Familial Hypercholesterolemia

Genotypic Identification of Human ApoB-100 R3500Q Mutation of a Hypercholesterolemia Patient Using S9 Epithelial Cell Lines and Allele Specific PCR

By: Ojie Alofoje, Taylor Moody, Baraa Osman, and Samantha Tauscher

The Jokers

Abstract:

Familial Hypercholesterolemia (FH) is a disease caused by a point mutation on the short arm of chromosome two on the APOB gene.  On the ApoB-100 locus a guanine is substituted into an adenine nucleotide at base number 10708 (Bonow et al, 2012). This genetic defect is crucial because it causes an increase in the low-density lipoprotein (LDL) levels in blood, leading to atherosclerosis (Havel 1984). Using allele-specific polymerase chain reaction (PCR) and gel electrophoresis, we amplified the mutation. Using published and custom primers that contained intentional mismatches to enhance binding specificity, and mutant and purified genomic DNA, results were seen in an aragrose gel.  During PCR, primers anneal to the DNA and replicate a sequence containing the mutation. Because PCR thermocycling will successfully amplify a target region of DNA, bands appeared in the gel that demonstrate a 628 base pair long PCR product of the target sequence (Keinanen,1990). Finally, an electric current was run through the agarose gel to interpret results from PCR. These methods together determine whether the DNA sequence carries the correct mutation causing FH. In performing the PCR assay we accumulated evidence indicating the presence of the mutation through the appearance of bands. (Kyi et al, 2000). The research team’s purpose of this experiment is to understand the specific aspects of PCR, primer design, and gel electrophoresis, in order to practice experimental design and procedure, as well as troubleshooting. Overall, being able to replicate the mutation in a DNA sequence, could make it possible for further research to be conducted on it, possibly resulting in a cure for the disease. To experience social aspects of this disease, each team member exercised daily and maintained low-cholesterol diets for a 30-day period. Also team members educated random citizens in two different locations of East Lansing. Sociologically, the two locations showed comparable opinions regarding the disease and its effects on individuals; also revealing how much different lifestyles take health into consideration in their lives.

Figure 1:

Legend: Gel Electrophoresis Results Using PCR with Mutant DNA and Mutant Published Primers

This is the gel displaying the PCR results for mutant published primers on mutant DNA. All the wells contained 3μl of mutant DNA, 2μl of published mutant forward primer, 2μl of reverse, 2μl of taq polymerase. Well #1 had an annealing temperature of 31⁰C, well #2 had a 33⁰C annealing temperature, well #3 was at 35.3⁰C, well #4 was at 39.6⁰C, and well #5 was at 42⁰C. For all the wells the initial was at 95⁰C for 5 minutes, denatured at 95⁰C for 1 minute, annealed at the specific temperature for 1 minute, elongated at 72⁰C for 2 minutes, and ran at 72⁰C for another 7 minutes for the final extension. This was done for 35 cycles. The 1Kb Plus ladder was used in the 2% agarose gel. The bigger bubbles in the picture is the zip lock bag in which the gel was in under the UV light. The specks in the gel could mean that the gel was refrigerated too fast, going from hot to cold too quickly. The bands near the 100 base pair mean primer dimers were present.  The streaking can mean that the DNA may be degraded or that there was nuclease contamination. The band in well #5 is at the 478 base pairs, which is exactly the length of the published primers target sequence.

Discussion:

Mutations in the APOB gene on chromosome two can lead to hypercholesterolemia by an SNP, a point mutation. This mutation causes a reduced function of the low density lipoprotein (LDL) ligand, which then affects the LDL receptors it binds to. The receptors, responsible for controlling bad cholesterol in the blood, have difficulty removing the LDL from the blood due to its misshape. This ultimately causes familial hypercholesterolemia (Jensen, 2002). The R3500Q mutation is the most common APOB gene mutation leading to familial hypercholesterolemia.

