Identification of C282Y Mutation on HFE gene of CALU-3 DNA by Allele Specific PCR and Gel Electrophoresis

By: Nicole Hanley, Paul Kelly, Raef Fadel, and Samantha Voorhorst

Abstract:

The C282Y mutation of the HFe gene accounts for 80% of Hereditary Hemochromatosis cases (Drakesmith and Townsend, 2000). This single base pair mutation involves a guanine-to-adenine substitution at nucleotide 845. This alters the protein sequence by substituting an amino acid cysteine for a tyrosine at position 282 of the protein product (Hanson et al, 2001). We used three uniquely designed primers in allele-specific PCR with gel electrophoresis analysis to determine whether desired strands of HFe DNA, ranging from nucleotides 4691 – 5710, contain the C282Y mutation. This specific sequence (nucleotides 4691 – 5710) is believed to contain the C282Y mutation (Drakesmith and Townsend, 2000). Two unique primers were constructed with intentional mismatches on the 3’ end and were coupled with a third unique primer to conduct allele-specific PCR on human DNA positive for the C282Y mutation, heterozygous for the C282Y mutation, and wild type human DNA without the C282Y mutation. We then ran gel electrophoresis analysis on the PCR products to determine whether the primers annealed at the correct base pair length supporting the presence or absence of the C282Y mutation with a band at approximately 1,020 base pairs. Samples of human wild-type DNA for C282Y mutation were acquired through the DNA purification of CALU-3 cells and through contact with the Life Sciences Building of Michigan State University both positive and carrier DNA for the mutation were obtained. We hypothesized that reverse primers 3' - cgg tcc acc tcg tgg - 5' and 3' - tgg tcc acc tcg tgg g - 5', along with forward primer 5' - ggt ggc tca ccc ctg - 3' would successfully identify the presence or absence of the C282Y mutation, due to an intentional base pair mismatch on the 3’ end of the reverse primers which will allow only the complimentary primers to anneal to the DNA, thermocycler temperatures ideally set to match the annealing temperatures of the primers, and agarose gel analysis of the AS-PCR product to visualize presence or absence of desired bands (Donohoe et al, 2000; Wu et al, 1989). The use of either sense strand primers Rprimer1 or Rprimer2, with anti-sense primer Fprimer, resulted in the failure to amplify a region of approximately 1,020 base pairs in length. In addition to primer design, we conducted a sociological experiment in order to better our understanding of Hemochromatosis, and to assess the general knowledge and awareness of university students about HH. A 5 question questionnaire was distributed to various halls and buildings across Michigan State University campus. Each student who took the assessment was placed in one of 4 categories depending on his/her major (Science, Math, Business, Fine Arts). The performance on the assessment (% correct out of 100) was plotted against number of students who received that score on the questionnaire. ANOVA statistical analysis was used to compare each major’s mean score on the assessment, to compare which major is generally more knowledgeable than the other 3. A p-value of 0.418048 was obtained, indicating no significant difference in knowledge of the disease among different majors in the university. However, an average of 25% on the assessment indicates that much more effort must be put into educating the youth on prevelant disorders such as Hereditary Hemochromatosis, allowing youth to more quickly recognize and diagnose the disease, leading to much more rapid treatment (Beutler et al, 2002).

Figure 3: Mutant primers with mutant DNA at varying annealing temperatures

Lanes 1 and 8 are 1Kb ladders which are used to provide a reference point for the amplified mutant Hemochromatosis DNA at base pair 1020. As seen by the presence of these bands it is evident that there is specific product rather than primer dimers, because primer dimers would be way lower along the gel. Lane 3 is lower along the ladder because PCR is not a perfect science and therefore there may be some discretion when running the same thing more than once. This lower band could be due to too much DNA placed into the well, or not enough MgCl2. Temperatures 50-55 degrees provide much higher intensity bands, showing that the annealing temperature should be closer to that rather than 45 degrees as seen in lanes 2 and 3. The bands throughout the whole gel are not super clear, showing possible nonspecific binding, providing evidence that the mutant DNA needs to run at a higher annealing temperature.

Discussion:

Experimental Summary

Hereditary Hemochromatosis is the most common autosomal recessive genetic disorder in the northern hemisphere with a prevalence of approximately 1/200 individuals of Northern European decent (Donohoe et al, 2000; Pietrangelo, 2006). HH is a hereditary iron loading disorder, caused by a genetically determined failure to prevent unneeded iron from entering the circulatory pool on every level, from the intestines, to the liver, to the bone marrow, and back through the blood stream (Anderson et al, 2009). The most common mutation causing HH is the C282Y mutation of the HFe gene on the short arm of chromosome 6, in which a guanine-to-adenine substitution at nucleotide 845 of the HFE gene (845GuanineàAdenine) which produces a substitution of amino acid cysteine for a tyrosine at position 282 of the protein product (Hanson et al, 2001). This substitution causes a malfunction in the binding of the protein complex of the HFe protein (Bennett et al, 2000). When the cysteine amino acid at position 282 of the HFE gene is lost, the disulfide bond that structures the alpha-3 domain cannot be formed, and the HFE protein is no longer able to complex with the beta₂-microglobulin. This results in the degradation of the mutant protein before it is properly incorporated into the cell membrane (Drakesmith and Townsend, 2000). Although PCR has proven effective for diagnosing genetic disorders such as Hereditary Hemochromatosis, the question we are addressing is whether or not a polymerase chain reaction (PCR) can be designed to specifically identify the C282Y mutation (Olynyk et al, 1999).

