| Case A | Case B | Case C |
Background: Cystic fibrosis (CF) is generally considered the most common severe autosomal recessive disorder in the Caucasian population, with a disease frequency of 1 in 2,000 and a carrier frequency of 1 in 20. The major clinical symptoms include chronic pulmonary disease, pancreatic insufficiency, and an increase in sweat electrolyte concentrations. The cause of the disease appears to be a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein involved in transporting ions across epithelial surfaces, such as the linings of the lungs and intestines. Several mutations have been identified as being associated with a non-functional CFTR protein. The most common mutation, accounting for about 50% of CF cases, is called delta F508; it is a three-base deletion resulting in the loss of a phenylalanine at position 508, in the ATP-binding portion of the protein. This mutation is detected by sequence analysis of PCR-amplified DNA, or by hybridization with mutation-specific probes (the latter method is illustrated in Cases B and C).
Click here to explore the nature of CFTR mutations using NCBI online tools, including the Genome Data Viewer and Structure (for 3D visualization), and links to information on prime editing as a potential tool to correct these and other mutations.
Rapid screening for cystic fibrosis is also done using RFLP markers linked to the CF gene on chromosome 7 (illustrated in Case A). Several RFLP analyses can be performed relatively quickly on PCR-amplified DNA from a blood sample or chorionic villus sample. Then, if a positive result is obtained with an RFLP marker, sequence analysis or mutation-specific probe hybridization can be done to confirm the CFTR mutation. An example of a linked RFLP marker is in the locus Mp6d.9, in which a point mutation linked to CF results in the loss of an MspI site.
Case A: As Sharon Brown browsed the local newspaper, she noticed the story about the five-year old boy with cystic fibrosis who lives on the next block. The article was mainly a human interest story about how the family was coping. There also was some background information about the disease and its inheritance patterns, including the statistics indicating that approximately 1 in 18 people in this part of Minnesota carried a cystic fibrosis mutation.
Sharon is two months pregnant. She realizes that she and her husband, Bob, should have been tested for the cystic fibrosis (CF) mutation since they each have some family history of the disease, but they really hadn’t expected to have a child so soon. She discusses this with her physician during her check-up the next day, and together they decide to test Sharon and Bob for a mutation in linked to the CF gene. They also decide to test the developing fetus. Two other families in the same town who also are in the first trimester of a pregnancy, Jill and Mike Jones and Carol and Ron Smith, also decide to be tested after reading the article.
Procedure: Blood is drawn from the parents, and a chorionic villus sample is taken from each fetus. DNA is isolated from the samples, and a small portion of chromosome 7 near the CF gene, a locus called Mp6d.9, is amplified by PCR. (Use the PCR function on the Data Screen, rather than 96-well PCR.) Control DNA samples with and without the CF mutation are also included. Digestion of the PCR fragments with the enzyme MspI is used to detect the RFLP linked to the mutated CF gene, which results in the loss of a MspI site. [Note: Small fragments are generated, so use shorter run times to see all of the fragments.]
DNA samples:
- Brown family:
- Bob
- Sharon
- Fetus
- Jones family:
- Jill
- Mike
- Fetus
- Smith family:
- Carol
- Ron
- Fetus
- Controls:
- DNA heterozygous control
- DNA homozygous mutation control
- DNA homozygous normal control
Note: The video links below show the analysis starting with digested PCR fragments. Click here to see a more detailed video showing the PCR and digestion procedures of Case It, using Case A for the Brown family as an example.
Results of PCR/RFLP analysis for Case A – Brown family
Results of PCR/RFLP analysis for Case A – Jones family
Results of PCR/RFLP analysis for Case A – Smith family
Click here to explore the nature of CFTR mutations using NCBI online tools
- What conclusions can you draw from the gel results?
- What options are available to the parents?
- What issues are raised by this type of testing?
Case B: (Contributed by Stephanie Dahlby, Dan Tally, and Janelle Veerkamp)
Lynda and Jim are expecting their first child. Recently, however, they learn that Lynda’s aunt died of CF and Jim’s uncle died of CF. They are worried that they might be carriers for the disease and pass cystic fibrosis on to their unborn child. They learn about a procedure which can determine whether they are carriers. They also learn about a procedure called amniocentesis which can detect if their unborn child has CF or is a carrier. However, amniocentesis is a very risky procedure. Jim and Lynda ultimately decide that they first want to be tested to see if they are carriers for the disease. If they learn that they both are carriers, they would like to go through with the amniocentesis to see if their child is affected.
DNA samples:
- Lynda
- Fetus
- Jim
- Control DNA with F508 mutation
- Control normal DNA, without mutation
Procedure: Run PCR on each of the DNA samples using the CF primers (NOTE: Use the PCR function on the Data Screen rather than the 96-well PCR). Then, using the dot blot screen, load the probes into the dots. Load the DNA samples into the corresponding wells. By comparing the dot blot patterns of Jim, Lynda, and the fetus to those of the two controls, determine whether these DNA samples are homozygous positive for the CF mutation, homozygous negative for CF, or heterozygous carriers for CF.
Results of PCR/dot blot analysis for Case B
- What conclusions can you draw from the gel results?
- What options are available to the parents?
- Should large-scale screening for CF carriers be done?
- How has the prognosis for children with CF changed and how might it change in the future?
- What other issues are raised by this type of testing?
Case C: The pre-marriage counseling session Carl and Maggie are having with Pastor Frank is not going at all as they had expected it to. After some of the anticipated discussion of relationship issues, the conversation turns to family planning. When both Carl and Maggie say they want to have children, Pastor Frank, instead of giving advise on how to properly rear children, begins to talk about genetic testing for Cystic Fibrosis! It turns out that Pastor Frank and his wife had two children affected with CF who died in their early teens. Because of the relatively high frequency of CF carriers and his opposition to abortion, Pastor Frank believes that all couples should be tested for the CF gene before getting married. Carl and Maggie are not sure they share Pastor Frank’s beliefs but decide to go along with being tested.
DNA samples:
- Carl
- Maggie
- Control DNA with F508 mutation
- Control normal DNA without mutation
Procedure: Run PCR on each of the DNA samples using the CF primers. (NOTE: Use the PCR function on the Data Screen rather than the 96-well PCR.) Then, using the dot blot screen, load the probes into the spots. Load the DNA samples into the corresponding wells. By comparing the dot blot patterns of Carl and Maggie to those of the two controls, determine whether these DNA samples are homozygous positive for the CF mutation, homozygous negative for CF, or heterozygous carriers for CF.
Results of PCR/dot blot analysis for Case C
- What conclusions can you draw from the gel results?
- What options are available to the parents?
- Should large-scale screening for CF carriers be done?
- How has the prognosis for children with CF changed and how might it change in the future?
- What other issues are raised by this type of testing?