Background: One form of inherited muscular dystrophy, Duchenne, is X-linked and therefore affects primarily males. The symptoms of Duchenne muscular dystrophy (DMD) include progressive and severe skeletal muscle weakness. A common mutation associated with DMD is a deletion of one or more exons in the dystrophin gene. These deletions can be detected by restriction enzyme digestion and Southern blotting using a combination of probes that will bind to multiple dystrophin exons.
Procedure: Digest each DNA sample with HindIII, then perform a Southern blot with the dystrophin gene probe (DMD probe). Based on the number and sizes of the fragments bound by the probe, determine the status of each of the individuals tested. Some fragments are small, so you will need to reduce the run time (use 50 minutes rather than 60). After running the Southern blot, click the Diff. display button above the gel and select the ‘on’ option. This will increase the transparency of the bands, enabling you to see if bands differ in the amount of DNA present.
Case A: Jean and Bill have three sons, ages 12, 8, and 7, and a daughter, age 6. The oldest son and daughter are healthy, but the two younger sons are exhibiting symptoms of muscle weakness consistent with early muscular dystrophy. Jean knows that she has a family history of muscular dystrophy, but she does not know whether she is a carrier of the disease gene. She seeks DNA testing to determine whether her younger sons may have inherited the form of the dystrophin gene associated with Duchenne muscular dystrophy (DMD).
DNA samples:
- Jean (mother)
- oldest son (unaffected)
- daughter
- 8-year-old son
- 7-year-old son
Results of Southern blot analysis for Case A
- What conclusions can you draw from these results?
- What is the molecular basis of this disease, and why does this result in the observed gel patterns?
- What issues are raised by this type of testing?
- Are the females heterozygous or homozygous? How can you tell?
Case B: Tabitha walked slowly to the kitchen table and sat down. She gazed out the window where her two daughters (aged 10 and 12) were playing in the sprinkler and chuckled at their antics. She thought of the son she and her husband lost nearly 2 years ago. He would have been 14 this year. And she just found out she was pregnant again – unplanned and unexpected. As she sipped her coffee she knew she could not endure a pregnancy wondering if the baby would turn out healthy, nor could she bear to lose another child to muscular dystrophy. She called the clinic to set up an appointment for DNA testing. She knew now that she was the source of the mutated gene and she wondered if she had passed it to her new baby. She also wondered if either of her daughters were carrying it, and hoped fervently they were not.
DNA samples:
- Tabitha (mother)
- fetus
- 10-yr-old daughter
- 12-yr-old daughter
Results of Southern blot analysis for Case B
- What conclusions can you draw from the results?
- What is the molecular basis of this disease, and why does this result in the observed gel patterns?
- What options are available to the family?
- What issues are raised by this type of testing?
- Is the fetus healthy?
- Is the fetus male or female? How do you know?
- Are the females heterozygous or homozygous? How can you tell?
Case C: Scott and Mary met as teenagers at the local MD telethon. Each was there volunteering their time in support of brothers they watched slowly suffer from progressive muscle degeneration. Now, years later, they were married to each other and ready to start a family of their own. Mary’s pregnancy test came back positive and the news filled them with both joy and dread. What if their child had muscular dystrophy? Mary decides to go in for DNA testing to find out is she is a carrier, and if the baby is affected.
DNA samples:
- Mary (mother)
- Scott (father)
- fetus
Results of Southern blot analysis for Case C
- What conclusions can you draw from the results?
- What is the molecular basis of this disease, and why does this result in the observed gel patterns?
- What options are available to the family?
- What issues are raised by this type of testing?
- Is the fetus healthy?
- Is the fetus male or female? How do you know?