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Teaching mitochondrial genetics & disease: a GENA project curriculum intervention.

Educational outreach by college and university faculty has been shown to have tangible benefits for participating students (National Science Foundation, 2007). In an effort to increase these activities, the American Society of Human Genetics partnered with the National Science Resources Center in 2006 to establish a professional development/research project called GENA (Geneticist-Educator Network of Alliances; gena.shtml), which was funded by the National Science Foundation. This initiative sought to promote educational outreach on a national level by establishing rigorous, long-term partnerships between university geneticists and high school biology teachers. The field of genetics was deemed an appropriate focus of this project because of the explosive growth of this discipline in recent years and its anticipated impact on healthcare in the future, making this an important area of educational emphasis. Here, we describe the results of one GENA partnership, a novel high-school-level learning plan focused on mitochondrial genetics and its important role in human disease.

One of the primary goals of GENA has been to produce original, freely available teaching plans designed to address a specific genetic concept; in this particular case, the focus was on patterns of inheritance. Inheritance is a fundamental genetics concept that likely receives appropriate attention in the science curricula of most states.

However, much of the emphasis in this regard tends to be directed toward Mendelian inheritance patterns, which are characteristic of genes found on chromosomes within the cell nucleus. By contrast, non-Mendelian inheritance, which includes (but is not limited to) traits associated with mitochondrial DNA (mtDNA), often tends to be overlooked. Therefore, the authors sought to create a learning plan that would address this topic, with a particular focus on human diseases associated with inherited mtDNA mutations. Ultimately, this concept led to the development of a curriculum piece designed to allow students to act as members of a virtual medical genetics laboratory. In this role, participants can utilize their newfound understanding of mitochondrial genetics to incorporate information about family history, clinical symptoms, and laboratory test results in order to establish an appropriate diagnosis for their "patients."

There are several reasons (both in terms of presenting new information and coordinating with an existing program of study) why this learning plan would be a useful addition within a high school biology curriculum. Beyond the fact that the plan emphasizes non-Mendelian inheritance, it also reveals the important association between mitochondria and human disease. Inherited mitochondrial diseases are a significant yet underappreciated source of morbidity and mortality that would likely benefit from increased attention from both scientists and clinicians. Therefore, in order to potentially increase awareness of these disorders among the next generation of biomedical specialists, it may be helpful to expose students (particularly those with a demonstrated aptitude) to these conditions within an appropriate context. In addition, although elements of mitochondrial genetics are integral to many basic biological concepts, mitochondrial disorders are typically not addressed in the general curriculum and are only briefly mentioned in the advanced/honors/AP biology curriculum (J. D. Sharer & R. A. Reardon, unpubl. data). This particular inquiry-based exercise fits effectively within the National Science Resources Center learning cycle ( resources/learning_cycle.html), and the diagnostic laboratory activities employed reinforce skills that may be acquired during other molecular biology activities. Finally, the pedigree analysis that is an integral component of the plan gives students an opportunity to make claims, use evidence, provide reasoning, and offer rebuttals to alternative claims (CERR model; McNeill & Krajcik, 2008).

** Mitochondrial Genetics

A single cell typically contains hundreds of individual mitochondria. Within each of these organelles are approximately 3-10 circular mtDNA molecules, which encode 13 of the ~80 proteins that constitute the electron transport chain (ETC), along with the transfer and ribosomal RNAs required for their synthesis. All the remaining ETC proteins are encoded by nuclear genes and must be transported to, and assembled within, the mitochondria (DiMauro & Schon, 2003).

The sequence of each mtDNA molecule is largely identical in most individuals. However, in some cases there is a variable mixture of normal and pathologically mutated mtDNA, a condition known as heteroplasmy (Smeitink et al., 2001). These mutations may be inherited (mtDNA is characterized by unique maternal inheritance, a classic example of non-Mendelian inheritance and an important element of this learning plan) or may arise randomly. Depending on the percentage of mutated mtDNA present and its tissue distribution, there may be significant impairment of normal function in the affected individual, which results in the symptoms of mitochondrial disease (DiMauro & Schon, 2003). These conditions tend to vary significantly in terms of the symptoms they produce and are often difficult to diagnose. This is largely because (1) the sensitivity of different tissues to damaged mitochondria is variable, (2) the relative levels of heteroplasmy can change over time (owing to random distribution of mtDNA during cell division), and (3) mitochondrial function can be significantly affected by environmental factors such as diet and medications (Smeitink et al., 2001; DiMauro & Schon, 2003).

