Hypertrophic Cardiomyopathy
Genotypic Identification of the MYH7 R403Q Mutation using PCR
On Human S9 Epithelial Cells yields negative results
http://www.youtube.com/watch?v=nQk9M2flJBI
Bhanu Swamy, Kevin Wheelock, Fletcher Smith, September Waites
On Human S9 Epithelial Cells yields negative results
http://www.youtube.com/watch?v=nQk9M2flJBI
Bhanu Swamy, Kevin Wheelock, Fletcher Smith, September Waites
Abstract
Our Story
Figure 6: Representation of psychosocial variables (stress level scores) measured by the developed Student Stress Scale Test to assess the psychosocial impacts of the holter monitor. In order to evaluate the level of stress created by wearing a holter monitor, a common cardiac test, an experiment was performed to measure the level of stress that each researcher endured on a daily basis by wearing a holter monitor. A 45-question Student Stress Scale Test was specifically designed with questions regarding the negative states of psychological and social stress brought upon by wearing the holter monitor. Each question response was based on the frequency of stress 0(Never), 1(Seldom), 2(Often), and 3(Regular). This test was given to each researcher as a pre-test before he or she wore the holter monitor, and then as a post-test after having worn the monitor in a 24 hour trial period. The graph shows the average pre and post-stress scores over a six trial period. Error bars represent standard deviation for each researcher. T-test analysis determined no significance between pre-test and post-test scores of all researchers with an average p-value of 0.40 and individually (p= 0.16), (p=0.19), (p=0.45), and (p=0.18). This illustrated that a holter monitor is not a significant source of increased stress levels for cardiac patients. Evaluation of the researchers’ pre-test and post-test scores provides implication that holter monitors do not diminish the quality of life in hypertrophic cardiomyopathy patients.
Hypertrophic cardiomyopathy (HCM) is a dominant genetic cardiac disorder that afflicts one in every 500 people. While there are numerous mutations which can lead to HCM, the R403Q mutation on the MYH7 gene leads to many of the most severe cases (Redwood et al, 1999). A diagnostic assay was created using polymerase chain reaction (PCR) for the R403Q mutation. DNA was extracted from the S9 human epithelial cell line. Primers were designed with an intentional pyrimidine-pyrimidine base pair mismatch near their 3’ ends. It was hypothesized that this would decrease the chance of a false positive by disallowing mutant primer to wild-type DNA or wild-type primer to mutant DNA extension due to a two base pair mismatch. Mutant-mutant/wild-type-wild-type extension was predicted to still occur because only one hairpin was present (Yaku et al, 2008). RPM and RPW were reverse primers designed only to amplify mutant or wild-type DNA, respectively. A third forward primer, FP, was designed to amplify either set of DNA. Gel electrophoresis analysis should have yielded a band 779 base pairs long when RPW was mixed with wild-type DNA and a band 780 base pairs long with RPM was mixed with mutant DNA. Electrophoresis showed no successful amplification of wild-type DNA. R403Q mutant DNA was never obtained as a positive control for RPM. To access the psychosocial impacts of wearing a holter monitor, stress levels were measured before and after wearing holter monitor over a 24 hour period with a pre-test and post-test from a generated Student Stress Scale Test. T-Test analysis presented no difference between pre-test and post –test scores with an average p-value of 0.40, and individually p-values of 0.16, 0.19, 0.45, and 0.18, which construed that holter monitors do not reduce the mental wellbeing or increase the stress of a HCM patient.
Discussion
Future Directions
Hypertrophic cardiomyopathy is an autosomal dominant disease that disrupts activity in the left ventricle, causing cardiac muscle hypertrophy (Perrot et al, 2005). The R403Q mutation is located on chromosome 14 and involves a base pair substitution of guanine to adenine at the 10172nd base pair in Exon 13, the locus of MYH7 that codes for βMHC proteins (Jaenicke et al, 1990). This is the most common genetic mutation leading to HCM, and the leading cause of sudden death in young athletes (Redwood et al, 1999). A recent case of HCM that made national headlines was that of a male high school basketball player who collapsed after a playoff win in western Michigan (Helms, 2011). An analysis of HCM patients revealed that mutations on the MYH7 gene contribute to 30% of cases where mutations were identified (Perrot et al, 2005). When the R403Q mutation is present, β-myosin heavy chain proteins malfunction and thus a decrease of force is exerted on the actin filament, leading to hypertrophy in the heart muscle (Redwood et al, 1999). This alteration of the β-myosin heavy chain protein causes a malfunction of the entire sarcomere protein (Redwood et al, 1999). A diagnostic assay is being created that aims to reliably diagnose the R403Q mutation in human genomic DNA. This assay is being created to prevent more sudden death incidents in young athletes or any HCM patient since no serious symptoms are exhibited.
