* This case study presents a fictitious scenario, but one that is based upon clinical observations.

† Background information on Acute Lymphocytic (Lymphoblastic) Leukemia modified from Satake, N., Acute Lymphoblastic Leukemia, http://www.emedicine.com/ped/topic2587.htm. Last accessed: 07/08/09.

“Pharmacogenetics” by Jeanne Ting Chowning Page 1

by

Jeanne Ting Chowning Department of Education, Northwest Association for Biomedical Research

Part I – Acute Lymphocytic (Lymphoblastic) Leukemia It’s called the children’s ward. For two teenagers who have been recently diagnosed with leukemia, it seems insulting to have their lives hijacked by doctors and nurses with stuffed animals clipped to their stethoscopes.

Laura is a forward on her school soccer team and leads the league in scoring. For the last four months, she has been really tired, but nothing seemed really wrong until her legs became covered with bruises. Just pressing her fingers on her skin was practically enough to make a bruise. It didn’t seem real when her doctor, Jane Ryder, diagnosed her with Acute Lymphocytic (or Lymphoblastic) Leukemia (ALL), or when she told her that ALL is the most common malignant (spreading) cancer found in children. She’s 14 years old; she’s not a child!

Beth is 13 and looks remarkably like Laura. Both have straight dark hair, large brown eyes, and tall slender builds. Beth has never been that athletic; she prefers reading and theater. She’s hoping to be part of the drama team next year when she goes to high school, even though she’ll only be a freshman. But she’s been missing a lot of school because of one virus after another, lots of fevers and night sweats, then that rash in the fall. Now she’s in a hospital, and it seems like the only people she sees are her parents, Dr. Ryder, and the nurses.

Laura and Beth both have ALL, which arises from the uncontrolled growth of immature lymphocytes (a type of white blood cell, or leukocyte). These cells, which are “stuck” in an early stage of development, become so numerous that they crowd out normal blood cells. Each year about 30 cases occur per million people, and most of those cases are in children aged 2–5 years. The cause of ALL remains largely unknown, although a small number of cases are associated with inherited genetic syndromes.† Both girls are suffering from anemia (low blood cell levels), fevers, bleeding, and are pale and thin. Dr. Ryder has decided to treat them as in- patients, keeping them in the hospital while treating them with a “thiopurine” drug called 6-mercaptopurine (6-MP) known to be highly effective in treating leukemia. Thiopurines are very similar to the regular purine nitrogen bases such as adenine and guanine that make up DNA and RNA. The only difference is that thiopurines have an extra sulfur group attached to them. They are similar enough to a regular purine base that our cells convert them to nucleotides (with the addition of a deoxyribose sugar and phosphate). These modified thioguanine nucleotides (TGN) are then incorporated into DNA.

The TGN nucleotides interfere with DNA replication and stop rapidly growing cells like cancer cells from further growth. Unfortunately, they also block the growth of other fast growing cells needed for good health, like the cells in the bone marrow that develop into erythrocytes (red blood cells) and leukocytes. As with

Pharmacogenetics: Using Genetics to Treat Disease*

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many drugs given as chemotherapy, it is important to give a high enough dosage to prevent cancer cells from replicating, while avoiding damage to the normal tissues. Too high a drug dose can be very toxic. Dr. Ryder knows that drugs are processed in various ways in the body. They must be absorbed by the blood, distributed throughout the body’s tissues, converted or transformed into forms that are easier to eliminate, and then removed from the body. Dr. Ryder gives both girls the same dosage of the drug before leaving the hospital for the night.

While making her rounds over the next few days, Dr. Ryder sees Laura’s vital signs plummet. Her anemia has worsened; her erythrocyte count is so low that her heart function could be compromised. Her fevers are spiking, and her breathing is becoming shallow and labored. She is not eating and is being hydrated intravenously. Her condition is life-threatening. In contrast, Beth’s anemia has decreased, she is free of fever, and is actually showing signs of an appetite and boredom, good indicators of improved health. Dr. Ryder had not anticipated that the drug could act so differently in two individuals. Even as she looks at Beth’s chart, she can picture Laura’s body struggling to hold its own just two private rooms away. Dr. Ryder knows she must find out why her patients are responding so differently. But where should she start, and will she find an answer in time to help Laura?

Questions 1a. Suggest a reason why the drug might affect the two girls differently.

1b. What tests might Dr. Ryder order to determine why the two girls are reacting as they are to the drug? Provide two or three appropriate examples of tests.

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Part II – Enzyme Activity Dr. Ryder learns that the difference in patient reaction to the drug probably has something to do with how the drug is naturally metabolized in the body to be removed as waste. After searching the scientific literature, she learns that the drug 6-MP can either be converted to the active form, TGN nucleotides, or can be inactivated with the help of the TPMT enzyme (thiopurine methyltransferase). Within each patient who takes the drug, both processes are occurring and they compete with each other.

Figure 1. Flow Chart Flow chart showing activiation and inactivation paths of the drug 6-MP.

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Since the therapy aims to harm rapidly replicating cells without overly impacting normal ones, it is important that excess drug is inactivated. Dr. Ryder decides to see how levels of the TPMT enzyme activity might vary between people.

She reviews the research papers that have been published about the TPMT enzyme and finds an interesting graph. From a study of 298 randomly selected Caucasian individuals, researchers found the following levels of TPMT enzyme activity:

Figure 2. Simplified Results. Simplified bar graph showing results from a study of 298 randomly selected Caucasian patients.

Questions 2a. If Dr. Ryder had 10 Caucasian patients in the next month, how many would you predict to have each

of the TPMT enzyme activity levels, based on the graph above?