In order for the research group to fully understand the conditions caused by this mutation, a 30-day plan was carried out: the group members added exercise into their daily routines, as well as a diet consisting of very little lipids (Bonow et al, 2012). Along with that, a sociological viewpoint of the genetic disorder was observed. Each researcher replicated conditions of high fat deposits in the body by wearing a fat suit for a day. Researchers tested to see if their anxiety levels increased whilst wearing the fat suit by taking the Social Anxiety Test. A short thirty second awareness movie also was produced and then taken to McDonald’s and IM-West  in East Lansing to survey multiple people of different backgrounds in their knowledge and opinion of Hypercholesterolemia and how it will affect their daily lives. It was found that researchers experienced higher levels of anxiety while wearing the fat suit. Also after issuing the surveys, people surveyed at IM-West tended to care more about their diet and overall cholesterol levels as opposed to people surveyed at McDonald’s who didn’t care much about their cholesterol levels.

It was found that the mutation lies on the 10708 base pair of the ApoB gene due to a guanine to adenine point mutation on R3500Q (Awad et al, 2011). Customized primers were produced using this information as well as the Yaku- Bonczyk method (Yaku et al, 2008). The custom primers, however, used an adapted version of the Yaku- Bonczyk method where two bases between the intentional mismatches, used in the Yaku- Bonczyk method, were designed to anneal to either type of DNA instead of the traditional one base pair. Despite the adapted Yaku-Bonczyk method used, we predicted that mutant primers still would not anneal to wild-type DNA and the wild-type primers still would not anneal to mutant DNA because Taq polymerase will rarely extend DNA sequences with two intentional mismatches. This method would prevent non-specific binding for increased accuracy of our PCR diagnostic assay. The published primers were obtained from (Kyi et al, 2000). We hypothesized that through the use of PCR and the adapted method of the Yaku- Bonczyk method for customized primers, we would genetically amplify the target sequence of 628 base pairs which will contain the R3500Q mutation of the APOB gene that will be supported by the gel electrophoresis analysis to determine its presence in DNA (Kyi et al, 2000).  We predicted our custom mutant primers would accurately amplify the 628 base pair target sequence, and the published primers would amplify the 478 base pair target sequence, which would be supported by gel electrophoresis analysis (Kyi et al, 2000). The presence would be prominent by a visible band on the gel, indicating that the specific mutant primer sequence is present and replicated. A gel of the lambda DNA PCR products, illustrates how successful our gel protocol was (Figure 1A). The same method, including the same concentration/ratio of reagents for PCR cocktails,  was used in hopes of obtaining visible bands at 628 base pairs for hypercholesterolemia mutant DNA run with custom designed primers.

We based our predictions off of a previously conducted experiment run in 1990, where the lab group conducted PCR and gel electrophoresis to determine heterozygous familial hypercholesterolemia (Keinanen, 1990). In the analysis of their gel electrophoresis, bands indicated the presence of the amplified target sequence. Our results, however,  refuted our hypothesis because we failed to accurately pinpoint the R3500Q mutation. Firstly, PCR reactions using published control primers were somewhat successful in amplifying DNA. A gel that analyzed a PCR reaction using mutant hypercholesterolemic DNA, obtained from the Cardiovascular Genetics Laboratory in Royal Perth hospital located in Perth, Australia, paired with published mutant and reverse primers showed a band that was approximately 480 base pairs long which indicated successful annealing, extension, and amplification of published mutant primers to mutant hypercholesterolemic DNA using an annealing temperature of 42°C (Figure 3). With this successful amplification, the presence of our mutant DNA was confirmed. However, PCR reactions using S9 epithelial cells, purified and used as wild-type DNA, paired with published wild-type primers demonstrated unsuccessful amplification of the targeted region. This suggests that there was non-specific binding as the published wild-type primer did not anneal and extend along the wild-type DNA. Upon conducting many failed attempts to successfully amplify the target region using the published wild-type primer with wild-type DNA using different temperatures and concentrations of PCR reagents, we proceeded to try using our custom wild-type primer with wild-type DNA. This resulted in non-specific annealing where the custom mutant primer failed to anneal to and extend along the wild-type DNA, most likely due to incorrect annealing temperatures.