Polymerase Chain Reaction, or PCR, involves using primers to bind to target areas of DNA and act as a starting point for taq polymerase to extend from, delineating a segment of DNA to be amplified. We hypothesized that by using primers designed with intentional base pair mismatches near their 3’ ends the accuracy of the assay could be increased by eliminating non-specific binding. An intentional pyrimidine-pyrimidine mismatch was built into the third base pair of the 3’ end of two primers, RPM and RPW. This was intended to prevent extension by Taq polymerase of the mutant primer when it annealed to wild-type DNA and vice versa, because the two base-pair mismatch will create a double-hairpin near the initial extension site (Yaku et al, 2008). This technique was designed to decrease the chances of false negatives or positives, yielding more reliable results to our diagnostic assay (Yaku et al, 2008). Two reverse primers, RPW and RPM, were used to differentiate between the binding of wild type and mutant DNA. Expectations were that a successful annealing of RPW to wild-type DNA and RPM to mutant DNA will occur due to the use of the Yaku primer design method. Predictions also included that RPM will not anneal to wild-type DNA and RPW will not anneal to mutant DNA because Taq polymerase will rarely extend DNA with two mismatches in the sequence (Yaku et al, 2008).
PCR of E. coli was run unsuccessfully, as primers did not anneal to the template DNA. The most likely conclusion is there could have been insufficient amounts of E. coli genome, which would explain the large bright bands under 100 base pairs, because there are insufficient amounts of DNA for primers to anneal to. Both calculated annealing temperatures for 8r and 1512f E. coli primers were between 50 and 52 degrees Celsius. PCR was run at 48, 50, and 52 degrees Celsius so this most likely does not offer an explanation for failure and bright bands near the bottom of the gel.
Successful PCR of Lambda virus was achieved. Predicted annealing temperatures for this reaction were 59 degrees Celsius for the forward primer and 55 degrees Celsius for the reverse primer. This reaction was run in a thermocycler at 50 degrees Celsius. Upon achieving the desired band of 500 base pairs in a gel, reaction conditions were analyzed and researchers indicated that running PCR at a lower temperature than its calculated temperature increases likelihood of primers annealing at the risk of having nonspecific binding. This success gave us a standard ratio of reagents to be used in cocktails of future PCR while also shows a reliable thermocycler was used.
PCR using control primers was successful in amplifying DNA, but not in the way it was supposed to. The gel gave a band that was approximately 150 base pairs long when a band of 295 base pairs would have indicated successful extension and amplification of wild-type control primers to S9 epithelial cells. This suggests there was nonspecific binding among wild-type primers and the S9 epithelial genome due to low annealing temperatures. With this amplification the presence of DNA is confirmed.
Both wild-type and mutant designed primers with wild-type DNA yielded unsuccessful amplification of the targeted region. Unsuccessful annealing of mutant primers to wild-type DNA resulted in a negative control for mutant primers as no band appearing in the gel. Mutant DNA was never attained so tests of wild-type and mutant primers with mutant DNA were not done. Successful annealing of RPM to mutant DNA would have affirmed the presence of the R403Q mutation at the MYH7 gene (Redwood et al, 1999). With this, our primers could then have been used to test any human cells and diagnose them with HCM. The results gathered from this PCR do not support our hypothesis since designed wild-type primers did not successfully anneal and extend along wild-type DNA. The negative control of designed mutant primers failing to anneal to wild-type DNA coincides with what was predicted and provides small support for the hypothesis. However, further reactions would be required to confirm predictions and strengthen the hypothesis.
It is important for the medical community to have reliable tests to discover and diagnose cardiac disorders. However, if these methods cause discomfort to the patient, it may decrease the likelihood of patients seeking treatment (Straub, 2007). In order to help develop an understanding of the psychological impact of wearing a holter monitor, team members wore a holter monitor for six 24 hour periods. A stress test was taken by each researcher before and after wearing the monitor to determine if the monitor contributed to elevated stress levels. An average P-value of .40 was calculated for all researchers, indicating that the holter monitor lead to no significant difference in increased stress. In general, it was found by researchers that while the monitors were inconvenient, they did not significantly impact emotional responses to daily routines.
Polymerase Chain Reaction, or PCR, involves using primers to bind to target areas of DNA and act as a starting point for taq polymerase to extend from, delineating a segment of DNA to be amplified. We hypothesized that by using primers designed with intentional base pair mismatches near their 3’ ends the accuracy of the assay could be increased by eliminating non-specific binding. An intentional pyrimidine-pyrimidine mismatch was built into the third base pair of the 3’ end of two primers, RPM and RPW. This was intended to prevent extension by Taq polymerase of the mutant primer when it annealed to wild-type DNA and vice versa, because the two base-pair mismatch will create a double-hairpin near the initial extension site (Yaku et al, 2008). This technique was designed to decrease the chances of false negatives or positives, yielding more reliable results to our diagnostic assay (Yaku et al, 2008). Two reverse primers, RPW and RPM, were used to differentiate between the binding of wild type and mutant DNA. Expectations were that a successful annealing of RPW to wild-type DNA and RPM to mutant DNA will occur due to the use of the Yaku primer design method. Predictions also included that RPM will not anneal to wild-type DNA and RPW will not anneal to mutant DNA because Taq polymerase will rarely extend DNA with two mismatches in the sequence (Yaku et al, 2008).
