an infant. And we had actually published an autopsy of her and three other patients recently with a pathologist. And we showed a really profound neuro degeneration in some of these patients. It's a primary neuro degeneration. The neurons just die. And there's no inflammatory reaction. And we don't know what the provoking agent is. It's obviously not sun exposure, but it may be some sort of oxidative damage that's poorly repaired with that speculation. Now, sometimes we'll get children that come where we, at an early age, and we don't know if they're going to, and the parents ask us, will they be developing neurologic disease or not? And we did a study with the audiologist, with the student Miriam Tutanchi, medical student plus Carmen Brewer in the audiology department. And again, looked at all the audiograms from the patients from that we've had since 1971. And we're able to plot the age of the patients and the audiograms. And a simple model came up. These are the patients with neurologic degeneration. They're hearing those. And acute burning on minimal sun exposure. So we found two markers that were very, very helpful. Hearing loss, acute burning on minimal sun exposure, and hearing loss, as detected by an audiogram. Now, even before there's clinical hearing loss at 20 decibels, we can see in the patients, if they're old enough, that they are maybe on the road to hearing loss. And that is a marker of progressive neuro degeneration. Whereas others, even some of them that have this burning phenotype, may have normal audiograms. And it's likely that they will still not have the profound neurologic degeneration. Another feature of this is that even the patients that don't have the neurologic degeneration are in the lower hearing levels. This is the medium. This is the 95th percentile on the other. And all of the patients are closer to the 95th percentile of hearing loss. Again, a suggestion of a premature aging of the auditory system. Zeroderma is a disease of premature aging. So I showed before that patient with metastatic melanoma who died. But in fact, the patients who have neurologic degeneration die at an earlier age than those that don't have the neurologic degeneration. And so in some of the patients, as they get older, they stop getting skin cancers, but they have the neurologic degeneration. So we move from the dermatologist and the oncologist to the neurologist taking care of them. I moved to the second disorder a little bit about the cocaine syndrome. Like Zeroderma, they're sun sensitive. They don't have the pigmentation, and they don't have cancer. But they can have ocular abnormalities in the retina, a different from the XP. They all have neurologic abnormalities. They have also a defective nucleotide excision repair. It's a different gene than the XP's. And it's mostly due to a developmental abnormality. So here's a patient we saw. She was three. She died at the age of 13. She was very sun sensitive and had developmental delay. Some of the patients, you can help with the diagnosis if you do a CAT scan of the brain. You can see calcification of the basal ganglia. This is a feature in some of the patients with cocaine syndrome. We don't know the pathophysiology of it, but it is sometimes helpful diagnostically. Now, the other disease is trichothiodystrophy. Tricho refers to hair. Thio is sulfur. So actually, they have low levels of sulfur containing amino acids. And dystrophy is abnormal. So some of them are sun sensitive. Some aren't. They don't have pigmentation. They do have ocular abnormalities, including congenital cataracts in many of them. They have neurologic abnormalities. They have a defect in the nucleotide excision repair. And some of them the exact same gene as in the XP patients. But the clinical phenotype is very different. They have different mutations in the same gene, but totally different phenotypes. They're mostly due to developmental defect, whereas in Zeroderma, it's the sun exposure. And so here's a child that we saw, Alan Joe, another medical student wrote this up. The children are born unusual with colloidal membrane on their skin. This is kind of, they've family described to this kind of a cellophane. And this appears after a week or so. Children often have low birth weight and neonatal abnormalities. So, patient has short spore spritle hair. Some of them never get haircuts because their hair break off, but they have long eyelashes. Here she is at four years. They have spoon shaped nails. These are things dermatologists love. The coil of Nikki is a spoon shaped nail. But the diagnosis is very simple. You just cut the hair and look at it on their polarizing microscope, the standard 100 in the past department. And you see dark and light alternating bands on the polarized microscope. And this is virtually diagnostic for tricothiodystrophy. It's a very simple diagnosis. It doesn't cost very much to do. And some, and it's one of those, if you don't think of it, you'll never diagnose it. But if you do think of it, it's a very simple diagnosis. In addition to the tiger tail banding, they have hair shaft abnormalities, including tricoschesis, and they break very easily. Now, one of the features of tricothiodystrophy is similar to cocaine syndrome. They both have a leukodystrophy. They have a, here's the normal myelin, the brain, and there's a child with tricothyodystrophy, has dysmyelination. With this particular MRI technique, the myelin shows up as dark and the absence of myelin is here. And so they have a developmental abnormality and some of them have absolutely no myelin in their brain. This is more amazing when you see them going, walking around and talking with them, and