Genetic Testing for Neurological Diseases – Kurt Fischbeck

Genetic Testing for Neurological Diseases – Kurt Fischbeck

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. 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 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; 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 co-inventor 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 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 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 disease-specific 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 pre-symptomatic
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 electro-physiologic 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, anti-coagulated 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 wheelchair-bound 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 X-linked recessive disease, so
it’s — I was thinking last night of putting together just a pedigree to show X-linked
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 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 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 three-quarters 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 self-pay 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 self-pay, 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 well-fitting 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, Charcot-Marie-Tooth disease. So 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 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 Charcot-Marie-Tooth disease, or
different types of Charcot-Marie-Tooth 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 Charcot-Marie-Tooth disease — the majority of patients with Charcot-Marie-Tooth
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 Charcot-Marie-Tooth 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 X-linked form of it, and there’s
type 2A. So 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 percent of patients. That’s caused by mutations in — that affect
a gene called PMP22. After that, probably the X-linked 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 Charcot-Marie-Tooth
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 Charcot-Marie-Tooth 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 Charcot-Marie-Tooth
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 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. 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 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 — or family where we found a rare form of Charcot-Marie-Tooth 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 genome-wide 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 Charcot-Marie-Tooth disease, axonal — so type 2 — and X-linked
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 re-consent 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 Charcot-Marie-Tooth 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 whole-genome 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 Charcot-Marie-Tooth disease. And he found that a variant in SH3TC2, which is — had
just been identified as a rare cause of axonal Charcot-Marie-Tooth 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
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 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 non-coating 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
tri-nucleotide 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 tri-nucleotide 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 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 poly-glutamine repeat in the Huntington protein. So it’s called
the poly-glutamine 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 pre-symptomatic
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, at-risk, to know for sure when they’re going to come down with
it, and there are lots of psycho-social risks with pre-symptomatic 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,
poly-glutamine 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 pre-symptomatic 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 genome-wide 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 Charcot-Marie-Tooth 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, 17-year-old 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 Charcot-Marie-Tooth 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
hypo-reflexia. 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, L-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 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 L-dopa 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 direct-to-consumer
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. 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 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 going on in that direction too, collecting
samples like from people in Italy that are in villages that 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. Male Speaker:
I’m sure Dr. Fischbeck would be delighted to answer your individual questions. Kenneth Fischbeck:
Yes. [applause] Kenneth Fischbeck:
Thanks, good to be here.


  1. Thankyou for this lecture. My family have multiple genetic defects including DRD as you discussed, Pernicious Anaemia, Ehlers Danlos Syndrome, heart problems with high blood pressure and high cholesterol; angina myocardial infarction, Cough Variant Asthma. Your information on the exom gene testing seems like a good overall test to identify these mutations? Plus knowing the cost which has always put us off because we were told it would cost thousands if pounds (we live in scotland, uk), is now achievable and affordable too. This lecture was very well explained and understandable for a layperson too. Thanks again.

  2. Hoping this will be seen by anyone who studies or researches the new gene tests available now. I'm curious about the comments from Angela below, for myself and family have the same illnesses she listed below, I too have Ehlers Danlos and I'm hopeful one day a cure or better treatment will be found for us. I'm here for genetic testing results Ive taken.
    The first was genome testing, the most recent is 23andme health. This one is fairly new because my doctor, Dr. John Christopher Caston is involved with researching and studying the results is when they're downloaded through the MTHFRSupport website. It's new so I know I can understand pieces like this result gtp cyclohydrolase with many others I don't, leave this to my doctor…The BIG deal for me is it's not common to see Ehlers Danlos in this type of forum…I'm always hopeful for us zebras with Ehlers Danlos syndrome…I hope this is not mere coincidence.

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