Peripheral Neuropathy Genetic Testing

Kenneth Fischbeck Thanks for the reminder. I was thinking on the way in that it’s easy just to walk across the street. I’m in the building 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 laughs and it was from.

This talk last year. I hadn’t noticed that I was on YouTube laughs. So hello out there laughs. Introducing this topic, also, I was thinking 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 was taking 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 into 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 laughs, you know, something we can just sort of plug in and get the answer, what the diagnosis is, what you know, what the risks are for developing different diseases.

Genetic Testing for Neurological Diseases Kurt Fischbeck

It’ll help us to manage those patients, kind of like in the old Star Trek series. McCoy or Beverly Crusher would take a 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, I’d just in the way of disclosure, as an NIH employee, I’m not allowed to take money for consulting, but that I think I mentioned last year, that doesn’t prevent people from asking me to consult. Foundations and companies are happy to get free advice. I tell them they get what they pay for, but it’s.

Laughter Kenneth Fischbeck laughs just to give 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 Distrophy Association, and then a variety of diseasespecific 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 Prosensa 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 coinventor on a patent based on the work that I was working there. So, that’s something is 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 genomewide 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 the 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 diseasespecific management and prognosis, and genetic counseling for the patient and then for family members, and then the risks that are involved. Things to watch out for as you enter into this whoa, a misprint there the risks of presymptomatic testing and incidental findings, which is a kind of thorny issue that there’s 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 or outpatient clinic. The first step is, of course, to characterize the disease, to see what are the what’s the history, the physical exam, lab test findings what’s the phenotype, is the way 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 Gene Passomany spelled phonetically was saying, we’ve been very successful at identifying disease genes. They’re now over 3,000 human disease genes that have been identified. Several hundred of these maybe 6 or 800 them affect the nervous system in one way or another. So we have now, you know, 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’ll 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 good neurologic history and examination and you know for a neurologic disease and just to stress the importance of getting family history. We all learned this in medical school, but I think with the daily pressure of seeing.

Patients and moving them through, we for whatever disorder, we’re 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 a neuropathy, for example You know, if you see a patient who has weakness, and atrophy, and sensory loss in their hands and feet, the signs of peripheral neuropathy, and you know, 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. Then a laboratory evaluation, and for the patients with neurologic diseases, there’s relevant blood work for neuromuscular diseases, in particular, the creatine kinase, CK and electrophysiologic tests, like EMG and nerve conduction and then imaging, you know, 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 histological examination. And you know, the thing is the genetic testing.

Has 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. You 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’s spit into a tube or do 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 you know, typically, we’ll throw it in the fridge at four degrees centigrade, but you know, the DNA’s been extracted from, you know remains of mammoths and Neanderthals.

It’s been out there for 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 DNA are 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 very well. I tried it on my wife last night. She didn’t like this laughs. It shows a woman with a mouth swab, you know, just collecting a sample of DNA to find out what kind of genetic.

Problems her husband or boyfriend might have. I’ve well, I’ll move on laughs. I used the 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 laughs laughter Kenneth Fischbeck Okay. Enough of that laughs. Okay laughs. 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, you know, 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 at which labs. It used to be it was started at the University of Washington as gene tests, and has been subsumed by the by NCBI, the 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 place is the labs that we’ve used recently are you know, there’re a number of good labs available. There’re actually dozens or hundreds of labs available around the world, but for different tests but for neurologic diagnosis, for neurologic diseases, Athena Diagnostics in Massachusetts is particularly has a lot of tests available in prevention diagnosis, and a lab in Atlanta, and GeneDx right here in Gaithersburg is good. And 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 you know, and how to get tested, which tests are appropriate for which patients. GeneReviews, I mentioned, was set up at the University of Washington, Seattle. OMIM, Online Mendelian Inheritance in Man, was started by Victor McKusick at Johns Hopkins University. It’s still maintained. It’s information about every hereditary disease human hereditary disease, organized in a way where you can scan it. If the 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. And then for neuromuscular disease, there’s a website we use I’ve used a lot laughs 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 Saint Louis, a comprehensive website about neuromuscular diseases that is very user friendly, I find. Okay. So, 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, you know, very characteristic clinical disease very clinic 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, and it affects about one in 3,000 boys. It comes on in the first few years of life, onset usually around age three or four, progresses gradually,.

