 Thanks for your reminder. I was thinking on the way in that it's easy just to walk across the street. I'm in the building 35 on the NIH campus, which is just on the other side of old Georgetown Road. And so it was good to come here. It's also, turning the mic on reminds me, just a couple of weeks ago I was interviewing a student, a medical student who wanted to come for a year to do research at the NIH and he told me, oh I saw you on YouTube. And it was from this talk last year. I hadn't noticed that I was on YouTube. So hello out there. Introducing this topic, also I was thinking of a few months ago I gave a talk like this up at Long Island Jewish North Shore Hospital, Long Island. And on the way there I flew up to LaGuardia and had a ride, a driver taking me to the medical center. And he was an engaging fellow. The driver had a business of three or four vehicles and he was telling me the real key to keeping these vehicles operating. The key to his business really was having a little device, which he showed me, a decoder device, which he can plug in to the car's computer system and it will tell him whatever the problem the car has and anticipate problems that that car is going to have in a way that he can do something about it. And I was thinking, boy, it'd be great to have something like that for people. Something we can just sort of plug in and get the answer, what the diagnosis is, what the risks are for developing different diseases that will help us to manage those patients. Kind of like in the old Star Trek series, McCoy or Beverly Crusher would wave a gadget over a patient and it would say exactly what the problem is. The thing is, we're approaching that. We're getting to that point with genetic diagnosis. It's really, the field is really moving quickly. We're being deluged with genetic information, kind of like the internet, but genetic information from patients. We have that capability. And what I'd like to talk about this morning is my take, and I'm not an expert on all aspects of this. Just my take, our experience with how, with genetic diagnosis, where we are now and where it seems to be going. So before I get much further, just in the way of disclosure, as an NIH employee, I'm not allowed to take money for consulting, but I think I mentioned last year that doesn't prevent people from asking me to consult. The foundations and companies are happy to get free advice. I tell them they get what they pay for, but it's just, it gives, it gives a sense of where I'm coming from. I do serve on advisory boards for a variety of disease foundations, the Muscular Dystrophy Association, the French Muscular Dystrophy Association, and then a variety of disease specific organizations. I've listed a couple here that are relevant to what I'm going to be talking about. And then I also consult for companies, Biogen-Idec and smaller biotech companies, Procensa in the Netherlands and Summit in England. They're developing treatment for Muscular Dystrophy. And then what I spoke about last year actually is an interesting experience of having done a sabbatical in industry at Novartis in Cambridge, and I found out subsequently that they listed me as a co-inventor on a patent based on the work that I was working there. So that's something, it's shared between Novartis and the NIH, and I may, the NIH may get some money from that, and I myself may get some money about that. I don't think it's relevant to what I'm going to be saying, but I think it's good to have up there as a way of disclosure. So what I'd like to talk about in this morning's lecture is genetic testing for neurologic diseases, how it's done. There are different approaches. Traditionally, we test for a specific gene that we think might have a mutation that would explain a patient's problem, but that's evolved to having gene panels to test for a number of different genes. And recently, over the last few years, to genome-wide analysis, to really look at all the genes, all 25,000 or so genes that we have to see which one has a mutation that explains a patient's problem. Now, as I go through this, I'll give some examples and talk about advantages. The advantages of having a diagnosis for a neurologic disease in terms of disease-specific management and prognosis and genetic counseling for the patient and for family members. And then the risks that are involved, things to watch out for as you enter into this misprint there, the risks of presymptomatic testing and incidental findings, which is a kind of thorny issue that we're having a lot of discussion about at the NIH now and elsewhere. So this is a modified version of a slide I used last year that shows just how we go about diagnosing patients with hereditary neurologic diseases. It's pretty straightforward, actually. You see the patient, the patient comes into the clinic, you see them in the hospital, outpatient clinic. The first step is, of course, to characterize the disease. To see what's the history, the physical exam, the lab test findings, what's the phenotype, as we refer to it, the disease manifestations. And then to collect samples, DNA samples, and to send those samples for DNA testing, and that will give you a genetic diagnosis. Over the last 25 years or so, as Jean Passomani was saying, we've been very successful at identifying disease genes. There are now over 3,000 human disease genes that have been identified. Several hundred of these, maybe six or 800, of them affect the nervous system in one way or another. So we have now 100 genes that cause deafness or more than 100 that cause epilepsy. Mutations in the genes cause epilepsy. More than 50 that cause neuropathy and ataxia and muscular dystrophy and so on. The challenge is to sort through all that information, to choose the tests appropriately and to try to get the information processed in a way that would be helpful to the patient's management. So just to work through these different steps in this process, in terms of characterizing the disease, the first thing is to get a good neurologic history and examination for a neurologic disease. And then this is the stress, the importance of getting family history. We all learn this in medical school, but I think with the daily pressure of seeing patients and moving them through, for whatever disorder, we oftentimes don't take the time to find out, well, you've got this problem. Is there anybody else in your family who has this problem? Which could really give insight into what the problem is, particularly for neuropathy, for example. You see a patient who has weakness and atrophy and sensory loss in their hands and feet, signs of peripheral neuropathy. And to know what the cause of that neuropathy is, it helps to find out, well, who else in the family is affected by this? Is anybody affected? If so, who? Map out the family history. In terms of laboratory evaluation and for the patients