Use of PCR to diagnose BMD/DMD in human cells by the detection of the deletion of exon 47 on the dystrophin gene

Megan Bergeron, Katie Demeuse, Raajan Naik, and Kim Vi

Abstract:

Duchenne and Becker muscular dystrophy (DMD/BMD) occur as a result of a mutation on the dystrophin gene, 65% of which come from deletion mutations (Forrest et al, 1988). PCR was used to detect the deletion of exon 47. DNA samples for this study were extracted from human cells using Generation Capture Column Kit (Qiagen 2013). It was hypothesized that by placing designed primers in the introns flanking exon 47, the presence or deletion of exon 47 could be determined (Chamberlain et al, 1988). A set of primers were obtained from the work of Beggs et al and used as a control for this study (Beggs et al, 1990). The DNA amplified from PCR was analyzed using agarose gel electrophoresis. For the designed primers, a band at 853 bp would signify the presence of exon 47, while a band at 703 bp signified mutant genotypes that lacked the exon (Chamberlain et al, 1988). Analysis of agarose gel electrophoresis for the designed primers showed non-specific binding. For the control primers, a band at 181 bp would signify the presence of exon 47, while the absence of a band would signify the lack of exon 47 (Chamberlain et al, 1988). During analysis of agarose gel electrophoresis for the control primers a band was observed at 181 bp with wild-type DNA, and no band was visible with mutant DNA. These results led to greater understanding of the genotypes of patients with BMD/DMD, and additional investigation would help increase the knowledge of how to treat the disease. To understand life with BMD/DMD, a sociological experiment was performed by comparing travel times in a wheelchair versus walking, comparing dominant and non-dominant handwriting, along with measuring the difficulty of being in a wheelchair during daily routines. It was found that transit via wheelchair takes slightly longer than by foot, and handwriting with the dominant hand was more legible than the non-dominant hand. The time it took to go through daily routines increased in the wheelchair, and times gradually improved because of our body’s ability to adapt to disadvantages (Bontje et al, 2004).

Figure 5: Amplification of wild-type and mutant DNA using control and designed primers. All samples included 8 mL of PCR cocktail were exposed to an initial denaturation of 95° for 3 minutes followed by 35 cycles of denaturation at 95° for 30 seconds, at the annealing temperature for 40 seconds and elongation at 72° for 45 seconds, with final elongation at 72° for 3 minutes. A 1% agarose gel was used with Glow-Green, which was run at 245V for 20 minutes. The 5 mL of 1 Kb Plus ladder in the first well of the gel showed that position of the band that was wild-type DNA with control primers (C-WT) was compatible with the predicted length of 181 bp of the DNA segments, showing that there was plausible evidence that PCR amplified exon 47 of the dystrophin gene. The third well containing control primers with mutant DNA (C-M) shows no band, positively diagnosing that exon 47 was missing. Wells 4 to 7 show the same designed primers with wild-type DNA (D-WT) with increasing annealing temperatures. The appearance of slight bands around 300 bp and 500 bp indicate non-specific binding, because no bands appeared around the expected length of 583 bp.

Discussion:

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive diseases most commonly caused by deletion mutations at position Xp21 on the dystrophin gene (Beggs et al, 1990). There are different severities of these types of muscular dystrophies, caused by mutations that occur at different places on the dystrophin gene. If one of the exons of the gene is deleted, a frame shift may occur that disturbs the reading frame, which will result in a truncated protein that will not function due to its immense instability. Depending on the location of the exon that is deleted, the deletion may only cause a slightly truncated and slightly more unstable protein, leading to a less severe phenotype (Beggs et al, 1990).

Exon 47 was investigated because it is part of the region on the dystrophin gene that is one of the most commonly deleted in patients of DMD (Bellayou et al, 2009). To test whether there was a deletion of exon 47 on the dystrophin gene, primers were designed to anneal to either side of the exon, fully in introns 46-47 and 47-48 (Figure 4). This approach allowed for more accurate results because it avoided the possibility that a mutant primer that does not contain complementary nucleotides to any part of exon 47, would anneal to wild-type DNA. This approach was also taken because a band was seen for both wild-type and mutant DNA, reducing the risk of a false-positive diagnosis of mutant DNA due to incorrect PCR procedure (Chamberlain et al, 1988).

