Muscular dystrophies: A case study of a genetic disease in humans

BIONB 325 Lecture 6
Lecture 6 Muscular dystrophies: A case study of a genetic disease in humans.
As I mentioned in my introductory lecture, I have included the muscular dystrophies,
because of the important lessons we can learn by discussing them, despite the fact that we now
know that they are primarily diseases of the skeletal muscle and not diseases of the nervous system.
There have been many surprises, however, as a result of these studies. It has turned out to be a very
complex gene with many interesting features for us to learn about. Several different promoters are
used to produce tissue specific transcripts, including a brain specific form. A similar case was
described for the MBP and not mentioned but it is also true for another myelin gene PLP. In the
assigned readings for the third lecture of this series you will find how these additional promoters
were found and described. Except in the specifics, the same discussion could be had for other
genes such as MBP.
For this lecture I will concentrate on Duchenne (DMD) and Becker (BMD) muscular dystrophies,
(see C.O.W. case 28 on Web page). Traditionally skeletal muscle diseases were included in any
discussion of neurodiseases, but it was only after the gene was identified for DMD that a better
understanding of the relative roles of skeletal muscle and the nervous system were clarified. A discussion
of these diseases, therefore, provides a good example of molecular diseases, and how one goes about
studying them, while at the same time, discussing the reasons why at one time it was thought that the
muscular dystrophies were neurodiseases. This discussion should help to understand some of the
difficulties that are encountered when interpreting clinical symptoms in terms of the underlying
mechanisms, and illustrate how identifying the responsible gene allows these issues to be better
understood.
One of the reasons skeletal muscle diseases are generally lumped together with neurodiseases is
because of the difficulties in determining whether their origin is in the muscle or in the nervous system. Up
until the time the gene was cloned for DMD, some people thought that it was a disease of the motor neuron,
that is, the neuron that innervates the skeletal muscle. Another hypothesis was that the primary site of the
disease was in the vascular system of the spinal cord, which resulted in damaging motor neurons.
Therefore, while no one doubted that the primary symptom of the disease was skeletal muscle weakness
and ultimately the loss of muscle mass, there was considerable doubt as to whether or not muscle was the
primary target of the disease.
Identifying the gene for DMD established the basis of the disease, and in the process, while
establishing the primary role of skeletal muscle, later studies suggested how other tissues, such as the
central and peripheral nervous systems, and cardiac muscle, might become involved. The identification of
the gene, however, implicated these tissues in ways that had not previously been considered. It was thought
that involvement of these other tissues were secondary to the skeletal muscle. Therefore, identification of
the gene established a totally new basis for the study of the disease. Initially the most important discovery
that resulted from the identification of the gene, was the deduced amino acid sequence for the protein, now
called "dystrophin", which was unknown at the time. In other words, in the two or three decades of very
serious research on these diseases, the protein product of the gene responsible for these diseases hadn't
even been identified. In addition, it became possible to show exactly what the relationship was between
DMD and BMD. After a few years it led to the identification of the molecular basis of a large number of
other less sever muscular dystrophies caused by mutations in other genes whose protein products interact
with dystrophin as well as how mutations in this gene involved other tissues such as the brain.
What are DMD and BMD and how are they recognized? They are the most common X-linked
recessive lethal diseases with an incidence of 1 in 3,500 male newborns. This represents a high frequency
and it is likely that this high rate is due to new mutations, previously estimated to account for about a third
of the cases of DMD. Before the gene was identified the primary physical tests for DMD were: (1) A sex
linked skeletal muscle weakness, and eventually a loss of muscle
mass. The disease affected primarily boys, had an early onset, in childhood, and death generally occurred
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BIONB 325 Lecture 6
before the age of 30. (2) High blood serum levels of creatine kinase (CK). The skeletal muscle is the
principle body source of this enzyme, and damaged muscle releases this enzyme into the blood. The
presence of high levels of this enzyme is indicative of damaged skeletal muscle. Carriers of the gene can be
detected because they also show elevated levels of CK, but not as high as symptomatic individuals but
higher than normal. (3) While the involvement of the skeletal muscle was clear the evidence for the
involvement of other tissues was less clear. There are some instances of effects on cardiac muscle, and in
approximately 33% of the patients there is a nonprogressive mental retardation. (5) In addition to DMD a
second category of muscular dystrophies called Becker type muscular dystrophy (BMD) was known to be
also X linked. These individuals have symptoms very similar to DMD but the muscle weakness and
progress of the disease is much less severe than DMD.
As I discuss the gene responsible for DMD and BMD, I will refer back to some of these symptoms
as we try to understand the linkage between the gene and the effect of its mutation. For example, you might
think from this discussion that the expression of the gene might be limited to skeletal muscle. Or, were the
cardiac problems and mental retardation significant clues to the involvement of nonskeletal muscle tissues?
