Genome 371, Autumn 2018 Quiz Section 9: Genetics of Cancer Worksheet All cancer is due to genetic mutations. However, in cancer that clusters in families (familial cancer) at least one of these mutations is being inherited within the family in Mendelian fashion. In cancer that does not cluster in families (sporadic cancer) the mutations are not inherited, but happen during a patient s lifetime. Genes in which mutations lead to cancer can be split into two general categories: Proto-oncogenes: Cancer arises from too much activity of the gene product. Tumor suppressor genes: Cancer arises from too little activity of the gene product. 1. Shown below is a greatly simplified diagram of cell signaling pathways, with the location of 6 proteins that are found to be mutated in cancer. Discuss within your group whether your assigned protein is encoded by a proto-oncogene or a tumor suppressor gene. Is the mutation in your gene that causes cancer dominant or recessive? Is the mutation Loss of Function (LOF) or Gain of Function (GOF)? A. growth factor that promotes division B. receptor for the growth factor in (A) C. protein kinase that stimulates division D. inhibitor of the protein kinase in (C) E. DNA damage sensor that halts division F. Chromosome damage sensor that kills the cell TS/PO? GOF/LOF? Dom/Rec? Genome 371 QS9 Worksheet Page 1
Breast cancer and the BRCA1 gene Breast cancer, the second most common type of cancer, usually affects women but in rare cases can affect men. It kills about 41,000 people a year in the US. Although most of these cases are sporadic, about 10% are familial and differ from the sporadic cases in that cancer develops at a younger age and both breasts are often affected. Mutations in a gene called BRCA1 are found in about half of the families with familial breast cancer. Women in these families also show a high rate of ovarian cancer. BRCA1 mutations are rare; about 1/3000 Europeans carries one. Consider a real family in which a mutation in BRCA1 is segregating; sons are designated M and daughters F. Those with no disease indication did not develop early onset cancer. Draw up a pedigree based on the following information: In the first generation are three sons, I-1M, I-3M and I-4M, each of whom lives to 77 or older. I-1M marries an unrelated woman (I-2F) with no prior familial history of breast/ovarian cancer, and they have seven children: II-1M, II-3F, II-4F (develops breast cancer at 32), II-5F (develops breast cancer at 45), II-6F (develops breast cancer at 36), II-7F (develops ovarian cancer at 48), II-8F. II-1M, who lives to be 91, marries an unrelated woman (II-2F) and they have four daughters: III-1F (develops breast cancer at 29), III-2F, III-3F and III-4F (develops breast cancer at 28). III-4F marries an unrelated man (III-5M) and they have one daughter, IV-1F, who develops breast cancer at 25. I. II. III. IV. What can you say about the mode of inheritance (dominant/recessive, etc.) of the BRCA1 mutation? Why? We wish to understand whether BRCA1 is a proto-oncogene or a tumor suppressor gene, and how it acts to cause breast and ovarian cancer. Consider the following experiments. What can they tell us about the action of BRCA1? Discuss within your group and be prepared to explain to the class as a whole. Genome 371 QS9 Worksheet Page 2
Experiment A: From mining databases such as OMIM as well as using data from newly discovered families, researchers assembled a large collection of BRCA1 mutations from families with early onset breast and ovarian cancer. Recall the conclusions from pre-worksheet Question 4: Allele 1: Allele 2: Allele 3: Allele 4: On the page 6 of the handout is a schematic of the BRCA1 gene with these mutations indicated. 1. What effect do you think these mutations might have on the activity of the BRCA1 protein? What does this collection of mutations suggest about the nature of cancer-causing BRCA1 alleles? 2. Is a cell with one healthy and one mutant copy of the BRCA1 gene a cancerous cell? Do you think at the cellular level that a mutant BRCA1 gene acts in a dominant or recessive fashion? 3. Do you think BRCA1 is more likely to be a proto-oncogene or a tumor suppressor gene? Why? Now consider another experiment that provides additional evidence pertinent to your conclusions above Genome 371 QS9 Worksheet Page 3
