Background: PKU is a genetic metabolic disease caused by a mutation in the phenylalanine hydroxylase enzyme. In the most common form of PKU, a C to T point mutation causes an arginine to be replaced by tryptophan at amino acid position 408, resulting in an inactive enzyme and incomplete metabolism of phenylalanine-containing compounds such as proteins. The resulting buildup of phenylalanine can cause mental retardation, eczema, loss of skin pigmentation, and other disorders. If detected early, the disease is treatable by excluding foods high in phenylalanine form the diet.
PKU is typically tested by measuring the blood level of phenylalanine in a blood sample taken at birth. The molecular test would be valuable as a follow-up to confirm the cause of high phenylalanine levels and to be better able to predict treatment outcomes. The mutation can be detected by dot blot analysis of PCR-amplified DNA from the blood sample. Probes that will bind either the mutated or non-mutated sequence are used in the dot blot to determine which form of the gene is present.
Procedure: To analyze these cases, use PCR with the PKU primers to amplify a portion of the phenylalanine hydroxylase gene from blood DNA samples. (NOTE: Use the PCR function on the Data Screen, rather than the 96-well PCR.) Then, using the dot blot, load the probes into the spots and add the PCR-amplified DNA samples into the corresponding wells.
Case A: Peter and Pam just had their first child. The PKU blood test performed at birth indicated a high level of phenylalanine in the blood. The physician suggests a follow-up DNA test immediately to confirm the PKU diagnosis and to determine the most appropriate treatment. She also suggests that Peter and Pam be tested to confirm their carrier status and predict the risk of PKU in subsequent offspring.
DNA samples:
- Peter
- Pam
- Infant
- Control DNA containing the PKU mutation
- Control normal DNA, without mutation
- What conclusions can you draw from the results of the DNA test?
- What is the molecular basis for the test, and how does this explain the test results?
- What issues does this type of testing raise?
Case B: Angie watched her little brother, Alan, grow up with PKU. She knows how wonderful it is that the dietary treatment that he has undertaken since being diagnosed by neonatal screening has prevented development of the worst PKU symptoms. But she has also seen that his life has not been an easy one. It is never easy being different and Alan’s strict dietary regimen has significantly affected his social interactions at school. Angie has always said that if she ever decides to have a child, she will be tested before she gets pregnant to see if she carries the PKU gene. This makes her current situation, an unplanned pregnancy by a man who was out of her life before either of them even knew, especially difficult. There are so many unknowns. Does she want to continue the pregnancy under these circumstances, even if she isn’t a carrier? If she is a carrier and the fetus is unaffected, is this her best chance to have child unaffected by PKU? Angie decides that the starting point for her difficult decisions must be to find out if she is a carrier for PKU.
DNA samples:
- Angie
- Control DNA containing the PKU mutation
- Control normal DNA, without mutation
- What is the chance that Angie is a carrier?
- What conclusions can you draw from the results of the DNA analysis?
- How would you counsel Angie based on the results of her test?
- Is the role of a genetic counselor different in a case in which an unaffected fetus may be aborted?
Case C: When Richard and Kathy’s first child, Robert, was tentatively diagnosed with Phenylketonuria on the basis of neonatal screening for high levels of phenylalanine, they were relieved to learn that appropriate dietary restrictions are an effective treatment for PKU. After some experience with maintaining the strict diet and the constant medical monitoring, they make some decisions about family planning. They know they still want to have a large family, but feel that they cannot handle the rigors of more children with PKU. When Kathy becomes pregnant again, they seek genetic testing to confirm the diagnosis and test the fetus for PKU.
DNA samples:
- Robert
- Richard
- Kathy
- Fetus
- Control DNA containing the PKU mutation
- Control normal DNA without mutation
- What conclusions can you draw from the results of the DNA analysis?
- How would you counsel Richard and Kathy based on the results of their tests?
- Is termination of the pregnancy appropriate in the case of a treatable disease?