One of the major controversies about the mechanism responsible for the disease was whether it was
primarily a muscle disease or was the muscle degeneration secondary to a malfunction in motor neurons, or
some other nervous system failure. I want to spend a few minutes discussing the nature of nerve-muscle
interactions that led people to think that perhaps muscular dystrophies were due primarily to a malfunction
of the motor neurons. This was a hotly debated topic until the gene was cloned. Why was this so?
Basically, it was due to the well-known fact that skeletal muscle degenerates when the motor neuron
innervating it fails. It had been shown, for example, that if you simply cut the nerve innervating skeletal
muscle, skeletal muscle rapidly begins to breakdown and muscle mass is lost. Polio might be a disease you
are familiar with, in which this occurs. The poliovirus affects primarily the motor neurons, but the primary
symptom is muscle weakness and paralysis. Anything that prevents the normal activation of skeletal
muscle will cause the muscle to begin to breakdown and degenerate. Weightlessness in space is another
way in which muscle has been shown to breakdown. The peripheral neuropathies (myelinopathies) that
result in demyelination also can cause muscle weakness because of the lost of myelin in motor axons. The
point for our present discussion is: Interfere with normal pattern of activation of skeletal muscle, it begins
to degenerate. Therefore, if the muscular dystrophies had been one of the motor neurons, the degeneration
of the skeletal muscle would have followed. This was why the conflict developed. Of course, the converse
is also true but more difficult to study. If you destroy the muscle, changes in the motor neurons occur.
Therefore, it was impossible to tell which came first. This problem was made particularly difficult to study
in humans because there was no good animal model of the disease. It was even worse than it seemed
because as it turned-out, the animal models in use for many years were not appropriate ones. They did not
share a common genetic cause.
This raises another important point, which you should be aware of. At the time of these studies,
there were animal models for inherited muscular dystrophies. They all caused muscle wasting and an
increase in CK. Several of them were used, but none of the mutations mapped to the X chromosome, until
relatively recently. The fact that there is an extraordinary synteny between the X-chromosomes in all
mammals was unrecognized as ruling out all the animal models as being valid for direct comparison to
DMD. This should alert you to the fact that many things can cause muscle degeneration and an increase
in CK levels and not be related to DMD. Eventually, a mouse with an inherited elevated serum CK level
was found and mapped to the X chromosome, but this animal showed little physical symptoms associated
with the dystrophies seen in humans. For reasons, that is not yet clear, these mice do not suffer nearly as
seriously from
the mutation as humans do. The most likely suggestion, although you will learn of other possibilities, is
that there is another protein, utrophin that is able to substitute for dystrophin more effectively in mice than
in humans. For example, if genes for dystrophin and utrophin are “knocked out” in mice, these animals
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BIONB 325 Lecture 6
have symptoms resembling DMD. On the other hand, several single gene inherited muscular
dystrophies in animals are more sever than the one that turned-out to be most related to DMD. If this
seems impossibly complex, the analysis that we will carryout in class will help to understand how these
complexities arise.
The application of recombinant DNA techniques to this problem about fifteen years ago very
quickly changed the entire approach to DMD and BMD, and subsequently other muscle diseases. Since it
too was the first illustration of the use of positional cloning, it changed the entire approach to many human
diseases. Much of what we talk about in this class evolved from these studies. The one fact that was
necessary, other than the techniques, and inspiration to do the experiments, was knowledge that the gene
was on the X chromosome; a fact that had been known for many decades. Genetic linkage analysis and
cytological abnormalities had further identified the gene to be located within chromosomal band Xp21.
These were the important observations, which initiated the cloning work. Therefore, none of the other
studies, of which their were probably thousands, played any direct role in cloning of the gene, and many
turned-out to be irrelevant for understanding the mechanism of the disease.
While the gene had been located to band Xp21, this constitutes a lot of DNA, and it contains an
unknown number of genes. How can we identify the specific gene responsible for DMD? Why not start
just as we did with Shiverer? We have in the case of DMD a patient with a disease, we know it is inherited
and is sex linked? What else do we need to know? IMPORTANT DIFFERENCE: WE DO NOT
HAVE A CANDIDATE GENE AS WE HAD IN SHIVERER. No gene that was known in this
area suggested that it might be a candidate gene.
It is difficult to decide where to begin this discussion because there is not enough time, to discuss
fully the procedures that were used. The details of the approach used 15-20 years ago would not be used
today, but I think that, because of the significance of this work, at least an outline of it is important. (In fact
I keep expecting these people to receive a Nobel Prize. Maybe this will be their year. I have included a
quotation below from Strachan and Read that emphasizes the significance of this work.)
