This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison
Future Directions
One of the most surprising findings I have made while reviewing the literature is the fact that FH heterozygotes are underdiagnosed (1). Although this is most likely due to the prevalence of high cholesterol, obesity, and subsequent heart disease throughout the developed world, having a genetic disorder that further predisposes an individual for atherosclerosis should not be underdiagnosed. Therefore, I propose to develop a genetic screen that can recognize the FH phenotype, without having to sequence the LDLR gene. I feel that performing microarray assays on a large number of individuals with varying degrees of the FH phenotype could allow us to discover gene expression patterns that would increase FH heterozygote diagnosis. If an acceptable screen can be established, individuals with a history of heart disease or even all individuals could be tested at an early age for FH. This could allow people who are shown to have the FH gene expression pattern to be put on drug treatments, like statins, and eat low cholesterol diets in order to curb their predisposition to atherosclerosis. It has been shown that FH heterozygotes are able to achieve normal LDL levels with a restricted diet and statin treatment (2).
In order to figure out an acceptable gene expression profile for diagnosing FH, numerous microarray assays need to be carried out on multiple individuals. Ideally, a pool of normal individuals with preferably no family history of heart disease or high LDL cholesterol will be used for comparison to individuals with both homozygous and heterozygous forms of FH. Also, the FH suffers should have varying degrees of LDLR dysfunction to get the full gene expression spectra. Although an eventual genetic screen will be more beneficial to FH heterozygotes, homozygotes need to be included in the microarrays because their gene expression profiles will be more constant. This is because homozygotes have very little, if any, LDLR function, meaning their cholesterol levels are much more genetically dependent. The FH heterozygote phenotype is much more dependent on environmental factors, which lead to a wider range of LDL cholesterol levels (2). Therefore, homozygous individuals with mutations that cause complete LDLR dysfunction would provide the best way to find out which genes are most differentially expressed in FH.
Also, multiple cell types need to be compared in order to gain the best expression profile. Perhaps certain cells give more consistent expression patterns, making diagnosis easier. I would suggest beginning with hepatocyte (liver) cell microarrays, since 70% of LDL clearance is done by these cells and LDLR is highly expressed (2). The only published study on FH gene expression using microarrays used monocytes for the comparison. The researchers found that the SREBF proteins were downregulated, indicating high levels of intracellular cholesterol (3). It is well known that atherosclerosis plaques are, in part, caused by the accumulation of foam cells, monocytes with high levels of intracellular cholesterol. The researchers found that this occurs in FH individuals as well, indicating other ways for LDL to penetrate monocytes (3). I feel that hepatocytes would tend to have upregulation of SREBF proteins in FH individuals, assuming the main pathway of LDL internalization in these cells is through LDLR. This is because if no functional LDLR is produced, as with some homozygous FH individuals, there would only be low basal levels of cholesterol from the cholesterol biosynthesis pathway, which would induce SREBF protein expression. There are most likely other cells that would also have different expression pathways, supporting my reasoning for assaying multiple cell types.
Also, multiple cell types need to be compared in order to gain the best expression profile. Perhaps certain cells give more consistent expression patterns, making diagnosis easier. I would suggest beginning with hepatocyte (liver) cell microarrays, since 70% of LDL clearance is done by these cells and LDLR is highly expressed (2). The only published study on FH gene expression using microarrays used monocytes for the comparison. The researchers found that the SREBF proteins were downregulated, indicating high levels of intracellular cholesterol (3). It is well known that atherosclerosis plaques are, in part, caused by the accumulation of foam cells, monocytes with high levels of intracellular cholesterol. The researchers found that this occurs in FH individuals as well, indicating other ways for LDL to penetrate monocytes (3). I feel that hepatocytes would tend to have upregulation of SREBF proteins in FH individuals, assuming the main pathway of LDL internalization in these cells is through LDLR. This is because if no functional LDLR is produced, as with some homozygous FH individuals, there would only be low basal levels of cholesterol from the cholesterol biosynthesis pathway, which would induce SREBF protein expression. There are most likely other cells that would also have different expression pathways, supporting my reasoning for assaying multiple cell types.
Hopefully, if enough combinations of microarrays are performed, a simple and reproducible expression pattern can be established for diagnosing FH. This type of diagnosis is definitely a reality in the near future as microarrays become more precise and cost effective.
