Determining Hereditary Nonpolyposis Colorectal Cancer(HNPCC)-Genetics Lab Report

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Abraham Baldwin Agricultural College Determining Hereditary Nonpolyposis Colorectal Cancer (HNPCC) and the Risks via DNA-Based Diagnostic Test Hannah Bryson Dr. Casimiro Abstract. Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominate inheritable disease that results in an elevated risk for cancer and can be diagnosed with genetic testing. This condition is linked to mutations in four caretaker genes that are responsible for DNA repairs. This study focused on determining the inheritance pattern of HNPCC within an affected family by the process of polymerase chain reaction (PCR) and gel electrophoresis. The results revealed genetic mutations are responsible for the disease, and that Susan, Stan, Marshall, Bob, and Claire carry the HNPCC mutation. This means that the patient, Bob, is at higher risk for developing colon cancer. Through genetic testing, it can provide a diagnosis for an individual predisposed to a mutation or cancer. This allows for the possibility of genetic counseling and intervention for those who possess the mutation, increasing survival chances. 1. Introduction What is HNPCC? Hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch Syndrome, is the most common autosomal dominate inherited cancer syndrome that predisposes individuals to a greater risk of developing colorectal cancer (CRC) by 60%-85%, endometrial cancer by 30%-50%, as well as various other cancers by 15% or less [1]. HNPCC accounts for 2%-7% of the total worldwide CRC [2] and is caused by gene mutations. There are four main identified genes that can cause HNPCC when mutated: MLH1, MSH2, MSH6, and PMS2. These four genes are referred to as caretaker genes. The purpose of caretaker genes is to scan the genome and repair damaged or mutated DNA. How does HNPCC occur? There are many cases in which HNPCC is due to germline mutations in the DNA mismatch repair (MMR) genes, thus leading to the tumor phenotype of microsatellite instability (MSI) [3]. This means that portions of repeating DNA change in their lengths, thus displaying instability. Approximately 1 in every 279 people carry a mismatch repair mutation [4]. When assessing MMR mutations of the caretaker genes, MLH1 and MSH2 are known to be the most common, accounting for nearly 90%; whereas MSH6 and PMS2 only account for approximately 10% of the mutations [4]. Germline mutations in the MLH1 and MSH2 genes commonly result in loss of heterozygosity (LOH) [5]. LOH is a frequent genetic event that can result in 1

Abraham Baldwin Agricultural College the growth of cancer cells due to function loss of tumor suppressors, such as the function loss of adenomatous polyposis coli (APC) [5]. There are two types of LOH; one type of LOH is a LOH with copy number losses [6]. A prime example of this would be a reduction in the copy number of wild type alleles, leading to insufficient DNA repair [7]. This insufficient DNA repair can increase the likelihood of tumorigenesis. In some HNPCC cancers, β- catenin mutations replace the APC mutations [8]. Molecular components behind colon cancer Colon cancer is a type of colorectal cancer that forms due to polyps forming on the intestinal walls and has the second highest mortality rate worldwide [9]. Cancer is the result of loss of control of the cell cycle. The mutation(s) that affect cancerous cell’s DNA typically leads to a change in the cell cycle regulation and the rate of miotic division in cells. Unlike normal cells, cancer cells do not need growth factors in order to reproduce. Rather, cancer cells require lipid synthesis for the formation of the cell and the tumor malignant progression [9]. Because of this, tumor cells heavily rely on de novo lipogenesis (DNL), which converts carbohydrates into fatty acids, in order to provide enough energy to meet the rapid proliferation demands of the cancer cells. ATP-citrate ligase (ACLY) is an enzyme that is responsible for the synthetization of lipids [9]. It promotes the migration of cancer cells due to synthesizing the necessary lipid requirements, ultimately resulting in colon cancer. Recommendations for those with HNPCC HNPCC is the most common inherited genetic mutation that increases the chances of developing CRC. It is crucial to take preliminary actions when thought to be and/or are at risk. CRC typically occurs at a relatively young age, approximately 44 years old, with right side predominance [4]. As a person’s age increases, the likelihood that the MSI frequency increases is high [9]. Getting regularly screened for HNPCC cuts the risk of CRC in half, potentially saving lives by identifying early possibly cancer [4]. Families with a pattern of colon cancer or individuals with clinical evidence and/or germline mutations are recommended to get annual colonoscopies starting around the ages 20-25 [4]. By doing this, it is possible to catch cancer growth in its early stages, allowing for treatment options. Purpose of this lab The primary focus of this lab was to verify the mode of inheritance for HNPCC within a family and determine who are carriers of the mutant allele. We constructed a pedigree based on the known family medical history provided by the consultand, Bob. Genetic testing was then performed by gel electrophoresis to determine whether the family had the MSH2 mutation or the wild type. It is proposed that the mutation does, in fact, follow the inheritance pattern of autosomal dominant, and Bob’s side of the family is more than likely the carrier of the mutant allele. 2. Materials and Methods 2

