Cystic Fibrosis pp. 303-305 CF is the most common autosomal-recessive disease

1. Cystic Fibrosis pp. 303-305 • CF is the most common autosomal-recessive disease • among people of Northern European descent • Average life expectancy ≈ 30 years • 1 of every 3200 births in the U.S. has the disease • 1 of every 31 Americans is a carrier • When 2 carriers have children = 25% chance of the disease 1/31 x 1/31 = 1/961 x ¼ = 1/3844

2. Cystic Fibrosis pp. 303-305 • First shows up in babies as: • Persistent diarrhea • Frequent pneumonia • Chronic coughing • Salty skin • Poor growth More states are now requiring CF tests for every newborn (in order to treat it earlier). …before Jan.1, 2006 Kentucky only tested for 5 inherited diseases….now 29 ! http://www.cff.org/publications/videos_by_request/#Faces_of_CF

3. Cystic Fibrosis • Average Life Expectancy ≈ 30 years • Chronic Pulmonary Disease (lungs) • ….lots of lung infections…breathing difficulty • Pancreatic Insufficiency (lack of digestive enzymes secreted by pancreas into intestine ….intestinal blockage) • Lots of chloride in sweat (not dangerous per se) • 4. Male Sterility

4. Cystic Fibrosis • Third gene to be cloned by “Positional Cloning” • (other two: Chronic Granulomatous & Muscular Dystrophy) • - Toronto, Canada • At a cost of $200 million (US) • Gene is huge = 190,000 bp (24 exons) • Codes for a Transmembrane Chloride Transporter • (CFTR = CF Transmembrane Conductance Regulator) • About 1000 known mutations, but 70% of the cases involve a single type, called F508

5. Cystic Fibrosis • Codes for a Transmembrane Chloride Transporter • Water transport follows chloride transport so this mutation • interferes with mucous secretions (water dilutes mucous). • In Homozygous Recessives = total Loss of Function. But • there are lots of interactions with other genes & the environment. • Up until the 1940s, death typically occurred in first few years. • In Heterozygotes, it might provide a selective advantage • where high rates of infantile diarrhea is common (Europe).

6. Life expectancy has risen from a few years to 30 years because of RESEARCH Anyone know why the rose is associated With this disease?

7. How was this gene cloned? The quickest and cheapest way to test for CF is the “Sweat Test” …collect perspiration & test for high chloride levels

8. In the old days, doctors used to lick newborn babies (to see whether they had salty skin) • Now they apply Pilocarpene • to the skin – wait 30 minutes • and measure chloride levels. Jaborandi (Pilocarpus pennatifolius) Native to Brazil….made the native Amazonians “jabber” Also, commonly used to treat glaucoma (high intraocular pressure)

9. How was this gene cloned? Pedigree Analysis …its more difficult with recessive alleles ! …and when it causes sterility ! …and when it is lethal ! Needs to be done hand-in-hand with molecular techniques

10. How was this gene cloned? 1) Which RFLP Marker is most closely linked to the mutation causing this disease? 2) Can you see where a ‘cross-over’ event has occurred? E+ E- E- E- B- B+ B- B- H+ H- X H- H+ S+ S+ S- S- CF CF Carrier Carrier E+ E- E- E- E- E- B- B- B+ B- B+ B- H+ H+ H- H- H+ H- S+ S- S+ S- S+ S- Cystic Healthy CF Fibrosis Carrier

11. How was this gene cloned? 1) Which RFLP Marker is most closely linked to the mutation causing this disease? 2) Can you see where a ‘cross-over’ event has occurred? E+ E- E- E- B- B+ B- B- H+ H- X H- H+ S+ S+ S- S- CF CF Carrier Carrier E+ E- E- E- E- E- B- B- B+ B- B+ B- H+ H+ H- H- H+ H- S+ S- S+ S- S+ S- The HindIII site Cystic Healthy CF Fibrosis Carrier

12. How was this gene cloned? RFLP Analysis ….they looked at 330 different RFLPs to find these that correlated with Cystic Fibrosis

14. Perhaps this larger band is correlated with being a “carrier”

15. Somatic Cell Hybridization pp. 131-136 http://www.mun.ca/biology/scarr/Somatic_Cell_Hybridization.htm

16. Somatic Cell Hybridization “Somatic cell hybrids are culture lines that contain the entire complement of the mouse genome and a few human chromosomes. These culture lines are developed by mixing human and rodent cells in the presence of the Sendai virus. The virus facilitates the fusing of the two cell types to form a hybrid cell. For a reason that is not entirely known, most, but not all, human chromosomes are lost from the hybrid cell lines. Usually a few human chromosomes are retained. Because the human and rodent chromosomes can be distinguished by chromosome staining techniques, it can be determined which human cells are retained with a specific cell line.” Phillip McClean. 1997. North Dakota State University http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/genomic/genomic3.htm

17. Somatic Cell Hybrids (human #3, #5, and #17)

18. With correct staining you can distinguish Rodent chromosomes from Human ones, and even observe translocation events.

19. 6 T 1 6 1T Translocation Exchange of chromosome parts Translocation Normal Normal Translocation

20. Which DNA region consistently hybridizes? Page 136 (your book) From page 136 here…

21. Which DNA region consistently hybridizes? Page 136 (your book) Region C From page 136 here…

22. This HindIII site correlates with CF Another group of researchers used this probe… E E E E E H* E E E | | 3.6 | 5.0 | 5.8 | 8.9 | | | Probe for Southern Blotting for CF

23. Lanes A, B, C, D, E has DNA from the people used to make the hybrid lines

24. Which human chromosome appears to consistently hybridize with the TCRB and DOCRI-917 probes?

26. Which human chromosome appears to consistently hybridize with the TCRB and DOCRI-917 probes?

27. Then used this probe (and others like it) to screen Genomic Libraries and cDNA Libraries to isolate the gene.

29. Normal

30. Normal Mutation Normal Mutation

31. Normal

32. < Link to animations about the disease.

38. R.B. Moss, New approaches to cystic fibrosis, 2001 Truncation NBD & R mutation Processing Hydrophobic Regions mRNA stability

39. R.B. Moss, New approaches to cystic fibrosis, 2001 What functional part of the protein is F508? Truncation NBD & R mutation Processing Hydrophobic Regions mRNA stability

41. The environment also affects • the severity & symptomology • of this disease: • Smoking • Second-hand Smoke • Diet • Pseudomonas exposure • Income (more severe in low-income patients)

42. Average 20 medications per day! • 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

43. Gene Therapy ? • Deliver a correct version of the gene • to the affected tissue using a vector. • Retroviruses = RNA viruses that integrate • their code into the genome of host. • Adenoviruses = DNA viruses that remain • in the nucleus of the host as a separate • entity (transcription of their genes occurs).