Utilizing Custom Oligonucleotides to Diagnose Hemophilia A

By Ryan Chandler, Evan Ballard, Emily Martinez, and Chamira Cole

Abstract

This study evaluated the effectiveness of diagnosing Hemophilia A through the use of properly configured primers, DNA extraction, PCR, and gel electrophoresis to detect the Hemophilia A mutation on the FVIII gene on the X chromosome. Additional data was gathered to assess the possibility and prevalence of genetic discrimination in society today. The viability of a genetic test for hemophilia was confirmed by our PCR assay. The mutant DNA presented a band at ~600 base pairs after gel electrophoresis, approximately 300bp shorter than the wild-type DNA, confirming the presence of the hemophilia A mutation. The genetic discrimination data was gathered utilizing a survey that illustrated the general consensus that discrimination is a very possible threat to those with genetic diseases. A genetic test for hemophilia A is not only possible, but feasible based on our results, but the results of such a test could pose a discriminatory risk to patients.

Discussion

Hemophilia A is a genetic disease caused by a mutation on the long arm of the X chromosome (Roosendaal, 2007). The decision to address the missense mutation at amino acid 10 in exon 1 was because it is a lesser-known mutation. When this mutation occurs, amino acid valine is replaced by a glycine because the new codon formed translates to a different amino acid (Markoff, A., V. Gerke, N, Bogdanova. 2009. It is important to know that missense mutations account for 10% of exon related mutations in Hemophilia A patients (Rosetti L. C., C. P. Radic, M. Candela, R. P. Bianco, M. de Tezandos Pinto, A. Goodeve, I. B. Larripa, C. D. De Brasi. 2007).

Testing was done to find out if an accurate diagnosis of Hemophilia A could be obtained through the use of PCR and gel electrophoresis. A genomic DNA extraction protocol was performed to obtain pure DNA, which was amplified with the PCR. Gel electrophoresis was then used to visualize the amplified DNA.

It was then hypothesized, that based on the primer design and a successful PCR, Hemophilia A could be diagnosed. A mutant DNA sample of the FVIII gene could be visualized at ~600bp on an agarose gel when the designed start and mutant primers were used (Figure 1 and 3). Furthermore, a wild-type DNA sample of the same gene could be visualized at ~600bp on the agarose gel when the mutant primer was substituted with the wild-type primer (Figure 1 and 3). Similar studies have been done on this type of mutation, simply on other exons of the gene (Youssoufian, H., S. E. Antonarakis, W. Bell, A. M. Griffin, H. H. Kazizian. 1988).

While the hypothesis was proven to be correct, it was originally predicted that the designed primers would definitely visualize bands at ~600bp with the wild type and start primer as well as the mutant and start. However, the bands did not show due to potential errors made in the research. With earlier gels, non-specific binding occurred due to low annealing temperature. When this was changed to higher annealing temperatures, not only then did bands not show, streaking also occurred. The PCR could be better if the right annealing temperature had been found, as well as doing the PCR sooner as this concern would have been noticed sooner.

The results discussed verify the hypothesis made because one was able to determine whether an unknown DNA sequence had the Factor VIII mutation or whether it displayed the wild type banding pattern. So running a PCR assay is indeed a useful process in determining the presence of the missense mutation in which the group was looking to identify. Also this work can be used to put into practice a more accurate way of testing for and deciphering between hemophilia A and hemophilia B. The current tests cannot specify which mutation a patient has which can affect which treatment is needed and implemented.

The results obtained aligned with additional scientific studies, one of which specialized in comparing the results of blood clot testing and DNA recombination (Higuchi, M., S. E. Antonarakis, L. Kasch, J. Oldenburg, E. Economus-Peterson, K. Olek, M. Arai, H. Inaba, H. H. Kazazian. 1991). The researchers of this article tested two different Hemphilia A patients with two different lesser known mutations. They also designed many of own primers to test against the DNA samples, which is what was done in this research. Using a similar method could have helped increase the chance of getting results because it would have diagnose the patients more easily as there would be two different bands for the mutant primers.

While using PCR is a good choice to diagnose a disease, they are not always reliable. It is not always possible or desirable to rely on PCR because a large number of samples are required to obtain bands in the gel. (Laird, P.W., A. Zijdervald, K. Linders, M. A. Rudnicki, R. Jaenisch, A. Berns. 1991.) Since ~300bp are barely large enough to show bands, a large DNA sample from a patient would be needed as any bp smaller than that will not show bands in a gel.

It was also hypothesized that a diagnosis of Hemophilia A and the disclosure of this information in a non-scientific setting may harbor negative consequences for an individual socially. I.e., an employer may overlook an individual due to increased on-the-job risks or higher medical insurance coverage costs. Looking into potential genetic discrimination was an important aspect of the project because it helps to connect the disease to something outside of the lab. It also strengthened the amount of knowledge the group has obtained about the mutation and the disease itself, teaching others about something they either had some knowledge on or none at all via surveys.

While the HPS project expanded on what the disease means to those in a non-scientific setting, there are some things that could have been improved. I.e., interviewing a person that was discriminated against because of having Hemophilia A. Doing this could have expanded the researchers understanding more of the effects this disease actually does to someone.

Figure 3. Gel electrophoresis of PCR amplified genomic DNA using designed primers. Lanes 1 and 7 contain a 1kb DNA ladder for comparison with the amplified banding patterns obtained. All other lanes contain normal genomic DNA extracted from IB3 cultured cells. Lanes 2 through 6 vary in the primer sets used in the PCR reaction. Lane 2 contains the start primer and control primer, producing a band at about 300bp. Lanes 3 and 4 contain start primer and wild-type primer producing a band at about 600bp. Lanes 5 and 6 contain start and mutant primer, producing no bands.