they're very friendly and active and sociable. But they have no problem in the brain. And then if you do an IQ test, it's about 50 or 60. Now, they're behaving at a much higher level. And we think it's a case where the IQ tests are not actually capturing the reality of the situation. And just to give you an idea, there's many different symptoms of clinical diversity in the patients. This is Dr. Salma Foghery, when she was a medical student, went through all of the literature. There are 112 patients with tricothyodystrophy described at that time, and they have symptoms of photosensitivity, a scaling skin, ichthyosis, brittle hair, intellectual impairment, short stature, birth characteristics, ocular abnormalities. They'll also have x-ray abnormalities on the bones. This means they can start out at many different specialists. And it takes somebody, usually a geneticist, to put it all together and realize just what's going on. And even that it's a genetic disorder. When we did the study, we were very shocked to find that the patients have a 20-fold increase in mortality before the age of 10 years. So we're trying to track down what that's due to, it may be due to infections or it may be other issues. And what also came out of this review is abnormal birth characteristics, which is present in 55%, but even more striking, there were pregnancy complications in the mothers of the patients who give birth to children with dracheotystrophy. And if they give birth to a normal child because it's a recessive disorder, they don't have problems in their pregnancy. And they may have mutations in what we call the XPD gene, and we have mothers of patients with seroderma pigmentosum who have mutations in that same gene. They don't have these pregnancy problems. So it's very specific to the particular mutations in the particular gene. And we learn about pregnancy abnormalities and complications, including help, helps in an intrauterine growth preeclampsia, eclampsia, help syndrome. All of these may be related to abnormalities in the DNA repair gene system. And we learned that this may be occurring in the general population in something we learned in studies of seroderma. So we're looking into the mechanisms of disease. These cells are hypersensitive to ultraviolet radiation. We see if we irradiate the cells in culture. This is a normal cell. The cells from the different patients are very sensitive to killing. The XP variant cells actually have a normal survival, but if you treat them with caffeine, then they become sensitive to killing. And the cocaine cells are just as sensitive of the XP except they don't get cancer. So this is a puzzle. Why these don't get cancer? Why the others do get cancer? And so the system has to do with DNA repair, the lifeguard of the gene pool. This is where we started with. Debate tomorrow, our research nurse came up with this. She's also a very good feeling with the patients and she's trained as a genetics counselor. So what does ultraviolet radiation do to the DNA? At adjacent pyrimidines, it forms two major types of photoproducts, CPD, cyclobutane, pyrimidine, dimer, and the 6-4 pyrimidone product. Most of these, these often involve adjacent pyrimidines or a thymine and a cytosine. And these have the same basis except it's a different type of bond that's formed. There's about three of these for every one of these. This is a more bulky, more abnormal lesion and it's more easily recognized and repaired than this. So the repair system is shown here. They're depending where the damage is occurring, either in an active gene or in the rest of the genome. You have what we call transcription coupled repair or a global genome repair. And different of the proteins recognize the damage here. The polymerase is stopped and the carcane's proteins accumulate right here. In the rest of the genome is the damages recognized by the XPB and XPE and XPC proteins. And then the path comes together. The DNA is unwound in the area of the damage and other proteins do the unwinding. And interestingly enough, the XPB and D proteins and the TTTA not only are involved in this step but they're also part of a basal transcription factor, TF2H transfector factor 2H. And this is a 10 subunit protein that works is to help transcribe all of the genes. And one of the things that mutations leading to Zeroderma have mutations that interrupt the nucleotide excision repair pathway whereas those that lead to tracheotide dystrophy more interrupt the transcription pathway. That's a theory but it's not proven yet. And then at the later stage, the XPF and G proteins will cut out the damage and that's why it's called excision repair and that leaves a gap of about 30 nucleotides and that's filled in by these other proteins. So this nucleotide excision repair, proteins that are in these boxes, if mutated will give human diseases. So we did initial studies with Dr. J. Robbins with valve fusing cells and if one corrected the defect in another one, they were in different complementation groups, if they didn't, they were in the same and we were finding these fusing a lot of pairs of cells and ended up with these complementation groups in collaboration with a group in the Netherlands that first found the complementation groups. We labeled them from A to E in terms of the lowest repair or the higher repair and then FG came along later. So these genes are located there. XP A is 9% of those. So over the years, we did about nearly 200 cells that we tested mostly. In recent years, Dr. Secunder Kahn in the lab did these tests and we found XP A was 9%, XP C was the most common in the United States, 37% XP D, 34%, some of them were XP, some of them