1. How To Use GCRC Genetic Resources John A Phillips III, MD Director, GCRC Genetics Vanderbilt Kelly A Taylor, MS, CGC Program in Human Genetics Cara B Sutcliffe, PhD Director, Genotyping Core Lab Dec 13, 2002

2. Goals of GCRC Genetics Provide genetic consultations to help GCRC Investigators Provide comprehensive genetic lab services to GCRC Investigators Provide education in human genetics to GCRC Investigators & trainees Evaluate repository of GCRC clinical data for future genotype phenotype studies

3. Organization of GCRC Genetics Initiative GCRC Advisory Committee GCRC Project Proposal GCRC Genetic Subcommittee GCRC Genetic Cores Subjects DNA Analysis Data Analysis Genetic Consultation

4. How To Use GCRC Genetic Resources Genetic approach to GCRC studies How to get help on your genetic IRB on GCRC How to have DNA isolated & genotyped on GCRC Putting it all together (AUG-TER)

5. Genetic Approach to GCRC Studies Obtain genetic information Familiy studies (linkage, sib pair &TDT) Association studies Drug metabolizing gene studies How to get started & educational materials

6. Obtain Genetic Information MIM2 MIM3 MIM4 MIM5 MIM6 MIM7 MIM8 MIM9 MIM10 MIM11 MIM12 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 196519701975198019851990199520002005 Year Number of Entries in Online Mendelian Inheritance in Man (OMIM) MIM1 OMIM 14,065 Dec 5, 2002 Number of M IM entries

7. Obtain Genetic Information OMIM RefSeq GenBank UniGene dbSNP http://www.gentest.com/human_p45 0_database/srchh450.asp http://medicine.iupui.edu/flockhart/in dex.html Midazolam?

8. Genetic Studies of Families

9. Family History Is the Key to: l Identify those at increased risk l Testing can be offered to clarify diagnosis l Preventative medicine can be available l Drug & other therapy can be offered

10. Genetic Studies of Families Linkage study Sib pair study Case Parent Triad TDT study

11. Linkage Studies

12. Association Studies SNP A is NOT Associated with Disease SNP B IS Associated with Disease

13. Drug Metabolizing Gene Scans ddA Trace ddC Trace A/A A/G G/G C/C C/T T/T GVS CYP 2D6 Exon 6 C > T DNA Sequencing CYP2D6 Exon 6 C > T

14. How to Get Started Obtain genetic information about your GCRC study from John Phillips Consider which genetic approach to use (linkage, sib pair, TDT or association) & discuss with John Consider which drug metabolizing genes are important

15. How to Get Started Submit IRB & GCRC study but ask for DNA collection Have GCRC isolate & store DNA for you Discuss genotyping with John & submit GCRC Genotyping Application

16. How to Get Started GCRC Genotyping Application Form: –study design –genotype(s) requested –Rationale for genotyping –Number of genotypes –Overlap with other support –Timeframe Educational materials: www.mc.vanderbilt.edu/GCRC

17. Genotyping Application Forms

18. How To Use GCRC Genetic Resources Genetic approach to GCRC studies How to get help on your genetic IRB on GCRC How to have DNA isolated & genotyped on GCRC Putting it all together (AUG-TER)

19. GCRC Genetics Ascertainment Services Kelly A Taylor, MS, CGC Program in Human Genetics Vanderbilt University Medical Center

20. Differences Between Genetic Research and Clinical Research Genetic Research Family is often study unit Altruistic benefits and motivations Psychosocial risks Clinical Research Individual is study unit Personal benefits and motivations Medical risks

21. Differences Between Genetic Research and Clinical Genetic Services Research Identify mutation No charge No results disclosure No direct benefit Education and referral Clinical Use mutations for diagnosis Fee for service Interpretation of results Clinically relevant information Genetic counseling

22. What Unique Issues Need to Be Considered in Developing an Ascertainment Protocol for a Genetic Study?

23. Issues To Be Addressed During the Informed Consent Process  Risks  DNA Ownership and Banking  Secondary Usage of DNA samples  Disclosure of Results  Incidental Findings Confidentiality  Recontacting Participants  Duty to Warn

