1. POSITIONAL CLONING TWO EXAMPLES

2. Inheritance pattern - dominant autosomal Entirely penetrant and fatal Frequency - about 1/10,000 live births Late onset - age 35 to 45 No biochemical defect known, until very recently No methods of treatment Because of late onset, many have had children before symptoms appear Huntington's disease

3. Huntington’s disease results from nerve cell degeneration in the basal ganglia

4. Influence of the Wexler family

6. Families with a history of Huntington's disease Indiana University maintains a National Research Roster for Huntington's Patients Large family with a history of Huntington's disease discovered living on shore of lake Maracaibo in Venezuela

8. For both families with a history of Huntington's disease: Blood samples taken from each member Permanent cell lines established Each family member analyzed by a neurologist for disease symptoms Paternity verified

10. Objective: Finding a probe that uncovered an RFLP that was linked to Huntington's disease. 1981 - Gusella's group started with a group of anonymous probes that uncovered RFLPs - very few available. They were incredibly lucky - the 12th probe they tried -called G8 - indicated linkage.

13. Disease associated with the A haplotype in the American family and the C haplotype in the Venezuelan family.

14. LOD Scores

15. 1983 - G8 (also called D4S10) mapped approximately 4 cM from the HD locus. It took 10 more years to clone the gene. Why?

16. 1986-87 DNA markers were used and D4S10 was localized by in situ hybridization and somatic cell genetics to chromosome region 4p16.3

18. Further linkage studies for isolating HD

20. Identification of Putative Coding Sequences Exon Trapping Use trapped exons to identify candidate genes from cDNAs

21. Four transcripts were analyzed.

23. IT15 - Huntingtin

24. Repeat Sequence Diseases

27. Implications No cure yet Testing is possible Ethical issues arise

28. Cystic fibrosis Autosomal recessive Not late onset Average life expectancy 27 years Treatment but no cure

29. The most common fatal genetic disease in the US today. It is an autosomal recessive, and it is found mostly in people of Northern European background. Affects multiple organs, all of which are involved with secretion. Mucus is very thick and sticky. Variable symptoms. Lungs: thick mucus harbors microorganisms, often leading to pneumonia. Also asthma, bronchitis, and other lung problems. Pancreas: decreased secretion of digestive enzymes lead to malnutrition, intestinal blockage, and other digestive problems male infertility abnormally salty sweat: often detectable in newborns. A sweat test is the standard method of diagnosis for CF.

31. About 1/20 to 1/25 Caucasians are carriers of the defective gene (two carriers have to mate to produce an affected individual). Why is the prevalence of this defect so high?

32. Biochemical work done before the gene was cloned pointed to salty skin as a primary clue. Problem in chloride ion transport. Chloride can't get out of the cell, so water stays in to dilute it-- can't come out to dilute the mucus. Work on cloning the gene started with a search in lots of families with many RFLP probes.

34. Pedigree Analysis A loosely linked RFLP marker was found. By in situ hybridization, this marker and therefore the CF gene were mapped to chromosome 7.

35. Further RFLP markers known to be located on chromosome 7 were then tested for linkage to CF. Two markers were found that flanked CF, met and D7S8, but they were 1600 kb apart. Eventually two more closely linked markers were found that narrowed the region to about 500 kb.

37. In the case of the CF gene, 7 jumps of 50 to 75 kb were made with each arrival point then serving as a new origin for chromosome walking.

39. The map position agreed with the linkage data Zoo blot The probe had a CpG island at the start of the coding sequence The gene was detected in Northern blots using RNA extracted from the "correct" tissue, in this case cultured epithelial (sweat gland) cells

46. Diagnostic tests are available, but extensive testing is necessary because there are so many different mutations.

47. Since the function of the gene is known, treatment strategies can be devised.

48. Conventional Therapy: Antibiotics Inhalers to reduce mucus blockage in lungs Medicines to reduce inflammation in lungs (e.g. corticosteroids, ibuprofen) Pancreatic enzymes Vitamins Extra calories

49. Gene therapy Insert CFTR gene in adenovirus vector. Virus is used to infect CF patient by inhalation. Virus inserts itself into lung cells, where gene functions normally.

50. Why is disease so prevalent? Selective advantage of heterozygotes? Big gene, many mutations