with neurologic diseases, there's relevant blood work for neuromuscular diseases, in particular the creatine kinase, CK, electrophysiologic tests like EMG, nerve conduction, and then imaging, brain imaging. But increasingly across the street, we're using muscle imaging as a way to help in the diagnosis of for neuromuscular diseases. And then, if necessary, nerve or muscle biopsy to get tissue for histologic examination. And the thing is that genetic testing is often made invasive procedures like that unnecessary because you can kind of cut to the chase and figure out what the genetic cause of the disease is without having to look at the tissue. But sometimes we still do. Okay, so then you've evaluated the patient, what's involved in sample collection. I just wrote this out last night. To make a couple of points, the samples are collected for DNA and that's remarkably easy to do. You can get DNA from any kind of cell or tissue. Typically, we can just draw one tube of blood, anticoagulated blood, to extract the DNA from the white blood cells. But it can also be done by saliva having the patient spit into a tube or do a kind of a mouthwash to collect DNA from the mouth, the cells in the mouth. Or from old, you can get DNA from old tissue samples from a microscope slide of a patient who died a long time ago if you need it. DNA is very stable at room temperature. We typically will throw it in the fridge at four degrees centigrade, but DNA has been extracted from remains of mammoths and neanderthals that's been out there in the environment for thousands of years. So the DNA you get from patients is very stable for weeks or months, years. And very small amounts are needed. You only, to make a genetic diagnosis, you can use DNA from one cell, picograms of the DNA can be used by amplifying the DNA and using it. So it's a remarkably stable substance and you need very little. I guess we all know that from crime stories now. And sometimes if there is a hereditary disease in the family, it's helpful to get samples from other family members to see whether the sequence variants you find in the DNA are tracking with the disease in the family. Let's see, okay, one joke slide. If nothing, it's nothing, go back to sleep. I was just getting a DNA sample. It shows a, I don't know, maybe this doesn't go over real well. I tried it on my wife last night. She didn't like this. It shows a woman with a mouth swab, just collecting a sample of DNA to find out what kind of genetic problems her husband or boyfriend might have. Well, I'll move on. I gave a talk at the University of Chicago some years ago. I don't know if anybody's been there, but afterwards, somebody came up to me and said, we don't do cartoons here. Okay, enough of that. Okay, so then you get the DNA sample, you get a blood sample, then the thing is to figure out, well, where do you send it? And I don't want to put in plugs for any particular lab, but one resource that's particularly useful is now run by the NIH, the genetic testing registry online. It's a listing of all labs that do genetic testing by what tests are done in which labs. It used to be, it was started at the University of Washington as gene tests, and it's been subsumed by NCBI and National Library of Medicine across the street here. So it's a good website. If you just Google on genetic tests, that'll come up as a way to figure out where to send samples. Just looking back over the last couple of months, the labs that we've used recently are the number of good labs available. There are actually dozens or hundreds of labs available around the world for different tests, but for neurologic diagnosis for neurologic diseases, Athena Diagnostics in Massachusetts particularly has a lot of tests available in prevention diagnosis and Lab in Atlanta, and Gene DX right here in Gaithersburg is good. In terms of knowing how to use these tests, there are resources available online that are quite good. Just to give information about genetic diseases and particularly neurologic, hereditary neurologic diseases and how to get tested, which tests are appropriate for which patients. Gene Reviews I mentioned was set up at the University of Washington in Seattle. OMIM, Online Mendelian Inheritance and Man, started by Victor McCusick at Johns Hopkins University, is still maintained. It's information about every human hereditary disease organized in a way where you can scan it. If a patient has deafness and vision loss, you can get the long list of diseases that would cause that combination of findings and know how to test for them. Then for neuromuscular diseases, the website we use, I've used a lot to see a patient then go to look on the computer to see what's going on, set up by Alan Pestronk at Washington University in St. Louis, a comprehensive website about neuromuscular diseases that is very user-friendly. I find. Just like to run through some examples now about how we do or how genetic diagnosis is done for hereditary neurologic diseases, I think a good place to start is with the disease I talked about last year in terms of about development of treatment is Duchenne muscular dystrophy. Okay, here's a very characteristic clinical disease, a characteristic clinical presentation for this disease that I described last year. I don't know if we see many children here at Suburban, but you'll see families who are affected by this disease fairly often. It affects about 1 in 3,000 boys. It comes on in the first few years of life, onset usually around age 3 or 4 progresses gradually. It causes weakness of the proximal muscles, so the shoulder and hip muscles, and then over a period of years it affects other muscles. Eventually the boys become wheelchair bound around age 10 or 12. It starts to affect respiratory and cardiac muscles, and the patients will die from the disease in their 20s usually. It's an excellent recessive disease. The thinking last night of putting together just a pedigree to show excellent recessive inheritance, but it is a disease that affects males. Boys are affected. Their mothers, sisters can carry the disease gene without showing manifestations. It can be passed down through families affecting only the males with women being carriers. It means that the mutation gene is on the X chromosome, and this is really one of the first, if not the first, gene to be identified by positional cloning back in the 1980s. It's a particularly large gene on the X chromosome and it codes a protein that has the name dystrophin. So the patients have mutations, usually deletions in the dystrophin gene that leads to a loss of dystrophin in the muscle, and this causes the muscle to degenerate. So there are characteristic clinical features of this disease. If you see a boy with this problem or see a family member, get the history of the affected individual, you can look for the characteristic features in terms of