Detection of Hereditary Breast Cancer

Breast Cancer Genes Two genes associated with inheritance of breast cancer Breast cancer gene 1 (BRCA1) – Chromosome 17 BRCA 2 – Chromosome 13 When normal… Both are tumor suppressor genes Repair damage and prevent cancer cells from forming When mutated… Lead to breast or ovarian cancer

BRCA 1 and BRCA 2 5 to 10% of all breast cancers are because of these genes Women… 12% chance of developing breast cancer in lifetime Goes up to 85% if said woman has mutated BRCA 1 or 2 If male has BRCA 2 mutation 1 in 10 ratio of getting breast cancer (100,000 jump)

Family Members Judy is worried!!!!! Jennifer has DNA sequencing… Jennifer had breast cancer Test positive of BRCA 2 (negative for BRCA 1) Laura Tests positive for same mutation as Jennifer

BRCA2 We will be looking at BRCA2 Contains 80,000 nucleotides 600 mutations associated with BRCA2 Most cause increased incidence of breast cancer (not all) Most of these mutations are insertion or deletion

Marker Analysis (Haplotyping) Due to expense of DNA sequencing… We will use marker analysis to test Jennifer and Diana Marker analysis… Genetic test Gene mutation is analyzed using a genetic marker Instead of analyzing genet itself Genetic marker: short sequence of DNA associated with a particular gene or trait with a known location on a chromosome

Short Tandem Repeats Genetic markers used in marker analysis are short DNA sequences Also called microsatellites STR = region of DNA composed of a short sequence of nucleotides repeated many times. Number of repeated STRs varies from person to person Different number of repeats = different alleles Most occur in introns (non-coding DNA) Do not affect gene function

STR for BRCA 2 Location = chromosome 13 STR analysis for this lab It is on 13, next to BRCA 2 gene

Gel Electrophoresis Different STRs have different repeats… Gel will separate alleles based on number of repeats More repeats travels less Less repeats travels more

Loading Samples 5 uL: 130 Volts for 30 minutes Lane #1: DNA Size Markers Lane #2: Helen’s DNA Lane #3: Harold’s DNA Lane #4: Susan’s DNA Lane #5: Adam’s DNA Lane #6: Negative Control

Determining Size of DNA Fragment Using a ruler Measure (ON EACH BAND!!!!) Distance of DNA fragment from origin (gel well) Distance from the origin (gel well) to the tracking dye

Calculate the Rf value Distance the DNA fragment has migrated from the origin (gel well) Rf = Distance from the origin (gel well) to the reference point (tracking dye)

Fragment Length in Base Pairs Distance Migrated (mm) A DNA Size Markers Fragment Length in Base Pairs Distance Migrated (mm) A Distance to Reference Point (mm) B Rf   A ÷ B Fragment 1 1353 Fragment 2 1078 Fragment 3 872 Fragment 4 603 Fragment 5 310 Fragment 6 281 Fragment 7 234 Fragment 8 194

Distance to Reference Point (mm) DNA Sample: Fragment: Distance Migrated (mm) A Distance to Reference Point (mm) B Rf   A ÷ B Diana Fragment 1 Fragment 2 Jennifer Laura Judy

DNA Sample: Fragment: Fragment Length (in base pairs) Allele Present: Diana Fragment 1   Fragment 2 Jennifer Laura Judy

Fragment Length in Base Pairs:   Allele: 200 Allele 1 300 Allele 2 400 Allele 3 500 Allele 4 600 Allele 5 700 Allele 6 800 Allele 7 900 Allele 8 1000 Allele 9

Questions Which allele is associated with the BRCA2 mutation? Explain your answer. Which family members have the BRCA2 mutation? Explain your answer. Explain whether you think Judy’s family occurrences of breast and ovarian cancers are sporadic, hereditary, or familial. Is Judy a good candidate for BRCA1 or BRCA2 genetic testing? Explain your answer.