Background: Breast cancer is the most common malignancy among women. Current estimates are that one in eight women born in 1990 will contract breast cancer by age 85. Many factors contribute to breast cancer risk. Inheritance of breast cancer susceptibility genes contribute to approximately 5-10% of all breast cancers. The breast/ovarian cancer susceptibility gene BRCA1 has been identified on chromosome 17. Women who inherit certain BRCA1 mutations have an 80% risk of breast cancer.
BRCA1 appears to encode a tumor suppressor protein. Mutations that affect the function of this protein cause increased rates of cell division and a predisposition towards the development of malignancy. Several BRCA1 mutations, including point mutations, deletions, and insertions, have been identified that may contribute to loss of tumor suppressor function. These mutations can be identified by amplifying portions of the BRCA1 gene by PCR and then using RFLP analysis, direct sequencing, or hybridization with specific probes to detect the presence of mutations. Large scale screening trials are underway to gain more information about the nature of the mutations responsible for increased cancer risk. One deletion mutation in exon 2, 185delAG, is highly prevalent among women of Eastern European Jewish descent, and screening efforts have targeted this population of women for further study.
For the screening, a small amount of blood is drawn. DNA is isolated from the blood, and part of the BRCA1 gene is amplified by PCR. The amplified DNA is run on a dot blot with specific probes corresponding to mutations known to be linked to increased breast cancer susceptibility. The probe will only bind to the DNA if that mutation is present. Probes corresponding to the normal sequence for that mutation site will help you determine whether the individual is homozygous or heterozygous for the mutation. Control DNA samples known to have specific mutation also are included. To analyze these cases, use the PCR function on the Data Screen, rather than the 96-well PCR, to generate DNA samples for the dot blot. Load probes in the spots and load the DNA samples into the corresponding wells.
Probes and primers for use in all three cases:
- probe: 185delAG (AG deletion in exon 2)
- probe: normal 185 (no mutation at this site)
- probe: 4184delTCAA (TCAA deletion in exon 11)
- probe: normal 4184
- probe: 5382insC (C insertion in exon 13)
- probe: normal 5382
- primers: forward and reverse PCR primers for the BRCA1 gene
Case A: While Elizabeth is reading the morning newspaper, she notices an ad for a free genetic screening for breast cancer at the clinic next week. The ad specifically invites women of Ashkenazi Jewish ancestry to participate. According to the newspaper ad, subjects will be tested to see whether they have mutations in the BRCA1 gene which would predispose them to breast cancer. Elizabeth, age 27, had heard about the discovery of the gene and about the mutation linked to Jewish women. Her paternal grandmother had been diagnosed with breast cancer at age 51 and died two years later, and Elizabeth worried that she had inherited the disease. She also worried about her mother, age 52 and apparently cancer-free so far, and her 7-year old daughter. Her daughter is not allowed to participate in the screening, but Elizabeth convinces her mother to go with her to get tested.
DNA samples:
- Elizabeth
- Mother
- 185delAG (DNA containing this mutation)
- 4184delTCAA (DNA containing this mutation)
- 5382insC (DNA containing this mutation)
- Normal BRCA1 (no mutations)
- What conclusions can you draw from the results of the DNA analysis?
- How would you counsel Elizabeth and her mother based on the results of the test?
- Who should have access to the test results?
- What other issues does this type of testing raise, and how should these issues be addressed?
Case B: The time passes slowly as Deborah waits for Aunt Millie to come out of surgery. It had come as no surprise when Aunt Millie was diagnosed with breast cancer. After all, for as long as Deborah could remember, her mother had talked about how breast cancer “ran in the family.” Deborah has already read the literature the doctor gave them about genetic testing for breast cancer susceptibility genes. It is one thing to know that several women in her mother’s family had developed breast cancer; it is quite another to learn that Aunt Millie has tested positive for such a gene and therefore, Deborah and her mother are at higher risk. Her mother has made it clear that she has no intention of being tested but as Deborah sits in the surgery waiting room, she comes to the inevitable conclusion that she has to find out if she carries the gene.
DNA samples:
- Aunt Millie
- Deborah
- 185delAG (DNA containing this mutation)
- 4184delTCAA (DNA containing this mutation)
- 5382insC (DNA containing this mutation)
- Normal BRCA1 (no mutations)
- What is the chance that Deborah carries the same breast cancer gene as Aunt Millie?
- What conclusions can you draw from the results of the DNA analysis?
- How would you counsel Deborah based on the results of the test?
- What are the implications for Deborah’s mother if Deborah’s test is positive?
- Does Deborah’s mother have a right not to know her status?
Case C: Cindy took the news very hard when her mother was diagnosed with breast cancer. The surgery and chemotherapy her mother has gone through have taken a significant toll on the whole family. Discovering that the breast cancer is related to the presence of a breast cancer susceptibility gene has only added to the concern. Cindy’s sister, Ellen, decided to have genetic testing done to determine if she carried the gene, but Cindy chose not to go with her for testing. The news that Ellen tested positive was devastating to Cindy and it has been even more difficult to accept Ellen’s subsequent decision to have a prophylactic double mastectomy. These events have caused Cindy to reevaluate her decision not to be tested; she almost feels an obligation to go through this experience for her sister. She schedules the appointment for testing, still undecided how she will react if the test is positive.
DNA samples:
- Mother
- Ellen
- Cindy
- 185delAG (DNA containing this mutation)
- 4184delTCAA (DNA containing this mutation)
- 5382insC (DNA containing this mutation)
- Normal BRCA1 (no mutations)
- What is the chance that Cindy carries the same breast cancer gene as her mother?
- Does Ellen’s positive test affect that chance?
- What conclusions can you draw from the results of the DNA analysis?
- Was Ellen’s decision to have a prophylactic double mastectomy appropriate?
- How would you counsel Cindy based on the results of her test?