This method of making the primers anneal completely in the introns was modeled after a successful study that tested deletions of exons 8, 17, and 19 (Chamberlain et al, 1988). Since the primers were intended to anneal outside of the exon 47, primers annealed to both wild-type and mutant genotypes and therefore only two primers were needed (Figure 4). The primers were also outside of the splice site regions, where the intron and the exon meet, to prevent a false negative in wild-type genotypes if a splice site was removed (Chamberlain et al, 1988). Annealing temperatures of 50°C were performed and then adjusted to greater temperatures to reduce the risk of non-specific binding occurring. It was hypothesized that if primers were attached on either side of the exon in question, PCR could diagnose patients with DMD/BMD by detecting the deletion of exon 47.

Results yielded no bands for wild type DNA and mutant DNA at the expected regions (Figures 5 & 6). It was expected that the wild type DNA would have a band at 853 bp and mutant DNA would have a band at 703 bp to reflect the deletion of the 150 bp exon 47. Non-specific binding was also experienced with the designed primers when annealing to wild type DNA. This is possibly due to using the annealing temperatures provided by IDT, where the primers were ordered from, rather than recalculating the annealing temperatures using a different formula to ensure that the most accurate temperatures were used. Non-specific binding would have likely occurred even after recalculating the temperatures due to incorrect primer design. Primers did not anneal to the wild type DNA due to incorrect primer design. This is indicated by non-specific binding causing bands to appear at 300 bp and 500 bp (Figure 5). Even if the primers annealed to the wild type DNA, it is believed that no band would appear for the mutant DNA due to primers being designed for only the deletion of exon 47 on the dystrophin gene. Mutant DNA obtained from Children’s National Hospital in Washington, D.C. contained the deletion of exons 45-47. It was hypothesized that introns 46-47 surrounding exon 47 that were chosen for the primers to anneal to were also deleted in the mutant DNA that was received. The mutant DNA is also believed to be degraded due to the presence of smears in gels (Figure 6).

A sociological experiment was performed, which allowed students to experience what it would be like to be inflicted with the symptoms of BMD. Patients lose the ability to walk early on in life, and muscle control in the arms gradually decreases throughout their lifespan. It was hypothesized that day-to-day activities such as writing and overall mobility would be greatly impaired. The impacts of having loss of muscular control in the arms and legs were tested by comparing the time it takes to travel a designated distance in a wheelchair versus walking. Results showed that there was a minimal increase in time for pushing someone in a wheelchair compared to walking (Figure 8). This means that although a person with BMD that has little control of their arms and legs is dependant on someone else, the time it takes for them to travel places is not significantly impacted. To show the difficulty in performing ordinary yet necessary tasks, decreased ability to write was measured. Writing sentences with the dominant and non-dominant hand were timed and compared in which writing with the non-dominant hand represented the loss of muscle control in the dominant arm faced by a BMD patient. Writing sentences with the non-dominant hand took approximately three times longer compared to writing with the dominant hand (Figure 8). This revealed the difficulty a BMD patient has when writing for everyday tasks.

To further study the life of a BMD patient, another experiment was conducted in which four subjects were required to perform five ventures in a wheelchair to emulate a month-long experiment in which each subject experienced a week in the life of a BMD patient. Subjects used the wheelchair in their residential halls at Michigan State University. A distinct pathway was chosen to be travelled by each subject in their residential hall. At the end of each venture a Quality of Life Scale (QOLS) was completed by the subject in order to measure the psychological and sociological impact that BMD can have on a person. Overall, subjects were found to have an improved sense of self worth with subsequent ventures (Figure 7). The three ventures that subjects conducted in Holmes Hall consisted of traveling to Sparty’s, the cafeteria, and the basement. The final venture was on the second floor of Landon Hall. The time required to complete each venture was recorded. A control was used where each member walked their predetermined route to compare the amount of time it would normally take them to travel the route versus the time it would take in a wheelchair and then completed the QOLS. It was hypothesized that the recorded times would decrease with each subsequent venture, demonstrating adaptations to using a wheelchair, and the subject’s self worth would improve.

In a larger sense, this experiment would represent the difficulty of life in a wheelchair after initial diagnosis, but also the increased facility and subsequent quality of life of a BMD/DMD patient with time. The lack of handicap-accessible ramps and elevators presented challenges for the subject in Landon Hall. In order to enter the building, one must walk up stairs but no ramps are available. Inside the building, there are no elevators; consequently, the venture in Landon Hall was restricted to one floor. It was concluded that it would be immensely difficult for a person with BMD to live in Landon Hall.