Three primers were used to conduct the polymerase chain reaction of the HFe DNA: Mutant primer, Wild type primer, and fprimer. The reverse primers will anneal to the sense strand of the HFe gene, and the fprimer will anneal to the antisense strand approximately 1020 base pairs away from the annealing site of the reverse primers, amplifying a region of approximately 1,020 base pairs in length (Wu et al, 1989). Rprimer1, with a nucleotide sequence of 5’-GGG TGC TCC ACC TGG T-3’, and an annealing temperature of 51.0°C, will anneal to the sense strand at nucleotides 5695 to 5710 if the C282Y mutation is present, in which case the mismatch will be present and the mutation located at the 3’ end of the primer will bind rather than annealing if the mutation is not present. Rprimer2, with a nucleotide sequence of 5’-GGT GCT CCA CCT GGC-3’, and an annealing temperature of 50.14°C, will anneal to the sense strand at nucleotides 5695-5709 if the mutation is not present. Fprimer1, with a nucleotide sequence of 5’-GGT GGC TCA CCC CTG-3’, and annealing temperature of 50.14°C, will anneal to the anti-sense strand of the HFe gene sequence approximately 1,020 base pairs away from reverse primers at nucleotides 4691-4705.

The single base pair mismatch between the two primers (rprimer1 and rprimer2) will allow for the allele specific PCR to be conducted, with rprimer2 annealing to a wild-type genotype and rprimer1 annealing to a mutant genotype due to the C282Y mutation (Hanson et al, 2001). By using the E. Coli DNA in our first PCR sample, it can be used as a positive control that the PCR machine has worked initially, as well as a positive control for gel electrophoresis because the band showed up at the given 500 base pairs where it was expected. This cannot act as the positive control of the AS-PCR of HFE gene because E. Coli PCR cocktail was not placed in every PCR machine every time a cocktail was run (Wu et al, 1989). Also a negative control will be used in which no DNA will be incorporated into the gel wells whatsoever. If after the gel has run there are no bands and no illuminated light in the wells, it supports our claim of a negative control. This also can only be used as a negative control for the first PCR test, but it can be used for a negative control for gel electrophoresis for the experiment as a whole (Wu et al, 1989).

The products of PCR were run and analyzed using gel electrophoresis. For both the wild types as well as the mutant each were run through a TBE gel using TBE buffer at 106 volts. The successful amplification of a sequence would have been at 1,020 base pairs in length using rprimer1 and fprimer1 will indicate the presence of the C282Y mutation at nucleotide 5695 (GàA). Conversely, the successful amplification of a sequence 1,020 base pairs in length using rprimer2 and fprimer1 will indicate the absence of the C282Y mutation at nucleotide 5695 (Wu et al, 1989). Despite designing primers that were said to amplify the given regions, the PCR did not amplify the desired section. The bands were too short to support our hypothesis, yet too long to be primer dimer. This could have been due to the design of the primer itself or also an array of other factors. The biggest factor that we analyzed during the PCR process was the annealing temperature (Wu et al, 1991). The sequences and annealing temperatures of the primers were designed and calculated using a modified version of the “rule of thumb” equation for annealing temperatures, and the LB145 Laboratory Guide section “Primer Design” for primer design (See Methods). Though the calculated temperatures were all around 51°C, we decided to start at 45°C in order to make sure that primers do not melt (Wu et al, 1991).

We hypothesize that with successful purification and extraction of human HFe DNA, accurate determination of the annealing temperatures of allele-specific primers, proper application of AS-PCR on human HFE gene, and successful PCR product analysis by gel electrophoresis, AS-PCR techniques could be coupled with gel electrophoresis analysis to successfully determine whether the C282Y mutation is present, or absent, on the HFE gene (Donohoe et al, 2000; Wu et al, 1989).

