CFH and Macular Degeneration
This web page was produced as an assignment for Gen677 at UW-Madison Spring 2010.
Conclusion
Based upon the data generated and evaluated and the literature read, I have devised a hypothesis. My hypothesis is that the mutation from a tyrosine to a histidine affects the ability of CFH to bind or interact with to other proteins. I would like to focus on proteins involved in the complement system, specifically complement factor I (CFI), C-reactive protein (CRP), and C3, because of the strong pathophysiological connection between AMD and the complement system. To test this hypothesis, I have designed the following yeast-2-hybrid (Y2H) experiment to explore the protein-protein interactions of CFH.
Y2H
To test whether the change from tyrosine to histidine affects ability of mutant CFH to bind to C3, CFI, and/or CRP, a Y2H will be conducted. The bait will the the mutant CFH and the bait will switch between C3, CFI, and CRP. To ensure that the bait and prey system is working correctly, the positive control will be wild-type CFH, seeing as it is already known that it interacts with C3, CFI, and CRP. The negative control will be a protein that has no interaction with C3, CFI, or CRP.
Expected Results
If the controls run correctly, a few conclusions can be made upon the results of this experiment. If there is no growth, there was not any or was not sufficient interaction between the respective proteins. If growth did occur, the respective proteins did interact. This, however, does not account for the situation in which the interaction has decreased, but is still sufficient for growth. This small change could still have a profound impact within the body. To measure whether the interaction of the proteins changed, the fluorescence of the betagalactosidase activity will be measured and compared between wild-type and mutant CFH.
Future Directions of Research
Several areas should be further explored to better understand CFH and AMD. Out of all the literature that I read, I did not find much about the heritablilty of AMD or CFH. All that I could find was the fact that if you have a family member with AMD, you have an increased risk of developing AMD. Knowing how AMD and CFH are passed down through the family is crucial to treating this disease. There are a few other genetic factors to AMD that have been recently discovered. Certain mutations of CFI and C5 greatly increase your risk of developing AMD. Researching these mutations would be important to fully understand the development of the disease and in turn treating it. A final area to look into is the area of drug development. There is no current cure for AMD, and the only treatments available are for treating the symptoms of wet AMD. There is no treatment for dry AMD. Therefore, identifying drug targets and development of a drug is necessary. Perhaps a replacement for CFH as a regulator of the complement system could be found or a drug that restores CFH's function could be created.
Y2H
To test whether the change from tyrosine to histidine affects ability of mutant CFH to bind to C3, CFI, and/or CRP, a Y2H will be conducted. The bait will the the mutant CFH and the bait will switch between C3, CFI, and CRP. To ensure that the bait and prey system is working correctly, the positive control will be wild-type CFH, seeing as it is already known that it interacts with C3, CFI, and CRP. The negative control will be a protein that has no interaction with C3, CFI, or CRP.
Expected Results
If the controls run correctly, a few conclusions can be made upon the results of this experiment. If there is no growth, there was not any or was not sufficient interaction between the respective proteins. If growth did occur, the respective proteins did interact. This, however, does not account for the situation in which the interaction has decreased, but is still sufficient for growth. This small change could still have a profound impact within the body. To measure whether the interaction of the proteins changed, the fluorescence of the betagalactosidase activity will be measured and compared between wild-type and mutant CFH.
Future Directions of Research
Several areas should be further explored to better understand CFH and AMD. Out of all the literature that I read, I did not find much about the heritablilty of AMD or CFH. All that I could find was the fact that if you have a family member with AMD, you have an increased risk of developing AMD. Knowing how AMD and CFH are passed down through the family is crucial to treating this disease. There are a few other genetic factors to AMD that have been recently discovered. Certain mutations of CFI and C5 greatly increase your risk of developing AMD. Researching these mutations would be important to fully understand the development of the disease and in turn treating it. A final area to look into is the area of drug development. There is no current cure for AMD, and the only treatments available are for treating the symptoms of wet AMD. There is no treatment for dry AMD. Therefore, identifying drug targets and development of a drug is necessary. Perhaps a replacement for CFH as a regulator of the complement system could be found or a drug that restores CFH's function could be created.
