Systematic Analysis of Genetic Variation of Duchenne Muscular Dystrophy and Implication for Cancer

Student: Hubert Chen
Table: MED1222
Experimentation location: Home
Regulated Research (Form 1c): No
Project continuation (Form 7): No

Abstract:

Background—Duchenne muscular dystrophy (DMD) is a rare, severe, progressive genetic disorder causing disability and premature death. Mutations in the DMD gene encoding the dystrophin protein lead to the dystrophinopathies DMD. Phenotypic variations in DMD may also occur in patients with the same primary mutation due to secondary genetic modifiers. Recent advances in molecular therapies for DMD have need of precise genetic diagnosis, since a large number of therapeutic approaches are mutation specific. Interestingly, it has also been reported that muscular dystrophy patients may be at increased risk of malignancy. In this study, we performed a systematic analysis of the DMD genetic variants via the Single Nucleotide Polymorphism Database (dbSNP) and explored protein-protein interactions (PPI) for genetic modifiers identified in DMD patients. In addition, DMD genetic alternations in different tumors have also been investigated.


Methods—The genetic variants of DMD genes were extracted from the dbSNP database with variant call format (VCF). wANNOVAR was used to annotate functional consequences of genetic variations. PPI map for genetic modifiers identified in DMD patients was constructed using search tool for the retrieval of interacting genes/proteins (STRING v11), then subsequently analyzed using Cytoscape 3.8.1 plugin Network Analyzer. Genetic alternations in the DMD gene with cancer was examined by using Cancer Genomics Portal (cBioPortal). Kaplan-Meier curves were stratified by genotype and comparisons were tested using the Log-rank test.


Results—We examined a total of 3,627 exonic SNPs in the DMD gene. SNPs distributed across all exons. The largest category was nonsynonymous account for nearly 64% of all mutations. Exon 19 appeared to have most density of pathogenic SNP distribution. Nonsense mutation (i.e. stopgain) or frameshift mutation likely lead to more pathogenic. Among the genetic modifiers identified in DMD patients, THBS1 has higher network topological parameters, followed by SPP1, ACTN3 and LTBP4. The network enrichment p-value was < 3.09e-08 and implicated in several interconnected molecular pathways regulating inflammatory response to muscle damage, regeneration, and fibrosis. In addition, we also observed significant poorer overall survival for cancer patients with DMD mutations.


Conclusion—We conducted a data mining study with a systematic genetic analysis of all variants, especially SNPs, in one of the largest known human gene. Network analysis highlighted non-random interconnectivity between the genetic modifiers identified in DMD patients, and potentially shed light on new genetic modifiers by their functional coupling to these known genes. Our results also suggest DMD gene may serve as a diagnostic and therapeutic target for certain types of cancer.


Keywords— Data mining, DMD, Dystrophin, Exon, Genetic, Mutation, Protein-protein interaction, SNPs, Survival, Tumor

Bibliography/Citations:

1. Mohammed, F., et al., Mutation spectrum analysis of Duchenne/Becker muscular dystrophy in 68 families in Kuwait: The era of personalized medicine. PLoS One, 2018. 13(5): p. e0197205.

2. Gadalla, S.M., et al., Cancer risk among patients with myotonic muscular dystrophy. Jama, 2011. 306(22): p. 2480-6.

3. Win, A.K., et al., Increased cancer risks in myotonic dystrophy. Mayo Clin Proc, 2012. 87(2): p. 130-5.

4. Lab, W.G. wANNOVAR. 2010-2020; Available from: http://wannovar.wglab.org/.          

5. cBioPortal for Cancer Genomics. 2020; Available from: https://www.cbioportal.org/.

6. Nelson, S.F., et al., Emerging genetic therapies to treat Duchenne muscular dystrophy. Current opinion in neurology, 2009. 22(5): p. 532.

7. Chang, X. and K. Wang, wANNOVAR: annotating genetic variants for personal genomes via the web. J Med Genet, 2012. 49(7): p. 433-6.

8. Institute, E.M.B.L.s.E.B. DMD ENSG00000198947 Transcripts. August 2020; Available from:http://Aug2020.archive.ensembl.org/Homo_sapiens/Gene/Summary?g=ENSG00000198947;r=X:31097677-33339441.

9. Szklarczyk, Damian et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic acids research vol. 47, D1 (2019): D607-D613.

10. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T Genome Res. 2003 Nov; 13(11):2498-504.