Abraham Baldwin Agricultural College To examine the DNA sequences for Bob’s family and determine whether each family member has inherited the HNPCC mutation, blood samples were collected from each willing family member and prepared to be loaded onto an agarose gel to be visualized by gel electrophoresis. Making the agarose gel To make 1.2% agarose gel, we weighed out 0.6g of agarose onto a scale and added it to an Erlenmeyer flask. 50mL of 1xTBE buffer was poured into a graduated cylinder and was added to the Erlenmeyer flask with the agarose. The Erlenmeyer flask was then placed into the microwave on intervals of 30 seconds. Note, it is important to use an Erlenmeyer flask to ensure that your solution does not boil over while microwaving it. Every 30 seconds, we would take the flask out and swirl the contents until the agarose was completely dissolved. From there, we allowed the solution to cool until it was comfortable to handle before adding 1 μL of ethidium bromide into the flask and gently swirling the contents together. We placed our agarose gel solution in a warm water bath to ensure the solution did not begin to solidify before we were ready to use it. Pouring the agarose gel Now that the gel was ready for use, we prepared our casting tray. To do this, we used 2 pieces of masking tape to seal off the open ends of the casting tray to make sure the contents of the agarose gel did not spill out of the casting tray. When the ends were sealed, we placed our combs in the casting tray. It was time to pour the 1.2% agarose gel into the casting tray. We poured approximately 100mL of the agarose gel into the casting tray using a graduated cylinder. Once it was poured, we let the agarose gel cool on our lab table for 15-20 minutes, or until the gel was completely solidified and ready to be used. Preparing the DNA samples As we waited for the agarose gel to solidify, we prepared our DNA samples for use. The DNA in this experiment has been increased by a polymerase chain reaction to create enough DNA for the process to occur. The DNA was then prepared for use by using a microfuge. The purpose of a microfuge is to be able to collect the totality of a small sample in the bottom of your test tube, so it is easy to collect the required quantity needed to perform the experiment. If an inadequate amount of sample is used, the results can become skewed or misrepresented. The samples were placed within the microfuge and ran for approximately five seconds, or until all the sample was at the bottom of the tube. The tubes were then placed into the test tube holders until it was time to collect the samples. Preparing the agarose gel Once our agarose gel was set, we carefully removed the masking tape from the ends of the casting tray and placed the tray into the electrophoresis chamber, ensuring that the cathodes and anodes line up. If the cathodes and anodes do not line up, the experiment will not work, and it could ruin your agarose DNA gel sample. 1 x TBE buffer was then poured over the agarose gel, ensuring that the top of the was completely covered in the buffer. Once the gel was covered with buffer, the combs were carefully removed, leaving wells for the DNA samples. If the combs are not carefully removed, you can 3