24. How Do I Add A Genetic Component to My Study? Get IRB approval Enroll patient Informed consent Blood sample Collect additional information Family history

25. How Can Kelly Help? IRB development Provide templates, answer questions Write IRB proposals Ascertainment and enrollment of participants Training of staff Blood collection kits

26. How Do I Contact Kelly? Kelly A Taylor, MS, CGC Phone: 322-7195 Email: ktaylor@phg.mc.vanderbilt.edu Website: http://phg.mc.vanderbilt.edu/fac

27. How To Use GCRC Genetic Resources Genetic approach to GCRC studies How to get help on your genetic IRB on GCRC How to have DNA isolated & genotyped on GCRC Putting it all together (AUG-TER)

28. DNA Isolation and Genotyping Cara Sutcliffe DNA Resources Core Vanderbilt University

29. An Overview Samples and Data Collected Experimental Coding Assigned Samples Processed Samples and Data Used in Project

30. Principles for Process Development Information Integrity –Confidentiality –Accuracy Sample Integrity –High yield of Appropriate Quality DNA –Controlled, Rapid, and Accurate Retrieval Practical Issues –Safety –Cost –Flexibility

31. Step One - Sample Arrives Sample Arrival - through delivery company or with a submitter from clinic or the field Record Keeping - information supplied by the submitter as well as qualitative determinations made by the core are recorded. Initial Sample Manipulations - print labels, initiate tracking forms, freeze aliquots, spot blood cards, complete log information

32. Step Two - DNA Extraction Possible Methods –Organic - “Classic” phenol/chloroform, simple but involves hazardous chemicals –Binding Column - Clean DNA, but not scalable –Silica Resin - scalable and inexpensive, but DNA is generally lower molecular weight –Lytic Preps - fast, inexpensive, and simple, but DNA is impure and thus subject to degradation –Salting Out –Magnetic Binding

33. Puregene The Puregene™ extraction kit by Gentra Systems is a modified salting out procedure - non- organic, scaleable, and extensively tested. In addition to being scaleable, the protocol easily lends itself to both automation and semi-automation for higher throughput and cost savings. DNA Stability data and additional protocols are available via PDF from the Gentra website.

34. MagnaPure –Surveyed to determine demand and price point for small (<1ml) volume extractions –System is capable of extracting DNA OR RNA from 32 samples in ~2 hours –In addition, the Magnapure sets up Roche Lightcycler reactions for real time PCR/SNP assays and can be programmed to set up 96 well plates for other genotyping assays.

35. MagnaPure II Nucleic acids can be isolated from whole blood, cultured cells, buccal cells. Samples are lysed, magnetic glass particles are added and the nucleic acids bind to them. Cellular debris is removed during washing steps. The purified DNA is eluted. Post-elution protocols provide for dilutions or assay set up.

36. Step Three - Quantitation Samples are quantitated using fluorometry. Fluorometry is initially performed using a Hoescht dye assay for dsDNA on the Hoefer DyNAQuant 200™. Sample readings <30ng/ul are confirmed by quantitative PCR using the RNaseP primer/probe set

37. Step Four - Storage Sample storage is secured. -80° and Liquid Nitrogen freezers are monitored by dial out alarm system for malfunctions. Orderly and intuitive racking. –VSN also serves as locator for DNA vials –Other samples are banked chronologically and tracked electronically with hardcopy backup.

