Diagnosing Niemann Pick disease, Type C

1. Diagnosing Niemann Pick disease, Type C MODIFIED FROM A SLIDE SHOW Developed by the Sanford PROMISE The Sanford PROMISE Program for the Midwest Initiative in Science Exploration

2. The Case Your summer job is as intern in a genetics lab at a Mount Blueberry Children’s hospital. A doctor comes to your team and says that he has a family in which he suspects three cousins all have Niemann-Pick type C disease. The family would like to know: 1) Do the children indeed have Niemann-Pick type C? 2) what are the risks of future children in the family developing the disease ?

3. Niemann Pick Type C • Niemann-Pick disease is an inherited condition in which patients have abnormal lipid metabolism causing harmful amounts of lipids to accumulate in the spleen, liver, lungs, bone marrow, and brain. • Caused by mutations in genes NPC1, NPC2, SMPD1 • NPC1 mutations account for 95% of type C cases. Video of Lysosomal Storage Diseases

4. Part 1 – Polymerase Chain Reaction (PCR) • PCR is a technique used to amplify specific regions of DNA • Start with one molecule of double stranded patient DNA and generate 2 after one cycle • Exponential increase in DNA

5. PCR movie http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/pcranimatie.gif

6. PCR primers • The primers are critical! • need to know a bit of sequence to make proper primers • primers can bracket target sequence • start with long piece of DNA & copy a specified shorter segment • primers define section of DNA to be cloned 20-30 cycles 3 steps/cycle 30 sec/step SLIDE FROM SLIDE SHOW BY KIM FOGLIA

7. Polymerase Chain Reaction (PCR) • What is in the PCR reaction mix? DNA Sample PCR Rxn Mix Thermocycler

8. Polymerase Chain Reaction (PCR) • What is in the PCR reaction mix? DNA Sample PCR Rxn Mix Thermocycler

9. SLIDE FROM SLIDE SHOW BY KIM FOGLIA PCR process • What do you need to do? • in tube: DNA, DNA polymerase enzyme, primer, nucleotides • denature DNA: heat (90°C) DNA to separate strands • anneal DNA: cool to hybridize with primers & build DNA (extension) What does 90°Cdo to ourDNA polymerase? play DNAi movie

10. The polymerase problem • Heat DNA to denature (unwind) it • 90°C destroys DNA polymerase • have to add new enzyme every cycle • almost impractical! • Need enzyme that can withstand 90°C… • Taq polymerase • from hot springs bacteria • Thermus aquaticus

11. SLIDE FROM SLIDE SHOW BY KIM FOGLIA Kary Mullis 1985 | 1993 • development of PCR technique • a copying machine for DNA

12. Part 1 – Polymerase Chain Reaction (PCR)

13. Polymerase Chain Reaction • Step 1: Denature DNA • Heat it up! • Step 2: Primer annealing • Get the first tracks laid out • Step 3: Extension • DNA polymerase fills in the gaps

14. Polymerase Chain Reaction (PCR) • Cycling Conditions • Initial Denaturation • 95˚C for 2 minutes • Denaturation • 95˚C for 30 seconds • Primer Annealing • 60˚C for 20 seconds • Extension • 72˚C for 1 minute • Final Extension • 72˚C for 3 minutes 20 Cycles

15. Part 2 – Family History Different types of genetic mutations

16. Punnett Square T t T t t t

17. Niemann Pick Type C

18. Part 2 – Family History

19. Part 2 – Family History

20. The Jones Family History

21. Part 3 – DNA Electrophoresis • DNA electrophoresis is a technique used to separate DNA by charge and size • DNA is a charged molecule – what charge?

22. DNA Electrophoresis • DNA is separated on an agarose gel based on size • TAE buffer is added to cover the gel • A power supply applies a current across the gel

23. DNA Electrophoresis Cathode (negative) Anode (positive)

24. DNA Ladder • Where do we expect to see the DNA bands from our PCR reaction?

25. Hypothesis DNA Ladder Carrier PATIENT Affected Unaffected 2000 bp X ? 1500 bp 1000 bp 750 bp 500 bp 250 bp

26. DNA Electrophoresis • Place micropipette tip into TAE bufferHOVER directly over the well in the agarose gel • Slowly pipet sample into the well

27. DNA Visualization • DNA cannot be visualized with the naked eye • GelRed will bind to DNA • GelRed is in the agarose gel • GelRed is excited by UV light and will give off visible light ***Dangers of UV light***

28. SET UP THE GELS CC UC AC 1 2 3 4 5 6 7 CC 8 9 10 11 12 13 AC UC

29. The Jones Family History #1 #2 #7 #10 #3 #4 Rick #8 Gina #11 #13 #12 #5 #6 #9

30. Career Pathways Careers • DNA Scientist • Biomedical lab • Clinical lab • Forensic analysis • Paternity testing • Clinical Geneticist Regional Groups • Identity Genetics Inc. • Sanford Health

31. Jolie Pitt carries a mutation in the BRCA 1 gene that gives her an 87% chance of developing breast cancer and a 50% chance of developing ovarian cancer. She lost her mother, grandmother, and aunt to cancer. BREAST CANCER GENE TEST BRCA gene test = blood test that uses DNA analysis to identify harmful mutations in either one of the two breast cancer susceptibility genes — BRCA1 and BRCA2. Women with mutations in these genes face a much higher risk of developing breast and ovarian cancer compared with the general population http://bloximages.chicago2.vip.townnews.com/idahopress.com/content/tncms/assets/v3/editorial/0/e7/0e7962f2-3ba8-11e3-a630-001a4bcf887a/526765798859f.image.jpg

32. BRCA gene test - not routine/offered only to people who are likely to have an inherited mutation, based on personal or family history, or who have specific types of breast cancer. BRCA gene mutation is uncommon. Responsible for about 5 %of breast cancers and about 10 -15 % of ovarian cancers. Genetic counseling= important part of the BRCA gene test process. Patients learn whether they carry the inherited BRCA gene mutation and receive an estimate of personal risk of breast/ovarian cancer. http://s3.amazonaws.com/nbcf-production-assets/attachments/000/000/385/standard_hires/57e514ad84f3780932dff41d5c63ee2c

33. SET UP THE GELS 1 2 3 4 CC UC AC 5 6 7 CC 8 9 10 11 12 13 AC UC

34. SET UP PCR TUBES & LOAD NIEMANN PICK GELS Holly H - #1 Grandpa Jenny- #2 Grandma /CC Sangah - #3 Terry / AC Ally- #4 Lisa / UC Cole- #5 Becky/ Raelyn #13 Mia- #6 Bobby / Ryan #12 Jesse- #7 Rita / Ryan #12 Tayler- #8 Rick / Gina #11 Sarah- #9 Phil / Paul #10 Jessica- #10 Paul / Phil #9 Mackenzie- #11 Gina / Rick #8 Erica- #12 Ryan / Rita #7 Heidi- #13 Raelyn / Bobby #6Jameson- Becky #5 / UCSydney- Lisa #4 / ACBlair- Terry #3 / CCHolly F- Grandma #2 Hannah-Grandpa #1

35. NO PCR FIRST!LOAD BRCA/CANCER TEST GELS Jenny - Healthy breast patient Holly H - Breast tumor patient Sangah - Patient blood (unknown) Ally- Control p53Cole- DNA standards Jameson- DNA standardsSydney- Control p53Blair- Patient blood (unknown)Holly F- Breast tumor patientHannah-Healthy breast patient