In addition to primer design, we conducted a sociological experiment in order to better our understanding of HH, along with the harsh reality of a patient diagnosed with the disease. Our experiment consisted of 2 members of our research team coating themselves with L’OREAL^tm Sublime Bronze Self Tanning Lotion in order to portray one of the most prevalent symptoms of the disease, bronze diabetes. They attended lectures within the Lyman Briggs Residential College of Michigan State University for two weeks, in order to gain perspective of the life in day of someone with the disease. Attending these lectures will allow the researchers to conduct their experiment amidst a more educated group of students, in hopes of attracting more controversy and attention to the “symptoms” of the disorder. After sitting in on lectures, a survey was dispersed in order to gain knowledge of how the students felt. Questions, such as, “Does it concern you that HH is the most common single nucleotide mutation among Caucasian Americans (1/200)?” were asked in order to present them with a statistic as well as understand if they realize the severity of the disease. By doing this part of the experiment it was apparent that people were staring and looking at us if we looked out of the ordinary. It made both of us understand how it may feel to actually have the disease and have to suffer in society, but also realize that there is pain that we can never understand such as testing our blood sugar and changing our diet for the rest of our lives. The other part of the 30 day DIY consisted of two members dispersing surveys to across campus, primarily in specific college buildings. The survey consisted of questions such as, “What is Hemochromatosis?” and “What are symptoms of HH?” These questions were aimed in order to test the knowledge of the campus about how prevalent the disease is and how severe it is if not treated. After collecting the surveys and scoring them an ANOVA was run across the groups of majors resulting in a p-value of 0.418048, meaning that there is not any significance across all the majors. In addition, p-values were obtained across grade levels in each major were all greater than the optimal .05, meaning that there is not significance or greater knowledge as students gain seniority. The average on the surveys was a 20% which is very low for such a prevalent disease in America. After calculating the p-value it is understood that there is not a great understanding of the disease and that there needs to be awareness in order to have people get tested so they can alter their lifestyles and prevent the future severity (Hanson et al, 2001). This sociological experiment will allow us to examine common university students’ awareness to the disorder, determining whether younger generations truly understand the prevalence and the complications that arise from this disorder.

Original Predictions and Ultimate Findings

We predicted a successful purification of the acquired HFe gene samples, providing a suitable HFe gene sequence to conduct the PCR test. The successful extraction and purification of the acquired DNA will provide the template DNA for which the designed primers will anneal upon (Takeuchi et al, 1997). After running gel electrophoresis with the supplied CalU-3 cells we were able to amplify product around the expected 1020 base pairs but there was an excess of non-specific binding so the precision of the band is not exact. The main way to help non-specific binding is to change annealing temperature, and this was supported by when we changed our annealing temperatures we went from having no bands, to non-specific binding making us believe that we were moving in the right direction.

We predicted that we will successfully amplify the desired region of the HFe gene DNA sequence (nucleotides 4691-5710) and this amplified DNA will be interpreted using gel electrophoresis to show the presence or absence of the C282Y mutation (Wu et al, 1989). By using the mutant primer as well as the forward primer and proper annealing temperatures will give us successful amplification. However during our research we were unable to successfully amplify bands for the mutant DNA around the expected base pair length. This can be caused due to the set annealing temperatures that were run with PCR (Wu et al, 1991). PCR is not an exact science, because we have run the same products multiple times and have received different results with the same PCR product.

Future Research

Despite its proven effectiveness in diagnosing patients with HH, the question we hope to address is whether or not the polymerase chain reaction (PCR) test can be applied specifically to the Cysteine282Tyrosine mutation, to not only diagnose those with HH, but to pin point the exact nature and location of the mutation (Olynyk et al, 1999). If PCR is successfully conducted on HFe DNA samples, targeting the C282Y mutation, a more distinctive understanding of the mutation, its origin, and its effects could be developed. Treating patients with HH is simple, affordable and efficient, and with the successful application of the polymerase chain reaction test to HFe genes with the C282Y mutation, a better understanding of the mutation along with more effective and cost efficient treatments for patients with HH could arise (Hanson et al, 2001).

In our findings throughout the semester, we were still unable to get bands at our desired location given out primers that were created. Some things that can be altered with keeping the same primers are the salt concentration. When the salt is increased in the solution it increases the clarity of the bands and preserves the DNA (Wu et al, 1991). However it is necessary that the temperature needs to be increase otherwise there will be a lot of non-specific binding (Roux, 2009). Since the primers came with salt already in them, and we also added salt to the PCR cocktail, we may have needed to start the annealing temperatures higher than 45°C.

During our research we initially ran PCR with E. Coli in order to test the PCR machines as well as gel electrophoresis to make sure that they were working properly. We expected to use this as a positive control; however since they were not run in every cocktail they are not technically a positive control. It goes for the negative control as well, we intended on not putting DNA in the cocktail and not expecting band; however since we only ran it once, it is not acceptable to use as a control through the whole project thought PCR, but rather just gel electrophoresis and PCR for the initial E. Coli results.

If the project could be extended for an addition six weeks there would be major things that would be changed. The first priority would be a change in our research would be the primer design. In our experiment we had a single nucleotide mismatch primer but for further research we would design Yaku primers due to the greater chance that they will anneal given the point mutation in our disease located at the 3’ end (Yaku et al, 2008). Since we had evidence of some binding the primers are the biggest concern, which coincides with the annealing temperature.