the age of onset, the distribution of weakness, the X-linked inheritance. They have very high creatine kinase, usually in the thousands. And then if they get an EMG or muscle biopsy, they show signs of myopathic features, so muscle degeneration and regeneration, and on the biopsy. But nowadays, we can just go from the clinical features, maybe the family, the pattern of inheritance, high CK, go directly to genetic diagnosis so we don't need to do muscle biopsies like we did in the old times or even in EMG. So the test here is targeted on a specific gene, the dystrophin gene, on the X chromosome, and as a first pass, you really look for deletions and sometimes duplications of parts of the gene. So it's a really big gene, more than 2 million base pairs, 2.3 million base pairs. It takes up about 1% of the X chromosome. The gene is broken up into coding regions called exons, separated by non-coding DNA called introns. And the patients are usually missing one or more of these exons. And the test is just to look to see by polymerase chain reaction, PCR, which of these exons, whether the exons are present or missing. And this test is present, it shows the abnormality in about three quarters of patients. To go beyond that, to get at the others, there's a more involved procedure of sequencing the whole gene, which used to be pretty laborious, but now it's pretty straightforward. It's kind of expensive. And that will add another 15%. So genetic testing will give you the cause of the disease, confirm, clinch the diagnosis in about 90% of patients on a blood sample or even a saliva sample. Now, the cost for self-pay patients, medical costs are all over the place according to what your insurance is and who's paying for it. Insurance will pay, in my experience, will generally pay for this kind of testing. If you have to do it as a self-pay, it's about $500 for the deletion testing. But it can run up to several thousand dollars, two or three, four thousand dollars to do sequencing the rest of the gene. But again, it's usually covered by insurance. For those patients, we're still wondering about the diagnosis and the genetic testing is negative. You can go ahead with a muscle biopsy and do dystrophin immunohistochemistry, and that will show the loss of dystrophin in nearly all, basically in all patients. So that's a backup if you really want to establish the diagnosis. So why do the diagnosis for this disease? I mean, you see the kid, it looks like Duchenne Muscular Dystrophy. What's the advantage of being sure about the diagnosis here, knowing exactly that this is a patient with Duchenne Muscular Dystrophy because there is an identifiable mutation in the dystrophin gene? Well, I think it helps in the clinical management. There is treatment. It's not an untreatable disease by any means. It's been well established that steroid treatment helps. It makes the kid stronger. It delays the progression of the disease. But steroid treatment comes with a lot of side effects. To know before you start the treatment that you're treating a disease that's known to respond to steroids, not something else, is important. So steroid treatment and then also kind of supportive care. The kid's optimal treatment of Duchenne Muscular Dystrophy involves cardiac and pulmonary and orthopedic support. Oftentimes they'll benefit. They develop scoliosis and benefit from spine surgery, physical and occupational therapy and assisted devices to have a well-fitting wheelchair. It helps to know with this particular patient that this is the diagnosis and this is what you have to look forward to in terms of the disease prognosis and to tap into the wealth of information about how to properly manage the patient. So then the other thing, as I mentioned earlier, is carrier genetic counseling to offer carrier testing. We oftentimes, over the years, have seen families with a patient who's affected where there's a sister or a mother who really wants to have more children and really does not want to have another child with this kind of condition. So we can see whether or not they're a carrier. Actually, this came up in my own family just a few weeks ago. My cousin's son married a woman who has a brother with what sounds like Duchenne Muscular Dystrophy and they're trying to talk with them about getting it diagnosed to see whether my cousin's wife is a carrier. It makes a lot of difference about how they go about planning their family. The same kind of testing that's used to diagnose the disease can be used to identify carriers and to do prenatal testing if someone becomes pregnant as a carrier to do the diagnosis very early in the pregnancy. Another advantage, I think, in knowing exactly what we're dealing with in a patient like this, with this kind of disorder, is to give them the opportunity to enroll, to connect up to the resources that are available, enroll in patient registries, MDA clinics, for example, to get involved in clinical trials and support groups. Not only the MDA, but Parent Project for Muscular Dystrophy. Each of these diseases has a group of committed patients and families to connect to if the patient you're seeing is so inclined. Okay, so that's Duchenne Dystrophy. I can go on to talk about another disease that's a bit more complicated. Actually, a set of diseases that goes by this fancy eponym, Charcot-Marie Tooth Disease. Basically, what's meant by Charcot-Marie Tooth Disease is hereditary motor and sensory neuropathy. This is what I was alluding to earlier. The names come from two French neurologists back in the 19th century, Charcot-Marie and a British fellow named Tooth. It's not a dental disease. That's just the names that stuck since they described it back in the 1880s. What this causes is progressive distal weakness and sensory loss. It causes the weakness of the hands and the feet, atrophy of the muscle, loss of sensation. I say that this is a pretty common disease for hereditary neurologic disorder. It affects about 1 in 1200 people overall in Europe where it's been studied. If that holds up in this area here in the Bethesda area, there are probably about 50 or 60 patients with this disease. You'll see them walking down the street if you're careful. They'll have a tendency for their feet to drop. One thing that really helps in a way of intervention is just to provide braces, a molded ankle foot orthosis that helps with the foot drop. Otherwise, it's a pretty benign disorder. A lot of people don't even know that they have it. It's a big family we had up from Pennsylvania, the Pachadi family, and said, oh, that's just the Pachadi foot problem. It's just the way their feet are. It usually doesn't affect life expectancy. They usually live out normally productive lives. This characteristic phenotype or characteristic pattern of disease manifestations has a broad variety of genetic causes. With Duchenne muscular dystrophy, or