38. Step Five - Requisition Must be authorized by the investigator using the Core’s database. The database records each requisition, tracking remaining amounts and recipients. VSN and Genetic Ids are matched to confirm delivery of the correct sample Color-coded storage vials aid in accurate retrieval

39. Quality Control DNA –Observed and Expected Yields are calculated and reviewed on every sample –Agarose gels are run on a subset of samples to confirm concentration and molecular weight –Bar Coded Labels are used on forms & sample containers –Color coded DNA storage vials Data –2X Proofreading –Daily Backup –Multiple levels of Password Protection

40. A Laboratory View Note the Presence and Use of Safety Devices –Face and Splash Shields –Nitrile gloves –Spill Tray –“One Hand” Tube Rack Color and Bar Coded Storage Tubes Bar Coded/Labeled Processing tubes

41. Genotyping Using the 7900HT The ABI PRISM® 7900HT: real-time PCR system that detects & quantitates nucleic acid sequences. Automation & 384-well plate capability allow for very high-throughput. Interchangeable formats (96 & 384 well) provide flexibility Hand-held and integrated bar code readers simplify sample tracking Wavelength detection from 500-660nm allows the use of multiple fluorophores in a single reaction Candidate gene/region association and linkage mapping studies are approachable with this technology

42. Assays-On-Demand Assays-on-Demand are approximately 200,000 “on the shelf” human SNP assays Designed around high allelle frequency SNPs identified in multiple databases, the assays use fluorogenic 5' nuclease chemistry and TaqMan probes. Validated on 90 individual DNAs to ensure biological relevance Allele frequencies are available for 4 ethnic groups Convenient online batch searching and ordering A sophisticated bioinformatics pipeline selects SNPs and designs assays

43. Assays-by-Design Assays-by-Design is a custom design service providing validated assay products for gene expression and SNP genotyping. You submit your target sequence, and ABI returns a QC-verified, all-in-one tube assay ready to use with TaqMan® Universal PCR Master Mix. Using Assays-by-Design eliminates the manual and technology-specific task of designing primers and probes Cost per data point comparable to "build your own" One-tube assays remove the steps of balancing primers and probes and optimizing reactions

44. Core Statistics Over 29,000 samples 75 Projects Ongoing for 40 Investigators Approximately 30 Services Offered 6 Multi-Center Projects ~130 liters of blood processed

45. For Further Information For A More Complete List of Services and Pricing See our Website (http://phg.mc.vanderbilt.edu) DNA Resources Core Section For Project Planning contact Cara Sutcliffe- 936-2744 or cara@phg.mc.vanderbilt.edu Thanks to the DNA Resources Core students and staff: Maria Comer, Derek Gatta, Lindsey Herrel, Elizabeth Matthews, Allison Mills and Kate Redding

46. How To Use GCRC Genetic Resources Genetic approach to GCRC studies How to get help on your genetic IRB on GCRC How to have DNA isolated & genotyped on GCRC Putting it all together (AUG-TER)

47. Putting It All Together (AUG-TER) Where to get application forms Types & size of studies that fit GCRC Who to contact: Genetic info & approaches: John Phillips IRBs & consents: Kelly Taylor DNA isolation: Nina Smith & Cara Sutcliffe Genotyping - Cara Sutcliffe

48. Genotyping Application Forms

49. Types & Size of GCRC Studies Familiy studies (linkage, sib pair &TDT) Association studies Drug metabolizing gene studies Linkage Sib pair TDT ddA Trace A/A A/G G/ G

50. Genetic Mapping Family Studies Chromosome Interval Met A A Met T T G G Val G G Val T T C C Ser T T Ser C C A A Leu C C Leu T T G G Gln C T A A Pro C C C C G G Cys T T G G T T STOP * 1.ESTs, unidentified 2. ESTs, unidentified 3.ESTs, highly similar to patched [Drosophila melanogaster] 4.Phosphofructokinase (PFK) BMPR2 Positional & Functional candidate for PPH 5. BMPR2 Positional & Functional candidate for PPH 6.ESTs, unidentified 7.Deleted in pancreatic cancer 1 (DPC1) 8.ESTs, unidentified Computer Search Mutation Detection Candidate Genes Disease Mutation Putting It All Together (AUG-TER) Genes in Interval

51. GCRC Genetic Resources GCRC Genetics Website: www.mc.vanderbilt.edu/GCRC Who to contact for help: Genetic info & approaches: John Phillips IRBs & consents: Kelly Taylor DNA isolation: Nina Smith or Cara Sutcliffe Genotyping - Cara Sutcliffe

52. GCRC Genetic Studies Can Help

53. Questions About GCRC Genetics?