Case Teaching Notes
for
“Tazswana’s Story:
How Alternative mRNA Splicing Leads to Genetic Disease and Cure”

by
S. Catherine Silver Key
Department of Biology
North Carolina Central University, Durham, NC


Introduction / Background

While students easily grasp the concepts of gene mutation, their effect on protein function, and their association with disease states, the concept of RNA processing is often foreign and less easily understood. To facilitate student learning of this concept, I have chosen to use a directed, interrupted case study method. This case engages students and allows them to ask questions and analyze information that is presented by the instructor in an efficient manner. During the class period, students use problem-solving skills as they are presented with increasingly complex information. Students are engaged in the story about a little girl with β-thalassemia, become curious in learning about how alternative pre-mRNA processing (splicing) causes her disease, and discover how gene therapy that targets the pre-mRNA splicing process may provide a cure for this life-threatening disease.

Tazswana’s case was created by integrating information from primary scientific publications on the molecular genetics of β-thalassemia and a type of gene therapy that specifically targets β-globin pre-mRNA processing. The innovative antisense gene therapy is also authentic. Importantly, this type of antisense gene therapy has been successfully applied in laboratory settings to correct particular defective splicing events that cause β-thalassemia. Thus, while the child, Taszwana, and her family are fictional, the cause of her β-thalassemia is based on mutations found in documented patients.

The case begins with an introduction to the importance of proper splicing to overall health, and from there leads students through the classical and then molecular genetics of the particular case. One feature of this case study is the series of handouts that allow students to use problem-solving skills as the material progresses from the relatively simple to the complex. Using the accompanying flow chart (see below), instructors will be able to implement this case study in a directed, interrupted manner.

Figure 1. Case Study Implementation Flow Chart

The case was originally developed at the June 2005 Case Studies in Science Summer Workshop at the University at Buffalo. At the workshop, it was designed to target non-majors, honors students. It was subsequently revised and tested in a junior level genetics course at North Carolina Central University. However, this case study could be modified for use in a cell, molecular genetics, or molecular biology course.

Objectives

Classroom Management

This case was designed for use in a 90-minute class period. We have used the case with student groups of three to five students per group. Students are introduced to β-thalassemia using a fictional story created from data found in the primary scientific literature. To implement this case study, students are provided with a series of handouts in a directed, interrupted style (see Figure 1). Basically, students receive a news article (Handout 1) and a pedigree worksheet (Handout 2) for homework. The instructor may assign additional questions to turn in as an incentive for completing all homework components. Students receive Parts I and II of Tazswana’s story in an interrupted style (Handouts 3 and 5). I separate the story by using PowerPoint slides to present information about hemoglobin structure and Tazswana’s blood test (see Handout 4). After reading Part II (Handout 5), students are given Handout 6 with Tazswana’s RNA test to complete. Handouts 7 and 8 allow students to analyze Tazswana’s sequence and evaluate the proposed gene therapy. During the case study, I also show the handout images as color PowerPoint slides to introduce each section of the case. A brief discussion on whether Tazswana should undergo the RNA gene therapy may ensue.

Prior Student Instruction

The study of RNA processing requires students to have had an introduction to the Central Dogma of Molecular Biology. Biology majors usually are familiar with the flow of information from DNA to RNA to protein and have prior knowledge about ribonucleotides, U G A C. However, they may not be aware that RNA can complementary base-pair to form the following two hybrids: (1) RNA-RNA (as in antisense oligo to pre-mRNA or snRNA to pre-mRNA) or (2) RNA-DNA (Northern blot and DNA oligonucleotides). As it is needed to discuss the therapeutic approach proposed in this exercise, these hybrid interactions can be introduced during the case study. One way to familiarize students with the concept of hybridization is to remind them that during DNA replication, complementary base pairing occurs between two DNA molecules: DNA-DNA. Spring-boarding from that familiar concept, students should easily understand that other nucleic acid combinations also form complementary base-pair interactions.

Further, it is assumed that genetics students will have been introduced to classical genetics including the interpretation of pedigree charts; thus, the homework assignment (Handout 2) will serve as a review, providing students the perspective needed for comprehension of this case.

In addition, students will require a basic introduction to gel electrophoresis and how to interpret “bands” on a gel in order to allow them to analyze the data. Building on the students’ prior knowledge, the course should introduce gel electrophoresis in lab or lecture activity before using this case study.

