Case Teaching Notes
for
“Pharmacogenetics: Using Genetics to Treat Disease”

by
Jeanne Ting Chowning, Director of Education, Northwest Association for Biomedical Research

Introduction / Background

This case study investigates the applications of genetics to medicine. Specifically, the case explores one of the first examples of a pharmacogenetic test to enter mainstream clinical practice. The discipline of pharmacogenetics examines how genetic variations in an individual correlate with their responses to a specific medication. The ultimate goal of pharmacogenetics is to develop medical treatments tailored to the individual.⁽¹⁾ Through a brief fictional scenario, students are introduced to the disease involved (acute lymphocytic leukemia) as well as the wide range of individual responses to the drug used to treat it. Students then interpret data similar to those initially published in scientific journals in order to construct an understanding of how genetic variation can be used to “tailor” medical care. Finally, students are asked to apply their understanding of the case material by making the appropriate medical recommendation based on a particular individual’s genotype.

This case is well-suited for undergraduate introductory biology, genetics, or molecular biology classes or professional programs such as nursing, pharmacy, or other health care educational programs. Although originally designed for a high school biology course, the case has also been used in high school biotechnology, advanced biology, and chemistry courses. The high school students who tested this case were very engaged with it and were able to follow the major concepts readily. It reinforced prior learning in genetics and provided a concrete example of how a pharmacogenetic test might be used in a health care setting. Students also enjoyed “puzzling” through the original data and drawing conclusions.

The case assumes some knowledge of DNA structure and function, protein structure, enzymes, and Mendelian genetics. The case is best conducted over a minimum of two 50-minute periods. An additional day could be spent exploring molecular structures and molecular models as well as the ethical considerations related to the use of genetic information to make medical decisions. The case can also be followed by a biotechnology unit where some of the tools related to the identification of specific DNA sequences can be taught.

Objectives

Upon completing the case, students will:

• Be able to use scientific/case study data and interpret graphs in order to draw conclusions.
• Understand how individual genetic variation can impact medical practice and clinical outcomes, using the example of leukemia and thiopurine methyltransferase (TPMT).
• Be able to predict how polymorphisms in the gene for TPMT indicate courses of medical care for particular individuals.

Enduring Understandings⁽²⁾

• Information about a patient’s genetic profile may allow for an individualized assessment of patient risk and customized design and delivery of therapies.

• Genetic information and environmental factors interact to produce disease states, influence treatments, and impact medical outcomes.

• The incorporation of new technologies to medicine raises ethical issues, such as those related to resource allocation, drug testing, and genetic privacy. (Extension)

National Science Standards: Grades 9–12

This case addresses a number of the National Science Standards established for grades 9 through 12 by the National Research Council.⁽³⁾

• Science as Inquiry
  • Abilities necessary to do scientific inquiry
  • Understandings about scientific inquiry
• Physical Science
  • Chemical reactions
• Life Science
  • Molecular basis of heredity
  • Biological evolution
  • Matter, energy, and organizations in living systems
• Science and Technology
  • Abilities of technological design
  • Understandings about science and technology
• Science in Personal and Social Perspectives
  • Personal health and community health
  • Science and technology in local, national, and global challenges
• History and Nature of Science
  • Science as human endeavor
  • Nature of scientific knowledge
  • Historical perspectives

Classroom Management

Teaching the Case

There are a variety of different approaches that could be used when teaching this case.

Suggested Approach

Part I: Acute Lymphocytic (Lymphoblastic) Leukemia (Estimated time: 25 minutes)

Have students read the case study and answer the questions in Part I. After they have written their answers, ask students to share them in pairs before eliciting answers from the whole group.

Part II: Enzyme Activity (Estimated time: 25 minutes)

Review the information in Part II with the whole class. Afterwards, allow students to interpret the data individually. Discuss the answers with the whole class.

Part III: TPMT Enzyme Activity Levels (Estimated time: 15 minutes)

Allow students to complete this section individually, and then review the material with the whole class.

Note: Instructors might want to break the case here, covering Parts I–III above in a single classroom session, and Parts IV and V below in a second session.