one gene you're talking about, the dystrophin gene, here the same disorder. There are about 78 different genes that can be mutated to cause this problem. Whoa, that's a diagnostic challenge. How do you approach this? Well, first, these different types of Charcomy-Tooth disease fall into two general categories according to what the underlying problem is. This problem is caused by degeneration of the nerves. Hereditary disorder that causes degeneration of the nerves. There are two basic ways that the nerves can degenerate. Here's a nerve cell axon in the peripheral nerve cut in cross-section. Here's the axon. It's wrapped in myelin by a Schwann cell. You can get Charcomy-Tooth disease. Some majority of patients with Charcomy-Tooth disease have a loss of myelin. It's a demyelinating disease. Then a minority, maybe 30 or 40%, have type 2 or axonal degeneration. By looking at the nerve, you can also tell the difference with less invasively by doing nerve conduction. Type 1, demyelinating Charcomy-Tooth disease or slowing of nerve conduction. Type 2, axonal form of the disease. There's a reduction in the amplitude. Anybody who can do a nerve conduction study can differentiate type 1 and type 2. In terms of the genetics, how did all these 78 different genes... Well, it turns out that there are really four that account for the majority of patients. There's type 1A, type 1B. There's an X-linked form of it, and then there's type 2A. Two dominantly inherited demyelinating diseases, an X-linked form which is kind of mixed demyelinating axonal and then an axonal form of type 2A. Actually, type 1A accounts for about 60% of patients. That's caused by mutations that affect a gene called PMP22. After that, probably the X-linked form is most common. Gapjunction protein, GJB1. Then, the type 1B and type 2A, which have mutations in myelin protein, zero and mitophucin. If you just look at these four, you're going to get most patients. The others get to be pretty rare. You say after these four, it falls off. The other mutations, at most, account for just 1% or 2% of patients. Then you get down to a lot of mutations that have only been identified in one family or two or three families. I'll give some examples here. Yeah, this slide doesn't show up real well, but this is the way you can see the mutation that causes the most common form of Charcomery II's disease, type 1A. What it is is a duplication, not an internal deletion or duplication like you see in the dystrophin gene, but here the whole gene is duplicated. It's having an extra copy of this PMP22 gene. You can do that by looking at blood cells under a microscope and using fluorescent labels for the gene. You can see that the patients have an extra copy of the gene. Normally, there's one copy on each chromosome. It's on chromosome 17. Each copy of chromosome 17 has one copy of this PMP22 gene, but in patients, there's an extra copy, so instead of seeing two red dots you see three. So it's a bit of an involved diagnostic test. It'll give you the answer most of the time, particularly if you know that the patient has a demyelinating form of Charcomery II's disease with a slowing of nerve conduction. The genetic diagnosis of all the others, the other relatively common forms and all of the rare forms is done by DNA sequencing. So how is that done? Well, companies, a number of the companies that offer genetic testing will offer gene panels so that you can test for, or they can test for a number of different 12 or 15 or 20 different known causes of Charcomery II's disease. If they hit the top four, then they're going to catch most of the patients, but the more they test for, the more comprehensive the diagnosis is. It helps, I think, if you're going to go with gene panels to know, first of all, whether it's type one or type two, this limits the options, but it's still possible to get genetic testing on a gene panel for all the known CMTs or the large majority of them. This has been really expensive. So like with Athena panel for type one or type two CMT, it costs more than $10,000, $12,000 to get all the CMTs, up to $18,000 or so. So it's a pretty expensive way of going about it, checking each gene individually. So the real approach here, which is gaining traction, is to do genome-wide analysis, to look at all the genes with new techniques that are available, to look at all of the genes, all 25,000 genes, and then to pull out of that information, the 78 genes that are known to be affected. And that's much less expensive. On a research basis, we do that test across the street here. It started out a few years ago, it cost us about $10,000 to do all 25,000 genes. Then over the last few years, it came down to $2,000, $1,500. Now, just in the last few weeks, it's come down to $500 to get sequence information on every one of the genes. So it's really amazing how the cost of this has come down, and it's done very efficiently. We do it on a research basis up at a center through the genome institute called NISC, but it's also becoming commercially available. So it's something you can get on any patient. The cost, the commercial costs are much higher because it has to meet CLIA standards, clinical grade standards. But genome-wide analysis is really changing the way we approach patients like this. So, oh, just here's an example of a patient where we found a family, where we found a rare form of shark and retooth disease, a family up from Pennsylvania. We collected samples from this family. I was at Penn before I came to the NIH, and we collected samples from this family back in the 1980s, I think, or a long time ago, at least more than 20 years ago, and had just had them. We didn't see any abnormality when we first collected them, and we just had them stored in the cold room here. When this new genome-wide analysis became available, we pulled the samples out, the DNA samples out, and sent them off for testing. So what this was is an usually severe form of shark and retooth disease, axonal, so type II, and X-linked recessive. And these patients in this family, and there were six or I think eight different males affected with this disease in the family, had the severe axonal neuropathy so that they barely could walk even as children, and with it they had deafness and cognitive impairment. We looked on the X-chromosome and mapped it to a particular region of the X-chromosome, just using markers, genetic markers in that region of the chromosome to a particular part of the chromosome that had about 40 or 50 genes. And then we used the new technology a couple of years ago to screen through all those genes and found a mutation in a gene called AIF-M1. It's a mitochondrial protein that induces apoptosis. So at the time it was great. Actually, it was a little interesting in dealing with the family. We found the mutation and then we said, oh boy, maybe we should re-consent the family