11. ANNOVAR Documentation. Utilize update-to-date information to functionally annotate genetic variants detected from diverse genomes, wANNOVAR supports only human genome annotation 2010-2018; Available from: https://doc-openbio.readthedocs.io/projects/annovar/en/latest/.

12.  Gao, J., et al., Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal, 2013. 6(269): p. pl1.

13. Chang, X. and K. Wang, wANNOVAR: annotating genetic variants for personal genomes via the web. J Med Genet, 2012. 49(7): p. 433-6.

14.  Gao, Q.Q. and E.M. McNally, The Dystrophin Complex: Structure, Function, and Implications for Therapy. Compr Physiol, 2015. 5(3): p. 1223-39.

15. Bello L, Pegoraro E. The "Usual Suspects": Genes for Inflammation, Fibrosis, Regeneration, and Muscle Strength Modify Duchenne Muscular Dystrophy. J Clin Med. 2019 May 10;8(5):649.


Additional Project Information

Project website: -- No project website --
 

Research Plan:

A.  Rationale:

Duchenne muscular dystrophy (DMD) is a rare, severe, progressive genetic disorder causing disability and premature death. Mutations in the DMD gene encoding the dystrophin protein lead to the dystrophinopathies DMD. Phenotypic variations in DMD may also occur in patients with the same primary mutation due to secondary genetic modifiers. Recent advances in molecular therapies for DMD have need of precise genetic diagnosis, since a large number of therapeutic approaches are mutation specific. Interestingly, it has also been reported that muscular dystrophy patients may be at increased risk of malignancy.

Although mutations in DMD genes have been widely studied, to our knowledge, a systematic genetic analysis of all variants, especially single nucleotide polymorphisms (SNPs), of the gene in human have not been reported. The roles of genetic alternations in the DMD gene with different tumor types were not yet fully understood.

B. Research Questions:

  1. What’s the functional distribution of SNPs by exonic regions in the DMD gene and association with the rare genetic disease?
  2. What’s DMD gene variants frequency from different geographical regions?
  3. What’s protein-protein interactions for genetic modifiers identified in DMD patients?
  4. What’s the roles of genetic alternations in the DMD gene with different tumor types, and how impact on clinically relevant outcomes, such as overall survival?

C.  Aim:

This study aims to

  1. Carry out a systematic analysis of the DMD genetic variants via Single Nucleotide Polymorphism Database (dbSNP) database.
  2. Explore protein-protein interactions (PPI) for genetic modifiers identified in DMD patients.
  3. Investigate potential relationships of genetic alternations in the DMD gene with cancer.

D.  Methods and Procedures:

  1. Carry out a systematic analysis of the DMD genetic variants via dbSNP database.

The source data for the DMD gene comes from the dbSNP database, which was established by National Center for Biotechnology Information (NCBI) in collaboration with the National Human Genome Research Institute (NHGRI) in 1998 for GenBank of publicly available nucleic acid and protein sequences. The genetic variants of DMD genes will be extracted from the dbSNP database with variant call format (VCF). Use wANNOVAR to annotate functional consequences of genetic variations.  The longest transcript which provided the most genetic annotation information will be used as reference. Data mining and visualization perform using R-4.0.2.

         2. Explore protein-protein interactions (PPI) genetic modifiers identified in DMD patients.

A genetic modifier is a genetic locus that positively or negatively changes the phenotype of a primary disease-causing mutation. PPI map for genetic modifiers identified in DMD patients will be constructed using search tool for the retrieval of interacting genes/proteins (STRING v11), then subsequently analyzed using Cytoscape 3.8.1 plugin Network Analyzer. Nodes with high degree and betweenness centrality (BC) value will be considered as key parameters to analyze the network.

     3. Investigate potential relationships of genetic alternations in the DMD gene with cancer.

Genetic alternations in the DMD gene with cancer will be examined by using Cancer Genomics Portal (cBioPortal). This web portal provides query interface combined with customized data enabled us to interactively explore genetic alterations across DMD samples, genes mutation site and frequency. Datasets include published cancer studies from The Cancer Genome Atlas (TCGA) and selected pediatric cancer studies. Kaplan-Meier curves will be stratified by genotype and comparisons will be tested using the Log-rank test. 

E.  Risk and Safety:

No statements of approval or informed consent will be required for this study as we obtained data from an open access database. No Identify any potential risks and safety precautions in this study.

