The R1141X mutation of ATP binding cassette sub-family C gene (ABCC6) is associated with Pseudoxanthoma Elasticum (PXE) (Bergen et al., 2000). A stop codon is generated in the 1141 amino acid caused by a C>T point mutation at the 3421 position on genomic DNA (Bergen et al., 2000). To determine whether the R1141X mutation was present, polymerase chain reaction (PCR) was applied to amplify target DNA sequences by running rapid heating cycles to replicate millions of strands of specific DNA (Schochetman et al., 1988). The sample DNA obtained was wild type, and was isolated and purified using the Generation Capture Column Handbook. An annealing temperature of 60°C was calculated for the PCR tests. We hypothesized that the PCR assay would serve as a genetic test to diagnose or clear a patient for PXE because either the DPXE or DWTF primer would anneal to the R1141X mutation or wild-type DNA respectively and produce an 894 bp fragment in gel electrophoresis, as shown in Figure 1. The RAND 36-Item Health Survey was used to evaluate the affect that PXE would have on a patient both physically and emotionally. The survey was used to determine overall functionality of an individual with PXE (Hays, 1993). Two members of the group took the survey while simulating the effects of PXE. This was achieved by wearing self-adhering bandages to simulate inelastic skin, wearing nylon-modified sunglasses to simulate vision loss, and the use of a random time-of-day generator to determine when the patient would express stroke-like symptoms for one hour. Through the experiment we found that PXE has a significant negative effect on physical functioning and general health of the patient but little effect on emotional/mental functioning. We found that a PCR assay performed using our designed primers will not yet serve as a functional genetic test for PXE. Also, we found that the symptoms of PXE have a significant negative impact on physiological and psychological functioning of the patient.
Experiment Summary:
Pseudoxanthoma elasticum (PXE) is an autosomal-recessive disorder (Nitschke et al, 2012), that primarily affects the eye, skin, and cardiovascular system and is generally defined as the calcification of the elastic fibers in these organs (Miksch et al, 2005). PXE is the result of a C?T (cytosine-to-thymine) single nucleotide polymorphism (SNP) at position 3421 on exon 24 of the ABCC6 gene, resulting in an Arg-to-Ter substitution known as the R1141X nonsense mutation (Le Saux, 2000). The R1141X mutation truncates ABCC6 mRNA rendering the protein non-functional. Le Saux speculated that ABCC6 deficiency perpetuates circulation metabolite imbalance, impairing the synthetic ability of elastoblasts to assemble elastic fibers (Le Saux et al, 2006). To determine if our hypothesis is supported or refuted, a PCR test will be run using our designed primers. We hypothesize that our PCR assay will diagnose or clear a patient for PXE because the DPXE or DWTF primer will anneal to the R1141X mutation or wild-type DNA respectively and produce a 894 bp fragment in gel electrophoresis (Ganguly et al., 1993).
In order to gather data, we have recorded the public’s response to the appearance of someone with PXE by performing a sociological experiment that includes reenacting the life of a PXE patient. To experience the life of a PXE patient, we loosely wrapped our neck, elbows, and knees with self-adhering bandage wrap to simulate loss of skin elasticity and excessive skin flaps, wore a modified pair of sunglasses to simulate progressive loss of vision, and limited right body functionality to simulate a stroke induced by vascular disease (Köblös et al., 2010). To quantify our physical and psychological functioning data, the pseudo patient took the RAND 36-Item health survey each week. This survey uses 36 items and 8 concepts to help identify overall health & perceived change in health (Hays et al., 1993). A standard vision test was also performed each week to quantify progressive vision loss. We are predicting that perceived change in health and overall (mental and physical) health will not be greatly affected by PXE due to its minimal physical effects on day to day life. (Roach et al., 2004).
Original Predictions
We will be testing our designed primers against DNA cells containing the R1141X mutation on the ABCC6 gene against wild-type DNA cells obtained from an unaffected patient. To seek the R1141X containing DNA, we have designed two primers: DPXE and DWTF. These primers were designed and calculated to anneal at 60°C and be run over 30 PCR cycles. The DPXE primer is designed to anneal to the R1141X mutated DNA, and the DWTF is deliberately created to anneal to wild-type DNA and therefore will not bind to the R1141X mutated DNA.