Finally, students need to have an introduction to β-globin and its role in forming hemoglobin and the shape of red blood cells either during the case study or in a previous class period. An explanation of the following six concepts are presented in the Detailed Case Analysis: the interactions of α-globin and β-globin in the formation of hemoglobin lattice, blood phenotypes, RNA analysis, splicing, the splicesome and pre-mRNA gene therapy.

Student Response

During the June 2005 Case Studies in Science Summer Workshop, test-students who were not biology majors were quite invested in the story and were anxious to discover how the oligonucleotide therapy would work. Genetics students at North Carolina Central University were equally engaged and progressed through the series of handouts and data analysis within the allotted 90 minutes. This case was not designed to introduce or discuss controversy; rather, its purpose is to lead students through a learning process in order to arrive at an understanding of the role of pre-mRNA processing in humans. Students have ample opportunity to interact within groups and use problem-solving skills as they strive to unravel the molecular cause and possible cure for the medical condition presented in “Tazswana’s Story.”

Other Possible Approaches and Follow-up Activities

As originally designed, the intent of the case is to focus on pre-mRNA processing in a directed, interrupted manner. However, instructors could broaden the scope of the case and increase discussion time by punctuating Tazswana’s story line with questions from Mrs. Williams including the following that explore evolutionary concepts (“Where did the disease come from?”), disparities in the health care system (“How could we avoid this disease as we have more children?”), and parental right issues (“Does her father need to agree to the informed consent for a clinical trial or can the mother be the sole guardian?”).

Intended to serve as a springboard for a unit on pre-mRNA processing in a genetics, molecular biology, or cell biology course, instructors may wish to pursue why the oligonucleotides are effective in blocking the splicing machinery by further explaining the RNA-protein nature of the splicesome. Alternatively, instructors may want to continue with the theme of “gene expression and genetic disease” since mutations that affect polyadenylation, transcription initiation, etc., cause many inherited diseases.

Follow-up activities may include investigating the differences between mutations that cause sickle cell anemia versus β-thalassemia, noting that β-thalassemia can also be caused by many mutations in the coding sequences of the β-globin gene, whereas sickle-cell is caused by one specific mutation.

Instructors may use the case to facilitate a discussion of treatment options for thalassemia patients in a human genetics course. Current options include frequent blood transfusions, bone marrow transplant, or hydroxyurea (HU) therapy. The latter therapy is a common and effective treatment for sickle-cell anemia. Hydroxyurea treatment increases expression of fetal hemoglobin production, bypassing the need for functional β-globin protein. Since there is only one sickle-cell allele in the β-globin gene and a plethora of thalassemia alleles in the same gene, it may not be surprising to learn that HU treatment is not as effective in treating all thalassemias. Recent clinical trials have identified specific patient genotypes that respond well to HU therapy, but most thalassemias do not (Alebouyeh, M., et al 2004). Certainly, this adds to the argument begun by Tazswana’s case that sequencing genes rather than merely observing blood samples is key to determining which treatment plan is most effective for each patient.

Alternative Implementation Plan

For shorter class periods or for general biology students, Handouts 1 and 2 may be used in a previous class along with an overview of the Central Dogma of Molecular Biology to orient students to the case study. Since student abilities are class dependent, questions and answers can be simplified per the instructor’s discretion. However, one suggestion for shortening the case for students in a course in General Biology or Human Genetics is to omit Handout 8, leaving students with the message that Tazswana will be treated with a gene therapy that will mask the mutated sites and force the cellular machinery to splice at the intact, appropriate sites. During the case study, I typically take class time to introduce a slide or two illustrating the action of the splicesome; however, it could be added to the homework assignment.

Interestingly, when an earlier version of this case was originally tested on non-majors, honors students, they really wanted to know what happened to Tazswana. Therefore, instructors will need to judge the ability of their students to comprehend the material in this case and may want to consider extending the case over multiple class periods to allow more discussion time.

Detailed Analysis

Detailed case analysis is provided in a separate file that is password-protected. To access this information, go to the detailed case analysis. You will be prompted for a username and password. If you have not yet registered with us, you can see whether you are eligible for an account by reviewing our password policy and then apply online or write to answerkey@sciencecases.org.