A BMD patient would be able to live in East Holmes Hall, but they would face many challenges. To get inside of East Holmes Hall, a ramp is available; however, there are no automatic doors. Once inside, a steep ramp must be used to get to the cafeteria and Sparty’s. In order to get up the ramp, assistance is needed. It is possible to go down the ramp without assistance, but this is dangerous. In one venture, going down the ramp unassisted led to crashing. Automatic doors were also unavailable to enter Sparty’s. This makes it difficult for someone with BMD to get around in Holmes Hall. However, it was observed that the West Holmes Hall entrance is more accommodating to those who are confined to a wheelchair. Automatic doors are available at the entrance, and an elevator allows one to travel to Sparty’s and the cafeteria without the use of a ramp. Despite the challenges in both residential halls, subsequent ventures displayed decreasing times and improved ratings on the QOLS as predicted. Consequently, this supported the hypothesis in which members improved in using a wheelchair after five ventures in each route and had improved QOLS ratings, illustrating the idea that patients with Becker muscular dystrophy can adapt to wheelchair use over time which also results in an improved sociological and psychological well being.

Times for the control ventures were approximately 16 seconds shorter than times recorded for the fastest venture when traveling to each location (Figure 7). This shows that the time taken when traveling by wheelchair compared to walking is not significantly different. A single-sample t test was performed for each subject and the decrease in time between the first and last venture for each subject was found to be statistically significant at an alpha value of 0.05. As a result, the data lend strong evidence to the hypothesis of adaptation of BMD patients in a wheelchair.While the overall quality of life was 10-20 points lower than the control at the end of the experiment, the overall quality of life score comparing ventures 1 and 5 had an increase of 10-30 points, showing that the quality of life does increase for a handicapped person with increasing time (Figure 7). Performing these social experiments created a greater understanding of the effects of this genetic disease, and was performed to understand the significance of the results obtained in the experiment. This research, and further research like this, contributes to the knowledge needed to create a cure for Becker muscular dystrophy and other similar disabling and fatal diseases.

Weaknesses in the diagnostic assay occur due to the use of only two types of control primers. The control primers used resulted in no band as expected when annealing to mutant DNA. Taking another approach that also detects a deletion of the 47th exon of the dystrophin gene will increase the certainty of a positive diagnosis. In recent studies, substitution mutations in intronic sequences, outside of the exon coding regions, have been found to result in Duchenne phenotypes. Therefore this alternative method would prevent the possibility that the primers would not anneal to DNA template in the introns due to the change or addition of intronic base pairs (Baskin et al, 2009). In addition, by only isolating one exon, the data obtained was limited because of the variation of mutations that can occur on the dystrophin gene. Those with DMD with point mutations or a deletion of another exon could not be detected using the method performed in this experiment (Hallwirth Pillay et al, 2007).

Future Directions:

Further investigations could be taken by designing different primers that annealed to the specific mutant DNA received from Children’s National Hospital in Washington, D.C. Due to lack of time, primers were not designed to anneal to diagnose a patient with deletions of exon 45 to 47 that was received. To get a correct diagnostic result using this mutant DNA, primers should be designed to anneal in the introns 44-46 and 47-48. The presence of a band would indicate a deletion of exons 45-47 while a lack of band would indicate that those exons are intact, because the segment that would result would be too large and would not get out of the well during gel electrophoresis.

Another investigation to further diagnose and better understand Duchenne and Becker muscular dystrophy include investigating deletion of all exons that cause the disease. The mutant DNA that was obtained could be tested to see how many more exons were deleted on the DMD gene. Testing the correlation between the location of the deletion and the severity of the symptoms could then be determined, which would help understand the dystrophin protein and its function even further. Since deletions to the DMD gene result in truncated, reduced production, or complete absence of the dystrophin protein, the amount of dystrophin protein expressed in the cells with the mutant DNA could then be measured and correlated to how many or which specific exons were missing. By studying other deletions of exons on the DMD gene, the effect that the deletions have on the dystrophin protein can be concluded. By doing this the phenotype that is expressed can be determined, and therefore diagnose whether the patient that the mutant DNA belongs to has DMD or BMD.

Moreover, biopsies could be taken from the mutant DNA patients in areas of dense muscle and be analyzed for adipose tissue to muscle tissue ratio in order to compare that ratio to the adipose/muscle ratio of other mutant DNA patients and also wild-type patients.  In this way, it could be determined if the absence of more or specific exons in the dystrophin gene would result in more severe forms of muscular dystrophy and the dystrophin proteins in these individuals could then be analyzed to see the extent at which they can function.

DIY 30 Days Experiment: http://youtu.be/XTamIrZxKng