A number of translocations had been identified in individuals with DMD, and they always involved
breakage of the X chromosome at site identified as Xp21. This means that the gene for DMD is located in
this region, but it still represented a large piece of DNA with no way of identifying the relevant gene. In
one of the DMD patient, a deletion of this region was identified cytogenetically. This observation led to
an effort to identify genes in the region of the deletion in the expectation that one might be responsible for
DMD. Note: It would be incorrect for you to conclude that the gene for DMD is deleted in this individual.
The hope was that a marker to the deleted region would prove to be close enough, and that it would be
useful for identifying the gene by further studies. As it turned out, as seen below, the deletion is in fact in
the gene for DMD.
Identification of the deleted region was done following a method loosely called “subtractive
hybridization”. I will not describe this technique, but those of you who are interested should read page
382 in the 1996 version of Strachan and Read. It is a technique that would not be used today. The result
was that they identified the genomic DNA from a normal person that was deleted in the boy. Using this
genomic DNA as a probe for Southern blots done on other DMD boys quickly showed that in a small
percentage (6.5%) had deletions in this region as well. This started a serious search for a gene in the
deleted region. By cutting the genomic DNA into smaller fragments, and using them to probe a c-DNA
library from skeletal muscle. This lead to the identification of a c-DNA which when tested against a large
number of DMD boys was partially deleted in about 50%. (This is using genomic DNA from these boys
and doing Southern blots using the c-DNAs as probes.) This strongly supported a deletion in this gene
was responsible for DMD. (What about the 50% that showed no deletions?) The size of the c-DNA
identified in this manner was eventually shown to be 16kb and the gene 2,400kb(2.4mb). These are
enormous sizes. You should realize that the 2.4mb piece of DNA is a factor of almost seventy times bigger
than the 37 kb initially identified in Shiverer as being the entire MBP gene, or twenty times as big as the
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BIONB 325 Lecture 6
105kb later found to be the correct size. This is one of the largest genes in the human genome and
one of the largest proteins.
Several important conclusions can be drawn from this work. Firstly, it should be absolutely clear
that DMD can result from deletions in this gene. (2) The deletions are not evenly spaced along the gene
but are clustered. (3) This is the largest human gene found to date. The c-DNA is spread over
approximately 2 million base pairs. The genomic locus is nearly 200 times the size of the transcript. This
one gene represents nearly 1/1000 of the total human genomic DNA. Ultimately, the protein was identified
as being 424 kD in size. This is an enormous protein, certainly one of the largest, if not the largest. The
protein was named dystrophin and I will spend my lecture dealing with the protein. One point I want to
emphasize to you is that protein was totally unknown until the gene and it c-DNA was identified and
characterized. (4) The size of the gene probably partially accounts for the number of mutations that occur in
it. The idea being, the bigger the gene the more likely mutations will occur. It might also, however, be
concluded that there may be, as well, hot spots within the gene that make it more susceptible to mutations
than it might be otherwise. Two probes covering only 2kb of the total c-DNA detected 80% of all the
deletions. This means that 80% of the deletions occurred in about 15% of the total DNA coding for the
protein. (5) 50% of the DMD patients could not be identified as having a deletion. Subsequently it was
found that about 30% of the DMD patients do not have deletions or duplications. (About 6% have
duplications.) Some have been identified now as having missense mutations. (6) Deletions commonly
result in much reduced if not total absence of dystrophin, the protein product of this gene in skeletal
muscle.
Shortly after the dystrophin gene was identified in DMD patients, mutations in this gene were also
found in patients with BMD. As you should recall, I mentioned that BMD was a similar to DMD but later
onset and slower in progress and it too was on the X chromosome. This raised the issue as to whether a
comparison between the mutations resulting in DMD would be more extensive than those found in BMD.
At least initially this did not appear to be clear-cut, and it was suggested that perhaps the mutations in BMD
resulted in the use of other start codons within the dystrophin gene. The assigned readings will make the
case for BDM being due to nonframeshift deletions and DMD being due to frameshift deletions. This
may account for the less sever symptoms seen in BDM.
What might be responsible for the 30% of the individuals with DMD who show no
deletions or duplications in the dystrophin gene? One possible hypothesis is that they don’t have DMD.
The second possibility is that they have mutations in the dystrophin gene, but the mutations are too small to
be detected with the tests used so far. For example, a missense mutation would not be detected with the
techniques I have described. This is the hypothesis I wish to test, i.e., with an individual classified as
having DMD but with no deletions nor duplications, does he have some other mutation in the dystrophin
gene? The procedure I am going to describe is PCR based. In discussion I will discuss this technique
briefly, but you should refer to any number of textbooks, if you have questions about how PCR is done. It
is extremely important that you understand the basic principles behind PCR.