Diagnostic methods for mitochondrial disorders have evolved rapidly in recent years and now include microarray and high-throughput sequencing techniques (Crimi et al., 2005; Li et al., 2010). However, older approaches such as polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis are still widely used for targeted mutation studies (Wong & Boles, 2005). This includes one of the most common mtDNA mutations: A3243G, in which an adenine residue is replaced by a guanine at position 3243 (Goto et al., 1990). This sequence change broadly disrupts mitochondrial protein synthesis by disturbing the transfer RNA for leucine ([tRNA.sup.leu]). Depending on the tissue distribution and level of heteroplasmy involved, A3243G may cause a variety of different clinical phenotypes, including diabetes or deafness. In particular, however, A3243G is often associated with a specific collection of symptoms that are known as MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes), which typically presents in childhood or adolescence (Shoffner & Wallace, 1995). When there is clinical suspicion of this disorder, PCR/RFLP analysis remains an effective means of identifying A3243G because this sequence change creates a novel restriction fragment pattern in mtDNA, thus revealing the presence of the mutation (Wong & Boles, 2005). Because the basic RFLP method can now be employed in many modern high school science classrooms, this technique was utilized in the learning plan. This approach can also be simulated effectively in settings where these methods are not currently available.

It is the authors' experience that many of the concepts described above are typically taught only in a postcollege, graduate setting--or often not at all. Furthermore, when these topics are taught in high school, they are often detached from other relevant curriculum components (e.g., DNA structure and function, organelle function, evolution, and the cellular basis for disease). Therefore, our intent was to reinforce molecular biology techniques and allow students to experience their relevance using an inquiry-based, mockclinical setting. Ultimately, utilizing concept mapping and guided by state and national teaching standards, an interactive learning plan (Mitochondrial Genetics and Disease Project; MGDP) was devised that simulates a clinical diagnostic laboratory. Using this approach, participants learn to appreciate the complexities of mitochondrial genetics and better understand the consequences when mitochondrial functions are disrupted.

** Standards & Objectives

Multiple state and national learning standards were incorporated into the development of the MGDP learning plan, and several specific educational objectives were also established (including content-specific concepts, identifying and addressing specific misconceptions, and achieving defined learning outcomes). These topics will be addressed in more detail in a future publication.

** Time Considerations

The optimal time frame for this module is 180 minutes of instruction, which allows students to focus on pedigree analysis and the complexities of mitochondrial genetics, participate in the laboratory analysis of "patient" DNA, and assimilate the various data to make reasonable claims about each patient's initial diagnosis of MELAS. The module can easily be used by teachers in traditional 47-minute periods, and also by teachers on a 94-minute block.

Teachers on a 47-minute period should teach the module during four class periods, in the following manner. On day 1, lead the students through analysis of three pedigrees (autosomal recessive, autosomal dominant, and maternal patterns of inheritance). End the first day with an overview of mitochondrial DNA and ask the students to read each patient's case history for homework. On day 2, the students perform the RFLP experiment (see below), followed by a discussion of the relationship between mitochondrial DNA and human health and disease, with a specific discussion regarding the relationship among the mitochondrial gene of interest, the electron transport chain, and metabolic dysfunction. At the end of the second day, gels are stained and set aside for day 3. For homework, the students should construct a pedigree for each patient (based on each patient's family history). On day 3, the teacher guides an analysis of the RFLP experiment and leads the students to the concept of heteroplasmy. The students draw their pedigrees on the board and leave them up until day 4; alternatively, they could tape pieces of paper containing their construct on a wall in the room. On day 4, the teacher coaches the students to reconcile the data from the electrophoresis experiment and the pedigrees and provide an overview of the synthesis questions.

Teachers on a 94-minute block should be able to utilize a two-period schedule. During the first day, the teacher leads the students through the pedigree analysis activity, focusing on mitochondrial genetics, the relationship between mitochondrial dysfunction and human health and disease, and the diagnosis of these patients. On day 2, the students perform the RFLP experiments and construct pedigrees while gel electrophoresis is underway; analyze the DNA patterns and reconcile the data from the RFLP experiment with the constructed pedigrees; and then make claims about each patient's initial diagnosis of MELAS. The students should then be assigned the synthesis questions as homework.