Low:

Medium:

High:

Would you expect the actual/observed number of patients to be different? Why might there be differences?

2b. Each individual inherits two copies of the gene for the enzyme, one from each parent. Dr. Ryder suspects that variation in enzyme activity level is controlled by two different versions (alleles) of that gene. Does this graph (and the number of phenotypes) suggest that enzyme activity levels are based on a dominant/recessive or a codominant pattern of inheritance? Explain your answer.

Source: Simplified graph patterned after the top panel of Figure 2 in: Weinshilboum, R.M., and S. Sladek (1980) Mercaptopurine pharmacogenetics: Monogenic inheritance of erythrocyte thiopurine methyltransferase activity. American Journal of Human Genetics 32:651–662.

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2c. Which bar (low, medium, or high) represents individuals who might be homozygous for a “low enzyme activity’” version of the gene? Which bar represents individuals who might be homozygous for a “high enzyme activity” version of the gene? Which bar represents heterozygotes?

2d. Answer the question: “How does enzyme activity level vary among the patients examined?” In your answer, be sure to include supporting data from the graph above. Explain how these data support your conclusion.

2e. Challenge question: The actual graph (below) showed much more detail. Why do you think that there is more variation between patients than shown in the simplified graph?

Figure 3. Histogram RBC TPMT frequency distribution histogram for 298 randomly selected Caucasian subjects.

Source: Histogram drawn after top panel of Figure 2 in: Weinshilboum, R.M., and S. Sladek (1980) Mercaptopurine pharmacogenetics: Monogenic inheritance of erythrocyte thiopurine methyltransferase activity. American Journal of Human Genetics 32:651–662.

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Part III – TPMT Enzyme Activity Levels Dr. Ryder tested Laura, who was very sick, and found that her TPMT enzyme activity level was extremely low.

Question 3a. Why would individuals with the lowest level of enzyme get the sickest when they take the drug?

Suggest one possible reason.

Investigating further, Dr. Ryder decides to look at drug levels in many patients who are all receiving the same standard doses of the thiopurine drug and compare them to enzyme levels. When she compares the level of thioguanine nucleotides (TGN) created by the thiopurine drug to the body’s level of TPMT enzyme in patients, this is what she finds:

Figure 4. Scatter Plot of TGN vs. Enzyme Activity Thioguanine nucleotide concentrations and TPMT enzyme activity levels in 95 Children with Acute Lymphoblastic Leukemia (ALL) who were being treated with standard doses of thiopurine drugs.

Source: Modified from Lennard L., J.S. Lilleyman, J. Van Loon, and R.M. Weinshilboum (1990) Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukaemia. Lancet 336:225–229.

Question 3b. Describe the relationship between TPMT enzyme activity levels and TGN levels. Be sure to include

supporting data from the graph.

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Part IV – Putting It All Together From her research, Dr. Ryder hypothesized that patients such as Laura (who became very sick upon receiving the drug) have very high / low TPMT enzyme activity and therefore very high / low levels of TGN nucleotides at normal doses. They easily became sick from the effects of the drug, and could even die. These patients are homozygous / heterozygous for the version of the gene encoding high / low enzyme activity. A better drug dose for these patients is 1/10th the level of other patients.

Patients such as Beth with high / low TPMT enzyme activity had high / low levels of TGN nucleotides. These patients would do well with the drug, and in some cases might even need a larger-than-normal dosage for the treatment to be most effective. These patients were either homozygous for the version of the gene encoding high / low enzyme activity, or were heterozygous.

Based on the graph in Part II, about 10% of the Caucasian population is homozygous / heterozygous.

Question 4. In the paragraphs above, circle the correct answer (high or low, heterozygous or homozygous).

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Part V – SNPs and TPMT DNA techniques reveal TPMT gene is located on chromosome 6, is about 34 kilobases in length (34,000 DNA bases), and has 8 exons. An exon is a region of a gene that is present in the final functional transcript (mRNA) from that gene. The diagram below shows a representation of the TPMT gene, showing the exons as boxes. The first “wild type” is the most common version. In our case, the second version of the TPMT gene is associated with low enzyme activity (TPMT*3A) and has two single nucleotide polymorphisms (SNPs), or changes in single DNA nucleotide bases (from “G” to “A” in one case and from “A” to “G” in another) that result in different amino acids being inserted in the enzyme. This, in turn, affects the enzyme’s function. Over 20 different gene variants have been found, three of which are shown below.

Figure 5. Selected Human TPMT Alleles. The wild-type human TPMT allele (TPMT*1) and variant alleles TPMT*3A, TPMT*3B, and TPMT*3C. Rectangles represent exons, with black coding areas and white untranslated regions.

Source: 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 this article.

Questions 5a. Dr. Ryder now has the ability to conduct a SNP genetic test on her patients to determine what level

of drug they should get. A new patient on the ward, Kevin, is homozygous for TPMT 3A*. The graph shown in Part III is reproduced on the next page. Circle the area of the graph that might likely corresponds to Kevin’s TGN and enzyme activity levels. Explain why you circled that region.

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Case copyright held by the National Center for Case Study Teaching in Science, University at Buffalo, State University of New York. Originally published February 4, 2010. Please see our usage guidelines, which outline our policy concerning permissible reproduction of this work. Title block illustration, licensed, ©Scott Maxwell | Dreamstime.com.

5b. What level of the drug (low, medium, or high) should Dr. Ryder give him? Explain your answer.

5c. In your own words, summarize how knowing someone’s TPMT DNA sequence could be used to determine what kind of medical care they should receive.

Postscript Dr. Ryder responded quickly to Laura’s drug reaction. She discontinued the drug while alternate treatment regimens were explored, and Laura’s condition began to improve.