Molecular Correlations and Epitope Mapping in Muscular Dystrophy: Advancing Detection and Treatment Strategies for DMD and BMD
By Katie Lee | Prosper High School, Prosper, Texas, United States
I. Introduction
Muscular dystrophies (MD) are a group of muscular diseases caused by a mutation in a person’s genes. Usually, a person will receive this genetic mutation from past family members as it runs through families and generations, or, in rare cases, a person may be the first to have it in their family. Many different genetic types can be affected to cause MD, meaning that even if two people have the same kind of muscular dystrophy, the symptoms may not be the same (“What Is Muscular Dystrophy? | CDC” 2022). MD most directly affects the majority of muscle fibers, organs, and body tissues. In turn, this genetic mutation causes mostly muscle degeneration and fiber death, with other symptoms being an ongoing weakness. There are multiple different approaches when treating MD. These methods consist of physical therapy, gene therapy, drug therapy, and surgery and help to give people who are affected by MD as much of an independent life as they can get (“Muscular Dystrophy,” n.d.). Although doctors know how to identify and treat MDs, they are still unable to find a cure for it. What drugs and treatments will we be able to use? These are all questions that researchers have been asking for several years. The real cure remains a mystery, but seeing as researchers have advanced as far as gene therapy, finding a cure is sure to happen in the times to come. To go through with both types of research present in the studies mentioned here, researchers utilized prior knowledge from multiplex PCR, southern blotting, and the reading frame theory (Nguyen et al. 1992) (Yoshida et al. 1993).
Keywords: Muscular Dystrophy; Duchenne Muscular Dystrophy (DMD); Becker Muscular Dystrophy (BMD); Diagnostic Mutation Detection; Epitope Mapping
II. Methods and Techniques
A study was conducted by Beggs et al. in which they uncovered the truth about the molecular and clinical correlations present in two common types of MD – Duchenne (DMD) and Becker (BMD). This discovery could lead to better detection of mutations within the DMD and BMD gene. By developing molecular methods for detecting these mutations, patients with atypical BMD can be identified quicker than they have been before. To conduct this research, PCR amplification was performed according to Sakuraba et al. in which the products were then electrophoresed on agarose gels further being stained with ethidium bromide. DNA must first be amplified through PCR to produce millions to billions of a segment of DNA, which helps with a more specific analysis during electrophoresis (Smith 2023). Electrophoresis, which is a laboratory technique used to analyze if a certain gene is present. To perform this technique, genomic DNA was isolated from leukocytes derived from four patients. Primers used in amplification derived from exon sequences of the DMD gene. To confirm the PCR results, Southern blot analysis was performed. By the use of PCR and gel electrophoresis, the researchers were able to identify which patients had deletions and in which exons with the bands present on the gel. Most DMD genes are expressed in cardiac muscle as well as skeletal muscle. Though these kinds of cardiac complications are rare in BMD patients, there have been reports of the fact. By utilizing this laboratory technique, the researchers were hoping to investigate the molecular basis of BMD’s phenotypic diversity. According to the results of the PCR analysis, two patients had a deletion involving exon 1 (both of which showed minimum muscular atrophy) and the other two had a deletion involving exon 47 (both of which had much more significant muscular atrophy). Further research confirmed that the first two patients’ deletion of exon 1 involved the 3’ end of exon 1 and that they also had a deletion in intron 1. Another patient in a separate study also showed the same features as the first two patients – with the addition of a typical deletion of the DMD gene. This led to the conclusion that deletions around exon 1 (though rare to be just exon 1) may impair the expression and function of cardiac muscles, but not so much in skeletal muscles (Yoshida et al. 1993). A separate study conducted by Nguyen et al. revealed that though a more detailed mapping is required, a minimum of 17 epitopes are involved in 44 of the monoclonal antibodies relating to MD. This information helps us understand different epitopes along the dystrophin molecule, which is the protein that is absent in patients with MD (MedlinePlus 2020). By knowing this information, we can better understand these monoclonal antibodies, which are of high value in research for alters dystrophins, relationships between dystrophin and protein (sequence and structure), alternative transcripts of the dystrophin gene, and especially for distinguishing successful transplants in MD patients. So as to go through with this research, Nguyen et al. utilized the laboratory technique, Western blotting. Western blotting is derived from Southern blotting, but the difference is that Western blotting makes use of proteins. In this process, a mixture of proteins is separated by weight/size, which in turn separates them by type. This process is through gel electrophoresis. To go through with this, you must load your protein samples into appropriate wells in the gels, apply a current to move the samples towards the positive side, transfer proteins from the gel to a membrane, and finally, detect the proteins of membranes (immunoblotting). Using this technique of western blotting, Nguyen et al. analyzed and mapped 44 monoclonal antibodies other than the 22 they studied in a previous lab. In doing so they hoped to reveal new information that could be of use in the future of treating and preventing MD. The Western blot showed the researchers that all C-terminal monoclonal antibodies recognized the complete 95 kDa fusion protein but only seven out of 19 recognized the last 126 amino acids. Because no antibodies recognized the trpE fusion protein, the researchers concluded that all epitomes lie between amino acids 1749 and 2102. Three different patterns were reviewed by the Western blots developed by rod antibodies, allowing them to classify them into three epitome groups. They concluded that at least two monoclonal antibodies – MANDYS105 and 110 – are specific for native dystrophin. Although they studied if all 44 monoclonal antibodies would recognize 44 different epitopes, the mapping was too broad to come to a consensus. However, they were able to learn that a minimum of 17 were involved (Nguyen et al. 1992).