It affects 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 wheelchairbound around age 10 or 12. It starts to affect respiratory and cardiac muscles, and patients will die from the disease in their 20s usually. And it’s an Xlinked recessive disease, so it’s I was thinking last night of putting together just a pedigree to show Xlinked recessive inheritance but it is a disease that affects males. So, boys are affected. Their mothers, sisters can carry the disease.

Gene without showing manifestation. So, it gets passed from can be passed down through families affecting only the males with women being carriers. And that means that the mutation gene is on the X chromosome and this was really one of the first, if not the first gene to be identified, by positional cloning back in the 1980s. It was it’s a particularly large gene on the X chromosome that encodes the 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 muscle, and this causes the muscle to degenerate. So there are characteristic clinical features to this disease. If you see a boy with this problem, or see a family member, get the history of the affected individual, you look for it you can look for the characteristic features in terms of the age of onset, the distribution of weakness, the Xlinked 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 generic diagnosis, so we don’t need to do muscle biopsies like we did in the old times, or even an EMG. So, the test here is targeted on a specific gene, the dystrophin gene on the X chromosome, and as a first pass, we 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.

takes up about 1 percent of the X chromosome. It’s broke the gene is broken up into coding regions called exons, separated by noncoding 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 shows the abnormality in about threequarters of patients. To go beyond that, to get at the others, there’s a more involved procedure, sequencing the.

Whole gene, which used to be pretty laborious, but now is pretty straightforward. It’s just kind of expensive. And that will add another 15 percent. So genetic testing will give you the cause of the disease, confirm clinch the diagnosis in about 90 percent of patients on a blood sample or even a saliva sample. Now, the cost for selfpay patients medical costs are all over the place, according to what your insurance is, and you know, 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 selfpay,.

It’s about $500 for the deletion testing, but it can run up to several thousand dollars 2 or 3 or $4,000 to do a sequencing of the rest of the gene. But again, it’s usually covered by insurance. For those patients still wondering about the diagnosis and the genetic testing is negative, you can go ahead with the 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. Why 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, there’s a it delays the progression.

Of the disease. But steroid 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 kids optimal treatment of Duchenne muscular dystrophy involves cardiac, and pulmonary, and orthopedic support. Often times they’ll benefit they develop scoliosis and benefit from spine surgery, physical and occupational therapy, and assisted devices to have a wellfitting wheelchair. It helps to know, you 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 often times, over the years, have seen, you know, families with a patient who’s affected where there’s a sister or a mother who really wants to have, you know who 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 get I talked with them about getting it diagnosed to see whether she 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. People want to if someone becomes pregnant who’s a carrier to do the diagnosis very early in the pregnancy. And 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 to talk about another disease that’s, you know, a bit more complicated actually, a set of diseases that goes by this fancy eponym, CharcotMarieTooth disease. So basically what’s meant by CharcotMarieTooth 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 nineteenth century, Charcot and Marie, and a British fellow named Tooth. It’s not a dental disease. It’s a.

Laughter Kenneth Fischbeck That’s just the names that stuck since they described back in the 1880s. What this causes is progressive distal weakness and sensory loss. It so it causes weakness of the hands and the feet, atrophy of the muscle, loss of sensation. I’d say, you know, that this is a pretty common disease for a hereditary neurologic disorder. It affects about one in 12,000 people overall in Europe where it’s been studied. So if that holds up in this area here in the Bethesda area, there’re 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. It one thing that really helps in way of intervention is just to provide braces molded ankle foot orthosis that helps with the foot drop. And otherwise it’s a pretty benign disorder. A lot of people don’t even know that they have it. It’s big family we had from Pennsylvania, the Pichotty spelled phonetically family, and said, oh, that’s just the Pichotty foot problem, you know. It’s just the way their feet are. It usually doesn’t affect life expectancy they.