We used the Yaku et al. primer method to design our primers. We predicted that these primers will be successful in locating the base pair mismatch on the mutated ABCC6 gene by amplifying the R1141X mutation in the DNA due to the higher accuracy of discrimination against non-complementary DNA (Yaku et al., 2008). We will be able to determine whether the R1141X mutation is present or absent in each set of DNA after it is run through gel electrophoresis.
We predicted that a band of 894 base pairs will result after the PCR reaction and gel electrophoresis has been completed, insinuating that the designed primers were successful in seeking and amplifying the specified DNA sequences (Figure 1). The data obtained from gel electrophoresis is predicted to support the conclusion that the designed PCR components will be successful in finding and replicating the specified DNA sequences if a band with a length of 800 base pairs or greater is formed (Shi et al, 2007). These bands will appear when a homozygous genotype is paired with its respective primer because our primers were designed to amplify a sequence of approximately 800bp (Shi et al, 2007). However, our designed primers did not produce the results that were originally predicted. There was no target amplification in either DPXE or DWTF, meaning our PCR assay will not yet function as a viable genetic test for PXE (Figure 2).
By reenacting the day to day life of a person affected by PXE, we will gain a better understanding of what life consists of with the disease. We predicted that PXE does not have a great negative effect on life functionality because it is a mostly cosmetic disease, and very minimally does it largely impede one’s life (Roach et al., 2004). However, our experimental data suggests that the progression of PXE into its advanced stages can noticeably impact the patient’s physical and psychological aspects of life (Figure 6). PXE is an intriguing disease to learn about and urges the question of what more can be done to overcome this disease: is this disease more preventable or even eliminable?
Future Directions:
If research were to be continued on the R1141X mutation causing PXE, genomic isolation experiments could be done to alter the gene in a way that prevents the mutation from occurring. Since the R1141X mutation is caused by a point mutation, R1141X is a good candidate for researching and developing a gene therapy. Upon further diagnosis and troubleshooting, our experiment would serve as a viable genetic test to be applied to amniocentesis. If the R1141X mutation can be identified in fetal development, gene therapy could be implemented early to prevent the development of PXE. Though minimal physical impediment is experienced by the patient, emotional trauma is still possible, and if a gene therapy existed to reduce/remove the cosmetic symptoms of this disease, many lives would be affected positively (Roach et al., 2004). Much experimentation will have to be done to accomplish these goals over a large span of time to increase ideas and decrease error.
Though our experiment design relied heavily on published works and literature written by experts who have performed these methods before, there is always room for error. Potential problems include miscalculation of annealing temperature leading to nonspecific annealing or destruction of the DNA if the temperature was too hot (Wittwer et al., 1993). We attempted to solve this problem by performing PCR on multiple identical PCR cocktails, except we introduced a range of annealing temperatures (52-62şC). The purpose of multiple temperatures was to ensure that annealing temperature was not the issue. Another problem that could be experienced pertaining to the PCR cocktails could be an improper Magnesium concentration. Since we were not seeing any results using the standard 2µM Mg concentration, we increased Mg concentration to 4µM because a higher concentration increases the likelihood for primer binding, including non-specific binding. Upon comparison between the two concentrations, we saw increased non-specific binding in the 4µM concentration, but still not our target band; therefore we can conclude that the Mg concentration was not our issue (Figure 2). We can also conclude that our procedure for creating agarose gels was not the issue because our published primer control appeared in the correct area (Figure 2). The issue with our experiment is most likely in the primer design itself. In the future, we would pick a smaller target DNA fragment around 500bp for amplification to decrease the likelihood of overlapping possible intron/exon junctions. More importantly, we would decide against using the Yaku primer design. Although the Yaku method may decrease false positives, the intentional mismatch may have been enough to prevent any primer binding. A possible error in our Yaku design is our choice of which intentional mismatch to use because the size and shape of the mismatched nucleotide can vary depending on the choice, so the effectiveness of primer binding may vary as well. Lastly, an issue that could be encountered is the faulty manufacturing of the designed primers, which can only be corrected if the specific flaw is known and the manufacturer can be addressed. By learning through these mistakes, research on this subject can continue.