Other Proposals
I also feel that there needs to be better treatment options for FH homozygotes. The most popular treatment, LDL apheresis, is expensive and requires multiple treatments per month. Unfortunately, this only slows atherosclerosis and death still occurs prematurely (2). Gene therapy techniques showed initial promise in the early 1990s, as reviewed in the science article, however the technique is fairly invasive and is not efficient enough to rectify the surgery. A recent study has been shown to provide functional LDLR for up to 9 months in a mouse model (4). This therapy is very exciting and could one day lead to a substitute to LDL apheresis. However, there still needs to be more testing done on animal models along with increased vector efficiency in order for a safe human treatment to be implemented. I think that gene therapy is probably one of the only ways in which FH homozygotes will effectively be cured of their disease. There has been relatively little gene therapy research done on FH and it could really prove to be very useful if more research is done.
Finally, it has been shown that viruses, like rhinovirus and the hepatitis C virus, utilize LDLR in order to infect cells (5, 6). I propose that experiments should be done comparing viral infection efficiency between FH and normal individuals. I believe that normal individuals will be more susceptible to viral infection compared to both heterozygous and homozygous FH individuals. Through this research we could gain valuable knowledge on viral infection, which could lead to new viral treatments.
References
1. Neil, H. A., Hammond, T., Huxley, R., Matthews, D. R., Humphries, S. E. (2000). Extent of underdiagnosis of familial hypercholesterolaemia in routine practice: prospective registry study. British Medical Journal, 321(7254), 148. PMID: 10894692
2. Rader, D. J., Cohen, J., & Hobbs, H. H. (2003). Monogenic hypercholesterolemia: new insights in pathogenesis and treatment. The Journal of Clinical Investigation, 111(12), 1795–1803. doi: 10.1172/JCI200318925
3. Mosig, S., Rennert, K. Buttner, P., Krause, S., Lutjohann, D., Soufl, M., Heller, R., Funke, H. (2008). Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism. BMC Medical Genomics, 1, 60. doi:10.1186/1755-8794-1-60
4. Hibbitt, O. C., McNeil, E., Lufino, M. M., Seymour, L., Channon, K. Wade-Martins, R. (2010). Long-term physiologically regulated expression of the low-density lipoprotein receptor in vivo using genomic DNA mini-gene constructs. Molecular Therapy, 18(2), 317-26. doi:10.1038/mt.2009.249
5. Mas Marques, A., Mueller, T., Welke, J., Taube, S., Sarrazin, C., Wiese, M., Halangk, J., Witt, H., Ahlenstiel, G., Spengler, U., Goebel, U., Schott, E., Weich, V., Schlosser, B., Wasmuth, H. E., Lammert, F., Berg, T., Schrerier, E. (2009). Low-density lipoprotein receptor variants are associated with spontaneous and treatment-induced recovery from hepatitis C virus infection. Infection, Genetics and Evolution, 9(5), 847-52. PMID: 19446659
6. Konecsni, T., Berka, U., Pickl-Herk, A., Bilek, G., Khan, A. G., Gajdzig, L., Fuchs, R., Blaas, D. (2009). Low pH-triggered beta-propeller switch of the low-density lipoprotein receptor assists rhinovirus infection. Journal of Virology, 83(21), 10922-30. doi:10.1128/JVI.01312-09
2. Rader, D. J., Cohen, J., & Hobbs, H. H. (2003). Monogenic hypercholesterolemia: new insights in pathogenesis and treatment. The Journal of Clinical Investigation, 111(12), 1795–1803. doi: 10.1172/JCI200318925
3. Mosig, S., Rennert, K. Buttner, P., Krause, S., Lutjohann, D., Soufl, M., Heller, R., Funke, H. (2008). Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism. BMC Medical Genomics, 1, 60. doi:10.1186/1755-8794-1-60
4. Hibbitt, O. C., McNeil, E., Lufino, M. M., Seymour, L., Channon, K. Wade-Martins, R. (2010). Long-term physiologically regulated expression of the low-density lipoprotein receptor in vivo using genomic DNA mini-gene constructs. Molecular Therapy, 18(2), 317-26. doi:10.1038/mt.2009.249
5. Mas Marques, A., Mueller, T., Welke, J., Taube, S., Sarrazin, C., Wiese, M., Halangk, J., Witt, H., Ahlenstiel, G., Spengler, U., Goebel, U., Schott, E., Weich, V., Schlosser, B., Wasmuth, H. E., Lammert, F., Berg, T., Schrerier, E. (2009). Low-density lipoprotein receptor variants are associated with spontaneous and treatment-induced recovery from hepatitis C virus infection. Infection, Genetics and Evolution, 9(5), 847-52. PMID: 19446659
6. Konecsni, T., Berka, U., Pickl-Herk, A., Bilek, G., Khan, A. G., Gajdzig, L., Fuchs, R., Blaas, D. (2009). Low pH-triggered beta-propeller switch of the low-density lipoprotein receptor assists rhinovirus infection. Journal of Virology, 83(21), 10922-30. doi:10.1128/JVI.01312-09