** Learning Plan

The pilot site for implementation of the MGDP learning plan was the Alabama School of Fine Arts (ASFA), an arts and sciences magnet school located in Birmingham, Alabama. The plan was adapted to the National Science Resources Center learning cycle rubric for teaching science and technology concepts and was integrated within the curriculum of two 11th-grade AP Biology classes composed of approximately 10 students each.

Part 1: Introduction & Pedigrees

The focus of the first, discussion-based session was to familiarize the students with pedigree analysis and introduce relevant aspects of mitochondrial genetics, particularly within the context of human disease. In terms of the former, the terminology and symbols used in a standard pedigree were first introduced. The students were then organized into small groups, which collaborated to identify the most likely inheritance pattern for a series of model pedigrees. These included examples of classic Mendelian inheritance patterns (e.g., recessive, dominant, X-linked); however, the final pedigree illustration demonstrated an unusual pattern: maternal inheritance. The introduction of this non-Mendelian inheritance pattern led to a discussion of mitochondrial genetics, with an emphasis on the contribution of mtDNA to oxidative phosphorylation and the important harmful consequences of mtDNA mutations on this process.

Part 2: Molecular Diagnostics

At the beginning of the second session, the students were engaged in the central problem of the learning plan: using virtual clinical and laboratory approaches to determine whether the presenting symptoms (described below) in two ersatz patients might be due to the presence of the A3243G mutation in their mtDNA.

* PATIENT 1: Seven-year-old female with a history of normal development until age 2. At this point she developed episodic vomiting, acidosis, epilepsy, general weakness, ataxia (stiff, unsteady gait), and dystonia (movement disorder characterized by involuntary muscle contractions).

* PATIENT 2: Fifty-year-old male with sudden-onset headaches and seizures. Patient has a history of diabetes and deafness. Magnetic resonance imaging (MRI) detected bitemporal lesions (dual areas of abnormal tissue within the brain).

In order to better understand the complexities of disorders associated with mtDNA mutations in general, and MELAS in particular, a significant amount of didactic effort was devoted to the underlying cause of this condition. Again, an important point of emphasis was the potentially devastating consequences of a seemingly minor, single-nucleotide change in the mtDNA sequence. Point mutations such as this may result in dysfunction of an isolated ETC protein, or can alter the structure of a transfer RNA, affecting the synthesis of multiple proteins (as in A3243G). In either case, if ETC function is compromised, this defect can be propagated throughout successive levels of complexity (organelles, cells, tissues, and organs), ultimately resulting in the symptoms associated with mitochondrial diseases such as MELAS. In this particular condition, organ systems such as skeletal muscles and the central nervous system are prominently involved (resulting in weakness, numbness, uncoordinated gait, seizures, stroke-like episodes, and deafness). In addition, lactic acidosis, a common but nonspecific systemic indicator of mitochondrial disease, is often (but not always) observed.

Following the discussion of mtDNA mutations and disease, the students were provided the opportunity to perform hands-on molecular diagnostic testing of "samples" from each patient. It was emphasized that, in combination with clinical and pedigree analysis, appropriate laboratory studies can provide compelling information regarding the diagnosis for each patient. The major elements of this exercise are shown in Figure 1 and described below.

In the ASFA setting, the students were provided two samples that had previously been prepared for gel electrophoresis. Supposedly, these represented processed blood samples collected from each patient; in reality, these samples were artificial and had been prepared to simulate RFLP analysis of each patient's mtDNA (Figure 1). If it was actually being performed, this technique would consist of PCR amplification of a 600-base-pair (bp) region spanning the mtDNA-[tRNA.sup.leu] coding region, followed by restriction enzyme digestion of the PCR product (Figure 1). Although not included in the standard version of the learning plan, PCR amplification and restriction digestion could be included as part of a more comprehensive laboratory exercise, if desired.

