Duchenne Muscular Dystrophy Medical Case
Overview: Duchenne muscular dystrophy is an inherited genetic disorder that causes progressive muscle
weakening. The symptoms rapidly worsen and death typically occurs around age 17. The onset of this disease
occurs by age 3 and the afflicted individual is usually in a wheelchair by age 12.
A couple, Sara and Daniel, comes to you because Duchenne muscular dystrophy runs in their family. Sara has
a brother who died from Duchenne muscular dystrophy and two nephews who are living with the disease.
Examine the pedigree below and answer the questions.
1. What type of inheritance pattern do you predict that Duchenne muscular dystrophy has?
2. Sara’s brother, Jack, is worried that if he has more children that they may end up with this disease even
though he has two healthy daughters. What do you tell him?
3. Sara’s sister, Joy, has two sons, Ben and Chris, with the disease and two healthy daughters, Ann and
Emma. What is the chance of one of these daughters being a carrier for the disease?
4. Joy’s son, Scott, is only 6 months old. Can you tell whether he has the disease or not?
5. In this pedigree, who was the original carrier of this disease?
Sara and Daniel want to find out if Sara is a carrier. Joy also wants her daughters tested as well as Scott. Read
the information below to help this family get the facts about their genes.
Duchenne muscular dystrophy is caused by a defective form of the protein dystrophin. Dystrophin has an
important structural role in muscle, heart, and brain. When dystrophin is absent or abnormal, the entire
muscle fiber complex is compromised. The gene that codes for dystrophin (DMD) is 2.6 Mb and contains 97
exons so it is quite large. Becker is a more mild form a muscular dystrophy and is also caused by a mutated
DMD gene. Both Becker and Duchenne are the result of deletions in the DMD gene (see diagram below). The
Duchenne deletion causes a frameshift mutation, but the Becker deletion does not.
The gene locus is Xp21.2, which means that the gene is considered sex-linked and recessive. As you found in
the pedigree, mothers who are carriers have a 50% chance of passing this abnormal gene on to their sons.
Daughters of carriers have a 50% chance of being a carrier themselves.
Image from: http://compbio.berkeley.edu/people/ed/rust/Dystrophin.html
6. Given this information, how could you test the people in Sara’s family?
7. How would gel electrophoresis be useful in determining whether someone is a carrier, afflicted with
Duchenne muscular dystrophy, or completely healthy?
8. Below is a gel. Predict the results and draw the bands for a carrier, someone completely healthy (not a
carrier), and someone with the disease
Now you will perform an experiment to test the members of Sara’s family. DNA was extracted from Sara, Ann,
Emma, Scott, Chris, and Jack. Jack’s DNA will act as a negative control (completely healthy) and Chris’s DNA
will act as a positive control (Duchenne). PCR was used to amplify the region of the DMD gene where the
deletion occurs. The PCR products are ready to be tested using gel electrophoresis. Follow the instructions
below to set up and run the gel.
Goal: to use gel electrophoresis to determine the presence or absence of mutated DMD gene. The mutated
DMD gene has a deletion.
Prepare the gel: Add 0.3g of agarose to 20ml of dH20 in an Erlenmeyer flask. Use a Kimwipe to stopper the
flask. Heat the solution in a microwave for approximately 30 secs. Allow the heated solution to cool for
approximately 2 minutes and pour the gel. Remove the comb after the gel has solidified. Pour 1x TBE buffer
so that it just covers the gel.
Load the gel: Add 10µl of each different PCR product to a separate well. Do not mix solutions and be sure to
use new tips each time that you pipette. The table below shows the labels used for each person. In the
diagram at the bottom of page, label which person’s sample is located in each well. You will have empty wells.
Run the gel: Close the chamber and attach the
red wire to the red (positive end) and the black
wire to the black (negative end) of the chamber
and do the same with the volt meter. Turn on
the power supply, set the voltage at ~140 and hit
start. Stop the gel after approximately 20
minutes. This should be plenty of time to see the
results.
Person
Sara
Ann
Emma
Scott
Chris
Jack
Label
A
B
C
D
E
F
Results
Draw the results: Draw the banding pattern that you get for each person on the diagram above. Determine
whether each person is a carrier, healthy, or has Duchenne muscular dystrophy and write it in the table above.