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Abraham Baldwin Agricultural College damage the agarose gel or the wells of the gel. Loading the agarose gel Now that the agarose gel was completely covered in buffer and the combs had been removed, it was time to load the wells with the DNA samples. To do this, a micropipette was used to transfer the DNA samples from the tube to the well. On the micropipette, we used disposable tips after each sample to avoid contamination. If you do not do this, your results can be inaccurate. For the 1-kb DNA ladder, we used the micropipette to add 10 μL of the DNA to the first lanes, on top and bottom, of the agarose gel. For the rest of the samples, we added 20μL of DNA to each lane. In the top row, the seven lanes include the 1-kb DNA ladder, the normal allele control group, the tumor DNA control group, the heterozygous DNA control group, Stan, Susan, and Marshall, in that order. The second row contains the 1-kb DNA ladder, Sara, Bob, Warren, Jane, Steve, and Claire, in that order. Agarose gel electrophoresis When all the samples were loaded and ready to go, the electrophoresis lid was placed onto the chamber with the cathodes and anodes lining up. If the cathodes and anodes do not line up correctly, the experiment will not work, and it could ruin the agarose gel samples. When we were sure the lid was correctly placed, the electrophoresis was connected to the power supply and set to 150V for 45 minutes. This allows plenty of time for the DNA to travel and separate into fragments so we can observe Bob’s families’ DNA and determine which family members inherited the HNPCC mutation. After 45 minutes, the gel was ready to be observed. Observing the gel electrophoresis To observe the DNA fragments in the agarose gel, we carefully removed the casting tray from the 1xTBE buffer; we held the casting tray by both open ends to ensure that our agarose DNA gel sample did not slip out of the casting tray. Once we got the casting tray completely removed from the 1xTBE buffer, we gently slid the agarose DNA gel sample into our hands. We were careful not to mess up the sample. From there, we placed our sample onto an ultraviolet gel imaging system that took a picture of our DNA fragments and digitalized it. With the digitalized image, we were able to compare the DNA fragments with the DNA ladder and determine whether each family member has inherited the HNPCC mutation. 3. Results By conducting this experiment, we were able to better understand the inheritance pattern of HNPCC and which of Bob’s family members are at a higher predisposition of developing colon cancer. To begin the process, a pedigree was drawn out (Figure 1) with the information provided. The filled figures represent individuals affected with colon cancer, the unfilled figures represent individuals without colon cancer, and the figures with lines through them represent deceased individuals. Because there was the potential of at-risk individuals, genetic testing was offered. 4

Abraham Baldwin Agricultural College The gel electrophoresis experiment allows for DNA to be cut by the length in their base pairs, allowing geneticists to determine mutations, such as HNPCC. It allows for a visualization of the genetic material; this can determine the likelihood of whether mutations may be inherited among family members. When performing the gel electrophoresis experiment, there were a total of four control groups used to compare Bob’s families’ DNA bands with. The control groups included the 1-kb DNA ladder, the normal allele DNA, the tumor DNA, and the heterozygous DNA. These control groups are essential when observing the DNA samples provided; without control groups it is nearly impossible to determine what variations are due to the experiment, opposed to other variables. Control groups allow for an accurate and reliable comparison when undergoing an experiment. The 1-kb DNA band is a highly purified DNA strand used to help distinguish the different DNA fragments based upon their molecular weight. The 1-kb DNA band had markers at 10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1.5kb, 1kb, and 0.5kb (see Figure 2). The normal allele is used to identify individuals with the normal alleles. The normal allele had a marker at approximately 5.3kb (see Figure 2). The tumor DNA marker is used to help identify changes in genes that may affect cancer growth. The tumor DNA had markers at approximately 9.1kb and 6.4kb (see Figure 2). Heterozygous DNA is used to determine the individuals carrying the mutant allele. The heterozygous DNA had markers at approximately 5.3kb, 6.4kb, and 9.1kb (see Figure 2). Using these controls allowed us to accurately compare the findings of the families’ DNA. Stan, Susan, Marshall, Bob, and Claire had markers at approximately 5.3kb, 6.4kb, and 9.1kb; Sara, Warren, Jane, and Steven had markers at approximately 5.3kb (see Figure 2). These genotype controls and the position of the markers in this experiment can be used to determine the consultand and his family’s disease genotypes. 5 Figure 1. Pedigree of Bob’s family. Solid figures represent known individuals diagnosed with cancer. Figures with lines through the center represent deceased family members. “Hollow” figures represent individuals not diagnosed with cancer. Stan (70 years old, diagnosed at 50) is located at II 17. Susan (50 years old, diagnosed at 42) is located at III 10. Marshall (47 years old, diagnosed at 46) is located at III 9. Sara (33 years old) is located at III 11. Bob (45 years old) is located at III 8. Warren (78 years old, diagnosed at 74) is located at II 11. Jane (42 years old) is located at III 7. Steven (19 years old) is located at IV 8. Claire (21 years old) is located at IV 7.

