Successful Diagnosis of Duchenne Muscular Dystrophy Using

PCR to Detect the Deleted 45^th Exon of the Human DMD Gene

By:

“La Toilette”

Buhlinger, Kaitlyn

Muresan, Steven

Parker, Andrea

Zamora-Sifuentes, Jose

Abstract:

Deletions within the dystrophin (DMD) gene cause 60% of all Duchenne muscular dystrophy (DMD) cases (Nowak and Davies, 2004). Diagnosis is essential as several gene therapies are being investigated. Our assay can detect the presence of the mutation, thereby assisting researchers in determining the type of therapy to follow (Burkin et al., 2005). Our experiment involved the deletion of the 45^th exon of the DMD gene. To amplify this segment of DNA with polymerase chain reaction (PCR), we designed two forward primers, one annealing at the intron 44-exon 45 junction of the DMD gene in wild-type DNA (T primer) and one at the intron 44-intron 45 junction in mutated DNA (M primer). A reverse primer (R primer) was designed to always anneal within the 45^th intron of the DMD gene (Beggs et al., 1990). We hypothesized that our unique primer design, with a forward primer annealing to the intron 44-intron 45 junction of mutant DNA, would allow us to detect a deletion of the 45^th exon by a difference in band length compared to wild-type DNA amplified with the same primers. Using a single blind study, we were able to detect the mutation using PCR and gel electrophoresis by the absence of a 765bp band when mutant DNA was amplified using the TR primer set. After completing a socio-psychological experiment accompanied by disability and socio-psychological questionnaires, R² values of 0.9052 and 0.9815, respectively (p<0.005) showed that the progressive symptoms of DMD were correlated with increasing disability and a deteriorating socio-psychological state.

Figure:

Figure 1. Single blind study for PCR amplifications of DMD gene with deletion of exon 45 from human skeletal muscle cells at annealing temperatures of 47ºC and 48ºC. After PCR amplification, gel electrophoresis was conducted in a 0.8% TBE gel run at 115V for 30 minutes to detect amplified fragments of DNA. M corresponds to 1.25ng of 1-Kb Plus ladder. A researcher loaded each lane without knowing the amount or type of DNA that was being loaded. Lanes 1, 2, and 4 had unknown DNA amplified with the wild-type forward and reverse primer set (TR). Lanes 3, 5, and 6 had unknown DNA amplified with the mutant forward and reverse primer set (MR). Non-specific binding is observed in each lane. No product is observed in Lane 1. Products of 765bp are observed in Lanes 2 and 4 while products of 589bp are observed in lanes 3, 5, and 6.

Discussion:

Experimental Summary

Duchenne muscular dystrophy (DMD), a disease resulting in degenerative muscle weakness and inevitable death, is the most common sex linked genetic disorder (Nowak and Davies, 2004). The most common genetic cause of DMD is a large deletion on the dystrophin gene (Beggs et al., 1990). We focused our research on diagnosing the disorder by detecting a deletion of the 45^th exon of the DMD gene (Chamberlain et al., 1988). We hypothesized that our unique primer design, with a forward primer annealing to the intron 44-intron 45 junction of mutant DNA, would allow us to detect a deletion of the 45^th exon on the DMD gene by a difference in band length compared to wild-type DNA amplified with the same primers (Figure 1). Adequate annealing temperatures were determined by trial and error using PCR (Innis et al., 1990). Ideal DNA concentrations were found by testing each sample with spectroscopy and analyzing the visual feedback from gel electrophoresis. The results obtained from PCR and gel electrophoresis do not oppose our hypothesis, but they suggest that an alternative primer design would have been more reliable in diagnosing the disease (Gurvich et al., 2008). A socio-psychological experiment was performed to illustrate a correlation between the degenerative nature of the disease and the deteriorating state of the patient’s socio-psychological well-being. To do so, the group mimicked the symptoms of muscular dystrophy in an additive and degenerative nature while assessing their reaction weekly using surveys to detect the physical and socio-psychological impact each new symptom presented (Bruce and Fries, 2003). Using linear regression as a statistical tool, the experiment showed both components, physical and socio-psychological, had a positive linear correlation to the degenerative nature of the disease.

Analysis of Results

The primers were designed to yield a band of 765bp when wild-type DNA was tested with the wild-type primer set (TR). A band of 589bp was expected when mutant DNA was used with the mutant primer set (MR). No bands were expected when mutant DNA was used with wild-type primers, nor when wild-type DNA was used with mutant primers. However, a band of 589bp was always observed when mutant primers were used, regardless if it was wild-type DNA being tested. This occurred because of an incorrect primer design. Eight nucleotides on the 3’ end of the mutant forward primer (M) annealed to intron 45 of the wild-type DNA, as well as to four other nucleotides within exon 45. This created a band equivalent to the one expected when using the MR primer set with mutant DNA. Since the MR primer set consistently produced a band of 589bp regardless of the type of DNA, it was deemed inconclusive in diagnosing Duchenne muscular dystrophy. Instead, the bands obtained were used as a positive internal control because of their constant presence. The constant bands not only confirmed that the PCR machine and gel electrophoresis were properly functioning, but also gave a comparative band length to better determine the presence of a 765bp long band.