before publishing it. And so I had the family names and they called back. I looked on the Internet to see if only one of about 20 or so family members could get their contact information on the Internet. And I called up this woman in Bucks County, Pennsylvania. Her husband answered the phone and he was a little skeptical about someone calling from the government about a genetic diagnosis. But he eventually handed the phone to his wife and she said, oh, Dr. Fischbeck, we've been waiting to hear from you all these years. They're really pleased to know exactly what the cause of the problem was. And there's some therapeutic implications here, possibility of treatment based on what's under the biochemistry here. So it just shows how the new technology gives us a new, a fresh look at diagnosis in these patients. It really enhances our capability. And then, you know, here's an interesting article in the New England Journal a few years ago from Jim Lubsky at Baylor College of Medicine, a geneticist there who is himself affected by shark and retooth disease. And this made an interesting story for the New England Journal. It was, he decided to do this new technology on his own DNA. He had never had a diagnosis before. It ran in his family, affected, you know, his siblings and mild manifestations in his father and his grandmother. And what he did is hold genome sequencing. So not just the coding regions, but he arranged at Baylor to have all of his DNA sequenced, all three billion base pairs, and sorted through all the variants. So in his own DNA, there were three million variants, and started to look to see which of those variants were shared by other family members, which of those variants could make sense as a shark and retooth disease gene. And he found a variant in SH3, TC2, which had just been identified as a rare cause of axonal shark and retooth disease, and that he and his other family members had variants in this gene that were tracking with the disease in his family. So this got some publicity. It was in New York Times and widespread publicity as a new approach to diagnosis, not by looking one by one at specific genes, but by looking at all the genes and extracting from that the information that gives you the diagnosis. So what about this new genome-wide analysis? And again, I'm not an expert on the technique, how it works exactly, but just how it's used or how it can be used. There are two different approaches. One is called whole genome sequencing, which is sequence all three billion base pairs of DNA that we carry, like was done with the human genome project. The cost of that has come down pretty dramatically, but it's still difficult in that it gives you a lot of variants that need to be sorted through. In Jim Lovsky's case, three billion variants to try to figure out which of those is causing the problem. So another approach that's used a lot more widely now is called exome sequencing. So that's just sequencing the coding regions, so the exons, all of the exons of all 25,000 genes. It's still a lot of information, but it's more likely that you're going to get a somewhat more manageable list of variants to sort through. The challenge here, it's a challenge in Jim Lovsky's family and the patient that we saw, the patient that I showed you and others that we see, is what it pushes the problem. So DNA sequencing is not limiting in this at all anymore. You can get sequence from all of the genes, the coding regions of all the genes, or all the non-coding regions, if you want. The challenge is to confirm the pathogenicity of the sequence variants that you find. So which of all these different variants is the cause of that patient's problem? And there are different ways we can go about doing that, but it's still pretty laborious at this stage. One is easiest is if the gene has already been reported, like in Jim's case with SH3, TC2, that the gene mutations in this gene are already known to cause the disease. So, okay, that solves the problem. But if you don't see that, if you want to see, well, you have a novel variant and you want to see if that's the cause, then it takes more work. And one thing is, and this is back to what I mentioned early on, that it may be helpful to have samples from other affected members of the family for comparison to see if the variants are what we say segregating with the disease in the family. That just means that the variant is tracking with the disease down through the family. All of the affected individuals have that variant and the unaffected siblings do not. That's called segregation. And then another thing that's useful here is to look to see whether the variants you're finding are present in healthy individuals because if they are, they're probably not causing the disease. And Les Beesicker at the Genome Institute has done some work to just collect samples from healthy individuals around Bethesda, over 500 healthy individuals to get control information for comparison. And something, you know, this is rapidly evolving and it's an important thing to do. The Heart Lung and Blood Institute has put together exome sequence data from over 6,000 individuals and it's available online in the nicely searchable forum on their website. So you can, if you see a variant, a list of variants, you can sort out those that are unlikely to be causing a disease because they're present in other people who don't have the disease. And then beyond that, you can look to see whether these variants, look at the equivalent gene in other species like in mice or rats or fish or worms, flies. And, you know, many genes are conserved across species and many sequences are conserved across species. If the variant that you see is in a, at a site in the DNA which is otherwise conserved, then it's more likely to be pathogenic. If it's not, then it's less. And then finally, and this can take a while, is to look to see what the effects of the variant are on the protein structure and function. So this is really done now on a research basis. I think, you know, the tests are clinically available. It's, it's a very powerful way to identify known, known variants. And, but beyond that, it gets to be more of a research thing at present. But this is evolving. I think this is going to become increasingly available on a clinical basis. And Nova Fairfax in Virginia is advertising exome sequencing on NPR, their ads that they'll, they'll do it for you. But it's, it's, it's still a lot of work to extract that information, extract from all the information you get, the information that's meaningful to that patient. So it really helps to have, you know, people who know, know how to use it, genetic counselor at least, or geneticist, or specialists in the, in the area of the disease to sort it through. But it, it's a rapidly evolving field. And I think the support will come to be able to do this. The more information