Abraham Baldwin Agricultural College 4. Discussion This experiment allowed us to visualize the DNA of Bob’s family to determine who possessed the mutant genotype. We began the process by drawing a family tree based on the family medical history provided by Bob. Bob (age 45), his wife Jane (age 42), their daughter Claire (age 21), and their son Steven (age 19) were all in good health. Bob has three siblings, two of which were diagnosed with colon cancer; his siblings are Susan (age 50; diagnosed at 42), Marshall (age 47, diagnosed at 46), and Sara (age 38). While Bob’s parents, Elizabeth and Robert, died at an early age, Elizabeth had a brother named Stan (age 70, diagnosed at 50), in addition to two other brothers and three sisters. Elizabeth’s mother died at the age of 42 from “female” cancer, and her father died at age 85. Robert had three brothers, all deceased, and three sisters, all living. Robert’s parents both died of unknown causes in 6 Figure 2. Results of gel electrophoresis of patients identified in Bob’s family pedigree. Upper gel: Lane 1: 1-kb DNA ladder, Lane 2: Control (normal allele) DNA, Lane 3: Control (tumor) DNA, Lane 4: Control (heterozygous) DNA, Lane 5: Stan (deceased at 70, with previous colon cancer diagnosis), Lane 6: Susan (age 50, with colon cancer), Lane 7: Marshall (age 47, with known HNPCC mutation). Lower gel: Lane 1: 1-kb DNA ladder, Lane 2: Sara (age 38), Lane 3: Bob (age 45), Lane 4: Warren (age 78, with previous colon cancer diagnosis), Lane 5: Jane (age 42), Lane 6: Steven (age 19), Lane 7: Claire (age 21).

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Abraham Baldwin Agricultural College their 60s. Jane has two brothers, one with two sons and one with a daughter. She also has a sister with three daughters. Jane’s parents, Roberta (age 67) and Herschel (age 70), are still living. Roberta three sister and three brothers, though one died in his 70s from a heart attack. Roberta’s mother died at age 75 with breast cancer, and her father died at age 65 from a heart attack. Herschel still has a living brother named Warren (age 78, diagnosed at 74) and two deceased sisters. Both Herschel’s parents are deceased. Other than that knowledge, Bob did not know of any other cancer on either side of the family. When initially looking at the pedigree, we can assume that both of Bob’s parents were carriers of the HNPCC mutation; this is due to both Susan and Marshall having been diagnosed with colon cancer. Because of this, it is highly likely that Bob is a carrier of the mutation, thus increasing his likelihood of developing colon cancer. To further determine this, genetic testing was done. Understanding the results Genetic testing allows geneticists to identify specific changes that may lead to a particular condition, such as colon cancer. Because Marshall was recently diagnosed with colon cancer, genetic testing was offered to him; a mutation was found on the MSH2 gene resulting in the recommendation of genetic testing on willing family members. DNA samples were collected from the willing family members and were loaded onto agarose gel and visualized by gel electrophoresis. The heterozygous DNA has markers in the same location as the tumor DNA and the normal allele. This means that, while anyone who is heterozygous will have a normal allele, they will also be a carrier of the mutation, increasing the likelihood of CRC. The results showed that Stan, Susan, Marshall, Bob, and Claire were all heterozygous; Stan, Susan, and Marshall were previously diagnosed with colon cancer. This means that Bob and Claire are carriers of the mutant allele, increases their chances of developing colon cancer. Sara, Warren, Jane, and Steven all had markers for the normal allele, meaning that they all have wild type alleles. While Warren may have had colon cancer, through the gel electrophoresis we were able to determine that he did not have the HNPCC mutation. We can determine that Jane’s side of the family are not carriers of the mutation, while Bob’s side of the family are carriers of the mutation. As stated previously, LOH, caused by mutation, causes the loss of the wild type allele in tumor suppressors, resulting in defective DNA repair. Because the wild type is lost, the cancerous cells within the tumor become the dominant genotypic allele. This is why we do not see the wild type allele within the tumor sample control group. The sizes of the wild type allele were approximately 5.3kb while the size of the tumor DNA alleles are approximately 9.1kb and 6.4kb. The variation in length displays the instability of the tumor cell. Determining the next steps We were able to use the samples provided by Bob’s family to determine the MSH2 mutation inheritance pattern; we were also able to tell who a carrier of the mutant allele is. Looking at the results, it is confirmed that HNPCC is inherited in an autosomal dominant fashion. If the mode of inheritance is autosomal dominance, that means that if Claire has children in the 7