Firstly, let us look at what the results would look like if there were a deletion for this individual. As
you should recall in order to perform PCR you must have two primers. The primers used in these studies
are ones based on the sequence of the dystrophin gene. One exon at a time is amplified by PCR. The
transparency shows several exons, which have the normal size, but two individuals are missing one exon.
This is a straightforward use of PCR. Let me remind you however, there are 79 exons in the dystrophin
gene. Clearly, to look at all 79 would not be practical except in a research environment. Fortunately, we
know from my earlier discussion that most deletions are found in a small number of exons. This means
that an initial survey of these exons would be a good strategy, and only then search the other exons if
nothing was detected in the most frequently affected exons.
The question now, however, is what about those individuals who don’t show any deletions?
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BIONB 325 Lecture 6
The assigned reading by Prior et al., describes one such boy who at the age of 8 appears to
have a sever form of DMD but with no deletions or duplications in the dystrophin gene. Does he in fact
have DMD? In order to test this question they amplified several exons, just as I described above, but in
addition to looking at the presence or absence of the exon they determined whether the exons carried a
missense mutation. They did this with a heteroduplex analysis. Heteroduplex analysis is done by
hybridizing the wild-type PCR product with the product from the patient. The product with the DNA from
the patient will bind to the wild-type DNA, but it will migrate anomalously on a special gel electrophoresis.
That is, because the two chains of DNA are very similar, but potentially with only one base substitution, the
heteroduplex will migrate differently from the homoduplex. Using this technique, this patient was found to
have a one base substitution, which results in the substitution of a leucine for an arginine in exon number 3.
This paper begins with the use of PCR to amplify specific sequences of DNA, i.e., make many
copies of single exon. The same exon is amplified from a control person as well as the boy with DMD.
Following the PCR reaction the DNA products are run in a special gel electrophoresis system called
“Hydrolink”. The DNA products are mixed, denatured at a high temperature causing the DNA strands to
come apart, and then the temperature is lowered allowing the strands to reanneal. When they reanneal, three
types of double strands can form: (1) homoduplexes of normal DNA, (2) homoduplexes containing the
missence mutation and (3) heteroduplexes formed from the DNA of the normal DNA and the DNA
containing the mutation. Base pairing in the heteroduplex will not be perfect, if there is one or more base
differences between the two DNAs. This difference in base pairing can be detected on the “Hydrolink”
gel following electrophoresis, see Fig 1 of Prior et al..
The PCR products must then be sequenced in order to establish the presence and nature of the
mutation. This is the way the missence mutation was identified in this young boy. You should pay special
attention to this paper, and its discussion of the relationship between mutation and phenotype. Also, notice
that despite this boy having DMD, he did have dystrophin in his skeletal muscle located in the correct
place; only the amount of dystrophin seems to be lower. Clearly, this mutation is seriously disrupting the
normal function of dystrophin.
The other aspect of this paper you should study is shown in Fig. 3. This describes the results of a
linkage study between the DMD gene and microsatellites, i.e., short CA repeats in the dystrophin gene
which can be used as polymorphic markers in the same way that restriction sites can be used. The
haplotype assignments were based on the molecular size of the PCR products. This is a similar analysis to
the haplotypes assignments based on the restriction endonuclease polymorphism. I have not described
RFLP haplotype analysis yet, but I will shortly, for those of you who are unfamiliar with it. Our first case
of such an analysis will appear in Alzheimer’s disease. The best description, however, is in a later paper by
Gusella et al., if you want to jump ahead.
Does the finding of the missense mutation prove that this boy has DMD? How do you
know that this substitution is not simply a rare polymorphism that has no effect on the function of
dystrophin? There are several points that must be made in answering this question. One, the
pedigree didn’t show the substitution to be a polymorphism. In fact the pedigree suggested that the
mutation was a new one and occurred between the grandmother and mother of the boy with DMD.
Secondly, the observation of reduced levels of dystrophin in this individual’s skeletal muscle
indicates that the mutation does affect the function of dystrophin, if only by reducing the amount
found in skeletal muscle. This result shows that the complete absence of dystrophin is not required
for an individual to have DMD.
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BIONB 325 Lecture 6
“Duchenne muscular dystrophy (DMD)
was a major test-bed for positional cloning
methods. Years of careful investigation of the
pathological changes in affected muscle had
failed to reveal the biochemical basis of DMD.
In the early 1980s, several groups competed
to clone the DMD gene, using different
approaches. The pioneering work of these
groups, overcoming formidable technical
difficulties to clone an unprecedented gene,
was probably the major inspiration for most
subsequent positional cloning efforts.”
Human Molecular Genetics (1999) Strachan and Read
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