we have, the more straightforward this process becomes, the easier it becomes. Well, I'd like to say a little bit here about repeat expansion diseases is something we've been involved in a long time, for a long time, identifying and characterizing these diseases. This isn't important. And when it comes to hereditary neurologic diseases, it's important to know about diseases that are caused not by deletions or point mutations, but by expanded simple sequence repeats, usually trinucleotide repeats. There are about 30 of these diseases that are now known, nearly all of them, neurologic. And in many cases, the expanded repeats are unstable so that as it gets passed down through families that there's a tendency for the repeat to become, which is already expanded to become longer from one generation to the next. And that results in increasing disease severity, a phenomenon we call anticipation. Now, one of the more famous of these diseases is Huntington's disease. This affects about one in 15,000 people. So there'll be a few people around Bethesda with this disease. I think I've seen them on the street. It causes Korea, these jerking movements, and psychological changes and cognitive decline. They become demented. They have a kind of impulsive behavior. They're prone to suicide, depression, which is important to keep in mind. Something like 15% suicide rate, I believe. It's caused by, it's a neurodegenerative disease caused by loss of neurons in the basal ganglia, or striatum, the caudate nucleus in particular, but also elsewhere in the brain. And the cause of this disease is an expanded trinucleotide repeat on chromosome 4. It's the cytosine adenine guanine. So three nucleotides are repeated in this gene, and the repeat becomes longer. The gene was given the name Huntington. So here's the Huntington gene. It's also a large gene, not as large, but similar to the dystrophin gene. And here in the first exon of this gene is the CAG repeat. In normal individuals, about 20 CAGs, CAG, CAG, CAG. And in patients with Huntington's disease, it's expanded to 40 or 60 or more CAGs. This CAG, so it's three nucleotides, include one amino acid. CAG encodes the amino acid glutamine. So this encodes a polyglutamine repeat in the Huntington protein. So this is called a polyglutamine expansion disease. Now in terms of families, and diagnosis, it's very easy to look at the length of the repeat in the DNA from a sample from a patient or family member. This is done by a PCR polymerase chain reaction to just amplify that part of the gene that has the repeat and then run the product out on a gel. The normal repeat varies in length. I said about 20, but it ranges from about, oh no, 13 to 30 or so CAGs. And here it's on chromosome 4. You see each copy of chromosome 4 has a different repeat length. It varies a lot in the normal range. But the patients who are affected here, so the shaded symbols are affected individuals in this family, squares are males and circles are females. The shaded symbols show those who are affected. And we see that they have a longer CAG repeat, which gives a band that runs higher on this gel. And you see, it's interesting, so this guy had an expanded CAG repeat of about 40 CAGs. And he passed it on to his children, his affected individuals, and it shifted in length. It got longer in some of these individuals, here up to about 60 or so in the youngest son. So you see the instability here. This guy, the youngest son had onset in childhood. The father had onset in the late 30s after he'd had most of his children, I guess. And so you see that even within this family, there's a correlation between repeat length and age of onset. The longer the repeat, the earlier the age of onset in this disease. But I want to focus on this person here. See this woman who has affected brothers and sister, she has a long CAG repeat, but she's not affected by the disease. At least she's not affected by it yet. So this is what we call a presymptomatic individual. Somebody who's got the mutation and is at risk of coming down with the disease most likely will or almost certainly will, but she doesn't know it yet. She doesn't have any signs of the disease. There's a correlation here, as I said, between repeat length and age of onset. But there's a lot of variability. You know, here's the normal repeat in these individuals. About 1,000 patients from Vancouver studied some years ago. You can see that as the repeat length, as the repeat length gets longer in this direction, the age of onset gets earlier. But there's a lot of variability. So in any given individual, it's hard to predict. I mean, you can predict that they will come down with the disease, but it's hard to predict when. They may not come down with it. They may come down with the disease in their 20s, or they may not come down with it until their 80s. We had a patient, a retired Washington DC police detective who was diagnosed in his 80s, started to develop these jerking movements. And his girlfriend bought him in. He said, he's just not dancing the way he used to. And it turned out to be a late onset form of Huntington's disease. So it's hard in any particular individual who's asymptomatic at risk to know for sure when they're going to come down with it. And there are lots of psychosocial risks with pre-symptomatic diagnosis. You're taking a healthy individual who has affected family members and offering, or you can offer a test to show whether or not they're carrying this gene. And it turns out that most people in that situation would opt not to know. Because there isn't any specific treatment here. They would opt not to know whether they're going to come down with it or not. And so I think when you encounter somebody like this, somebody said, oh, my father died of Huntington's disease. And I'd kind of like to know whether I got it. Some people it's very empowering to know. Other people don't want to go there. But it's important for somebody to sit down with them and talk it through. And genetic counselors are made for this. I think it's really good to engage. I mean, it's important to engage a genetic counselor before you send the test. I mean, it's easy to just send the test off to one of these companies to get it done. But to get counseling, genetic counseling, psychological counseling to make sure the patient knows what they're getting into before. And then when the test results come back, whether they're positive or negative to kind of help them through this process. Now, this is notorious for Huntington's disease. And it's particularly important for Huntington's disease because of the high suicide risk and other psychological problems these patients can get. But the same kinds of considerations really apply to other late onset neurodegenerative diseases. And there are a lot of late onset neurodegenerative diseases where this could apply. So we call Huntington's