Part IV: Putting It All Together (Estimated time: 10 minutes)

Allow students to complete this section individually, and then review the material with the whole class.

Part V: SNPs and TPMT (Estimated time: 15 minutes)

Students must apply their understanding and make recommendations about the best course of treatment for a third patient.

Variations to the Above

Part I

If time is limited, students may read the case and answer the questions in Part I for homework. Class can begin with a quick quiz, perhaps using personal response system (“clickers”). Students can also role-play the parts of Laura, Beth, and Dr. Ryder and have the actors ask questions at the end to the class.

Parts II–IV

You may wish to have students complete these section in pairs rather than individually.

Overall

Instead of having students work through the questions in the sheets provided, you may wish to use the data for presentation slides and use the questions as prompts for discussion.

Additional Components

Molecular Modeling (Estimated time: 30–60 minutes)

This component is recommended for upper-level college undergraduates. Walk students through the steps outlined on the sheet as they work at a computer, or demonstrate the models to them with a projector attached to a computer. The models can be shown when the molecules in question are reviewed with the class. Note that the structure files can be downloaded and then viewed off-line provided you have the Cn3D software on your computer.

Ethical Implications (Estimated time: 60 minutes)

Utilizing background information in these teaching notes (see Pharmacogenetics and its Social Context below), discuss some of the ethical implications of pharmacogenetics with students. For example, students could brainstorm the main ethical issues associated with the case. One ethical question can be chosen for further analysis. Then, students can identify the relevant facts and who or what stands to be impacted by how the ethical question is resolved (“the stakeholders”). Lastly, students can identify several possible solutions to the ethical issues, selecting the one solution they believe is strongest and justifying their position. A variation would be to assign students to single-stakeholder groups and have students identify the values and concerns of their stakeholder before moving to mixed-stakeholder groups to share their values and concerns and to discuss potential options. For example, students could role-play different stakeholders related to FDA approval of the genetic test for TMPT variation, and then decide as a group whether or not the test should be approved. Alternatively, after group discussion, students could write their own position paper. Afterwards, students can compare their position with the actual FDA position. Further ideas for discussion structures related to bioethical issues can be found at http://www.nwabr.org/education/primer.html.

Flow Chart—Comparison of Drug Metabolism

The flow chart (also available as either a PDF or a MS Word document) summarizes how the thiopurine drugs are metabolized differently by individuals who are tolerant to the drugs compared to those who are not. Instructors may also wish to ask students to develop a concept map that shows the relationships between key concepts in the case.

Blocks of Analysis

Thiopurine Drugs

• Thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine are used to treat Acute Lymphocytic Leukemia (ALL), autoimmune disorders, inflammatory bowel disease, and organ transplants.

• They are guanine analogs, and have a sulfhydryl group attached to C6 (see below).

• Thiopurines are “prodrugs” and are inactive until they are processed by the body into active form.

• Thiopurines are converted in the body to thioguanine nucleotides, which can be incorporated into DNA during replication.

• When incorporated into replicating DNA, the altered nucleotides stop the process of replication from continuing. Thiopurines can also impact the purine biosynthesis pathway.

• Thiopurines especially affect cells that are frequently replicating (such as cancer cells).

• Thiopurines have a “narrow therapeutic index,” which means that they can be very toxic if not administered correctly. [The “therapeutic index” compares the amount of a drug that causes a therapeutic effect to the amount that causes death (by dividing the lethal dose for 50% of the population by the minimum effective dose for 50% of the population). If there is little difference between lethal and therapeutic doses, the drug is termed to have a “narrow therapeutic index.”] The ideal dosage is high enough to damage frequently dividing cancer cells, but low enough that excess drug can be cleared with the aid of the TPMT enzyme.

• When the thiopurine drug levels are too high, a life threatening condition called myelosuppression can occur. This is a decrease in the production of white blood cells, red blood cells, and plasma cells in the bone marrow.