III. Conclusion
In both studies, the conclusions align with the results they were given. However, there were some weaknesses present in the research. For instance, in the second study mentioned, there were many possible errors mentioned. First, because there was an incorrectly folded end, the antibodies could have failed to recognize the intact dystrophin molecule. There was little surprise in the research, but the first study did have an unexpected result. For the first patient, there was an unexpected band during the Southern blot used to confirm the results. Instead of there being a band correlating with exon 1, a unique abnormal band of 4 kb (which was not detected by controls) was present. However, it was further investigated and found that this was due to a novel junctional Hindi III fragment (which includes exon 2) (Yoshida et al. 1993). Some questions naturally arose from both of these studies. For the first study, it was a mystery to the researchers regarding the role of exon 1 in skeletal and cardiac muscles. This can be looked into by studying the makeup of both skeletal and cardiac muscles, and studying how the addition or subtraction of exon 1 affects each. For the next study, the researchers found that they could not yet find out if a-actinin or another sarcoplasmic protein was responsible for some of the results, as they did not bind to anything, even the dystrophin itself. A study that could help with this is something that focuses specifically on these proteins. A more specific mapping and analysis would be required.
IV. References
“What Is Muscular Dystrophy? | CDC.” 2022. Centers for Disease Control and Prevention. November 21, 2022. https://www.cdc.gov/ncbddd/musculardystrophy/facts.html.
“Muscular Dystrophy.” n.d. National Institute of Neurological Disorders and Stroke. https://www.ninds.nih.gov/health-information/disorders/muscular-dystrophy#:~:text=Although%20MD%20can%20affect%20several.
Nguyen, T M, I B Ginjaar, G J B van Ommen, and G E Morris. 1992. “Monoclonal Antibodies for Dystrophin Analysis. Epitope Mapping and Improved Binding to SDS-Treated Muscle Sections.” Biochemical Journal 288 (2): 663–68. https://doi.org/10.1042/bj2880663.
Yoshida, Kunihiro, Shu-Ichi Ikeda, Akinori Nakamura, Mitsuru Kagoshima, Shin’ichi Takeda, Shin’ichi Shoji, and Nobuo Yanagisawa. 1993. “Molecular Analysis of the Duchenne Muscular Dystrophy Gene in Patients with Becker Muscular Dystrophy Presenting with Dilated Cardiomyopathy.” Muscle & Nerve 16 (11): 1161–66. https://doi.org/10.1002/mus.880161104.
Smith, Mike. 2023. “Polymerase Chain Reaction (PCR).” Genome.gov. National Human Genome Research Institute. 2023. https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction.
MedlinePlus. 2020. “DMD Gene: MedlinePlus Genetics.” Medlineplus.gov. August 18, 2020. https://medlineplus.gov/genetics/gene/dmd/.
The Scientific Review Article published, “Molecular Correlations and Epitope Mapping in Muscular Dystrophy: Advancing Detection and Treatment Strategies for DMD and BMD,” was received on July 17, 2023, and was reviewed and accepted on July 18, 2023. To contact editors and reviewers please click here.