PCR of E. coli was run unsuccessfully, as primers did not anneal to the template DNA. The most likely conclusion is there could have been insufficient amounts of E. coli genome, which would explain the large bright bands under 100 base pairs, because there are insufficient amounts of DNA for primers to anneal to. Both calculated annealing temperatures for 8r and 1512f E. coli primers were between 50 and 52 degrees Celsius. PCR was run at 48, 50, and 52 degrees Celsius so this most likely does not offer an explanation for failure and bright bands near the bottom of the gel.
Successful PCR of Lambda virus was achieved. Predicted annealing temperatures for this reaction were 59 degrees Celsius for the forward primer and 55 degrees Celsius for the reverse primer. This reaction was run in a thermocycler at 50 degrees Celsius. Upon achieving the desired band of 500 base pairs in a gel, reaction conditions were analyzed and researchers indicated that running PCR at a lower temperature than its calculated temperature increases likelihood of primers annealing at the risk of having nonspecific binding. This success gave us a standard ratio of reagents to be used in cocktails of future PCR while also shows a reliable thermocycler was used.
PCR using control primers was successful in amplifying DNA, but not in the way it was supposed to. The gel gave a band that was approximately 150 base pairs long when a band of 295 base pairs would have indicated successful extension and amplification of wild-type control primers to S9 epithelial cells. This suggests there was nonspecific binding among wild-type primers and the S9 epithelial genome due to low annealing temperatures. With this amplification the presence of DNA is confirmed.
Both wild-type and mutant designed primers with wild-type DNA yielded unsuccessful amplification of the targeted region. Unsuccessful annealing of mutant primers to wild-type DNA resulted in a negative control for mutant primers as no band appearing in the gel. Mutant DNA was never attained so tests of wild-type and mutant primers with mutant DNA were not done. Successful annealing of RPM to mutant DNA would have affirmed the presence of the R403Q mutation at the MYH7 gene (Redwood et al, 1999). With this, our primers could then have been used to test any human cells and diagnose them with HCM. The results gathered from this PCR do not support our hypothesis since designed wild-type primers did not successfully anneal and extend along wild-type DNA. The negative control of designed mutant primers failing to anneal to wild-type DNA coincides with what was predicted and provides small support for the hypothesis. However, further reactions would be required to confirm predictions and strengthen the hypothesis.
It is important for the medical community to have reliable tests to discover and diagnose cardiac disorders. However, if these methods cause discomfort to the patient, it may decrease the likelihood of patients seeking treatment (Straub, 2007). In order to help develop an understanding of the psychological impact of wearing a holter monitor, team members wore a holter monitor for six 24 hour periods. A stress test was taken by each researcher before and after wearing the monitor to determine if the monitor contributed to elevated stress levels. An average P-value of .40 was calculated for all researchers, indicating that the holter monitor lead to no significant difference in increased stress. In general, it was found by researchers that while the monitors were inconvenient, they did not significantly impact emotional responses to daily routines.
Unsuccessful amplification of E.coli resulted in very large bright bands under 100 base pairs. Given the 8F and 1512R primers used, the resulting band should have been 1504 bp. Not only were the bands not the correct length, they were unusually bright. Bright bands were most likely a result of increased volume of primers and DNTP’s. For this trial, primers and DNTP’s were increased from 1 L to 2 L. The bands were representative of the primer lengths as primer dimers were found under 100 bp. In future experiments, more E.coli genome would be used along with hotter temperatures to increase binding specificity.
PCR using designed primers was the most significant assay through our experimenting. Inability to obtain mutant DNA did not allow us to test wild-type and mutant primers with mutant DNA, and in the future, acquisition of a genome containing the R403Q mutation would be essential to obtain a successful assay to test for the mutation. Testing wild-type and mutant primers against a wild-type genome yielded negative results. The gel shows fuzzy bands near its bottom suggesting primer dimers. Increasing annealing temperatures would increase binding specificity allowing primers to anneal in the targeted area and yield a positive result. In future PCR of designed primers, redesigning primers to decrease potential binding sites would increase chances of positive results.
In testing control primers obtained according to those used by Stravopodis’ paper, results were slightly positive. Some annealing occurred, but at 150 base pairs instead of the desired 295 bp. This non-specific binding occurred due to the use of low annealing temperatures. Increasing annealing temperature would increase chances of binding specificity, yielding a positive result.
PCR using designed primers was the most significant assay through our experimenting. Inability to obtain mutant DNA did not allow us to test wild-type and mutant primers with mutant DNA, and in the future, acquisition of a genome containing the R403Q mutation would be essential to obtain a successful assay to test for the mutation. Testing wild-type and mutant primers against a wild-type genome yielded negative results. The gel shows fuzzy bands near its bottom suggesting primer dimers. Increasing annealing temperatures would increase binding specificity allowing primers to anneal in the targeted area and yield a positive result. In future PCR of designed primers, redesigning primers to decrease potential binding sites would increase chances of positive results.
In testing control primers obtained according to those used by Stravopodis’ paper, results were slightly positive. Some annealing occurred, but at 150 base pairs instead of the desired 295 bp. This non-specific binding occurred due to the use of low annealing temperatures. Increasing annealing temperature would increase chances of binding specificity, yielding a positive result.
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