Matthew Cable-Fabiszak and Dr. John Kauwe, Biology Department
Review of Purpose of Project
The planned objective of this mentored research opportunity was to identify families in the Cache County Aging Study that harbored mutations in three specific genes known to cause familial Alzheimer’s disease: APP, PS1, and PS2. This knowledge would have been beneficial because it would have provided important information concerning novel variation that causes disease as well as identify specific families who could be important resources for future studies of Alzheimer’s disease.
As will be discussed in further detail, experiment design was altered to focus on a different gene: ABCA7. ABCA7 is another gene with known mutations that correlate with familial Alzheimer’s disease and late-onset Alzheimer’s disease. Instead of observing known mutations in samples to provide information on specific families, over 500 individuals had a specific portion of their ABCA7 gene sequenced in effort to find novel variants that altered protein function.
Review of Importance of Project
Alzheimer’s disease (AD) is a major disease in the current population. In 2000, there were 4.5 million people diagnosed with AD in the US. Due to rapid growth of the oldest age groups in the US population, in 2050 that number will almost triple to 13.2 million. In short, the number of people with AD will continue to rise unless research leads to the prevention of AD.1 As AD patients live from 8 to 20 years after onset of illness, both emotional and fiscal costs are high. In fact, it was estimated that the direct and indirect cost of caring for people with AD was more than 100 billion dollars in 1994 alone.2 Research leading to a 5 year delay in the onset of AD is suggested to cut AD cases in half in just one generation.3 Thus, genetic and environmental factors contributing to the development of AD need to be identified and utilized as diagnostic mechanisms.
Alterations to Planned Experiments
As discussed above, experiment design did change from the original ORCA grant proposal. For several months, our collaborate researchers at Utah State University were undergoing discussions with our lab to decide which lab would have the privilege of sequencing the genome of the twelve individuals referenced in the proposal. At the time of writing the proposal, it appeared that our lab would spearhead the sequencing of these individuals. After several months, however, our collaborators discussed their desire to perform this work, and we reached a standstill. Having already been awarded an ORCA grant, Dr. Kauwe and I agreed to change experiment design and proceed to identification of novel mutations that altered protein structure in different individuals. Our experiment design greatly changed in regards to number of individuals, their relationship, and the specific gene region to be targeted, but all other methods except primer design were virtually unchanged (PCR, cleaning, and sequencing protocol).
Because common variants at ABCA7 are associated with AD4, we decided to use power of so many DNA samples from the Cache County Aging Study to try and find novel mutations in this gene. Successful primer design was achieved after accessing NCBI’s reference sequence for the ABCA7 gene. After studying the region, we used Primer3Plus and our own intuition to design appropriate primers for the region to be sequenced in the 500 individuals. In order to test the primer sets, Polymerase Chain Reaction (PCR) was first completed with a DNA control already in use in the Kauwe Lab, and agarose gel electrophoresis was used to determine PCR’s success. PCR was also used to amplify the region at question.
Successful PCR products were cleaned, underwent Big Dye Sequencing Reaction, were filtered by Sephadex and then sent to the BYU Sequencing Center (DNASC). Upon arrival of data from DNASC, Sequencher 4.8 was used for analysis to determine significant genetic variants. Bioinformatic software such as PolyPhen, Pupasuite 3 and SNPSeek were used to generate hypothesis concerning which genetic variants were involved in the function of the genes.
Results
Without differentiating between introns and exons (exons being the regions that end up directly coding for a specific protein) we found 9 novel mutations in the sequenced region of our samples. However, upon differentiating between introns and exons, two mutations remained. After analysis with the above mentioned bioinformatics software, one mutation was found to alter protein function. Because this mutation was found in only one sample, however, we could conclude that it did not significantly correlate with the population and development of AD. Future implementations of our experiment design should be used with the Cache County Aging Study samples to continue to sequence further regions of ABCA7, as well as MS4A6A/MS4A4E, EPHA1, CD33 and CD2AP.
Scholarly Sources
- Herbert LE, Scherr PA, Bienias JL, Bennet DA, and Evans DA (2003). Alzheimer disease in the US population: prevalence estimates using the 2000 census. Archives of Neurology 60, 1119-22.
- Ernst RL and Hay JW (1994). The U.S. economic and social costs of Alzheimer’s disease revisited. American Journal of Public Health 84, 1261-4.
- Brookmeyer R, Gray S, and Kawas C (1998). Projections of Alzheimer’s disease in the United States and the public health impact of delaying disease onset. American Journal of Public Health 88, 1337-42.
- Hollingworth P, Harold D, Sims R, et al. (2011). Common variants at ABCA7, MS4A6A/MS4A4E, EPHA1, CD33 and CD2AP are associated with Alzheimer’s disease. Nature Genetics 803, 10.1038.