Background: Huntington’s chorea is a neurodegenerative disease characterized by motor, cognitive, and emotional symptoms. The age of onset for symptoms is generally 30-50 years. The genetic basis of the disease is an amplification in a gene with an (as yet) unknown function. A triplet (CAG) is repeated 35 or fewer times in asymptomatic individuals; having more than 35 repeats is associated with disease symptoms. Huntington’s disease is considered a dominant disorder, since one copy of the amplified gene appears to be sufficient to cause disease symptoms.
Procedure: This amplification can be detected by restriction enzyme digestion and Southern blot analysis, since the size of the fragment bound by the probe is increased as a result of the amplification of the triplet repeat. Alternatively, PCR can be used to isolate the region containing the triplet repeats; the relative size of the repeat region can be determined by running the PCR products on a gel. Further analysis can be conducted via bioinformatics procedures, as described below.
- Southern blot: Digest the DNA samples with EcoRI, and then perform a Southern blot with the Huntington’s probe. By comparing the sizes of the fragments bound by the probe, determine the Huntington’s gene status of Susan and her brother.
- PCR: Use the HD primers to perform PCR on the DNA samples. Run the PCR products on a gel (note that the DNA fragments are small, so shorter gel run times may be needed)
- Bioinformatics (1): Use BLAST to identify the gene associated with the larger gel fragments. Click on the large gel fragment from one of the samples that is positive for Huntington’s disease. The sequence of that DNA should be visible in the lower window (if not, check the Sequence box above the window). The entire sequence, just a portion containing the repeat region, can be sent for BLAST analysis (the latter will work better if using fragments from the Southern blot). See the Bioinformatics tutorial for more detailed instructions.
- Bioinformatics (2): Compare the lengths of the repeated regions by aligning sequences. How many triplet repeats does each individual have? Select the PCR fragments from the Opened & Processed window or, if Southern blot was used, highlight the region containing the repeats from each sample and export for alignment (the Search feature can be used to find the CAG repeats in the original DNA file). Note that trees built using these sequences are not meaningful. Use the Alignment Viewer window in MEGA to locate the repeat regions.
Case A: Susan is a 23-year-old whose father, age 55, and paternal aunt, age 61, have been diagnosed with Huntington’s chorea. A paternal uncle, age 66, appears to be unaffected by the disease. Susan wants to know if she inherited the mutated gene from her father so that she can prepare for that future if necessary. She arranges to undergo DNA testing for Huntington’s disease. Her 17-year old brother, John, also decides to be tested after talking with Susan.
DNA samples:
- Susan (patient)
- Father (affected)
- Aunt (affected)
- Uncle (unaffected)
- John (brother)
- Control DNA with HD mutation
- Control DNA, normal (without HD mutation)
- 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?
- How would you counsel Susan and her brother based on the results of the test?
- What issues are raised by this type of testing?
Case B: Josiah and Eldrea were worried about their 52-year-old father. He was starting to act sometimes like this older brother, their uncle Theo. Theo was 15 years older than their father and he had been recently diagnosed with Huntington disease. After speaking with the family physician they learned a diagnostic DNA test was available. They wanted to their father to have the test, and they decided they should take it themselves so that they can better prepare for their future.
DNA samples:
- Father
- Uncle Theo
- Josiah
- Eldrea
- Control DNA with HD mutation
- Control DNA, normal (with no mutation)
- 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?
Case C: Forty-four year old Jerry is haunted by the specter of Huntington disease. It took his grandmother, a favorite uncle, and now he sees signs of motor impairment in his 67-year-old mother, Sophie. He worries that he might have inherited the disease and wonders, too, if he may have passed it to any of his 3 children. After several late night family discussions, a date is set for them to provide samples for DNA testing. While he is certain he and his mother should be tested, he wonders if his children are making the right choice.
DNA samples:
- Sophie (mother)
- Jerry (father)
- 22-year-old son
- 19-year-old daughter
- 18-year-old son
- Control DNA with HD mutation
- Control DNA, normal (without HD mutation)
- 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?