a polyglutamine expansion disease. There are other diseases with the same kind of mutations, same kind of mechanism that affect other parts of the nervous system, like the spinal cerebellary ataxias, cause loss of coordination, Kennedy's disease, a motor neuron disease that has the same kind of mutation. That same kind of concerns about presymptomatic diagnosis apply to these. But also to other late onset neurodegenerative diseases with known genes like Alzheimer's disease or Parkinson's disease or ALS. Each of these diseases, the most patients, we still don't know what the genetic cause is, but there are genes that have been identified. Frontotemporal dementia is another one. We saw a patient a few years, or a person, not a patient, a few years ago, a woman who found out that she was at risk for frontotemporal dementia. Her father died of it. And somebody just, somebody out in Nebraska sent her a letter saying, oh, you know, you've got, you could have this genetic defect that causes you to become demented. A lawyer from Charlottesville, she was, she was really kind of distraught about that. And we did testing for her, found out that she did not carry it. She was very, very happy to know she wasn't. But I think in dealing with patients, people like this, it's good to make sure they get good genetic counseling or psychological counseling as they go through the process because it can be, it can be a challenge. Okay, the last thing I wanted to talk about here are incidental findings. We've, we've had a lot of talk about this recently, as I said before, but one thing you encounter as you get into genome-wide analysis is you've, you come up with mutations in genes that you're not looking for that could be important. So called these incidental, unexpected incidental mutations. If you're looking at all 25,000 genes, all of us are carrying mutations in genes. And some of them are important to know about. So some of them could have therapeutic implications. For example, breast cancer or colon cancer. If you have a mutation, even, you know, so you get tested for, I don't know, Charcot-Marie-Tooth disease and you find out that you have a gene that predisposes to breast cancer or colon cancer. It's, it's arguably, it's good to know because it, it affects whether you get mammography, whether you get mass, you know, prophylactic mastectomy. For colon cancer, in fact, it has an effect on how often you get colonoscopy. There are therapeutic implications with these findings. And so there's a lot of discussion about how this should be handled recently. The American College of Medical Genetics, ACMG, last year published a list of 56 genes where mutations should be reported to the patients. And mutations in these particular 56 genes, mostly cancer genes like these, have been showing up in about 2 to 3 percent of exomes. So, you know, it, it's something, you know, we're still struggling with exactly how this should be handled. Should every patient who gets exome, should, should somebody look at these 56 genes? Should that be required or should it be encouraged? We're having a series of meetings to try to work this through. But I think, I think standards, this, this is the start on establishing standards on how to deal with this situation. So, so it's important to be aware of if, if you're going to go to ANOVA Fairfax and order exome sequencing to know that this could happen, that you could find something you're not looking for. It's, in some ways it's analogous to getting an MRI scan of the brain and looking for one thing and finding something else. And, you know, we're kind of learning from the radiologist as we go along to some extent. But, you know, there's a lot that needs to be worked out in terms of the strategy here. So, in closing, I'd like to, I think as clinicians, I'm still very much a clinician at heart, we like to trade, tell stories. And my, I was saying, my wife, my wife will sometimes say to me afterwards, you know, you really didn't like it. You start to talking, you forget that as a physician, what you're talking about may not be interesting or pleasant for somebody else to hear about this came up. I was talking about metastatic prostate cancer. And my wife said afterwards, you know, that's not really dinner table conversation. But, you know, we learned from each other, I think, in sharing stories. And I, when I was putting this talk together, I went back to a story that is from several years ago that I think is worth retelling. So, this is a patient we saw at the NIH, you know, some years ago. 17-year-old girl complained of progressive difficulty walking. It started when she was little. At age five, her right foot turned inward. At age seven, she was seen by a physician. She had mild weakness of the arms and right leg and deformity of the right foot. She got MRI scans of her head and spine were normal. And she had EMG. It looked like it showed some changes of myopathy in the leg, the perineus muscle in the leg and the biceps muscle in the arm. And then later, she required braces, like I was talking about for shark and retooth disease, for progressive deformity of the feet. And she fell frequently, difficulty throwing a ball. Exam showed that she had a kind of a funny smile, a transverse smile, normal muscle tone. She had winging of her shoulder blades or scapula and proximal weakness of the arms, foot deformities, normal sensory exam, and hyporeflexia. They got a muscle biopsy. She was seen at a, my wife said I shouldn't say which one. She was seen at a major academic medical center by a real expert in neurogenetics. And she was given the diagnosis of fascios, scapula, humoral muscular dystrophy. So it's a form of muscular dystrophy that affects the muscles of the face, the shoulder blades and the upper arms. There's a picture of a patient she didn't look like this. But this is from, I think from Alan Pestronk's website that's Washington University St. Louis showing FSH dystrophy causes weakness and atrophy of the face and the shoulders and the upper arms. And that's what she was thought to have. So later at age 15, she could still walk a short distance from Carter School in the morning. She could no longer walk that distance by the end of the day. So it's varying over the course of the day, which you would not expect for muscular dystrophy. At dinner, she had difficulty raising her head to eat and was extremely slow to complete her meal. She used a wheelchair for all but short distances and she began to have episodes where her legs stiffened up and locked. And then a diagnostic test was performed. Anybody have any ideas? This is a hard one. After all, the expert at an unnamed major medical center couldn't figure this one out. But somebody, an astute clinician at the NIH did. It wasn't me, one of the fellows, I think, thought of the test to do. And what the test was was to give her a low