After testing various annealing temperatures, 48^oC was clearly the best temperature when TR primer set was used. At this temperature, a band was seen around 765bp, as expected for wild-type DNA (Figure 2a). The curved nature of the wells made it difficult to determine the exact band lengths of the amplified products, so a semi-log plot was made to more accurately determine the band lengths. For lanes 1 and 2, the lengths obtained were 757.83 ± 8.75bp and 775.33 ± 8.75bp respectively (Figure 2b). These band lengths confirmed the idea that the DNA sample used was wild-type; it does not have a deletion of the 45^th exon of the DMD gene. A band length of 765bp indicates healthy DNA for future diagnosing. Lane 3 shows a band of approximately 589bp, more accurately determined by the semi-log plot to be 584.43 ± 5.21bp (Figure 2). This band was not originally expected, as the mutant primers should not have worked on wild-type DNA. The presence of the band when using the MR primer set on mutant and wild-type DNA can be explained by the flawed primer design previously discussed. However, its constant appearance served as an internal positive control.

Originally, no band was observed when using mutant primer set. Consequently, the amount of DNA found on each PCR tube was increased from 10.55ng to 21.1ng to increase the likelihood of observing bands (Etlik et al., 2008). Bands were observed in lanes 2 and 3 at approximately 589bp when PCR’s annealing stage temperature was set to 47^oC (Figure 3a). These bands were more accurately determined to be 589.87 ± 5.21bp and 572.16 ± 5.21bp using a second semi-log plot (Figure 3b). As expected, no defined band was observed in lane 1 from using the TR primer set with mutant DNA. For future diagnosis, the result of seeing no bands for mutant DNA tested with TR primers allows the researchers to determine the absence of the 45^th exon.

In addition, a single blind study was performed to determine if the diagnosis of DMD was possible by using the designed assay (Figure 4). One researcher loaded unknown PCR products into a 0.8% agarose gel while a second one was aware of the type of the samples being loaded. When diagnosing the disease, the presence of bands 589bp long produced by the MR primer set were treated as an internal positive control. The other group members successfully determined which lanes contained wild-type DNA and mutant DNA based on the presence of a band of 765bp (wild-type DNA) or absence of bands (mutant DNA). In lane 1 no band was observed, indicating that the DNA used in that lane was mutated and that that individual would be sick. Lanes 2 and 4 have a band of 765bp long that indicates that exon 45 was present and that the individual was healthy. Even though the deletion of the 45^th exon was successfully identified, this assay would not be reliable in actually diagnosing the disease, since an absence of bands is never a dependable diagnosis. A diagnosis based on false negatives is unreliable and could potentially lead to problematic treatments (Gillet et al., 2010). A positive result needs to be obtained when mutant primers are used with mutant DNA to actually detect the deletion of the 45^th exon, thereby diagnosing the patient (Innis et al., 1990).

Socio-psychological Experiment

A socio-psychological experiment was completed alongside the laboratory research. We hypothesized that we would be able to successfully show the correlation between the degenerative nature of Duchenne muscular dystrophy and the deteriorating socio-psychological state associated with it. We mimicked symptoms that were progressive, accumulating, and that replicated the severity of the disease for a period of five weeks. After calculating R² values of 0.9052 (p<0.05) and 0.9815 (p<0.05) for the physical and socio-psychological impact of the disease, respectively, it was concluded that our hypothesis was supported by these results. Although some studies, such as the one conducted by Bird et al., have findings similar to those of our experiment, others have discovered that severely disabled patients have a positive outlook on life and their socio-psychological state is positively affected (Bach et al., 1991). In contrast to their research, our results from our personal experiences reproducing the symptoms of the disease illustrate that our hypothesis is correct: there is a correlation between the degenerative nature of Duchenne Muscular Dystrophy and the deteriorating socio-psychological state associated with it.

Future Directions

Non-specific binding was observed in most of our gels. However, the presence of clear bands allowed us to still identify the presence or absence of exon 45. To avoid non-specific binding in the future, we would raise the temperature by increments of 0.5ºC (Mendel and Mendel, 1985). This would allow us to get definite bands in a more specific temperature region.

In addition to non-specific biding, there was a flaw in our primer design because the mutant forward primer undesirably annealed to wild-type DNA. To keep our original idea of having the mutant forward primer anneal at the intron 44-intron 45 junction but avoiding improper annealing to wild-type DNA, the mutant forward primer could be redesigned for the majority of the primer to anneal in intron 44 so that it would be less likely to extend over the present 45^th exon in wild-type DNA. The new primer would have the sequence: 5'-CCTTTTTGGTATCTTACAGG-3' (Tm=54.3^oC) with the only nucleotide in intron 45 being the final G on the 3’ end. However, since the first nucleotide in exon 45 is also a G, the primer could still possibly anneal in wild-type DNA. Nevertheless, after reviewing the DNA sequence, we concluded that having a forward primer annealing at the intron44-intron45 junction is not the best approach to detect this particular deletion. As a result of these difficulties, we would suggest carrying out a double primer assay.

Double primer assays have been used by other scientists and have been proven successful on identifying this mutation (Chamberlain et al., 1988). By following this procedure, we could design a different forward primer, 5'-TGCTCTTGAAAAGGTTTCC- 3' (Tm=53^oC), to completely anneal within the 44^th intron. The original reverse primer would be kept completely annealing in intron 45. If used on wild-type DNA a product of 1061bp would be expected. If used on mutant DNA, where exon 45 is deleted, a product of 885bp would be amplified.