Abraham Baldwin Agricultural College future, there is a 50% chance that she will pass on the mutated gene. By undergoing genetic testing, Bob’s at-risk family is now able to take precautionary steps in order to reduce the likelihood of developing CRC. While Stan, Susan, and Marshall already developed colon cancer, Bob and Claire are recommended to get annual screenings done. This allows for early detection to make sure that the mutated gene does not become activated, resulting in possible cancer formation. If cancer has developed, it allows for early intervention. The earlier the cancer is caught, the more options for treatments are available and the lower the mortality rate. Part 1 Questions 1. Describe Bob’s risk for colon cancer. Describe Jane’s risk. Because we know that the pattern of inheritance is autosomal dominate, and Stan, Susan, and Marshall all have colon cancer, it is likely that Bob is at risk for colon cancer. There is a 50% chance that Bob has the mutation. It is unlikely that Jane will develop colon cancer from the HNPCC mutation. There is only one person in her family, Warren, with colon cancer; however, it is not consistent with the pattern of inheritance. Thus, it is likely that Jane is not at risk for colon cancer. 2. As Bob’s genetic counselor, what issues might you choose to discuss with Bob? With the quantity of family members with colon cancer on Bob’s side of the family, it would be recommended for Bob to receive genetic testing. Because the majority of his siblings were diagnosed with colon cancer, Bob is at risk, as well. Screening for colon cancer allows for premature actions to be taken. 3. Is there anyone in either family you would like to find out more about? There is not a lot of information about Bob’s side of the family. Specifically, I would like to know more about Bob’s maternal grandmother. It was mentioned that she died of “female cancer.” Female cancer could very likely be ovarian or endometrial cancer, both which are associated with HNPCC. If we could confirm what cancer she had and whether she had the mutation, we would be able to extend the pattern to another generation which strengthens the evidence of the mutation across the generations. 4. What is the relationship between cell cycle regulation and cancer? What makes a cancerous cell different from a normal cell? Cancer is the result of loss of control of the cell cycle. The mutation(s) that affect cancerous cell’s DNA typically leads to a change in the cell cycle regulation and the rate of miotic division in cells. Unlike normal cells, cancer cells do not need growth factors in order to reproduce. They cannot communicate with neighboring cells, unlike normal cells. All of these factors allow cancer cells to multiply at a high rate in a short amount of time, unlike normal cells. Discussion Questions 1. Does having the mutation mean that an individual will definitely develop cancer? Why or why not? Having the mutation does not mean that the individual will definitely develop cancer. In this instance, an individual would need two mutated alleles to completely inactivate the HNPCC tumor suppressor gene, MMR. Having only one mutation simply means that the person is more likely to develop cancer in comparison to someone who does not have the mutation. 2. According to the genetic test results, what is the risk that Bob’s daughter Claire will pass the HNPCC mutation along to her children? The likelihood that Claire will pass on the mutation to her offspring is 50%. Because 8