Both custom mutant and wild-type primers ran with wild-type DNA yielded no results or bands on the gel. This means there was unsuccessful amplification of the targeted region using custom designed primers. However, the unsuccessful annealing of the custom mutant primer with the wild-type DNA resulted in  negative control for the mutant primer. The designed wild type primer failed to anneal and extend along wild-type DNA. With this, our hypothesis was not supported. However, the negative control for the custom mutant primer not annealing to the wild-type DNA partially supported the hypothesis.

Both custom mutant and wild-type primers ran with R3500Q mutant DNA yielded no results as well. This means there was non-specific annealing and unsuccessful amplification of the 628 base pair target region. The unsuccessful annealing of the custom wild-type primer with the mutant DNA resulted in a negative control for the custom wild-type primer. This provides small support for the hypothesis, but given the fact that the custom mutant primer did not anneal to and extend along the mutant DNA after many attempts, our hypothesis cannot be fully supported.

Some weaknesses in our experiment include: failure to create a custom primer set targeting the mutation, non-specific annealing and incorrect annealing temperatures, or incorrect amounts/concentrations of ingredients for the PCR solution. Since our custom primers did not target the mutation, the PCR failed to amplify the specific genomic sequences needed to support the presence of the mutation. If the denaturing temperature is not correct, the DNA strands may not pull apart enough for the primers to bind to the DNA, and if the annealing temperature is incorrect, the primers may not be activated to bind to the separated DNA strands. Finally, if the PRC reagents of the PCR solution are in disproportional amounts, the temperatures necessary to denature the DNA could be different, possibly melting the cocktail or not heating it up enough for denaturation to occur. If our custom primers fail to result in the replication of 628 base pairs we will create new primers containing more guanine and cytosine base pairs in order to have a more stable annealing temperature. If the DNA PCR solution fails to contain the proper amount of components then we will more closely resemble the published PCR solution used in previous experiments on the APOB gene.

For future directions, we would troubleshoot each and every unsuccessful assay and propose another procedure to resolve the problems faced. The unsuccessful amplification of the control published primers, excluding the published mutant primer, obtained from Kyi’s paper (Kyi et al, 2000) could have been a result of using low annealing temperatures. Increasing the annealing temperatures for the PCR assay using the published primers could increase binding specificity, thus resulting in positive results when analyzing a gel. In one gel where we tested published mutant primers with mutant DNA, fuzzy bands under 100 base pairs were noticed. Another gel where we tested custom and published wild-type primers with wild-type (genomic) DNA shows the same fuzzy bands at around 100 base pairs (Figures I & H). These fuzzy bands could suggest primer dimers, where primers anneal to and extend along each other. To troubleshoot this problem in future experiments, higher annealing temperatures would increase binding specificity. This should allow primers to only anneal to and extend along the targeted region yielding in positive results. The unsuccessful amplification of the custom mutant primers could have been a result of using high annealing temperatures because the custom primers used were shorter than most primers and require a lower annealing temperature to increase binding specificity. One other reason as to why the custom primers yielded no positive results could be because of the adapted Yaku-Bonczyk primer design method used in designing the custom primers. We hypothesized that the adapted method of the Yaku-Bonczyk would yield results like the normal Yaku-Bonczyk method would. According to the results we gathered, this refuted our hypothesis as we did not get positive results using our primer set. In future experiments, we would design our primers using the normal Yaku-Bonczyk method by allowing only one base pair, between the intentional mismatches, to anneal to both types of DNA, mutant and wild-type.

Sociological Movie: http://youtu.be/7Xty2m2kFIE