When the nucleotide sequence at position 3243 is normal (occupied by an adenine residue), the resulting PCR product is cleaved by the restriction endonuclease to produce two fragments of 400 and 200 bp (Figure 1). However, A3243G destroys the restriction site and prevents digestion of the PCR product (resulting in a single ~600 bp fragment), thus providing a simple yet effective means of identifying the presence of this mutation (indeed, this technique remains a commonly employed approach for the detection of this and other specific mutations in many diagnostic laboratories).

Following an explanation of the RFLP method, the students were divided into approximately three groups in order to perform duplicate gel electrophoresis on the patient samples. The samples to be analyzed included a reference molecular weight marker and appropriate controls, in addition to the two patient samples (Figure 1). Once electrophoresis was completed, the separated DNA fragments were visualized and photographed to facilitate further examination of the results. If preferable, gel electrophoresis can be simulated, providing another point in which the learning plan can be modified to suit different educational settings.

Part 3: Associate Gels with Pedigrees

The goal of the final session was to allow the students to reflect on and interpret the data they had collected. First, the consequences of changing the DNA sequence of a restriction enzyme recognition site was reviewed in terms of the target A3243G mutation and in the context of the RFLP analysis performed in lesson 2. Then the students were provided with family histories for each patient, allowing them to construct specific pedigrees that would aid diagnostic evaluation (Figure 2).

With all available data now in place, the students were asked to provide a diagnosis for each patient and defend these assertions if alternative explanations were proposed. Finally, in order to complete the learning plan, each student was required to formally document their findings and interpretations using the CERR paradigm of summative assessment (McNeill & Krajcik, 2008).

Part 4: RFLP Analysis

A refined, user-friendly version of the MGDP learning plan is now commercially available through the scientific supply company PASCO (Mitochondrial Genetics Kit, PASCO catalog no. BP-6946; the associated users' manual is freely available at pascostore/showdetl.cfm?&DID=9&Product_ID=60978&groupID= 701&page=Manuals).

The RFLP analysis can also be performed using readily available scientific supplies. For example, to produce appropriate DNA fragments, the common cloning vector pUC19 can first be digested with BsaXI + Pfo I, which will yield a 613-bp fragment that simulates the original mtDNA PCR product. Following purification of this fragment, digestion with Hind III will yield fragments of 207 and 406 bp (thus mimicking the normal [tRNA.sup.leu] digestion). Several other potential strategies for producing the appropriate DNA pieces are likely to exist. If desired, actual PCR amplification of an appropriate DNA sequence can be performed prior to treatment with restriction analysis.

Once the necessary DNA fragments have been prepared, they can be analyzed using standard agarose gel electrophoresis materials and techniques. We have achieved consistent results using 0.8% agarose gels run at 150 volts for approximately 20-30 minutes. The gel should include an appropriate DNA molecular weight marker (e.g., Sigma catalog no. D5042), along with a normal control sample (400 + 200 bp), abnormal control (600 bp only), patient 1 (normal pattern), and patient 2 (600 + 400 + 200 bp). Once electrophoresis has been completed, the DNA fragments can be visualized using a variety of methods (e.g., acridine orange) and photographed for analysis.

** Assessment Techniques

Two methods were utilized to evaluate the MGDP learning plan: a student questionnaire (employed in years 1-3) and a pre- and post-intervention examination (year 3 only).

The student questionnaire was administered immediately after completion of the MGDP learning plan. Based on an initial prompt ("This series of laboratories and learning cycles helped me better understand ..."), the students were asked to rate different topics addressed in the intervention (see Figure 3; strongly agree = 10 pts; agree = 5 pts; neutral = 0 pts; disagree = -5 pts; strongly disagree = -10 pts). Results were quantitated by assigning these numerical values to each response as indicated. Impact scores were then calculated for each topic by determining the total points for each category and dividing by the number of respondents; the higher the impact score, the greater the perceived educational benefit of the learning plan for the category.

Pre- and post-intervention examinations were utilized in the third iteration of the MGDP plan (Figure 3). Each student in the two participating AP Biology classes was given a short quiz immediately before participation in the plan, and then the same questions were asked again immediately thereafter. The test consisted of 11 short-answer questions that addressed knowledge of pertinent information covered by the MGDP learning plan.