Usually live out normally productive lives. Now so this characteristic phenotype, or characteristic pattern of disease manifestations has a broad variety of genetic causes. So with Duchenne muscular dystrophy, one gene you’re talking about, the dystrophin gene here, the same disorder, they’re about 78 last count, 78 different genes that can be mutated to cause this problem. Whoa laughs. That’s a diagnostic challenge. So, how do you approach this Well, first, these different causes of CharcotMarieTooth disease, or different types of CharcotMarieTooth disease, fall into two general categories according to whether the problem is what the underlying.

Problem is. So this problem is caused by degeneration of the nerves, a hereditary disorder that causes degeneration of the nerves, and there’re two basic ways that the nerves can degenerate. Here’s a nerve cell, axon in the peripheral nerve, cut in cross section, and here’s the axon. It’s wrapped in mylan by a Schwann cell. And you can get CharcotMarieTooth disease the majority of patients with CharcotMarieTooth disease have a loss of myelin. It’s a demyelinating disease. And then, the minority, maybe 40 30 or 40 percent, have type 2 or axonal.

Degeneration. So, you can tell the difference by looking at the nerve. You can also tell the difference with less invasively by doing nerve conduction. Type 1, demyelinating CharcotMarieTooth disease, there’s slowing of nerve conduction. Type 2, axonal form of the disease, there’s a reduction in the amplitude. So, anybody with who can do a nerve conduction study can differentiate type 1 and type 2. So then, in terms of the genetics, how do all the 78 genes well, it turns out that there’re really four that account for the majority of patients. There’s type 1A, type.

1B, there’s an Xlinked form of it, and there’s type 2A. So two dominantly inherited demyelinating diseases, an Xlinked form, which is kind of mixed demyelinatingaxonal, and then an axonal form of type 2A. Actually, type 1A accounts for about 60 percent of patients. That’s caused by mutations in that affect a gene called PMP22. After that, probably the Xlinked form is most common, gap junction protein, GJB1. And then, the type 1B and type 2A, which have mutations in myelin protein 0 and mitofusin. So you know, if you just look at these four, you’re going to get most patients. The others.

Get to be pretty rare, you know. So you say after these four it falls off, so that the other mutations most account for, you know, just 1 or 2 percent of patients and then you get down to a lot of mutations that have only been identified in one family or two or three families. And 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 CharcotMarieTooth disease, type 1A, and what it does 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, and you can do that by looking at blood cells under the 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, you know, a bit of an involved diagnostic test. It’ll give you the answer most of the time, particularly if there is a if you know that the patient has a demyelinating form of CharcotMarieTooth 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, you know, companies a number of the companies that offer genetic testing will offer gene panels so that you.

Can test or they can laughs test for, you know, a number of different 12 or 15 or 20 different known causes of CharcotMarieTooth disease. If they hit the top four then they’re going to be catch most of the patients, but the more they test for, the more comprehensive the diagnosis is. It helps, I think, in if you’re going to go with gene panels, to know first of all whether it’s type 1 or type 2. This limits the options, but it’s still possible to get genetic testing on a gene panel for all of the known CMTs or the large majority.

Of them. This has been really expensive, you know. It costs so, like with Athena panel for type 1 or type 2, CMT will cost more than $10,000 $12,000 to get all the CMTs $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 genomewide 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. 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 do 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 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 be meet CLEA standards, clinical grade standards, but genomewide analysis is really changing the way we approach patients like this. So oh, just here’s an example of a patient where we found or family where we found a rare form of CharcotMarieTooth disease, a family up from Pennsylvania. We’d 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, but I think 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. And you know, when this new genomewide analysis became available, we pulled the samples out, the DNA samples out, and sent them off for testing. We had previously well, so what this was is an unusually severe form of CharcotMarieTooth.

Disease, axonal so type 2 and Xlinked recessive. And these this patient’s in this family and there were six, or I think, eight different males affected with this disease in the family had this 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 the gene called AIFN 1. It’s a mitochondrial protein that induces apoptosis. So you know 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 reconsent the family before publishing it, and so it looked had the family names and called back. I looked on the internet to see if only one of about 20 or so family members was 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. Fishbeck, we’ve been waiting to hear from you all these years. They really were pleased to know exactly what the cause of the problem was, and there were some therapeutic implications here, a possibility of treatment based on this what’s.