were trichothiadistrophy. And here is a complex relationship between the genetics and the clinical features. So for instance, an XP C defect gives you Zeroderma, whereas an XP A defect, depending what it is, you'll either have Zeroderma or without neurologic abnormalities or with neurologic abnormalities. XP D is the most complex one because depending on the mutation, you could either get just Zeroderma or just trichothiadistrophy or Zeroderma with neurologic abnormalities or a combination of two diseases, Zeroderma with Cocaine syndrome. So the genetics is very challenging. And it also gives you very tremendous insights into the relationship between these proteins and mutations in clinical disease. We move to treatment methods using, these people are tremendously high risk of cancer so we can do studies that give you significant differences just using small numbers of patients. The management of XP, there's support groups, several of them now are available in the United States and actually all over the world. Sun protection, skin examination, removal of lesions can prevent them with retinoids and some of these others I'll talk about. So here's a child with XP actually from Texas. At the age of four, she was diagnosed because of sun sensitivity and they were able to get some special cloth that was lightweight and sun protected from NASA and they made these NASA suits. This was very good at sun protecting and she was diagnosed very early and protected very early. But we ran into a problem related to what? In terms of not going outside and growth and nutrition. Vitamin D, yeah. She wasn't getting sufficient vitamin D. Vitamin D is the sunshine vitamin and if these people don't go out in the sun, they don't get it. We had done a previous study on patients who had already been out in the sun a lot and had many skin cancers and they had normal levels of vitamin D. They were adults and they were active but this child never was out in the sun. It's very well protected and actually developed fractures of her bone till we realized what was going on. Now we recommend supplementing all of the patients with vitamin D tablets and getting vitamin D levels. Now here's an unusual patient who had many skin cancers on his face and then he had massive surgery when he was a teenager. They took all of the skin off his face and then replaced it with skin from a sun shielded skin from his abdomen and these are the areas that were grafted to his face and he's now in his 60s and he has cancers elsewhere in his body but not in the grafted sites. In addition, after he had the surgery he protected his skin very well. He used some cosmetics that cover the face so it can't even see that type of thing and his cosmetics are also good at sun protection. Now we did a study a number of years ago with Dr. John DiGiovanna and Dr. Gary Peck with again a small number of patients who had many skin cancers and we treated them with isotretinoin or Accutane and we found that it was very effective in preventing new skin cancers in these XP patients and so if you look at one of these patients in the two years before treatment she had 43 skin cancers. During the treatment with high dose Accutane she had only three tumors but then when we stopped the treatment she had 18 tumors in a year. So you see having the medicine was essential of her keeping it up and here's another patient where these are individual tumors. This is before treatment and then during treatment and then when we stopped. So we resumed treatment in him at a lower dose and we're able to successfully control his skin cancers. Unfortunately he also had an asterisytoma of his spinal cord and then a metastatic melanoma and this patient died of those other tumors. However this was very effective chemo prevention and it was actually one of the first studies showing chemo prevention in humans unfortunately a lot of side effects for the oral isotretinone, for the Accutane, the ectropion, dry skin, chylitis, pyogenic granulomas, they can get a calcification of the ligaments and tendons of the body, of the bones. So there is a scale of toxicity. This is still useful in some XP patients under the right circumstances but you wouldn't want to give this to a child for instance who is still growing and these may interfere with the normal growth. Another study we did with Maria Turner where we injected interferon alpha into melanoma in situ lesion. This is before treatment and then you can see this area was injected and then went away 50 days after a single injection. It was very localized. Only the area that was injected resolved and this area that wasn't injected didn't resolve. So this showed that there may be a new modulation that might be helpful. And here's a family that came to us that was living in Denver, Colorado a number of years ago. They had two children. Here's a one year old and a five year old. And they said, we can go. At this point, we want to move and see if we can protect our children. And they came to us, they were living in Denver. At that time, the UV maps were just coming out and they said that the Denver, Colorado had the same UV as Jacksonville, Florida. And so they said, well, that's not good because they were so high up. And they said, where could we go in the country? Our job will permit me to go. And so what do you think the advice was we gave them? Name is a good one, but that wasn't the one. There's still a lot of sun in there. Seattle, that's where they did. They moved to Seattle, Washington. Here she is now 18 years later and here he is 23 years later. Actually, I was out there and visited with them a couple years ago, brought them to a DERM conference, similar to