Metabolism

The following points address how the thiopurine methyltransferase (TPMT) enzyme found in the body metabolizes the thioguanine nucleotides (TGN) created by the thiopurine drug

• The TPMT enzyme adds a methyl group (CH₃) to the sulfhydryl group (SH), which prevents the incorporation of the thioguanine drugs into replicating DNA.

• The enzyme helps the body metabolize/process the thiopurine drugs by limiting their potency (“deactivating”).

• Several different genetic polymorphisms of the gene result in an enzyme that has low activity, probably because the enzyme is more susceptible to degradation.

• In a large population of Caucasians, 89% were homozygous for “high activity” alleles, 11% heterozygous (one “high” and one “low,” resulting in intermediate activity), and 1/300 were homozygous for “low activity.” (Later reports of East Asians found no such distribution among them).⁽⁴⁾

• Those individuals homozygous for low activity alleles are not able to process the thioguanine nucleotides well, and when the thiopurine drug is administered at the usual level it can result in death.

• The natural substrate for TPMT has not yet been identified.

Thiopurine Methyltransferase (TPMT) Gene

Genetic variation in the TPMT gene

• The TPMT gene is 34kb in length and consists of 10 exons.

• It is located on the short arm of chromosome 6.

• While several variant alleles exist, TPMT 3*A is the most common variant allele for low TPMT activity in Caucasians. It is currently thought that 6 variants exist.⁽⁵⁾

• TPMT*3A has two single nucleotide polymorphisms (SNPs) that alter the encoded amino acids (i.e., they are nonsynonymous SNPs).

• In Asians, TPMT*3A is either not present or occurs at very low frequency, and TPMT*3C is the most common variant allele.

• In addition, the 5’ region of the TPMT gene includes a variable number tandem repeat (VNTR) in which a 17 or 18 base pair element is repeated from four to eight times. Studies have suggested an inverse relation between the total number of VNTRs on both chromosomes and TMPT activity.

• Researchers at several institutions, including St. Jude’s Children’s Hospital in Memphis, Tennessee, and at the Mayo Clinic in Rochester, Minnesota, screen patients to determine their TPMT genotype before administering chemotherapy with thiopurine drugs.

Pharmacogenetics—Other Examples

Students may also be interested in some of the other SNPs associated with medical or health-related outcomes. These examples take some time to explain and could be their own cases. Students should realize that some pharmacogenetic effects are due to a few genetic differences, but many are caused by multiple genes and more complex interactions. The additional examples may be most appropriate for advanced undergraduates.

• Cytochrome P-450 Enzymes (CYP) and Drug Processing—There are over a thousand different CYPs, found mostly in the liver. CYP enzymes play an important role in helping the body to process harmful substances by making them more water-soluble. A 1997 study of over 315 drugs found that 56% of them were cleared by enzymes in this family.⁽⁶⁾ Sometimes they play an important clinical role in converting a drug into a more active form. Many important CYP genes are found to vary between individuals (either in the number of copies of the genes or in their sequence), and some of this variance accounts for different responses to drugs. Some extensively studied CYP genes include 2C9 and 2D6. CYP2D6, CYP2C9, CYP2C19, and CYP1A2 testing is currently offered by the Seattle-based Genelex company.⁽⁷⁾

• Aldehyde Dehydrogenase and Alcohol Metabolism—Alcohol dehydrogenase and aldehyde dehydrogenase together help detoxify a wide range of organic compounds, toxins, and pollutants. Population studies have indicated that about half of certain Asian populations, including Chinese, Japanese, and Koreans, have a deficiency in the enzyme aldehyde dehydrogenase (ALDH2), which processes acetaldehyde. Acetaldehyde is a product of alcohol metabolism. The mutant ALDH2*2 allele, which is incompletely dominant, reduces enzyme activity and increases enzyme turnover. Asians possessing one or more of the ALDH2*2 alleles become visibly flushed after drinking alcohol, in contrast to those who are homozygous for ALDH2*1.⁽⁸⁾