9.
9. Which allele (normal DMD or mutated DMD) moved further into the gel and why?
10. Why are most patients with DMD boys?
11. Can a boy be a carrier for Duchenne muscular dystrophy?
12. What will you tell Sara? If you were Sara or married to Sara, would you have children?
13. In the process of in vitro fertilization embryos are created in test tubes by the combining the mother’s
egg and the father’s sperm. Babies created in such a manner get the nick name “test tube babies”. The
first test tube baby, Louise Brown, was born in 1978 in Britain. The embryos are then implanted into
the mother’s uterus. The mother is then pregnant and gives birth to the embryos that survive.
Preimplantation genetic diagnosis (PGD) is a test that screens for genetic flaws among embryos used
before in vitro fertilization. Fertility specialists can use the results of this analysis to select only
mutation-free embryos for implantation into the mother's uterus. The embryos with mutations are
destroyed.
If you were Sara or married to Sara, would you want to use in vitro fertilization and PGD to make sure
that your child does not have Duchenne muscular dystrophy? What are the advantages of doing this
testing? What are the disadvantages of doing this testing?
Teacher notes:
Two dyes will be used to represent the mutated DMD and normal DMD genes. Bromphenol blue travels faster
in agarose gel than xylene cyanole; therefore bromphenol blue will represent the mutated gene with a
deletion and xylene cyanole will represent the normal DMD gene.
Make stock solutions of the two dyes. Add 0.25g of bromphenol blue to 10ml of dH2O and 1ml of glycerol.
Repeat for xylene cyanole.
For 6 gels, you will need 10µl of each sample to load for a total of 60µl. I always make extra solutions (due to
pipetting errors) so I recommend making group tubes that hold at least 80µl. I make 50:50 mixed solution of
1ml total for the carriers. The healthy and DMD samples can be taken directly from the straight dye (stock). If
you teach several classes or want more groups, you can multiply by the number of groups.
Person
Label
Results
Dye Sample Used
Stock totals
Group total
Sara
Ann
Emma
Scott
Chris
Jack
A
B
C
D
E
F
Carrier
Carrier
Healthy
Healthy
DMD
Healthy
BB and XC
BB and XC
XC only
XC only
BB only
XC only
40µl BB + 40µl XC
40µl BB + 40µl XC
80µl XC
80µl XC
80µl BB
80µl XC
80µl
80µl
80µl
80µl
80µl
80µl
Class Totals
(with 6 groups)
480µl
480µl
480µl
480µl
480µl
480µl
The xylene cyanole bands
are purple and the
bromphenol bands are
blue.
10X TBE Buffer Preparation:
Mix components listed to make 1 liter 10X TBE. To make a 1X TBE, add 100ml of 10X TBE to 900ml of dH2O. 10X
TBE buffer = 108g Tris Base, 55g Boric Acid, and 40ml EDTA (pH 8.0)
CHEAP AND FUN REPLACEMENT: Use any brand clear Gatorade (watermelon is clear) instead of buffer. Since
you are not using DNA, Gatorade provides the electrolytes!
Answers to questions:
1. The disease is sex-linked and recessive.
2. Jack does not have the mutated DMD gene or else he would be sick. He cannot pass the mutated DMD
gene to his daughters or sons! It is highly unlikely that his wife is a carrier because she is not related by
blood to this family.
3. The chance of her son having DMD is 50% and the chance of either daughter having the disease is 50%.
4. Scott is too young to show symptoms so there is no way to know whether he has DMD or not until he is
older or gets tested.
5. The grandmother (Sara’s mom) was a carrier.
6. Gel electrophoresis
7. The gene with a deletion will be smaller than the normal gene. Gel electrophoresis can separate the
genes based on their sizes.
8. The diagram should look something like this. The normal alleles should be closer to the wells and the
mutated alleles farther along in the gel. A carrier is heterozygous.
9. The mutated allele moves farther because it is smaller due to the deletion.
10. Most patients with DMD are boys because girls would need to inherit two alleles (one from dad and one
from mom). This means the dad would have the disease. Sadly, boys do not usually live long enough to
pass the gene to daughters.
11. Boys only get one X chromosome so they only get one copy of the DMD gene.
12. Opinion
13. Opinion