Abraham Baldwin Agricultural College the pattern of inheritance is autosomal dominant and she is heterozygous, 50% of her offspring would inherit the mutation. You can confirm this by doing a Punnett Square. 5. Conclusion Genetic testing allows for the early detection of individuals with possible predispositions for mutations or cancers. In our experiment, we were able to determine that the mode of inheritance is autosomal dominant. Our hypothesis that Bob’s side of the family carried and passed on the mutant allele was proved true through this experiment. Susan, Stan, and Marshall all previously had colon cancer before the genetic testing. Through the gel electrophoresis, we were able to determine that Bob carries the HNPCC mutation, in addition to his daughter, Claire. This means that these two individuals are at a higher risk of developing colon cancer in the future; Claire has the potential to pass the mutation down to her offspring. Because of this, it is important for them to regularly monitor their health and take preventative measures when necessary. Early detection of cancer is crucial for survival rates but often goes unnoticed until the cancer has become too advanced. Annual screenings, involving colonoscopies, stool samples, and blood tests, help protect individuals predisposed to things such as colon cancer in patients. 9

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Abraham Baldwin Agricultural College References 1. Bröcker-Vriends, A.H.J.T., de Jong, A.E., Hendriks, Y., …Winjnen, J.Th. (2004). Identification of HNPCC by Molecular Analysis of Colorectal and Endometrial Tumors . Diseases Markers, 20(4-5), 207-213. 2. Arroyave, A.J., Good, A.W., Ward, A.J., Orucevic, A.L., McLoughlin, J.M. (2023). When to Consider Lynch Syndrome in Non-Colon and Non-Endometrial Malignancies. The American Surgeon, 89(5), 1912-1922. 3. Boardman LA., Schmidt S., Lindor NM., et al. (2001). A search for germline APC mutations in early onset colorectal cancer or familial colorectal cancer with normal DNA mismatch repair. Genes Chromosomes Cancer, 30(2), 181- 186. 4. Lubiński, J., Lynch, H.T., Lynch, J.F., Shaw, T.G. (2003). HNPCC (Lynch Syndrome): Differential Diagnosis, Molecular Genetics and Management - a Review. Hereditary Cancer in Clinical Practice, 1(1), 7-18. 5. Hadziavdić V., Pavlović-Calić N., Eminović I. (2009). Molecular analysis: microsatellity instability and loss of heterozygosity of tumor suppressor gene in hereditary non- polyposis colorectal cancers (HNPCC). Bosnian Journal of Basic Medical Science , 9(1), 10-18. 6. Zhang X., Sjöblom T. (2021). Targeting Loss of Heterozygosity: A Novel Paradigm for Cancer Therapy. Pharmaceuticals (Basel) , 14(1), 57. 7. Beck NE., Tomlinson IP., Homfray TF., Frayling IM., Hodgson SV., Bodmer WF. (1997). Frequency of germline hereditary non-polyposis colorectal cancer gene mutations in patients with multiple or early onset colorectal adenomas. Gut , 41(2), 235-238. 8. Johnson V., Lipton LR., Cummings C., et al. (2005). Analysis of somatic molecular changes, clinicopathological features, family history, and germline mutations in colorectal cancer families: evidence for efficient diagnosis of HNPCC and for the existence of distinct groups of non-HNPCC families. Journal of Medical Genetics , 42(10), 756-762. 9. Han Y, Hua Q, Huang G, …Zhao X. (2019). ACLY Facilitates Colon Cancer Cell Metastasis by CTNNB1. Journal of Experimental and Clinical Cancer Research , 38(1), 401. 10

Determining Hereditary Nonpolyposis Colorectal Cancer(HNPCC)-Genetics Lab Report

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