dose of Sinemet, al-Dopa. So her family history, I think also good to, as I mentioned earlier on, to get a family history here. She did have an affected sister with this disorder, which turned out to be what we called Dopa-responsive dystonia, rare disorder, but remarkably treatable hereditary neurologic disease. With the one dose of Sinemet, this girl who had been severely disabled by the disease, she was 17 since she was five, gradually progressive, with one pill, she was normal. It completely did away with all of her disease manifestations and it was a sustained improvement. So in her family history, a sister who was affected with the same problem than other family members who were affected with Parkinson's disease or other clinical symptoms consistent with this diagnosis. So what is Dopa-responsive dystonia? I hadn't really heard of it so much before we made this diagnosis, but it's not that uncommon. It's a childhood onset disease that causes dystonia or abnormal stiffness of the muscles, usually involving the legs, but it can affect other parts of the body. And other family members, other people carrying this gene can have Parkinsonism, spastic parapheresis, or what looks like a myopathy. Characteristically, it varies over the course of the day, as this patients did, and it can respond dramatically and with a sustained response to low doses of Sinemet, the drug that we use for Parkinson's disease. Now the mutations are known, the genes are known, I haven't updated this slide, but I went and gave a talk about this at the famous academic institution where this diagnosis was missed. They didn't seem to appreciate it very much. But it's the GTP cyclohydrolase, autosomal dominant disease. Back when we saw this patient had about 85 different mutations, and we found a mutation in the family, or tyrosine hydroxylase, both genes that are involved in the synthesis of dopamine. So these are both important enzymes in dopamine synthesis. So giving L-dopa, as in Parkinson's disease, but much more dramatically helps patients with this disease. Whoops, where am I here? Okay. Just the important thing to remember here is that, or to be aware of, I guess, is that doporesponsive dystonia is an important disease to diagnose and it's a treatable. So when you look at all these genes, sometimes you come on to something like this that's imminently treatable and really does a lot of good for the patient or family. I'm very appreciative when you can do this. Okay. So this is the kind of thing, a kind of treatable disorder that could come out of whole exome sequencing if you're doing it, you know, if you thought she had muscular dystrophy or even you thought you did whole exome for some other reason, it's good to know that there are mutations in this gene because it can really mean a lot in terms of the management. Take home lessons. Genetic testing is rapidly evolving, is a diagnostic tool. We're entering into a new age here where we're going to have this information available. Whether we ask for it or not, people have their direct-to-consumer testing services like 23andMe that are offering genetic testing and it's really an evolving, a changing playing field. People are going to come to us with a list of mutations say which of these fits with my diagnosis. The testing allows comprehensive diagnosis of hereditary neurologic diseases with important implications for clinical management. Pre-symptomatic diagnosis should be done with care and related to that, incidental findings are going to arise from genome-wide analysis and it's important to have a strategy. We're working on that, but it's important to have a strategy for dealing with this kind of thing. I like this quote from a Shakespeare quote. It applies, I think, to genetics in general and particularly genetic diagnosis, the which observed a man may prophesy with the near aim of the main chance of things as yet not come to life, which in their seeds and weak beginnings lie in treasure. We've come a long way. I think in genetic diagnosis and things are moving very rapidly, it is hard to prophesy where things are going to be five or 10 or 20 years from now, but it's going to be different I think in terms of diagnosis. I think we're going to be a lot closer to Beverly Crusher with her scanner or the car mechanic with his decoder that he can plug in and we have to learn how to deal with that. Thanks. Yeah, yeah, so I'm not the MS expert. There's some other good people across the street. You could bring over to talk about MS in general, like B.B. Bilacoba, for example, but retroviral integration can cause mutations and can cause problems and it's something that was a problem with gene therapy in Europe and France, for example, that in trying to deliver genes, if the delivery system is going to put that gene into the genome, it can cause problems by integrating into the genome in such a way that it could cause a genetic problem, particularly cancer, integrating into a tumor suppressor gene or an oncogene can bring out a malignancy and so there's been a lot of work in gene therapy field to try to avoid that, to try to use viruses that don't integrate. I don't know if that answers your question. I'm not sure about the MS explanation. Yeah, yeah, yeah, yeah. So it can have an effect on something that's already there. Actually, it's interesting, the FSH dystrophy story in terms of the mechanism is interesting because what the mechanism has just been worked out in the last year or two as being a kind of activation of genes that are latent on chromosome 4 that may have arisen from retroviral insertion. So the Dux4 gene is left over from an ancient retroviral insertion that gets activated in patients with the disease and causes muscle degeneration. So it's an interesting mechanism. Yeah, it gets more severe. Yeah, it's not as good a correlation but the disease is definitely more severe the longer the repeat. That's true for the other repeat expansion diseases. Ah, yeah, yeah, yeah. Yeah, yeah, you can use the genes in both directions. Actually, on that list of 56 genes to watch out for, for incidental findings, it's not the HDL but the LDL receptor where mutations cause high cholesterol. But for HDL, I think the whole idea of identifying good genes or good variants is something that's, well, it came up at a symposium we had across the street here just a few days ago. One way to do that is to look at elderly, healthy people to see what genes they're carrying. Sort of the opposite of looking at patients with the disease. What are the variants that predispose to health and longevity? And there's some projects going on in that direction too. Collecting samples from people in Italy and villages who are in their 90s without showing any kind of physical disability. And that can lead to a number of different approaches to treatment for the rest of us. Thanks. Good to be here.