** Results

Diagnostic Laboratory Exercise

In lesson 3, having collected information about each patient's clinical symptoms, family history, and laboratory results, the students were asked to explain their diagnoses. As shown in Figure 1, lane 4, the restriction pattern from patient 1 was determined to be normal (digested), indicating the absence of the A3243G mutation in circulating mitochondria. This provided evidence against MELAS due to A3243G in patient 1. It was emphasized that this result did not rule out the possibility that another mitochondrial mutation (or a nuclear DNA mutation) could be causing the patient's symptoms, or that the mutation might be present in other tissues.

Patient 2 (Figure 1, lane 5) was found to have an unexpected result, namely three discrete DNA bands as opposed to one (consistent with the presence of the A3243G mutation) or two (normal digestion), a result that was not previously considered. This situation offered an opportunity for the students to apply their newly acquired knowledge to provide an explanation. Ultimately, using inquiry and deductive reasoning, the students realized that this result was suggestive of heteroplasmy with respect to the mtDNA in patient 2's blood cells--that is, there exists a mixture of both normal and A3243G sequences (which is typical of MELAS and other mtDNA disorders). This result, along with the family history and clinical findings, was suggestive of MELAS in this patient. It was noted that this would represent an atypical presentation of the disease in an older individual, consistent with a progressive increase in levels of heteroplasmy over time.

Student Survey

The survey was designed to assess the perceived educational impact of the plan across a range of different concepts and was administered immediately after the learning plan in each of the 3 years that it was employed (total participants = 56).

The cumulative impact scores for each category are shown in Figure 3. Based on these data, it is evident that the greatest perceived benefit was achieved regarding pedigrees and pedigree analysis, structure and function of mitochondria and mtDNA, and Mendelian and non-Mendelian patterns of inheritance. Lower scores were observed regarding the impact of the intervention on structure and function of DNA and RNA, which were not covered in the plan, and thus essentially represented controls for the survey. It was noted that impact scores for the concepts cellular respiration, oxidative phosphorylation, use of restriction enzymes in biotechnology, and biotechnology skills were in an intermediate range, consistent with the fact that these areas had been described to the students previously; hence, the educational benefit in these areas was not perceived to be particularly high.

The results of the pre- and post-intervention examinations given in year 3 are shown in Figure 3, which indicated, for each class, the pre- and post-exam performance for each question (percentage of correct scores) and the average scores. Not surprisingly, the post-exam scores demonstrated significant improvement in relation to the student's performance before the learning plan. Although not unexpected, these data indicate a positive educational impact in terms of understanding relatively advanced genetic and biological concepts.

** Conclusions

This report describes the development, implementation, and assessment of a high-school-level learning plan that was created as part of the GENA Project, a national collaborative program designed to encourage educational outreach by college and university genetics faculty. As members of the pilot GENA cohort, the authors addressed the concept of non-Mendelian inheritance by developing a learning plan that simulates a diagnostic laboratory setting, with a focus on mitochondrial genetics and disease. The pilot version of the MGDP learning plan was first implemented in the curriculum of an AP Biology course in fall 2007; the third and final iteration of the plan took place in 2009. At least one assessment technique was utilized in all 3 years, including a pre-and post-intervention exam that demonstrated conceptualization of learning-plan elements by participants. Finally, in addition to the written description in the online GENA database (, an optimized version of the plan has been developed and made commercially available through a partnership between the authors and Edvotek and PASCO Scientific (Roseville, CA; This kit includes thermostable DNA samples, teacher background information, student protocols, and a summative assessment form.

DOI: 10.1525/abt.2012.74.4.4

** Acknowledgments

We thank Bo Yuen and Val Zvereff for their assistance with DNA manipulations in years 1 and 2. We also acknowledge past and present investigators of the GENA Project (Kenna Shaw, David Marsland, Henry Milne, Toby Horn, and Michael Dougherty) as well as the American Society of Human Genetics, the National Science Foundation, and the National Science Resource Center for their support. Finally, we thank Nassim Lewis, PASCO Scientific, and EDVOTEK for their assistance.


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RYAN A. REARDON is Science Content Director, A+ College Ready, Birmingham, AL 35294; e-mail: J. DANIEL SHARER is Assistant Professor of Genetics at the University of Alabama-Birmingham, Kaul 652A, 720 20th Street South, Birmingham, AL 35294; e-mail:
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Author:Reardon, Ryan A.; Sharer, J. Daniel
Publication:The American Biology Teacher
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2012
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