• Herceptin® and Breast Cancer—Breast cancers are varied; each has its own set of genetic mutations, tissue characteristics, and optimal treatment. Women who have metastatic breast cancer (cancer which can spread to other organs) and also overexpress the HER2/neu gene have an aggressive form of the disease. Approximately 25–30% of metastatic breast cancer tumors overexpress the HER2/neu gene, which codes for a cell surface receptor. The receptor is thought to play a role in normal cell growth by signaling the cell to divide. When the HER2 gene is overexpressed, extra receptors are produced on the cell surface, triggering uncontrolled growth and cancer. Herceptin® is an artificially developed monoclonal antibody specifically targeted against the HER2 receptor. Herceptin® is thought to work by binding to receptor sites on the cell surface, limiting cell division and growth.⁽⁹⁾

• Warfarin and Blood Clotting—Warfarin is a widely used blood anti-coagulant. It has a “narrow therapeutic range,” meaning that the dosage is very important (too much of the drug leads to excess bleeding, too little results in blood clotting). The main enzyme involved in warfarin metabolism is CYP2C9, and research indicates that two common variant alleles (polymorphisms) of the CYP2C9 gene appear to influence patient outcomes. The *2 and *3 variants appear to have reduced activity and patients with these genotypes require lower doses of the drug.⁽¹⁰⁾ Recent research indicates that variants in the VKORC1 gene encoding the actual protein target of warfarin (vitamin K epoxide reductase, a protein involved in vitamin K metabolism) also critically impacts drug response.⁽¹¹⁾ The VKORC1 genotype explains approximately 20–25% of variation in response to warfarin, and the CYP2C9 genotype explains approximately 6–10% of the variation. Studies also suggest that persons of Asian, European, and African ancestry tend to require on average lower, intermediate, and higher doses of warfarin, respectively.

Pharmacogenetics and its Social Context

The use of genetic information to determine how medicine is practiced involves many ethical, legal, and social issues. For example:

• Resource Allocation Issues—Developing treatments based on genetic information is costly and time-consuming. Is it “fair” use of our resources as a society to focus on developing such technologies, especially when they may benefit a limited number of people or be available only to those who could afford them? How is such research situated in the larger context of health care disparities in our country?

• Genetic Privacy Issues—How can we be sure that genetic information is used to benefit patients and not discriminate against them? What are the implications of having genetic information about your health? Who should have access to that information? Should that information be available to prospective employers and insurance agencies? Should laws be enacted to protect genetic privacy? Is it fair to insurance agencies if you know you are at risk for a certain disorder and you don’t divulge that information? In certain cases, such as the TPMT one described in this lesson, knowing one’s genetic status can lead to specific medical actions. Also, since TPMT is not currently known to cause disease, the genetic information is not stigmatizing.

• Ethnic/Racial Differences and Allele Types—Different alleles are often found to be associated more frequently with particular ethnic/racial groups. In the TPMT example, *3A allele is most frequently found in white European populations, is less frequently found in African Americans, and has never been seen in East Asians. The *3C allele is the most common variant in East Asian populations. How will the discovery of genetic information about populations and the utilization of that information for medical purposes impact our understanding of race?

• TPMT and the FDA—In 2003, the FDA analyzed information available about the TPMT enzyme and its role in processing thiopurine drugs, in order to decide whether or not to require testing of patients prior to administration of those drugs. They decided against mandatory testing for several reasons:

  • Testing would be costly, especially given that there are so few people who are strongly negatively affected by the drug (1 in 300 Caucasians).
  • Patients are already watched closely when the drugs are administered.
  • Doctors may shy away from using a potentially life-saving drug because of the added burden of testing.
  • The information about whether heterozygotes (people in the intermediate range of enzyme activity, with one copy of the normal activity and one copy of the low activity alleles) benefited from testing was not conclusive.

  Instead of requiring patient testing, the FDA required labeling of the drug packaging. While a few institutions test their patients, they are not required by law to do so.

  In 2005, the FDA announced the first drug that would require testing before it could be prescribed (Aczone, an acne drug).⁽¹²⁾

Drug makers will be greatly influenced by the new developments in this field—they will be able to sell their drugs to potentially fewer people, but those people are more likely to benefit from the drugs and less likely to file damaging lawsuits.