the ones they had here, put her in a room and said, what's the diagnosis? And even dermatologists couldn't tell she had Zeroderma. So she's very, she's actually very sun sensitive, but she's also very well protected, doing very nicely. So that's a success story. So Zeroderma, all of these are sun sensitive. Zeroderma, pigmentation, increased skin cancer, cocaine syndrome, short stature, neurodegeneration. Triadosteia, dystrophy, sulfur deficient brittle hair, short stature, just myelination of the brain. DNA repair plays a major role in protection against skin cancer. Inherited defects in DNA repair may result in several recessive diseases with very different clinical features and DNA repair genes are also important for normal growth and development. Now we move to the future. So I'll talk a little bit about the genetics of melanoma. More and more I've been coming out about this in the general population. It's really very exciting. Now they found mutations that target treatment to particular mutations. And so this is a paper by Dr. Henson Sow. I took that. And several of the genes are important. This is in the PI3 kinase pathway. BRAF is an oncogene that is mostly mutated and the V600E mutation is now the target of some therapies that are fairly effective at least for short periods of time. So 50 to 60% of the tumors in the general population have BRAF mutations, 20% NRAS, 40% KIT. And then P10 is a tumor suppressor gene and approximately 10% of those in the general population have mutations in P10. We did a study with Dr. Masaki and Dr. Wang who were the postdocs in the lab successful, successively, and also Dr. Horma, Hormac. And looked at P10 mutations in Nevae and melanomas in the Zerodermin patients. Again, the pathology department never throws anything out. So we went to the department, had them recut the slides. And here is the slide. Then we did laser capture micro dissection. This is really neat. You can just look through the microscope and mark out the area that you're interested in and then press a button and cut out. It's a little lazy that goes around and cuts it out. And then this is on a special slide that it's over a well and the tissue just falls into the well. And so you can get a few hundred cells, 700 to 1,000 that we use, and then take that away and extract the DNA sequencing. The advantage of that is that you can specifically look at the tumors that you're interested in and not the rest of the tissue that might not contain the mutations. And so what does UV do? We started out before there were adjacent pyrimidines giving you cyclobutane pyrimidine dimers, CPDs. A normal cell has this. If the damage is there, it's repaired and then you're back to where you started. If it's not repaired, you can get a T changing to a C, for instance, and this is a mutation. And it's a UV type mutation because it's occurring at adjacent pyrimidines. And so we looked at the mutation, the P10 tumor suppressor gene in melanomas in the Xeroderma patients. And 51, or 88% of them had mutations and they were UV type. And in the XP Nevi, there was similar high frequency and even in the melanomas of non-XP patients. So was this due to ultrasound exposure? We looked at another gene, another type of cancer that's where P10 is mutated. The cancer endometrium clearly not UV exposed site. Only 59% of the mutations in this Sanger database show as UV type. And this cancer is the central nervous engine, 61%. So this is significant. And the sequence itself has 54%. And so these are occurring at almost the random sequence whereas here there's a bias towards the UV. So the higher frequency of UV type or A substitution mutations in P10 in XP Nevi and melanomas compared to internal tumors in the general population. Focus is on, it's more molecular evidence of the UV damage causing the mutation of the tumors. Now what about these other genes? Well, we've talked about P10 and in the melanoma in situ and invasive melanoma. Overall, about half of them had mutations in P10. But in BRAF, only 11% and RAS 21% kit at 21%. And so these numbers are much lower or this is much higher. There's a greater frequency of mutations in P10 than in BRAF and RAS or kit in the XP compared to the general population where it's the other way around. So that's, and the melanomas themselves histologically are a little different than those in the general population. They're more lentiginous and the molecular level there also are different. And so this says that the frequency is much higher and these are clearly due to UV. And so the P10 mutations may be more UV generated than the other ones. In fact, that BRAF hotspot that I mentioned before is not a UV type lesion, type mutation. So this suggests that we might be able to treat some of the XP patients. Do I have it here? Oops, here we go. Study promotes strong support, provides strong support for the role of UV in induction of P10 mutations in pre-malignant and malignant pigment lesions in XP patients and the clinical and histologic appearance of the molecular pathology of XP, Neva and melanomas is different from that of the general population. The increased frequency of P10 mutations in the XP patients may offer an opportunity for control and prevention of these lesions by use of P10 pathway inhibitors such as rapamycin, serolimus. Rapamycin is used in immune suppression and kidney transplants and other situations. We might be able to use it topically. It's used now topically in tuberous sclerosis patients and we might be able to use it topically without inducing immune suppression in the XP patients who are ready. We know how immune suppression itself increases skin cancers. So we might be able to