Answer Key

Answers to the questions posed in the case study are provided in a separate answer key to the case. Those answers are password-protected. To access the answers for this case, go to the key. You will be prompted for a username and password. If you have not yet registered with us, you can see whether you are eligible for an account by reviewing our password policy and then apply online or write to answerkey@sciencecases.org.

References

Alebouyeh, M., et al. 2004. Hydroxyurea in the treatment of major beta-thalassemia and importance of genetic screening. Annals of Hematology 83(7): 430–3.
Antonarakis, S.E., et al., 1984. Beta-thalassemia in American Blacks: novel mutations in the “TATA” box and an acceptor splice site. Proceedings of the National Academy of Sciences 81(4): 1154–8.
Boletini, E., et al. 1994. Sickle cell anemia, sickle cell beta-thalassemia, and thalassemia major in Albania: characterization of mutations. Human Genetics 93(2): 82–7.
Cartegni, L., and A.R. Krainer. 2003. Correction of disease-associated exon skipping by synthetic exon-specific activators. Nature: Structural Biology 10(2): 20–5.
Dimovski, A.J., et al. 1996. A large beta-thalassemia deletion in a family of Indonesian-Malay descent. Hemoglobin 20(4): 377–92.
Gorman, L., et al. 1998. Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs. Proceedings of the National Academy of Sciences 95(9): 4929–34.
Ho, P.J., and S.L. Thein. 2000. Gene regulation and deregulation: a beta globin perspective. Blood Reviews 14(2): 78–93.
Kole, R., M. Vacek, and T. Williams. 2004. Modification of alternative splicing by antisense therapeutics. Oligonucleotides 14(1): 65–74.
Kwiatkowski, D.P. 2005. How malaria has affected the human genome and what human genetics can teach us about malaria. American Journal of Human Genetics 77(2): 171–92.
Lodish, H., et al. 1999. Molecular Cell Biology. New York: W. H. Freeman and Company.
Michalowski, J. 2005. Alternative splicing. HHMI Bulletin 18(2): 22–27.
Panigrahi, I., et al. 2005. Hemoglobin E-beta thalassemia: factors affecting phenotype. Indian Pediatrics 42(4): 357–362.
Schrier, S.L. 1997. Pathophysiology of the thalassemias. The Albion Walter Hewlett Award presentation. Western Journal of Medicine 167(2): 82–9.

Suggested Online Textbook Resources

For more details on pre-mRNA processing, Entrez Pubmed Bookshelf (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books) provides free access to The Cell: A Molecular Approach, 2nd Edition. A search for “RNA processing and turnover” within the textbook’s search engine will pull up Chapter 6 hyperlinks complete with figures. An excellent resource can also be found in the Entrez Bookshelf: Molecular Biology of the Cell, 4th Edition by Alberts et al. A search in this online book for “RNA processing” will result in hyperlinks to pertinent book sections, one of which is “RNA Splicing Removes Intron Sequences from Newly Transcribed Pre-mRNAs,” which, coincidentally, is also Chapter 6. Recommended figures include alternative splicing, two types of splicing errors, and abnormal processing of β-globin. There are also nice figures of the overall splicing reaction and a summary of the steps of gene expression in the Alberts online text. Finally, Chapter 14 in Genetics: A Conceptual Approach, 2nd Edition by Benjamin Pierce contains free comprehensive, interactive animations for learning RNA processing: see http://bcs.whfreeman.com/pierce2e/.

Go back to the case

Acknowledgements: Initial work was supported by grant GM-000678 from the Minority Opportunities in Research Division of the National Institute of General Medical Sciences through the Seeding Post-doctoral Innovators in Research and Education (SPIRE) Program at the University of North Carolina at Chapel Hill and through a travel award from the National Center for Case Study Teaching in Science to attend the Case Studies in Science summer workshop held at the University of Buffalo, State University of New York, on June 6–10, 2005. I appreciate the insight and support of the following summer workshop colleagues: Gita Krishnaswamy, Christine Holler-Dinsmore, and Sarah Spinette. Additionally, I am grateful to Brian Rybarczyk and Mitch McVey for their valuable comments that helped to shape the teaching notes. Finally, I greatly appreciate the comments of the reviewers, who provided valuable suggestions for the refinement of this case study.

This case was developed with support from the National Science Foundation under CCLI Award #0341279. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Date Posted: 12/12/06 nas

Originally published at http://www.sciencecases.org/tazswana/tazswana_notes.asp

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