Questions and Answers

1.    What was the major objective of your project and what was your plan to achieve it? 

The major objectives of this project: 

  1. )    Carry out a systematic analysis of the DMD genetic variants via Single Nucleotide Polymorphism Database (dbSNP) database. 
  2. )    Explore protein-protein interactions (PPI) for genetic modifiers identified in DMD patients. 
  3. )    Investigate potential relationships of genetic alternations in the DMD gene with cancer.

       I developed a project research plan based on project goals, then actually executed the planned project steps to achieve goals. 

       a. Was that goal the result of any specific situation, experience, or problem you encountered?  
           Duchenne muscular dystrophy (DMD) is a fatal genetic disease without a cure. Recent advances in molecular therapies for DMD have need of         precise genetic diagnosis, since a large number of therapeutic approaches are mutation specific. Gene therapy is a promising experimental                         approach that uses genes to treat disorders that result from genetic mutations. There are huge unmet medical needs for the treatment of DMD.                   Leveraging biology to understand human diseases using advanced biotechnology has always fascinated me and the importance of understanding it has never been more important than it is today. I am very interested in gaining some insight about the disease that addresses real-world  challenges and look for innovative ways to solve them.
       b. Were you trying to solve a problem, answer a question, or test a hypothesis?
         I tried to answer some research questions. 

  1. )    What’s the functional distribution of SNPs by exonic regions in the DMD gene and association with the rare genetic disease?
  2. )    What’s DMD gene variants frequency from different geographical regions? 
  3. )    What’s protein-protein interactions for genetic modifiers identified in DMD patients?
  4. )    What’s the roles of genetic alternations in the DMD gene with different tumor types, and how impact on clinically relevant outcomes, such as      overall survival?

2. What were the major tasks you had to perform in order to complete your project?

The major tasks I had to perform in order to complete the project include develop a research plan, literature search, analysis data and report writing.

       a. For teams, describe what each member worked on.

        I independently conducted the research with supervising from my mentor.

3. What is new or novel about your project?

There are currently no curative therapies for DMD. Huge unmet medical needs still remain for the treatment of DMD. Historically, DMD gene mutations have been associated with the development of dystrophinopathies. However, the involvement of DMD gene in tumorigenesis is emerging. To our knowledge, this is first data mining study with a systematic genetic analysis of all variants, especially SNPs, of the DMD gene in human.

       a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before?

         The DMD disease is a new field to me. I haven't done the project before.

       b. Is your project's objective, or the way you implemented it, different from anything you have seen?

       To our knowledge, this is first data mining study with a systematic genetic analysis of all variants, especially SNPs, of the DMD gene in human.

       c. If you believe your work to be unique in some way, what research have you done to confirm that it is?

        I conducted a thorough literature search to confirm.

4. What was the most challenging part of completing your project?

DMD gene is one of the largest of the identified human genes. Interdisciplinary researches also require more quantitative skills in mathematics and computer sciences, along with a thorough knowledge of the associated biology. I need build my quantitative skills to analysis such complex disease.

      a. What problems did you encounter, and how did you overcome them?

There is a huge amount of stuff I need to learn, when starting on the new project. First, I needed to read literature to understand the disease, and to propose my hypothesis and research plan. However, sometimes the original hypothesis was not always supported by data or didn’t capture the full picture. I need rethink to modify the hypothesis and plan based on emerging data and then to test the modified hypothesis. This cycle of designing, testing, and failure is extremely important and also challenge to science because it makes sure that the end explanation is supported by data and encapsulates the complete picture.

      b. What did you learn from overcoming these problems?

The only way to make new scientific discoveries is by stepping into the unknown and to not be afraid of failure because I know that part of the journey is failure.

5. If you were going to do this project again, are there any things you would you do differently the next time?

When facing a new project, there appears to be a huge amount of stuff I need to know to tackle the problem properly. I will prioritize the most important steps and work on a regular basis.

6. Did working on this project give you any ideas for other projects? 

Yes. The current project demonstrates that the relationship between DMD genetic status and prognosis may be tumor-type specific. Its biological function in tumorigenesis as well as prognosis is complicated and remains to be further investigated.

7. How did COVID-19 affect the completion of your project?

I completed the research at home. I obtained data from an open access database, conducted qualitative data analysis, and discussed with my mentor via zoom meetings. Covid-19 has had no major impact my work.