Understood of 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 it was in the New England Journal a few years ago from Jim Lupski at Baylor College of Medicine, a geneticist there, who his himself affected by CharcotMarieTooth 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 the 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 wholegenome sequencing. So not just the coding regions, but he arranged at Baylor to have all of his DNA sequenced, all 3 billion base pairs, and sorted through all the variants. So in his own DNA there were 3 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 CharcotMarieTooth disease. And he found.

That a variant in SH3TC2, which is had just been identified as a rare cause of axonal CharcotMarieTooth disease, and that he and his other family members had variance in this gene that were tracking with the disease in his family. So this got some publicity it was in the New York Times and widespread publicity as a new approach to diagnosis, not by looking onebyone 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 genomewide analysis And again, I’m not an expert on the technique.

Or 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 3 billion base pairs of DNA that we carry, like what’s done with the human genome project. The cost of that has come down pretty dramatically, but it’s still it’s still difficult in that it gives you a lot of variance that need to be sorted through. In Jim Lupski’s case 3 billion variants to and try and 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. That’s still a lot of information, but it’s more likely that you’re going to get a more manageable list of variants to sort through. The challenge here it’s a challenge in Jim Lupski’s family, and in the patient we saw the patient I showed you, and others that we see is what it 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 noncoating regions, if you want. The challenge is to confirm the pathogenicity of the sequence variance that you find. So which of all these different variants is the cause of that patient’s problem So you know it’s and there’s 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 SH3TC2. 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 may want to see, well, you have a novel variant 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 family members for comparison to see if the variance.

Are, what we say, segregating with the disease, in the family. That just means that they’re the variant is present, 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 then they probably aren’t causing the disease. And Les Biesecker at the Genome Institutes have done some work just to 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 an exon sequence data from over 6,000 individuals and it’s available online in a nicely searchable form on their on their website. So if you see a variant, a list of variants you can sort out ones that are unlikely to be causing the 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, and 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 a 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 function. So this is really done now on a research basis, I think, you know, the tests are clinically available. It’s a very powerful way to identify known variance, 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. INOVA Fairfax in Virginia’s advertising excellent sequencing on NPR there are ads that they’ll do it for you, but it’s still a lot of work to extract that information, extract from.

All the information you get the information that meaningful to that patient. So it really helps to have, you know, people who know how to use it, a genetic counselor, at least, or geneticist to or specialist in the area of the disease to kind of sort it through. But 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 here about repeat expansion disease. This is something.

We’ve been involved in a long time for a long time, identifying and characterizing these diseases. This is important when it comes to hereditary neurological 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, there’s a tendency for the repeat to become which has 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. Now, this affects about one in 15,000 people, so they’ll be a few people around Bethesda with this disease. I think I’ve seen them on the street. It causes chorea. It’s 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.

A 15 percent 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 cytosineadenineguanine. 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.

Dystrophine gene. And here in the first exon of this gene is a 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. The CAG so it’s three nucleotides that encode one amino acid CAG encodes the amino acid glutamine, so this is encodes a polyglutamine repeat in the Huntington protein. So it’s called the polyglutamine expansion disease. Now, in terms of families, it’s 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 PCR, pulmonary chains 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 13 to 30 or so CAGs, and so 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 are affected here, so the shaded symbols are affected individuals.

In this family squares are males and the 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, see here. So this guy had an expanded CAG repeat of about 40 CAGs, and he passed it on to his children, his affected individuals, and they it shifted in length. It got longer in some of these individuals, up to 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. He the father had onset in, you know, the late 30s after he had most of his children, I guess, and so you see that that even within his family there is a correlation between repeat length and age on onset the longer the repeat, the earlier the age of onset in this disease. I want to focus on this person here. See this woman, who has affected brothers and a 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 presymptomatic individual, somebody’s who 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, and she doesn’t have any signs of the disease. There’s a correlation here, as I said, between repeat length and age of onsets, so 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 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 in their 20s, or they may not come down with it until their 80s. We had a patient, a retired Washington, D.C. police detective, who was diagnosed in his 80s, started to develop these jerky movements, and his girlfriend brought him and she said.