treat the patients lesions topically without inducing systemic immune suppression. And then the last topic has to do with an exciting new work by Christine Kuchow who is right here in the audience. Christine, where are? There you are. She did this. And she's a postdoc from Germany and we're looking at the nice genetic, we're ending up with genetics again. Induce DNA Repair and Zeroderma Group C cells by a read-through of stop codons. So what does that mean? There's a premature termination. A codon or stop codons can cause elongation. A rest of the mRNA is degraded by nonsense mediated decay. And so this is the normal elongation of the RNA. If you have the wrong, stop codons are normally at the end of a sequence of a protein. Whereas if you have a mutation that puts a stop codon in the middle, it will stop. And then you'll get a shorter protein or no protein or what'll happen is the message will be degraded and you'll get no message there. And that if we have many XP patients, XP-C patients who have this type of mutation and you can use aminoglycosides like gentamycin, which kill bacteria by messing up the ribosome. But in mammalian cells with the right dose, you may be able to get it to read through a certain extent of the stop codons. And this might be useful for treating some of the patients. Aminoglycosides blind to ribosomal RNA of the small ribosomal subunit leading to conformational changes that can result in transfer RNA mispairing to the message. And so Chris Dan did a study with gentamycin and geneticin G418 and inducing XP-C protein in cells. So it's a neat assay. The XP-C is, as I mentioned before, the first protein that goes to the side of damage. So this study involves radiating cells through a millipore filter that has very small holes in it and gives you small areas of UV damage throughout the cell. But if it shows up as the nuclei, they then stain with antibodies to the XP-C protein and the protein goes to where the damage is using confocal microscopy to watch it. And so these are areas of UV that went through the millipore filter that damaged the DNA that then the XP-C protein accumulated. Well, in the cells from the XP patients, when you do it, they have mutations in the XP-C gene that gives you stop codon. They have no protein and the protein doesn't accumulate. However, if you treat the normal cell with geneticin, well, you still get it. But if you treat the XP cells with geneticin, you've induced a small amount of the XP-C protein. And again, but however it depends which cell you use, some of them do it, some of them don't do it. So the trick is getting the right combination of cell and drug. We did other studies from families with different levels of residual repair. And if there's no XP-C protein, the patients get a lot of cancer. If there's only a few percent, like three to 5%, it's a very much milder disease. So we may not have to actually correct fully to get some effective thing. Gentamicin itself is very toxic systemically. But again, we're working with skin cancers and we're dermatologists and we may be able to use topical formulations that just put on the skin. Let's see if we can induce repair. There's gentamicin, similar. So treatment with topical amino-glaucosides may increase DNA repair in some XP patients with premature stop codons. And we're currently looking into that. So here's the study just across the street in building 37. You can see it from the parking lot here. We have lots of collaborators in the clinical studies I mentioned over here. We have a whole series of postdoctoral fellows and very talented medical students spending summers in the lab working with others. And my final slide wishes you all happiness. For Dr. Kramer, I have a couple of, what happens when XP patients smoke? Well, smoking is like putting a sun in their lungs that causes them as the carcinogens in the cigarette smoke bind to the DNA. They form adducts that are again poorly repaired by the same system. One of our patients was, I think in his teens, he started smoking and he died in his early 20s of lung cancer, who's the only, actually we have that. His second patient also has lung cancer, who's a smoker. So we encourage, when we talk with the parents, we tell them not to smoke and not to expose the children to secondhand smoke. Other comments or questions, yes? I was curious about the degree of accelerated aging in the XP. Is the hearing similar to the presbytuses of the elderly individual with high frequency losses? Correct. Could you? Yeah, the question was, do the hearing loss in the XP patients mimic that in older individuals with a high frequency hearing loss? And the answer is yes. It is, some of them, it's like a 40 or 50 year reduction. Yes. Correct. Yeah, this is in a planning stage right now. And we're not sure that even the gentamized somebody, the one we would use, not testing a whole series of other amino glycosides, and we would hope that we could find a concentration that wasn't absorbed, that wouldn't preclude these hearing problems. That's one of the reasons we're moving towards topical. But we wanted to pick, the advantage is that it's on the market now and we may be able to use it without having to develop a whole new drug and a health that go through that whole process. Other comments or questions? Could you comment on the nails? Is that sort of nail change also present in telemedicine disease? And patients with the dyscarotosis congenital, it's slightly different. I don't know that you can actually distinguish the diseases just based on the appearance of the nails. Other comments or questions? If not, thank you very much for your very thorough presentation. Thank you.