Experiment B: Recall the gene that you worked with on the pre-worksheet exercise. Let us assume that what was shown was a portion of the BRCA1 gene. In BRCA1 the microsatellite repeat is highly variable. Therefore, there are more alleles than you considered in the pre-worksheet exercise. Intron VNTR site 5 1 2 3 4 5 Exon (CAG) n The gel below shows PCR products from amplifying this repeat using DNA from healthy and cancer tissues of four unrelated women with familial breast cancer. Lanes marked Healthy contain DNA from healthy tissue; lanes marked Cancer contain DNA from cancer tissue from the same patient. + and - below the gel image indicate the presence or absence of functional BRCA1 protein, respectively, in the indicated tissues. 1 1 2 2 3 3 4 4 Lanes: Marker Healthy Cancer Healthy Cancer Healthy Cancer Healthy Cancer Marker Size standard in bp 300 200 150 100 50 300 200 150 100 50 Functional BRCA1 protein + + + + Genome 371 QS9 Worksheet Page 4
The cartoons below represent the BRCA1 gene as a framework for you to explain the PCR results. Woman #: Healthy tissue Cancer tissue Indicate the size of the PCR product (if any) here. 4. On a DNA level, what could be happening in cancer cells to give these results? List as many mechanisms as you can think of. 5. Does this experiment provide additional support for your conclusions on the previous page with regard to whether the mutant BRCA1 allele is dominant or recessive at the cellular level, and whether BRCA1 is a proto-oncogene vs. a tumor suppressor gene? The BRCA1 Paradox: 6. What did our pedigree suggest about whether mutant BRCA1 is dominant or recessive in families? Is a heterozygous woman at high risk of cancer? 7. What do experiments A and B suggest about whether mutant BRCA1 is dominant or recessive in a single cell? Is a heterozygous cell generally going to reproduce out of control? 8. How can these two observations be made to fit together? Going back to the pedigree you constructed earlier (p. 2) most unusually, II-8F (who at 81 is still cancer-free) married an unrelated man* whose family had no prior history of breast/ovarian cancer and they had a daughter, III-6F, who developed breast cancer at age 33. Why might some women who inherit a mutant BRCA1 gene not develop cancer over a long lifetime? [*that s not the unusual part] 9. Can we make any guesses about the probable function of normal BRCA1 protein in healthy cells? Genome 371 QS9 Worksheet Page 5
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Genome 371, Autumn 2018 Quiz Section 9: Homework Questions for you think about and discuss For those interested in more detail on cancer genetics, the following book section is recommended. For exams you are responsible only for material covered in lecture and quiz sections, but the additional material may be helpful in clarifying areas of confusion. http://www.ncbi.nlm.nih.gov/books/nbk26816/ 1. Mice are a useful model of many human diseases, but they live only two years, and have other significant differences from humans. Genetically normal mice almost never get breast cancer, so researchers attempted to create a mouse model by genetic engineering. Mice have a gene similar to BRCA1. Genetic engineering was used to make knock-out mice with no functional copies of BRCA1. The resulting mice (both males and females) died as embryos. What do these deaths suggest about the normal function of BRCA1? Is it likely to be specific to breast tissue? Women who carry mutant alleles of BRCA1 but never develop cancer are very rare. However, female mice that have no functional copies of the BRCA1 gene in their mammary gland tissue develop cancer less than 50% of the time. What might be different between humans and mice with regard to development of breast cancer? 2. Is BRCA1 likely to be a tumor suppressor gene or a proto-oncogene? What is its loss doing to these cells? 3. Why might some women who inherit a mutant BRCA1 gene not develop cancer over a long lifetime? 4. Only one report of a human homozygous for a BRCA1 mutation has been published, but it is now believed that this may have been an error due to one of her alleles being much more difficult to amplify with PCR than the other, leading to the false appearance that she was homozygous for the easy-toamplify allele. What does this suggest about the role of BRCA1 in human development? 5. Cancer-causing viruses often carry mutant alleles of proto-oncogenes. No cancer-causing virus has been found to carry a mutant allele of a tumor suppressor gene. Why might this be? 6. BRCA1 is sometimes described in newspapers as a gene for breast cancer. However, the gene is highly conserved in zebrafish, which have no breasts. What might this gene actually do? 7. (The answer to this question is not fully known, but it is interesting to think about.) Quite often, a human disease will affect only one or a few tissues (breast and ovary tissue for BRCA1-related cancer, certain nerve cells for Huntington's Disease, etc.) However, when the genetic basis of the disease is discovered it proves to be damage to a function carried out by most or all cells in the body. How might a mutation in a generally used function lead to disease only in specific tissues? Genome 371 QS9 Worksheet Page 7