He’s just not dancing the way he used to, and laughs it turned out to be a late onset form of Huntington’s disease. So it’s hard in any particular individual, who is asymptomatic, atrisk, to know for sure when they’re going to come down with it, and there are lots of psychosocial risks with presymptomatic diagnosis. You take 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. They’d.

Rather not, 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, you know, I’d kind of like to know whether I’ve 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. You know, 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 the companies to get it done, but they 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 you know, we call Huntington’s, polyglutamine expansion disease they’re other diseases with the same kind of mutations, same kind of mechanisms that affect other parts of the nervous system, like the spinocerebellar ataxia, plus 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, most patients we still don’t know what the genetic cause is, but there are genes that have been identified. Frontal temporal 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 frontal temporal dementia. Her father had died of it. Somebody just somebody out in Nebraska sent her a letter saying, oh you know, you could have this genetic defect that causes.

You to become demented a lawyer from Charlottesville and 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 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, incidental findings. We’ve had a lot.

Of talk about this recently, as I’ve said before, but one thing you encounter as you get into genomewide analysis is you come up with mutations and genes you’re not looking for that could be important. So we call 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 CharcotMarieTooth Disease, and find out that you have a gene that predisposes to breast cancer or colon cancer. It’s arguably, it’s good to know, because it affects whether you get a mammography, whether you get prophylactic mastectomy. For colon cancer it has an effect on how often you get colonoscopy. There are therapeutic implications with these findings, and so 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’s something you know, we’re still struggling with exactly how this should be handled. Should every patient who gets exomed, should somebody look at these 56 genes, should that be required or should that be encouraged We’re having a series of meetings to try to work this through. But I think standards, this is a start on establishing standards on how to deal with this situation. So it’s important to be aware.

Of, if you’re going to go INOVA Fairfax and order exome sequencing, to know that this could happen, to know that you could find something you’re not looking for. In some way it’s analogous to get an MRI scan of the brain, and looking for one thing and finding something else, and we’re kind of learning from the radiologist as we go along to some extent, but there’s a lot that needs to be worked out in terms of the strategy here. So in closing, I would like to I think as clinicians I’m still very much a clinician.

At heart we like to trade tell stories, and I was saying, my wife will sometimes say to me afterwards, you know, you really didn’t 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. laughter Kenneth Fischbeck But you know, we learn from each other, I think, in sharing stories, and I when I was putting this talk together, I went back.

To a story that from several years ago that I think it’s worth retelling. So this is a patient we saw at the NIH some years ago, 17yearold girl complained of progressive difficulty walking. It started when she was little. At age five, her right foot turned inward age seven she was seen by a physician, she had mild weakness of the arms, right leg, and deformity of the right foot. She got an MRI scan that said her head and spine were normal, and she had an EMG that looked like it showed some changes of myopathy in the.

Leg the peroneus muscle in the leg, and the biceps muscle in the arm. And then later she required braces, like I was talking about for CharcotMarieTooth Disease, for progressive deformity of the feet, and she fell frequently, difficulty throwing a ball. Exam showed that she had kind of a funny smile, a transverse smile of normal muscle tone. She had winging of her shoulder blade, her scapula, and proximal weakness of the arms, foot deformities, normal sensory exams, 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 facioscapulohumeral 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 at 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 car to 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 from 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 idea Well, 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, Ldopa. 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 call, dopa responsive dystonia, rare disorder, but remarkably treatable, hereditary.

Neurologic disease. With the one dose of Sinemet, this girl who’d been severely disabled by the disease since 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 is affected with the same problem, and then 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 Parkinson’s, spastic paresis, or what looks like a myopathy. Characteristically it varies over the course of the day, as this patient’s did, and it can respond dramatically and with the sustained response to low doses of Sinemet, the drug that we use for Parkinson’s disease. Now, the mutations are known, the genes are know 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 though. But the it’s the GTP cyclohydrolase autosomal dominant disease, back when we saw this patient, 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 you know, giving Ldopa as in Parkinson’s disease, but much more dramatically.