The fear of lawsuits may also drive doctors to recommend tests. Ethical dilemmas may arise for doctors whose patients want drugs that may not be recommended for them based on their genetic profile. There are private companies such as GeneLex that now offer tests privately to individuals who are interested in learning their genotypes in genes related to drug processing.

Answer Key

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Footnotes and References

(1) Genetic Science Learning Center

Pharmacogenetics definition: http://gslc.genetics.utah.edu/units/pharma/phwhatis/

TMPT Animation: http://gslc.genetics.utah.edu/units/pharma/phmedcare/

(2) Jay McTighe and Grant Wiggins define “enduring understandings” in the book, The Understanding by Design Handbook, as “… the big ideas, or the important understandings, that we want students to really ‘get inside of’ and retain after they’ve forgotten many of the details. Put differently, the enduring understandings provide a larger purpose for learning the targeted content: They implicitly answer the question: Why is this topic worth studying?” Thus, “… enduring understandings … anchor the unit and establish a rationale for it.”

(3) National Research Council (NRC). (1996) National science education standards. Washington, DC: National Academy Press.

(4) Weinshilboum, R. (2001) Thiopurine pharmacogenetics: Clinical and molecular studies of thiopurine methyltransferase. American Society for Pharmacology and Experimental Therapeutics 29:601–605. Available online at http://dmd.aspetjournals.org/. This case is based on the above article.

(5) OMIM: Online Mendelian Inheritance in Man

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=OMIM&dopt=Detailed&tmpl=dispomimTemplate&list_uids=187680

(6) http://www.anaesthetist.com/physiol/basics/metabol/cyp/cyp.htm

Accessed 07/08/09

(7) http://www.healthanddna.com/ConsumerGeneTests_%20HelporHarm_.pdf

Accessed 07/08/09

(8) http://www.annals.org/cgi/content/full/127/5/376

Accessed 07/08/09

(9) http://www.genetics.com.au/factsheet/fs25.html

Accessed 07/08/09

(10) Higashi MK, Veenstra DL, Kondo LM, Wittkowski AK, Sriouanprachanh SL, Farin FM, Rettie AE. Association between CYP2C9 genetic variants and anticoagulation-related outcomes during warfarin therapy. JAMA. 2002; 287: 13: 1690–1698.

(11) Rieder MJ, Reinier AP, Gage BF, Nickerson DA, Eby CS, McLeod HL, Blough DK, Thummel KE, Venstra DV, Rettie AE, Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose. NEJM. 2005; 352; 22: 2285–2293.

(12) Pollack, A. A Special Drug Just for You, at the End of a Long Pipeline, New York Times, November 8, 2005.

http://www.nytimes.com/2005/11/08/health/08phar.html

Additional Resources

http://discoverysedge.mayo.edu/pharmacogenomics/

Brief overview of the TPMT case and other pharmacogenetic examples in layperson’s terms.

Mancinelli, L., M. Cronin, and W. Sadee (200) Pharmacogenomics: The promise of personalized medicine. AAPS PharmSci. 2 (1): Article 4. DOI: 10.1208/ps020104 Good overview of pharmacogenetics and pharmacogenomics with table providing examples of inherited or acquired variations in enzymes and receptors that affect drug response.

http://www.aapspharmsci.org/view.asp?art=ps020104

http://www.ornl.gov/sci/techresources/Human_Genome/medicine/pharma.shtml

General primer on pharmacogenomics.

Krynetski, E.Y., and W.E. Evans (1998) Pharmacogenetics of cancer therapy: Getting personal.

Am J Hum Genet 63:11–16. Available online via PubMed at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1377260/

More scientific background on TPMT

GeneTests and GeneReviews

http://www.genetests.org

Weinshilboum, R., and L. Wang. (2004) Pharmacogenomics: Bench to bedside. Nature Reviews: Drug Discovery 3:739–748.

This comprehensive review article provides background on the development of pharmacogenetics/genomics and highlights important aspects that need to be considered in order to bring the scientific developments into clinical practice. TPMT is a featured example.

Date Posted: February 4, 2010.

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