Helps patients with this disease and whoops, where am I here Okay, just the important thing to remember here is that or to be aware of, I guess is that dopa responsive dystonia is an important disease to diagnose, and it’s treatable. So when you look at all these genes sometimes you come on to something like this that’s imminently treatable and really, really does a lot of good for the patient or the family are 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 a whole exome sequencing if you’re doing it you know, if you thought she had muscular dystrophy, or even if you thought you did the whole exome for some other reason, it’s good to know that there are mutation in this gene, because it can really mean a lot in terms of the management. Take home lessons, genetic testing is rapidly evolving as a diagnostic tool. We’re entering into a new age here, where we can have this information available whether we ask for it.

Or not. People have their directtoconsumer testing services, like 23andMe, they’re offering, you know, genetic testing and it’s really an evolving, a changing playing field. People are going to come to us with a list of mutations, saying which of these fits with my diagnosis The testing allows comprehensive diagnosis of hereditary neurological diseases with important implications for clinical management. Presymptomatic diagnosis should be done with care, and related to that, incidental findings are going to arise from genomewide analysis, and it’s important to have the strategy we’re working on that but it’s important to have the.

Strategy for dealing with this kind of thing. So I like this quote from a Shakespeare quote applies, I think, to genetics in general, in particular genetic diagnosis. The witch observed a man made prophecy with a near aim of the main chance of things as yet not come to life, which in their seeds and weak beginnings by in treasure. We’re we’ve come a long way, you know, I think, in genetic diagnosis and things are moving very rapidly. It is hard to prophecy 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 and we’ll be a lot closer to a Beverly Crusher with her scanner, or the car mechanic with his decoder that you can plug in, and we have to learn how to deal with that. Thanks. applause Male Speaker Dr. Fischbeck has received multiple teaching awards, and if you’ll agree with me inaudible questions or comments. Kenneth Fischbeck Yes. Male Speaker In Europe there’s a concept of treating MS with a retroviral drug inaudible a theory retroviral incorporation as genome as being.

Expressed. Do you have any thoughts on that, not the genome, but the nonsense area inaudible. Kenneth Fischbeck Yes. Yes, I’m not the MS expert there are some good people across the street you could bring over to talk about MS in general, like Bibi Bielekova, for example, but you know, retroviral integration is some it can cause mutations, and can cause problems. You know, it’s something that was a problem with gene therapy in Europe in 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, it can cause problems by integrating into the genome in such a way that it could cause a genetic problem, particularly cancer. You know, integrating into a tumor suppresser gene or a inaudible gene can bring out a malignancy, and so there’s been a lot of work in gene therapy field to try to avoid that, you know, 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.

Male Speaker unintelligible retroviral components have been there and incorporated in the genes eons ago. It’s an expression. Kenneth Fischbeck Yes, yes, so it can have an effect on something that is already there. Actually, you know, it’s interesting the FHS dystrophy story in terms of the mechanism is interesting, because what the mechanism is just been worked out in the last year or two as being a kind of activation of genes that are latent in on chromosome 4 that may have risen from retroviral insertion. So the dux4 gene is.

Left over from an ancient retroviral insertion that gets it’s activated in patients with the disease, and causes muscle degeneration. So it’s an interesting mechanism. Male Speaker A quick question on inaudible the length of the repeats and also inaudible and truncation on the inaudible onset to death is. Kenneth Fischbeck Yes, it gets more severe. Yes, it’s not a good a correlation, but the disease is definitely more severe the longer the repeat that’s true for the other repeat expansion diseases. Male Speaker In addition to finding, say, a gene that can.

Be responsive to a medication, did they ever find like there’s a family of land factors inaudible with a very high inaudible HDL that they don’t get cornered are they able to emasculate the genes so you can use that preventing part of the gene Kenneth Fischbeck Yes, yes. Male Speaker focus not the treatment, but change genetic expansion to make it not be able to get a certain disease. Kenneth Fischbeck Yeah, yeah. You can use the genes in both directions. Actually, you know, on that list of 56 genes to watch out for, for incidental.

Findings is not the HDL, but the LDL receptor, where mutations cause high cholesterol, but for HDL, you know, I think the whole idea of identifying good genes or good variants is something that 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, you know, to see what genes they’re carrying. It’s sort of opposite of looking at patients with the disease. What are the variance that predisposed to health and longevity, and they’re some projects.

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